21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, H1983, M1983, 38555 or 593 molecules and uses therefor

The present application is a continuation of U.S. patent application Ser. No. 10/423,543, filed Apr. 25, 2003 (abandoned), which is a continuation-in-part of U.S. patent application Ser. No. 10/278,036, filed Oct. 22, 2002 (abandoned), which is a continuation of U.S. patent application Ser. No. 09/711,216, filed Nov. 9, 2000 (abandoned), which claims the benefit of U.S. Provisional Application Ser. No. 60/205,447, filed May 19, 2000 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 10/012,055, filed Nov. 13, 2001 (abandoned), which claims the benefit of U.S. Provisional Application Ser. No. 60/248,325, filed Nov. 14, 2000 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 10/003,690, filed Nov. 15, 2001, now U.S. Pat. No. 6,787,345, which claims the benefit of U.S. Provisional Application Ser. No. 60/248,893, filed Nov. 15, 2000 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 09/797,039, filed Feb. 28, 2001, now U.S. Pat. No. 6,730,491, which claims the benefit of U.S. Provisional Application Ser. No. 60/186,061, filed Feb. 29, 2000 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 10/217,168, filed Aug. 12, 2002, now U.S. Pat. No. 6,929,938, which claims the benefit of U.S. Provisional Application Ser. No. 60/312,539, filed Aug. 15, 2001 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 09/929,218, filed Aug. 14, 2001 (abandoned), which claims the benefit of U.S. Provisional Application Ser. No. 60/257,511, filed Dec. 22, 2000 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 09/963,159, filed Sep. 25, 2001 (abandoned), which claims the benefit of U.S. Provisional Application Ser. No. 60/234,922, filed Sep. 25, 2000 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 10/121,911, filed Apr. 12, 2002, now U.S. Pat. No. 6,607,892, which is a divisional of U.S. patent application Ser. No. 09/412,210, filed Oct. 5, 1999, now U.S. Pat. No. 6,403,358. U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 10/105,989, filed Mar. 25, 2002 (abandoned), which is a continuation of U.S. patent application Ser. No. 09/392,189, filed Sep. 9, 1999 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 10/336,153, filed Jan. 3, 2003 (abandoned), which is a continuation of U.S. patent application Ser. No. 09/845,044, filed Apr. 27, 2001 (abandoned), which claims the benefit of U.S. Provisional Application Ser. No. 60/200,688, filed Apr. 28, 2000 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 09/928,531, filed Aug. 13, 2001 (abandoned), which claims the benefit of U.S. Provisional Application Ser. No. 60/235,035, filed Sep. 25, 2000 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 09/920,346, filed Jul. 31, 2001 (abandoned), which claims the benefit of U.S. Provisional Application Ser. No. 60/221,925, filed Jul. 31, 2000 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 10/008,016, filed Nov. 8, 2001 (abandoned), which claims the benefit of U.S. Provisional Application Ser. No. 60/260,166, filed Jan. 5, 2001 (abandoned) and of U.S. Provisional Application Ser. No. 60/246,669, filed Nov. 8, 2000 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 09/909,743, filed Jul. 20, 2001 (abandoned), which is a divisional of U.S. patent application Ser. No. 09/448,076, filed Nov. 23, 1999, now U.S. Pat. No. 6,300,092, which is a continuation-in-part of U.S. patent application Ser. No. 09/276,400, filed Mar. 25, 1999, now U.S. Pat. No. 6,140,056, which claims the benefit of U.S. Provisional Application Ser. No. 60/117,580, filed Jan. 27, 1999 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 10/336,489, filed Jan. 2, 2003 (abandoned), which is a continuation of U.S. patent application Ser. No. 09/608,921, filed Jun. 30, 2000 (abandoned), which is a continuation-in-part of U.S. patent application Ser. No. 09/163,821, filed Sep. 30, 1998 (abandoned). U.S. patent application Ser. No. 10/423,543 is also a continuation-in-part of U.S. patent application Ser. No. 10/060,763, filed Jan. 30, 2002 (abandoned), which is a continuation of U.S. patent application Ser. No. 09/365,162, filed Jul. 30, 1999 (abandoned). The entire contents of each of the above-referenced patent applications are incorporated herein by this reference.

The enormous variety of biochemical reactions that comprise life are nearly all mediated by a series of biological catalysts known as enzymes. Enzymes are proteins which possess specific catalytic activities that enable them to catalyze a series of reactions, hence enabling metabolic pathways to degrade and to reconstruct products needed to maintain organisms. By the binding of substrates through geometrically and physically complementary reactions, enzymes are stereospecific in binding substrates as well as in catalyzing reactions. The stringency for this stereospecificity varies as some enzymes are more specific to the identity of their substrates, while others are capable of binding multiple substrates and can catalyze numerous types of reactions.

Examples of enzymes include, for example, guanylate kinases, phophatidylinositol 4-phosphate 5-kinases, kinases, transferases, aminopeptidases, adenylate cyclases, calpain proteases, oxidoreductases, neprilysin proteases, AMP binding enzymes and lysyl oxidases. Such enzymes have the ability to, for example: (1) modulate ATP-dependent phosphorylation of GMP, dGMP, or cGMP; (2) catalyze the formation of phosphoinositol-4,5-bisphosphate via the phosphorylation of phosphatidylinositol-4-phosphate; (3) mediate the phosphoinositide signaling cascade; (4) convert a substrate or target molecule to a product (e.g., transfer of a phosphate group to a substrate or target molecule, or conversion of ATP to ADP); (5) interact with and/or phosphate transfer to a second protein; (6) modulate intra- or intercellular signaling and/or gene transcription (e.g., either directly or indirectly); (7) modulate the phosphorylation state of target molecules (e.g., a kinase or a phosphatase molecule) or the phosphorylation state of one or more proteins involved in cellular growth, metabolism, or differentiation, e.g., cardiac, epithelial, or neuronal cell growth or differentiation; (8) convert a substrate or target molecule to a product (e.g., transfer of a methyl group to or from the substrate or target molecule); (9) interact with and/or methyl transfer to a second target molecule e.g., a nucleic acid molecule (e.g., DNA or RNA), a small organic molecule (e.g., a hormone, neurotransmitter or a coenzyme) or a protein; (10) cleave a protein precursor to maturation; (11) catalyze protein degradation; (12) catalyze the formation of a covalent bond within or between an amino acid residue (e.g., a serine or threonine residue) and a phosphate moiety; (13) modulate the cAMP signal transduction pathway; (14) modulate a target cell's cAMP concentration; (15) modulate cAMP-dependent protein kinase activity, such as protein kinase A; (16) modulate a calpain protease response; (17) modulate metabolism and catabolism of biochemical molecules, e.g., molecules necessary for energy production or storage; (18) modulate betaine synthesis from choline; (19) modulate methionine synthesis from homocysteine; (20) modulate the activity of a bioactive peptide, (21) cleave a neprilysin substrate, e.g., enkephalin; (22) modulate membrane excitability, (23) influence the resting potential of membranes; (24) modulate acetyl-CoA ligase activity; (25) promote activation of acetate; (26) promote acetate utilization; (27) enhance uptake of acetate into fatty acids and biochemical products made from fatty acids (e.g., lipids and hormones such as sterol hormones); (28) crosslink an extracellular matrix component; (29) regulate bone resorption and/or metabolism; and (30) regulate copper metabolism. Accordingly, there exists a need to identify additional human enzymes, for example, for use as disease markers and as targets for identifying various therapeutic modulators.

The present invention is based, at least in part, on the discovery of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules, referred to herein as “21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593”. The 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 nucleic acid and protein molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes, e.g., including cell proliferation, differentiation, growth and division. In particular, these nucleic acid molecules will be advantageous in the regulation of any cellular function, uncontrolled proliferation and differentiation, such as in cases of cancer. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593-encoding nucleic acids.

The nucleotide sequence of the cDNA encoding 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 and the amino acid sequence of 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides are depicted in Table 1.

Accordingly, in one aspect, the invention features a nucleic acid molecule which encodes a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein or polypeptide, e.g., a biologically active portion of the 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein. In a preferred embodiment, the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, 6, 11, 19, 22, 25, 32, 40, 44, 47, 50, 55, 58, 64, 67, 72, 89, 105, 108 or 112. In other embodiments, the invention provides isolated 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:1, 3, 5, 7, 10, 12, 18, 20, 21, 23, 24, 26, 31, 33, 39, 41, 43, 45, 46, 48, 49, 51, 54, 56, 57, 59, 63, 65, 66, 68, 71, 73, 88, 90, 104, 106, 107, 109, 111 or 113. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:1, 3, 5, 7, 10, 12, 18, 20, 21, 23, 24, 26, 31, 33, 39, 41, 43, 45, 46, 48, 49, 51, 54, 56, 57, 59, 63, 65, 66, 68, 71, 73, 88, 90, 104, 106, 107, 109, 111 or 113. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringent hybridization condition as described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 10, 12, 18, 20, 21, 23, 24, 26, 31, 33, 39, 41, 43, 45, 46, 48, 49, 51, 54, 56, 57, 59, 63, 65, 66, 68, 71, 73, 88, 90, 104, 106, 107, 109, 111 or 113, wherein the nucleic acid encodes a full length 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acid constructs which include a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included are vectors and host cells containing the 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing polypeptides.

In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593-encoding nucleic acids.

In still another related aspect, isolated nucleic acid molecules that are antisense to a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 encoding nucleic acid molecule are provided.

In another aspect, the invention features 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593-associated disorders. In another embodiment, the invention provides 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides having a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 activity.

In other embodiments, the invention provides 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides, e.g., a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptide having the amino acid sequence shown in SEQ ID NO:2, 6, 11, 19, 22, 25, 32, 40, 44, 47, 50, 55, 58, 64, 67, 72, 89, 105, 108 or 112; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2, 6, 11, 19, 22, 25, 32, 40, 44, 47, 50, 55, 58, 64, 67, 72, 89, 105, 108 or 112; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringent hybridization condition as described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 10, 12, 18, 20, 21, 23, 24, 26, 31, 33, 39, 41, 43, 45, 46, 48, 49, 51, 54, 56, 57, 59, 63, 65, 66, 68, 71, 73, 88, 90, 104, 106, 107, 109, 111 or 113, wherein the nucleic acid encodes a full length 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acid constructs which include a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 nucleic acid molecule described herein.

In a related aspect, the invention provides 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides or fragments operatively linked to non-21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides to form fusion proteins.

In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically or selectively bind 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides.

In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides or nucleic acids.

In still another aspect, the invention provides a process for modulating 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptide or nucleic acid expression or activity, e.g., using the compounds identified in the screens described herein. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides or nucleic acids, such as conditions or disorders involving aberrant or deficient 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 expression. Examples of such disorders include, but are not limited to cellular proliferative and/or differentiative disorders, brain disorders, platelet disorders, breast disorders, colon disorders, kidney (renal) disorders, lung disorders, ovarian disorders, prostate disorders, cervical disorders, spleen disorders, thymus disorders, thyroid disorders, testis disorders, hematopoietic disorders, pancreatic disorders, skeletal muscle disorders, skin (dermal) disorders, disorders associated with bone metabolism, immune, e.g., inflammatory, disorders, cardiovascular disorders, endothelial cell disorders, liver disorders, viral diseases, pain disorders, metabolic disorders, neurological or CNS disorders, erythroid disorders, blood vessel disorders or angiogenic disorders.

The invention also provides assays for determining the activity of or the presence or absence of 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.

In a further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptide or nucleic acid molecule, including for disease diagnosis.

In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Human 21910

The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as “membrane-associated guanylate kinase”, “MAGK” or “21910” nucleic acid and protein molecules. Guanylate kinase molecules are novel members of a family of enzymes possessing kinase activity. Guanylate kinases are essential enzymes in nucleotide metabolism pathways catalyzing the ATP-dependent phosphorylation of either GMP to GDP or dGMP to dGDP. Guanyate kinase molecules also function in the recovery of cGMP (cGMP→GMP→GDP→GTP→cGMP) thereby serving to regulate the supply of guanine nucleotides to signal transduction pathway components (Brady et al. (1996) J. Biol. Chem. 271(28):16734-40; Kumar, et al. (2000) Eur. J. Biochem. 267(2):606). Guanylate kinases are essential to a wide range of cellular processes including but not limited to nucleotide metabolic processes (e.g., supplying the building blocks for nucleic acids), phototransduction processes (e.g., regulating the opening and/or closing of cGMP gated-channels), cellular growth and proliferation, and signaling pathways (Fitzgibbon, et al (1996) FEBS Letters 385:185-188).

Membrane-bound forms of guanylate kinase molecules have also been discovered. Members of the membrane-associated guanylate kinase family interact with the cytoskeleton of the cell and regulate cell proliferation, signaling pathways, and intercellular junctions. (Kim, et al. (1996) Genomics 31(2):223). These molecules participate in the assembly of multiprotein complexes on the inner surface of the plasma membrane and cluster ion channels, receptors, adhesion molecules and cytosolic signaling proteins at synapses, cellular junctions, and polarized membrane domains (Fannin and Anderson (1999) Curr. Opin. Cell Biol. 11(4):432; Dobrosotskaya, et al. (1997) J. Biol. Chem. 272(50):31589). In addition, membrane-associated guanylate kinases have recently been found to have a transcriptional regulatory function (Hsueh, et al. (2000) Nature 404(6775):298). Typically, these molecules contain multiple protein-protein interaction motifs including a PDZ domain in the N-terminal portion of the protein, followed by a SH3 domain, followed by a guanylate kinase domain at the C-terminus (Dobrosotskaya, et al., supra). Membrane-associated guanylate kinases have been found to be localized to tight junctions in epithelial cell membranes and more notably in neuronal cells (Wu, et al. (2000) Proc. Natl. Acad. Sci. USA 97(8):4233); Hsuesh, supra).

In humans, guanylate kinases are used as targets for cancer chemotherapy and have been found to be inhibited by the antitumor drug, 6-thioguanine. In addition, guanylate kinase activity is required for the activation of antiviral drugs such as acyclovir and ganciclovir in virus-infected cells (Brady et al., supra).

Members of the guanylate kinase family have been identified in many organisms, including E. coli , yeast, mouse, and human. Greater conservation has been found between mammalian guanylate kinases than between mammalian and yeast or E. coli . However, the overall structure of the molecule is conserved, including conservation of a “giant anion hole” active site which functions to bind nucleoside triphosphates (Brady et al., supra; Stehle and Schulz (1992) J. Mol. Biol. 224(4):1127).

The MAGK molecules of the present invention, through association with cell surface signaling complexes involved in cellular growth and proliferation, may play a role in the modulation of cellular growth signaling mechanisms. As used herein, the terms “cellular growth signaling mechanisms,” “cell signaling,” or “cell growth signaling” includes signal transmission from a cell surface signaling complex which regulates, for example, 1) cell transversal through the cell cycle, 2) cell differentiation, 3) cell survival, and/or 4) cell migration.

In a preferred embodiment, the MAGK molecules of the present invention are involved in metabolic processes of the cell and in the modulation of cellular growth signaling mechanisms. Thus, the MAGK molecules may modulate cellular growth, differentiation, or migration, and may play a role in disorders characterized by aberrantly regulated growth, proliferation, differentiation, or migration. Accordingly, in one aspect, the present invention provides methods and compositions for the diagnosis and treatment of a cellular growth or proliferation disease or disorder, e.g., cancer, including, but not limited to, lung cancer and colon cancer.

The term “treatment” as used herein, is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

A “cellular growth or proliferation disease or disorder” includes those diseases or disorders that affect cell growth or proliferation processes. As used herein, a “cellular growth or proliferation process” is a process by which a cell increases in number, size or content, by which a cell develops a specialized set of characteristics which differ from that of other cells, or by which a cell moves closer to or further from a particular location or stimulus. Such disorders include, but are not limited to, cancer, e.g., carcinoma, sarcoma, or leukemia, examples of which include, but are not limited to, colon, lung, liver, ovary, and breast; tumorigenesis and metastasis; skeletal dysplasia; hepatic disorders; and hematopoietic and/or myeloproliferative disorders.

The novel MAGK molecules of the present invention have increased expression in tumor cells, e.g., lung tumor cells and colon tumor cells, as compared to normal lung and colon cells. Increased expression of MAGK in tumor cells results in an increase in cell growth signaling, thereby increasing the cellular growth and proliferation of tumor cells. Accordingly, the MAGK molecules of the present invention provide novel diagnostic targets and therapeutic agents to control MAGK-related disorders, e.g., cellular growth or proliferation diseases or disorders, e.g., cancer, including, but not limited to colon cancer or lung cancer. Accordingly, the present invention further provides methods for identifying the presence of a MAGK nucleic acid or polypeptide molecule associated with a cellular growth or proliferation disease or disorder. In addition, the invention provides methods for identifying a subject at risk for a cellular growth or proliferation disease or disorder, by detecting the presence of a MAGK nucleic acid or polypeptide molecule, or by detecting aberrant or abnormal MAGK expression or activity.

The invention also provides a method for identifying a compound capable of treating a cellular growth or proliferation disease or disorder, characterized by aberrant MAGK nucleic acid expression or MAGK protein activity by assaying the ability of the compound to modulate the expression of a MAGK nucleic acid or the activity of a MAGK protein. Furthermore, the invention provides a method for treating a subject having a cellular growth or proliferation disease or disorder characterized by aberrant MAGK protein activity or aberrant MAGK nucleic acid expression by administering to the subject a MAGK modulator which is capable of modulating MAGK protein activity or MAGK nucleic acid expression.

Moreover, the invention provides a method for identifying a compound capable of modulating cellular growth and/or proliferation and cellular signaling by modulating the expression of a MAGK nucleic acid or the activity of a MAGK protein. The invention provides a method for modulating cellular growth and/or proliferation and cellular signaling comprising contacting an endothelial cell with a MAGK modulator.

The present invention is directed to novel members of the guanylate kinase family of enzymes, e.g. the MAGK proteins, biologically active fragments thereof, homologues thereof, and/or nucleic acid molecules encoding such proteins, homologues and/or biologically active fragments, and the use thereof for treating and/or diagnosing a cellular growth or proliferation disease or disorder. The term “family” when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin, e.g., mouse or monkey proteins. Members of a family may also have common functional characteristics.

Accordingly, in one embodiment, a MAGK molecule of the present invention is identified based on the presence of a “ATP/GTP-binding site motif A (P-loop)” in the protein or corresponding nucleic acid molecule. As used herein, the term “ATP/GTP-binding site motif A (P-loop)” includes a protein motif having an amino acid sequence of about 8 amino acid residues. Preferably, a P-loop has about 5-8 residues and the following consensus sequence: [AG]—X(4)-G-K—[ST] (SEQ ID NO:4) (Saraste M., Sibbald P. R., Wittinghofer A. (1990) Trends Biochem. Sci. 15:430-434). To identify the presence of a ATP/GTP-binding site motif A (P-loop) in a MAGK protein, and make the determination that a protein of interest has a particular motif, the amino acid sequence of the protein may be searched against a database of known protein motifs (e.g., the ProSite database). The ATP/GTP-binding site motif A (P-loop) has been assigned ProSite accession number PS00017. A search was performed against the ProSite database resulting in the identification of a ATP/GTP-binding site motif A (P-loop) in the amino acid sequence of human MAGK (SEQ ID NO:2) at about residues 404-411 of SEQ ID NO:2.

In another embodiment, a MAGK molecule of the present invention is identified based on the presence of a “guanylate kinase domain” in the protein or corresponding nucleic acid molecule. As used herein, the term “guanylate kinase domain” includes a protein domain having an amino acid sequence of about 50-200 amino acid residues and a bit score of about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220 or more. Preferably, a guanylate kinase domain includes at least about 100-200, or more preferably about 109 amino acid residues, and a bit score of at least 139.4. To identify the presence of a guanylate kinase domain in a MAGK protein, and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein may be searched against a database of known protein domains (e.g., the HMM database). A search was performed against the HMM database resulting in the identification of a guanylate kinase domain in the amino acid sequence of human MAGK (SEQ ID NO:2) at about residues 515-624 of SEQ ID NO:2.

A guanylate kinase domain can further be characterized based on the presence of a guanylate kinase consensus sequence in the protein or corresponding nucleic acid molecule. As used herein, the term “guanylate kinase domain” includes a protein motif having an amino acid sequence of about 18 amino acid residues. Preferably, a guanylate kinase domain has about 15-20 residues. To identify the presence of a guanylate kinase domain in a MAGK protein, and make the determination that a protein of interest has a particular motif, the amino acid sequence of the protein may be searched against a database of known protein motifs (e.g., the ProSite database). The guanylate kinase domain has been assigned ProSite accession number PS00856. A search was performed against the ProSite database resulting in the identification of a guanylate kinase domain in the amino acid sequence of human MAGK (SEQ ID NO:2) at about residues 514-531 of SEQ ID NO:2.

In another embodiment, a MAGK molecule of the present invention is identified based on the presence of a “PDZ domain” in the protein or corresponding nucleic acid molecule. As used herein, the term “PDZ domain” includes a protein domain having an amino acid sequence of about 50-200 amino acid residues and a bit score of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 or more. Preferably, a PDZ domain includes at least about 50-150, or more preferably about 79 amino acid residues, and a bit score of at least 52.4. To identify the presence of a PDZ domain in a MAGK protein, and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein may be searched against a database of known protein domains (e.g., the HMM database). A search was performed against the HMM database resulting in the identification of a PDZ domain in the amino acid sequence of human MAGK (SEQ ID NO:2) at about residues 256-335 of SEQ ID NO:2.

In another embodiment, a MAGK molecule of the present invention is identified based on the presence of a “SH3 domain” in the protein or corresponding nucleic acid molecule. As used herein, the term “SH3 domain” includes a protein domain having an amino acid sequence of about 50-150 amino acid residues and a bit score of about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more. Preferably, a SH3 domain includes at least about 50-100, or more preferably about 67 amino acid residues, and a bit score of at least 5.2. To identify the presence of a SH3 domain in a MAGK protein, and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein may be searched against a database of known protein domains (e.g., the HMM database). A search was performed against the HMM database resulting in the identification of a SH3 domain in the amino acid sequence of human MAGK (SEQ ID NO:2) at about residues 348-415 of SEQ ID NO:2.

In a preferred embodiment, the MAGK molecules of the invention include at least one, preferably two, more preferably three or more or more of the following domains: an ATP/GTP-binding site motif A (P-loop), a guanylate kinase domain, a PDZ domain, and a SH3 domain.

In yet another embodiment, isolated proteins of the present invention, preferably MAGK proteins, have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2, or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:1 or 3. As used herein, the term “sufficiently identical” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains have at least 30%, 40%, or 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical. Furthermore, amino acid or nucleotide sequences which share at least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95% homology and share a common functional activity are defined herein as sufficiently identical.

As used interchangeably herein, an “MAGK activity”, “biological activity of MAGK,” or “functional activity of MAGK,” refers to an activity exerted by a MAGK protein, polypeptide or nucleic acid molecule on a MAGK responsive cell or tissue, or on a MAGK protein substrate, as determined in vivo, or in vitro, according to standard techniques. As used herein, a “membrane-associated guanylate kinase activity” includes ATP-dependent phosphorylation of GMP (or dGMP) into GDP (or dGDP) involved, for example, in the production of molecules necessary for signal transduction, cell signaling, cellular growth, cellular proliferation, and the like. In one embodiment, a MAGK activity is a direct activity, such as an association with a MAGK-target molecule. As used herein, a “target molecule” or “binding partner” is a molecule with which a MAGK protein binds or interacts in nature, such that MAGK-mediated function is achieved, e.g., modulation of cellular signaling, growth, and/or proliferation. A MAGK target molecule can be a non-MAGK molecule or a MAGK protein or polypeptide of the present invention (e.g., ATP). In an exemplary embodiment, a MAGK target molecule is a MAGK ligand (e.g., GMP, dGMP). Alternatively, a MAGK activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the MAGK protein with a MAGK ligand. The biological activities of MAGK are described herein. For example, the MAGK proteins of the present invention can have one or more of the following activities: i) interaction of a MAGK protein molecule with a non-MAGK protein molecule (e.g. GMP, ATP), ii) modification of a MAGK substrate (e.g. GMP or dGMP), iii) assembly of protein complexes at cell-junctions, iv) interaction with the cellular cytoskeleton, and v) interaction between a membrane-bound MAGK protein and a non-MAGK protein. In yet another preferred embodiment, a MAGK activity is at least one or more of the following activities: 1) modulation of ATP-dependent phosphorylation of GMP, dGMP, or cGMP 2) modulation of cellular signal transduction, 3) modulation of metabolism or catabolism of metabolically important biomolecules (e.g., nucleotides), 4) modulation of cellular growth and differentiation, 5) modulation of cellular proliferation, a 6) modulation of cell signaling mechanisms, e.g., cellular growth signaling mechanisms, 7) modulation of intercellular junctions, 8) modulation of transcription, and 9) modulation of paracellular pathways.

Accordingly, another embodiment of the invention features isolated MAGK proteins and polypeptides having a MAGK activity. Other preferred proteins are MAGK proteins having one or more of the following domains: an ATP/GTP-binding site motif A (P-loop), a guanylate kinase domain, a PDZ domain, a SH3 domain, and, preferably, a MAGK activity.

Additional preferred proteins have one or more of the following domains: an ATP/GTP-binding site motif A (P-loop), a guanylate kinase domain, a PDZ domain, a SH3 domain, and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3.

The nucleotide sequence of the isolated human MAGK cDNA and the predicted amino acid sequence of the human MAGK polypeptide are shown in SEQ ID NOs:1 and 2, respectively.

Isolation of the 21910 or “MAGK” cDNA

The invention is based, at least in part, on the discovery of a human gene encoding a novel protein, referred to herein as 21910 or MAGK. The entire sequence of human clone Fbh21910 was determined and found to contain an open reading frame termed human “21910” or “MAGK”, set forth in SEQ ID NO:1 and 3. The 74.36 kD MAGK protein comprises about 675 amino acids and is shown in SEQ ID NO:2. The coding region (open reading frame) of SEQ ID NO:1, is set forth as SEQ ID NO:3.

Analysis of the Human 21910 or MAGK Molecule

The amino acid sequence of human MAGK was analyzed using the program PSORT to predict the localization of the protein within the cell. This program assesses the presence of different targeting and localization amino acid sequences within the query sequence. The results of the analysis predict that human MAGK (SEQ ID NO:2) is intracellular (e.g. nuclear, cytoplasmic, cytoskeletal).

A search of the amino acid sequence of MAGK was also performed against the ProSite database. This search resulted in the identification of a “ATP/GTP-binding site motif A (P-loop)” in the amino acid sequence of MAGK (SEQ ID NO:2) at about residues 404-411 and a “guanylate kinase signature” in the amino acid sequence of MAGK (SEQ ID NO:2) at about residues 514-531. This search also resulted in the identification of a potential N-glycosylation site at about residues 82-85 of SEQ ID NO:2, a number of potential protein kinase C phosphorylation sites at about residues 84-86, 130-132, 253-255, 270-272, 432-434, 514-516, 517-519, 562-564, 569-571, 576-578, 581-583, and 584-586 of SEQ ID NO:2, a number of potential casein kinase II phosphorylation sites at about residues 14-17, 25-28, 97-100, 137-140, 143-146, 383-386, 422-425, 465-468, 517-520, 558-561, and 646-649 of SEQ ID NO:2, a tyrosine kinase phosphorylation site at about residues 586-593 of SEQ ID NO:2, a number of potential N-myristoylation sites at about residues 205-210, 247-2525, and 405-410 of SEQ ID NO:2, and a potential amidation site at about residues 72-76 of SEQ ID NO:2.

A search of the amino acid sequence of MAGK was also performed against the HMM database. This search resulted in the identification of a “guanylate kinase domain” in the amino acid sequence of MAGK (SEQ ID NO:2) at about residues 515-624 (score=139.4), a “PDZ domain” in the amino acid sequence of MAGK (SEQ ID NO:2) at about residues 256-335 (score=52.4), and a “SH3 domain” in the amino acid sequence of MAGK (SEQ ID NO:2) at about residues 348-415 (score=5.2).

Other HMM hits of interest that were identified in the HMM database include, for example, a “NAD-dependent DNA ligase domain” at about residues 529-535 of SEQ ID NO:2 (score=2.3), an “X-Pro dipeptidyl-peptidase domain” at about residues 642-658 of SEQ ID NO:2 (score=−0.0), and a “caulimovirus movement protein domain” at about residues 420-673 of SEQ ID NO:2 (score=−184.0).

Tissue Distribution of 21910 or MAGK by In situ Analysis

For in situ analysis, various tissues, e.g. tissues obtained from normal lung and colon and lung and colon tumors, were first frozen on dry ice.

In situ hybridization results indicated no expression in 2 normal lung samples. By contrast, expression was detected in 2 of 4 lung tumor samples. Results further indicated no expression in 3 normal tumor samples and strong expression in 4 of 4 primary colon tumors tested and 3 of 3 colon metastases tested. Breast and ovary tissue also showed tumor specific expression.

Tissue Expression Analysis of 21910 or MAGK mRNA Using TaqMan™ Analysis

This example describes the tissue distribution of human MAGK mRNA (huMAGK) in a variety of cells and tissues, as determined using the TaqMan™ procedure.

The expression levels of human 21910 or MAGK mRNA in various human cell types and tissues was first determined in an array profiling experiment comparing the expression of genes in lung tumor cell lines versus normal bronchial epithelium. These experiments demonstrated that MAGK expression is increased 2-fold in a small cell lung tumor line as compared to normal epithelium.

The RNA used in the array profiling experiment was isolated from the following cell lines: NHBE (available from Clonetics®) and NCI-H69 (available from ATCC®). NHBE cells were grown in BEGM (bronchial epithelium growth) Bulletkit® medium. The cells were grown to 80% confluency in a T175 flask and harvested for RNA by the Qiagen® Midi RNA preparation method. NCI-H69 cells were grown in suspension in T175 flasks in RPMI+2% Hyclone FBS, 2 mM L-Glutamine, 10 mM HEPES, and 1/100 Gibco® Selenium/Insulin/Transferrin supplement medium. RNA was prepared with the Qiagen® RNA Midi Kit, as directed by the manufacturer.

The expression levels of human 21910 or MAGK mRNA in various human cell types and tissues were analyzed in detail in a second experiment using the TaqMan™ procedure. As shown in Table 2, the highest 21910 or MAGK expression was detected in brain, epithelial cells, and fetal heart.

As shown in Table 3, increased expression of human 21910 or MAGK was detected in 6 of 8 lung tumor samples (T) versus normal lung tissue samples (N). As shown in Table 4, increased expression of huMAGK was detected in 4 of 7 colon tumor samples (T) versus normal colon tissue samples (N).

These data reveal a significant up-regulation of MAGK mRNA in colon and lung carcinomas. Given that the mRNA for MAGK is expressed in a variety of tumors, with significant up-regulation in carcinoma samples in comparison to normal samples, it is believed that inhibition of MAGK activity may inhibit tumor progression by inhibiting cell growth signaling and cellular growth and proliferation.

Human 56634

The present invention is based, at least in part, on the discovery of a novel phosphatidylinositol 4-phosphate 5-kinase termed 56634. The human 56634 sequence (SEQ ID NO:5), which is approximately 3224 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1266 nucleotides, including the termination codon. The coding sequence encodes a 421 amino acid protein (SEQ ID NO:6).

Human 56634 contains the following regions or other structural features: a phosphatidylinositol 4-phosphate 5-kinase domain (PFAM Accession Number PF01504) located at about amino acid residues 72 to 421 of SEQ ID NO:6; one predicted N-glycosylation site (PS00001) at about amino acids 165 to 168 of SEQ ID NO:6; seven predicted Protein Kinase C phosphorylation sites (PS00005) at about amino acids 28 to 30, 79 to 81, 208 to 210, 229 to 231, 239 to 241, 338 to 340, and 391 to 393 of SEQ ID NO:6; ten predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino 58 to 61, 132 to 135, 155 to 158, 229 to 232, 239 to 242, 294 to 297, 307 to 310, 327 to 330, 349 to 352, and 377 to 380 of SEQ ID NO:6; one predicted tyrosine kinase phosphorylation sites (PS00007) from about amino acid 114 to 122 of SEQ ID NO:6; and four predicted N-myristoylation sites (PS00008) from about amino acid 54 to 59, 221 to 226, 323 to 328, and 397 to 402 of SEQ ID NO:6.

For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420.

Signal transduction through phosphoinositol lipids plays an important role in various cellular processes, including vesicular secretion, cytoskeletal organization, and cell growth and differentiation. The phosphatidylinositol (PI) signal transduction pathway is regulated, in part, by the conversion of PI, a membrane lipid bearing a sugar moiety attached via an intermediate phosphate residue, into singly, doubly, and triply phosphorylated products (Carpenter and Cantley (1996) Curr Opin Cell Biol 8:153-158). A crucial step in the pathway occurs when phosphatidylinositol 4-phosphate (PIP) is phosphorylated to become phosphatidylinositol 4,5-bisphosphate (PIP2), a step catalyzed by phosphatidylinositol 4-phosphate 5-kinase (Boronenkov and Anderson (1995) J Biol Chem 270:2881-2884.). The hydrolysis of PIP2 by phospholipase C (PLC) produces the second messengers diacylglycerol (DAG) and inositol tris-phosphate (IP3). DAG is an activator of protein kinase C (PKC) and IP3 plays an important role in the release of intracellular calcium. In addition, PIP2 is converted into phosphatidylinositol 3,4,5-tris-phosphate, which activates some PKC isoforms. Thus, the phosphatidylinositol 4-phosphate 5-kinase family of proteins plays an important role in the regulation of the phosphoinositide signaling cascade by catalyzing key phosphorylation events.

The 56634 protein contains a significant number of structural characteristics in common with members of the phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family. The phosphatidylinositol 4-phosphate 5-kinase family comprises a number of related enzymes that share a common catalytic mechanism. PIP5K catalyses the formation of phosphoinositol-4,5-bisphosphate via the phosphorylation of phosphatidylinositol-4-phosphate, a precursor in the phosphinositide signaling pathway. Phosphatidylinositol 4-phosphate 5-kinase has been shown to be required for vesicular secretion and trafficking of a wide variety of cells (Hay et al. (1995) Nature 374:173-7; Yamamoto et al. (1995) Mol Biol Cell 6:525-39). In addition, there is evidence that phosphatidylinositol 4-phosphate 5-kinase is involved in signal transduction and regulation of the actin cytoskeleton via the interaction with the Rho family of small G proteins (Chong et al. (1994) Cell 79:507-13; Ren et al. (1996) Mol Biol Cell 7:435-442), suggesting a role in cell movement and metastasis.

Thus, this (PIP5K) family includes enzymes critical for the proper function of many physiological systems, including vesicle secretion and trafficking, cell signaling, and cellular proliferation and differentiation.

A 56634 polypeptide can include a “phosphatidylinositol 4-phosphate 5-kinase domain” or regions homologous with a “phosphatidylinositol 4-phosphate 5-kinase domain”.

As used herein, the term “phosphatidylinositol 4-phosphate 5-kinase domain” includes an amino acid sequence of about 200-500 amino acid residues in length and having a bit score for the alignment of the sequence to the phosphatidylinositol 4-phosphate 5-kinase domain profile (Pfam HMM) of at least 100. Preferably, a phosphatidylinositol 4-phosphate 5-kinase domain includes at least about 200 to 500 amino acids, more preferably about 250 to 450 amino acid residues, or about 300 to 400 amino acids and has a bit score for the alignment of the sequence to the phosphatidylinositol 4-phosphate 5-kinase domain (HMM) of at least 100, preferably at least 200, 300, 400 or greater. The phosphatidylinositol 4-phosphate 5-kinase domain (HMM) has been assigned the PFAM Accession Number PF01504. The phosphatidylinositol 4-phosphate 5-kinase domain (HMM) has been assigned the SMART identifier PIPK — 2. An alignment of the phosphatidylinositol 4-phosphate 5-kinase domain (amino acids 72 to 421 of SEQ ID NO:6) of human 56634 with the PIPK — 2 consensus amino acid sequences derived from a hidden Markov model derived from SMART yielded a score of 586.8 (E=1.4e-172). The PIPK — 2 sequence is depicted as SEQ ID NO:9. An alignment of the phosphatidylinositol 4-phosphate 5-kinase domain (amino acids 124 to 420 of SEQ ID NO:6) of human 56634 with the PIP5K consensus amino acid sequences derived from a hidden Markov model derived from PFAM yielded a score of 530.2 (E=1.5e-155). The PIP5K sequence is depicted as SEQ ID NO:8.

In a preferred embodiment 56634 polypeptide or protein has a “phosphatidylinositol 4-phosphate 5-kinase domain” or a region which includes at least about 200 to 500, more preferably about 250 to 450, or 300 to 400 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “phosphatidylinositol 4-phosphate 5-kinase,” e.g., the phosphatidylinositol 4-phosphate 5-kinase domain of human 56634 (e.g., residues 72 to 421 of SEQ ID NO:6).

To identify the presence of a “phosphatidylinositol 4-phosphate 5-kinase” domain in a 56634 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against the Pfam database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a “phosphatidylinositol 4-phosphate 5-kinase” domain in the amino acid sequence of human 56634 at about residues 124-420 of SEQ ID NO:6. The sequence of the identified Pfam “phosphatidylinositol 4-phosphate 5-kinase” domain is depicted in SEQ ID NO:8.

To identify the presence of a “phosphatidylinositol 4-phosphate 5-kinase” domain in a 56634 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against a SMART database (Simple Modular Architecture Research Tool) of HMMs as described in Schultz et al. (1998), Proc. Natl. Acad. Sci. USA 95:5857 and Schultz et al. (200) Nucl. Acids Res 28:231. The database contains domains identified by profiling with the hidden Markov models of the HMMer2 search program (R. Durbin et al. (1998) Biological sequence analysis: probabilistic models of proteins and nucleic acids. Cambridge University Press). The database also is extensively annotated and monitored by experts to enhance accuracy. A search was performed against the HMM database resulting in the identification of a “phosphatidylinositol 4-phosphate 5-kinase” domain in the amino acid sequence of human 56634 at about residues 72 to 421 of SEQ ID NO:6. The sequence of the identified SMART “phosphatidylinositol 4-phosphate 5-kinase” domain is depicted in SEQ ID NO:9.

A 56634 polypeptide can include a “phosphatidylinositol 4-phosphate 5-kinase domain” or regions homologous with a “phosphatidylinositol 4-phosphate 5-kinase domain.” A 56634 polypeptide can optionally further include at least one N-glycosylation site; at least one, two, three, four, five, six, preferably seven protein kinase C phosphorylation sites; at least one, two, three, four, five, six, seven, eight, nine, preferably ten, casein kinase II phosphorylation sites; at least one tyrosine kinase phosphorylation site; at least one, two, three, preferably four, N-myristylation sites.

As the 56634 polypeptides of the invention may modulate 56634-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 56634-mediated or related disorders, e.g., cancer, as described below.

As used herein, a “56634 activity”, “biological activity of 56634” or “functional activity of 56634”, refers to an activity exerted by a 56634 protein, polypeptide or nucleic acid molecule. For example, a 56634 activity can be an activity exerted by 56634 in a physiological milieu on, e.g., a 56634-responsive cell or on a 56634 substrate, e.g., a protein substrate. A 56634 activity can be determined in vivo or in vitro. In one embodiment, a 56634 activity is a direct activity, such as an association with a 56634 target molecule. A “target molecule” or “binding partner” is a molecule with which a 56634 protein binds or interacts in nature. In an exemplary embodiment, 56634 is an enzyme for converting phosphatidylinositol 4-phosphate (PIP) to phosphatidylinositol 4,5-bis-phosphate (PIP2).

A 56634 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 56634 protein with a 56634 receptor. The features of the 56634 molecules of the present invention can provide similar biological activities as phosphatidylinositol 4-phosphate 5-kinase family members. For example, the 56634 proteins of the present invention can have one or more of the following activities: (1) catalyses the formation of phosphoinositol-4,5-bisphosphate via the phosphorylation of phosphatidylinositol-4-phosphate; (2) mediates the phosphoinositide signaling cascade; (3) mediates vesicular trafficking; or (4) mediates organization of the cytoskeleton. As a result, the 56634 protein may have a critical function in one or more of the following physiological processes: (a) vesicular secretion; (b) phosphoinositide signaling; or (c) cell proliferation and differentiation.

Several lines of evidence have shown coordinate increases in phosphatidylinositol and PIP kinase activities in human cancer cells, suggesting an increased capacity for signal transduction. Among PIPKs, two major subtypes (types I and II), each comprising two isoforms (Ia, Ib, IIa, IIb), have been identified to date. Type II phosphatidylinositol phosphate kinase (PIPKII) is an enzyme responsible for the synthesis of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)) from phosphatidylinositol-5-phosphate (PI-5-P). Mitogenic stimulation, such as by serum, EGF, and PDGF treatment, results in phosphorylation in vivo of rat PIPKIIg (JBC 273:20292, 1998). In addition, PIPKIIb isoform has also been show to interact not only with the EGF receptor, but also selectively with other members of the ErbB tyrosine kinase family (Cell Signal 11:171, 1999).

As described below, expression of 56634 is increased after the treatment of mitogens, including EGF and serum. In addition expression of 56634 is increased in many clinical tumor tissues when compared to normal tissue controls, suggesting an increased capacity for PIP kinase mediated signal transduction. Therefore, inhibition of 56634 may reduce the signaling potential of cancer cells, thereby halting and possibly reducing the growth of tumor cells. Thus, the 56634 molecules can act as novel diagnostic targets and therapeutic agents for controlling proliferation and differentiation related disorders.

Examples of such disorders include cancer, e.g., ovarian, breast, lung or colon cancer. Thus, the 56634 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders.

Identification and Characterization of Human 56634 cDNA

The human 56634 sequence (SEQ ID NO:5) is approximately 3224 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TAA). The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 1266 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO:5; SEQ ID NO:7). The coding sequence encodes a 421 amino acid protein (SEQ ID NO:6).

Tissue Distribution of 56634 mRNA by TaqMan Analysis and In Situ Hybridization

Endogenous human 56634 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology.

To determine the level of 56634 in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues and several cell lines shown in Tables 5-12, below.

TaqMan analysis revealed that the expression of 56634 was increased with addition of the growth factor EGF to serum free culture media of the SKOV3 ovarian cancer cell line for 15, 30 or 60 minutes (Table 5). The expression of 56634 was also similarly increased when the breast cancer cell line MCF10A was treated with EGF for comparable time points (Table 6). 56634 was also shown to be induced in the HEY ovarian cell line with the addition of serum following overnight serum starvation (Table 7). When normal human ovarian epithelial cells (NOE) are compared with clinical ascites samples from several patients, 56634 was found to be upregulated in the ascites samples compared to the NOE (Table 8). Clinical data comparing expression of 56634 in solid tumor vs. normal tissue counterpart (Table 9), and expression in Phase I normal and diseased tissues (Table 10), all indicate that this gene is upregulated in tumor tissues compared to normal tissue counterparts. 56634 is also expressed in several xenograft friendly cell lines (Table 11).

In Situ Hybridization (ISH):

56634 was found to be expressed by ISH in ovarian, breast and colon tumor clinical samples. 56634 was localized to 0/3 normal ovary samples, 6/12 ovarian tumors, 2/2 normal breast, 4/4 breast tumors, 0/1 normal colon, 0/3 colon primary tumors, and 0/2 colon to liver metastases. See Table 12.

Human 55053 (EPK-55053)

The present invention is based, at least in part, on the discovery of novel members of a family of molecules, referred to herein as “Eukaryotic Protein Kinase-55053” or “EPK-55053” nucleic acid and polypeptide molecules. Members of this family of molecules are able to participate in the modulation of the phosphorylation state of EPK-55053 substrate molecules. By doing so, these molecules are able to contribute to the regulation and/or modulation of the activity of these substrate molecules, and, hence, the biochemical pathways with which the substrates are associated.

Protein kinases and phosphatases play critical roles in the regulation of biochemical and morphological changes associated with cellular growth and division (D'Urso, G. et al. (1990) Science 250:786-791; Birchmeier, C. et al. (1993) Bioessays 15:185-189). They serve as growth factor receptors and signal transducers and have been implicated in cellular transformation and malignancy (Hunter, T. et al. (1992) Cell 70:375-387; Posada, J. et al. (1992) Mol. Biol. Cell 3:583-592; Hunter, T. et al. (1994) Cell 79:573-582). For example, protein kinases have been shown to participate in the transmission of signals from growth-factor receptors (Sturgill, T. W. et al. (1988) Nature 344:715-718; Gomez, N. et al. (1991) Nature 353:170-173), control of entry of cells into mitosis (Nurse, P. (1990) Nature 344:503-508; Maller, J. L. (1991) Curr. Opin. Cell Biol. 3:269-275) and regulation of actin bundling (Husain-Chishti, A. et al. (1988) Nature 334:718-721).

Protein kinases and phosphatases can be divided into different groups based on either amino acid sequence similarity or specificity for either serine/threonine or tyrosine residues. A small number of dual-specificity kinases and phosphatases have also been described. Within the broad classification, kinases and phosphatases can be further sub-divided into families whose members share a higher degree of catalytic domain amino acid sequence identity and also have similar biochemical properties. Most protein kinase and phosphatase family members also share structural features outside the kinase and phosphatase domain, respectively, that reflect their particular cellular roles. These include regulatory domains that control kinase or phosphatase activity or interaction with other proteins (Hanks, S. K. et al. (1988) Science 241:42-52).

In one embodiment, the EPK-55053 molecules of the present invention include at least one “transmembrane domain.” As used herein, the term “transmembrane domain” includes an amino acid sequence of about 20-45 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, alanines, valines, phenylalanines, prolines or methionines. Transmembrane domains are described in, for example, Zagotta W. N. et al. (1996) Annu. Rev. Neurosci. 19:235-263, the contents of which are incorporated herein by reference. Amino acid residues 214-231 of the human EPK-55053 polypeptide (SEQ ID NO:11) comprise a transmembrane domain.

To identify the presence of a transmembrane domain in an EPK-55053 protein, and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein may be subjected to MEMSAT analysis. A MEMSAT analysis of the EPK-55053 protein set forth as SEQ ID NO:11 results in the identification of a transmembrane domain in the amino acid sequence of human EPK-55053 (SEQ ID NO:11) at about residues 214-231 (having a score of 4.1). Two other potential transmembrane domains were also identified at about amino acids 624-640 and 681-697 or SEQ ID NO:11.

In another embodiment, the EPK-55053 molecules of the present invention include at least one “eukaryotic protein kinase domain”. As used herein, the term “eukaryotic protein kinase domain” includes a protein domain having at least about 150-350 amino acid residues and a bit score of at least 150 when compared against a eukaryotic protein kinase domain Hidden Markov Model (HMM), e.g., PFAM Accession Number PF00069. Preferably, a eukaryotic protein kinase domain includes a protein having an amino acid sequence of about 190-320, 210-300, 250-260 or more preferably about 252 amino acid residues, and a bit score of at least 150, 210, 250, 290, or more preferably, 323.4. To identify the presence of a eukaryotic protein kinase domain in an EPK-55053 protein, and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein may be searched against a database of known protein domains (e.g., the HMM database). The eukaryotic protein kinase domain has been assigned the PFAM Accession No. PF00069 (see the PFAM website, available through the University of Washington at St. Louis) and InterPro Accession No. IPR000719 (see the website for the European Bioinformatics Institute). A search was performed against the HMM database resulting in the identification of a eukaryotic protein kinase domain in the amino acid sequence of human EPK-55053 (SEQ ID NO:11) at about residues 34-285 of SEQ ID NO:11. The identified eukaryotic protein kinase domain is depicted as SEQ ID NO:14.

In another embodiment, the isolated nucleic acid molecules of the present invention encodes at least one “ubiquitin-associated domain” or “UBA domain”. As used interchangeably herein, the terms “ubiquitin-associated domain” and “UBA domain” include a protein domain having at least about 10-70 amino acid residues when compared against a UBA domain Hidden Markov Model (HMM), e.g., PFAM Accession Number PF00627. Preferably, a UBA domain includes a protein having an amino acid sequence of about 10-70, 20-60, 30-50, 35-45 or more preferably about 40 amino acid residues, and a bit score of at least about 7.7. UBA domains (described in, for example, Diekmann et al. (1998) Nat. Struct. Biol. 5:1042-1047) are domains that belong to an extensive family of proteins which share a conserved sequence and which have associations with ubiquitin and the ubiquitination pathway. To identify the presence of a UBA domain in an EPK-55053 protein, and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein may be searched against a database of known protein domains (e.g., the HMM database). The UBA domain has been assigned the PFAM Accession No. PF00627 (see the PFAM website, available through the University of Washington at St. Louis) and InterPro Accession No. IPR000449 (see the website for the European Bioinformatics Institute). A search was performed against the HMM database resulting in the identification of a UBA domain in the amino acid sequence of human EPK-55053 (SEQ ID NO:11) at about residues 315-356 of SEQ ID NO:11. The identified UBA domain is depicted in SEQ ID NO:15.

To elucidate the substrate specificity of the HPK-55053 proteins of the present invention, further HMM analyses were performed using a proprietary database of Markov models, referred to herein as the SMART HMM database. This analysis resulted in the identification of a serine threonine kinase (“serkin — 6”) domain at about amino acids 34-285 of the human EPK-55053 amino acid sequence set forth as SEQ ID NO:11. Notably, this serine/threonine kinase domain overlaps almost exclusively with the protein kinase domain identified by HMM searching of the PFAM database, identifying the instant proteins as serine/threonine kinases as compared to tyrosine kinases. This analysis also resulted in the identification of a tyrosine kinase domain (“tyrkin — 6) at about amino acid residues 34-286 of SEQ ID NO:11. The identified serkin — 6 and tyrkin — 6 domains are depicted in SEQ ID NO:16 and 17, respectively.

Moreover, a signature sequence which is specific for serine/threonine kinases (consensus sequence given as SEQ ID NO:13) was identified at about residues 152-164 of SEQ ID NO:11. This signature sequence occurs in the central part of the kinase catalytic domain of serine/threonine kinases and contains a conserved aspartate residue which is important for the catalytic activity of the enzyme (Knighton D. R. et al. (1991) Science 253:407-414). The consensus signature sequence described under the Prosite accession number PS00108 and is given as:

(SEQ ID NO:13)

(2)-N-[LIVMFYCT](3)

A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28:405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Methods Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference.

In a preferred embodiment, the EPK-55053 molecules of the invention include at least one transmembrane domain and/or at least one eukaryotic protein kinase domain, and/or at least one UBA domain.

Isolated EPK-55053 polypeptides of the present invention, have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:11 or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:10 or 12. As used herein, the term “sufficiently identical” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains having at least 60%, 65%, 70%, 75%, 76%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more homology or identity across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical. Furthermore, amino acid or nucleotide sequences which share at least 60%, 65%, 70%, 75%, 76%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more homology or identity and share a common functional activity are defined herein as sufficiently identical.

In a preferred embodiment, an EPK-55053 polypeptide includes at least one or more of the following domains: a transmembrane domain, a eukaryotic protein kinase domain, a UBA domain, and has an amino acid sequence at least about 60%, 65%, 70%, 75%, 76%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more homologous or identical to the amino acid sequence of SEQ ID NO:11. In yet another preferred embodiment, an EPK-55053 polypeptide includes at least one or more of the following domains: a transmembrane domain, a eukaryotic protein kinase domain, a UBA domain, and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:10 or SEQ ID NO:12. In another preferred embodiment, an EPK-55053 polypeptide includes at least one or more of the following domains: a transmembrane domain, a eukaryotic protein kinase domain, a UBA domain, and has an EPK-55053 activity.

As used interchangeably herein, “EPK-55053 activity”, “biological activity of EPK-55053” or “functional activity of EPK-55053”, includes an activity exerted by an EPK-55053 polypeptide or nucleic acid molecule on an EPK-55053 responsive cell or tissue, or on an EPK-55053 polypeptide substrate, as determined in vivo, or in vitro, according to standard techniques. In one embodiment, an EPK-55053 activity is a direct activity, such as an association with an EPK-55053-target molecule. As used herein, a “target molecule” or “binding partner” is a molecule with which an EPK-55053 polypeptide binds or interacts in nature, such that EPK-55053-mediated function is achieved. An EPK-55053 target molecule can be a non-EPK-55053 molecule, for example, a non-EPK-55053 polypeptide. Additional, exemplary EPK-55053 target molecules can include lipid moieties, a lipid-associated moiety, or a nucleic acid. In another embodiment, an EPK-55053 activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the EPK-55053 polypeptide with an EPK-55053 ligand.

In a preferred embodiment, an EPK-55053 polypeptide has one or more of the following activities: (1) interaction with an EPK-55053 substrate or target molecule (e.g., a non-EPK-55053 protein); (2) conversion of an EPK-55053 substrate or target molecule to a product (e.g., transfer of a phosphate group to a substrate or target molecule, or conversion of ATP to ADP); (3) interaction with and/or phosphate transfer to a second non-EPK-55053 protein; (4) modulation of intra- or intercellular signaling and/or gene transcription (e.g., either directly or indirectly); (5) modulation of the phosphorylation state of EPK-55053 target molecules (e.g., a kinase or a phosphatase molecule) or the phosphorylation state of one or more proteins involved in cellular growth, metabolism, or differentiation, e.g., cardiac, epithelial, or neuronal cell growth or differentiation, as described in, for example, Lodish H. et al., Molecular Cell Biology (Scientific American Books Inc., New York, N.Y., 1995) and Stryer L., Biochemistry (W.H. Freeman, New York), the contents of which are incorporated herein by reference; (6) modulation of the activity of one or more proteins involved in cellular growth or differentiation, e.g., cardiac, epithelial, or neuronal cell growth or differentiation; (7) modulation of expression of one or more genes (e.g., a transcription factor); (8) modulation of signal transduction; and (9) participation in immunoregulation.

In other preferred embodiments, the EPK-55053 polypeptides of the present invention have one or more of the following activities: (1) modulation of cancer or tumor progression; (2) modulation of cellular proliferation; (3) modulation of tissue development (e.g., embryogenesis); (4) modulation of differentiation; (5) modulation of apoptosis; (6) modulation of energy metabolism; and (7) modulation of a ubiquitination pathway. Thus, the EPK-55053 molecules of the present invention can participate in: (a) the regulation of transmission of signals from cellular receptors, e.g., growth factor receptors; (b) the modulation of the entry of cells into mitosis; (c) the modulation of cellular differentiation; (d) the modulation of cell death; (e) the regulation of cytoskeleton function, e.g., actin bundling; and (f) metabolic pathways and the regulation of metabolic pathways.

The EPK-55053 molecules, by participating in the regulation of phosphorylation states, provide novel diagnostic targets and therapeutic agents for controlling or treating a variety of kinase associated disorders. As used herein, the term “kinase associated disorder” include disorders, diseases, or conditions which are characterized by aberrant, e.g., upregulated, downregulated, or misregulated, protein kinase levels. In a preferred embodiment, a kinase associated disorder includes the inhibition or over-stimulation of the activity of kinases involved in signaling pathways associated with cellular growth can lead to perturbed cellular growth, which can in turn lead to cellular growth-related disorders. As used herein, a “cellular growth-related disorder”, includes a disorder, disease, or condition characterized by a deregulation, e.g., an upregulation or a downregulation, of cellular growth. Cellular growth deregulation may be due to a deregulation of cellular proliferation, cell cycle progression, cellular differentiation and/or cellular hypertrophy. Examples of cellular growth related disorders include cardiovascular disorders such as heart failure, hypertension, atrial fibrillation, dilated cardiomyopathy, idiopathic cardiomyopathy, or angina; proliferative disorders or differentiative disorders such as cancer, e.g., melanoma, prostate cancer, cervical cancer, breast cancer, colon cancer, or sarcoma.

Other examples of EPK-55053 associated disorders include CNS disorders, cardiac-related disorders (cardiovascular disorders), disorders of the musculoskeletal system, hormonal disorders, immune disorders, such as autoimmune disorders or immune deficiency disorders, e.g., congenital X-linked infantile hypogammaglobulinemia, transient hypogammaglobulinemia, common variable immunodeficiency, selective IgA deficiency, chronic mucocutaneous candidiasis, or severe combined immunodeficiency.

EPK-55053 associated or related disorders also include disorders affecting tissues in which EPK-55053 protein is expressed.

Isolation of the Human EPK-55053 cDNA

The invention is based, at least in part, on the discovery of a human gene encoding a novel 85.6 kD polypeptide, referred to herein as human EPK-55053. The entire sequence of the human clone 55053 was determined and found to contain an open reading frame termed human “EPK-55053.” The nucleotide sequence of the human EPK-55053 gene contains 2980 nucleic acids and is set forth in the Sequence Listing as SEQ ID NO:10. The amino acid sequence of the human EPK-55053, containing 778 amino acids, is set forth in the Sequence Listing as SEQ ID NO:11. The coding region (open reading frame) of SEQ ID NO:10 is set forth as SEQ ID NO:12.

Analysis of the Human EPK-55053 Molecules

A search using the polypeptide sequence of SEQ ID NO:11 was performed against the HMM database in PFAM resulting in the identification of a eukaryotic protein kinase domain in the amino acid sequence of human EPK-55053 at about residues 34-285 of SEQ ID NO:11 (score=323.4). Searching the SMART HMM database resulted in the further identification of this domain as a serine threonine kinase domain. The identified eukaryotic protein kinase domain and serine threonine kinase domain are depicted as SEQ ID NO:14, 16 and 17.

This search also resulted in the identification of a UBA domain in the amino acid sequence of human EPK-55053 at about residues 315-356 of SEQ ID NO:11 (score=7.7). The identified UAB domain is depicted as SEQ ID NO:15.

A search using the polypeptide sequence of SEQ ID NO:11 was also performed against the MEMSAT database, resulting in the identification of potential transmembrane domains (score=4.1) in the amino acid sequence of human EPK-55053 (SEQ ID NO:11) at about residues 214-231, 624-640, and 681-697.

Searches of the amino acid sequence of human EPK-55053 were further performed against the Prosite database. These searches resulted in the identification in the amino acid sequence of human EPK-55053 of a potential cAMP/cGMP-dependant protein kinase phosphorylation site (ProSite Accession No. PS00004) at about residues 272-275 of SEQ ID NO:11. A glycosaminoglycan attachment site (ProSite Accession No. PS00002) was also identified at about residues 682-685 of SEQ ID NO:11. Fifteen potential protein kinase C phosphorylation sites (ProSite Accession No. PS00005) were identified at about residues 129-131, 417-419, 427-429, 447-449, 472-474, 496-498, 508-510, 523-525, 555-557, 563-565, 619-621, 643-645, 676-678, 699-701, and 758-760 of SEQ ID NO:11. Twelve potential casein kinase II sites (ProSite Accession No. PS00006) were identified at about residues 114-117, 129-132, 142-145, 185-188, 311-314, 341-344, 363-366, 404-407, 575-578, 586-589, 668-671, and 715-718 of SEQ ID NO:11. Eleven potential N-myristoylation sites (ProSite Accession No. PS00008) were identified at about residues 4-9, 10-15, 57-62, 435440, 485490, 507-512, 530-535, 541-546, 597-602, and 681-686 of SEQ ID NO:11. Three amidation sites (ProSite Accession No. PS00009) were identified at about residues 208-211, 300-303, and 390-393 of SEQ ID NO:11. Most notably, a serine/threonine protein kinase active site signature (ProSite Accession No. PS00108) was identified at about residues 152-164 of SEQ ID NO:11.

The amino acid sequence of human EPK-55053 was analyzed using the program PSORT (available online; see Nakai, K. and Kanehisa, M. (1992) Genomics 14:897-911) to predict the localization of the proteins within the cell. This program assesses the presence of different targeting and localization amino acid sequences within the query sequence. The results of the analyses show that human EPK-55053 may be localized to the cytoplasm, nucleus, or mitochondria.

Further homologies of interest were identified by using the amino acid sequence of EPK-55053 (SEQ ID NO:11) to search the ProDom database (available through the Institute National de la Recherche Agronomique, France). This search resulted in the identification of homology in the amino acid sequence of human EPK-55053 to a yeast probable serine/threonine protein kinase, a hypothetical 169.2 kD protein, a transmembrane kinase protein, a putative NPK-1 kinase, a C. elegans serine/threonine protein kinase, and HRPOPK-1 protein.

Human 2504, 15977 and 14760

The present invention is based, in part, on the discovery of novel protein kinase family members, referred to herein as “2504, 15977, and 14760”. The nucleotide sequence of a cDNA encoding 2504 is shown in SEQ ID NO:18, and the amino acid sequence of a 2504 polypeptide is shown in SEQ ID NO:19. In addition, the nucleotide sequence of the 2504 coding region is depicted in SEQ ID NO:20. The nucleotide sequence of a cDNA encoding 15977 is shown in SEQ ID NO:21, and the amino acid sequence of a 15977 polypeptide is shown in SEQ ID NO:22. In addition, the nucleotide sequence of the 15977 coding region is depicted in SEQ ID NO:23. The nucleotide sequence of a cDNA encoding 14760 is shown in SEQ ID NO:24, and the amino acid sequence of a 14760 polypeptide is shown in SEQ ID NO:25. In addition, the nucleotide sequence of the 14760 coding region is depicted in SEQ ID NO:26.

Human 2504

The human 2504 sequence (SEQ ID NO:18), which is approximately 2297 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1503 nucleotides (nucleotides 154-1656 of SEQ ID NO:18; SEQ ID NO:20). The coding sequence encodes a 501 amino acid protein (SEQ ID NO:19).

This mature protein form is approximately 501 amino acid residues in length (from about amino acid 1 to amino acid 501 of SEQ ID NO:19). Human 2504 contains the following regions or other structural features: a eukaryotic protein kinase domain (PFAM Accession PF00069) located at about amino acid residues 37 to 286 of SEQ ID NO:19; and a serine/threonine kinase domain located at about amino acid residues 24 to 286 of SEQ ID NO:19.

The 2504 protein also includes the following domains: twelve predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acids 21 to 23, 46-48, 51-53, 91-93, 103-105, 118-120, 138-140, 292-294, 422-424, 482-484, and 495-497 of SEQ ID NO:19; ten predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino 7-10, 91-94, 103-106, 118-121, 276-279, 341-344, 364-367, 470-473, 483-486, and 495-498 of SEQ ID NO:19; two predicted tyrosine kinase phosphorylation sites (PS00007) located at about amino acids 127-135 and 484-491 of SEQ ID NO:19; two predicted N-myristoylation sites (PS00008) located at about amino acids 288-293 and 349-354 of SEQ ID NO:19; and one predicted amidation site located at about amino acids 59-62 of SEQ ID NO:19.

For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420.

Human 15977

The human 15977 sequence (SEQ ID NO:21), which is approximately 4417 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1377 nucleotides (nucleotides 337-1713 of SEQ ID NO:21; SEQ ID NO:23). The coding sequence encodes a 459 amino acid protein (SEQ ID NO:22).

This mature protein form is approximately 459 amino acid residues in length (from about amino acid 1 to amino acid 459 of SEQ ID NO:22). Human 15977 contains the following regions or other structural features: a eukaryotic protein kinase domain (PFAM Accession PF00069) located at about amino acid residues 44 to 276 of SEQ ID NO:22; and a serine/threonine kinase domain located at about amino acid residues 44 to 329 of SEQ ID NO:22.

The 15977 protein also includes the following domains: two predicted N-glycosylation sites (PS00001) located at about amino acids 370-373 and 388-391 of SEQ ID NO:22; two cAMP- and cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about amino acids 270-273 and 451-454 of SEQ ID NO:22; nine predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acids 14-16, 137-139, 199-201, 214-216, 229-231, 258-260, 269-271, 355-357, and 373-375 of SEQ ID NO:22; eight predicted Casein Kinase II sites (PS00006) located at about amino 96-99, 124-127, 150-153, 229-232, 258-261, 273-276, 355-358, and 411-414 of SEQ ID NO:22; two predicted N-myristoylation sites (PS00008) located at about amino 30-35 and 422-427 of SEQ ID NO:22; one predicted amidation site (PS00009) located at about amino acids 46-49 of SEQ ID NO:22; and a Serine/Threonine protein kinase active-site signature (PS 00108) located at about amino acids 160-172 of SEQ ID NO:22.

For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420.

Human 14760

The human 14760 sequence (SEQ ID NO:24), which is approximately 2046 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1788 nucleotides (nucleotides 119-1906 of SEQ ID NO:24; SEQ ID NO:26). The coding sequence encodes a 596 amino acid protein (SEQ ID NO:25).

This mature protein form is approximately 596 amino acid residues in length (from about amino acid 1 to amino acid 596 of SEQ ID NO:25). Human 14760 contains the following regions or other structural features: a eukaryotic protein kinase domain (PFAM Accession PF00069) located at about amino acid residues 285 to 540 of SEQ ID NO:25; and a serine/threonine kinase domain located at about amino acid residues 285 to 540 of SEQ ID NO:25.

The 14760 protein also includes the following domains: two predicted N-glycosylation sites (PS00001) located at about amino acids 278-281 and 416-419 of SEQ ID NO:25; three cAMP- and cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about amino acids 140-143, 317-320, and 583-586 SEQ ID NO:25; eleven predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acids 17-19, 49-51, 59-61, 107-109, 159-161, 203-205, 224-226, 235-237, 247-249, 320-322, and 460-462 of SEQ ID NO:25; eight predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino 157-160, 184-187, 203-206, 247-250, 301-304, 320-323, 351-354, and 379-382 of SEQ ID NO:25; one predicted tyrosine kinase phosphorylation sites (PS00007) located at about amino acids 370-376 of SEQ ID NO:25; nine predicted N-myristoylation sites (PS00008) located at about amino acids 83-88, 116-121, 135-140, 178-183, 241-246, 277-282, 293-298, 308-313, and 589-594 of SEQ ID NO:25; one predicted amidation site (PS00009) located at about amino acids 128-131 of SEQ ID NO:25; a protein kinases ATP-binding region signature located at about amino acids 291-299 of SEQ ID NO:25; and a Serine/Threonine protein kinase active-site signature (PS 00108) located at about amino acids 402-414 of SEQ ID NO:25.

For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420.

The 2504, 15977, and 14760 proteins contains a significant number of structural characteristics in common with members of the protein kinase family. The term “family” when referring to the protein and nucleic acid molecules of the invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

A 2504, 15977, or 14760 polypeptide can include a “protein kinase domain” or regions homologous with a “protein kinase domain”.

As used herein, the term “protein kinase” includes a protein or polypeptide which is capable of modulating its own phosphorylation state or the phosphorylation state of another protein or polypeptide. Protein kinases play critical roles in the regulation of biochemical and morphological changes associated with cellular growth and division (D'Urso, G. et al. (1990) Science 250: 786-791; Birchmeier. C. et al. (1993) Bioessays 15: 185-189). They serve as growth factor receptors and signal transducers and have been implicated in cellular transformation and malignancy (Hunter, T. et al. (1992) Cell 70: 375-387; Posada, J. et al. (1992) Mol. Biol. Cell 3: 583-592; Hunter, T. et al. (1994) Cell 79: 573-582). For example, protein kinases have been shown to participate in the transmission of signals from growth-factor receptors (Sturgill, T. W. et al. (1988) Nature 344: 715-718; Gomez, N. et al. (1991) Nature 353: 170-173), control of entry of cells into mitosis (Nurse, P. (1990) Nature 344: 503-508; Maller, J. L. (1991) Curr. Opin. Cell Biol. 3: 269-275) and regulation of actin bundling (Husain-Chishti, A. et al. (1988) Nature 334: 718-721).

Protein kinases can have a specificity for (i.e., a specificity to phosphorylate) serine/threonine residues, tyrosine residues, or both serine/threonine and tyrosine residues, e.g., the dual specificity kinases. As referred to herein, protein kinases preferably include a catalytic domain of about 200-400 amino acid residues in length, preferably about 200-300 amino acid residues in length, or more preferably about 250-300 amino acid residues in length. Specificity of a protein kinase for phosphorylation of either tyrosine or serine/threonine can be predicted by the sequence of two of the subdomains (VIb and VIII) in which different residues are conserved in each class (as described in, for example, Hanks et al. (1988) Science 241:42-52) the contents of which are incorporated herein by reference). These subdomains are also described in further detail herein.

Protein kinases play a role in signaling pathways associated with cellular growth. For example, protein kinases are involved in the regulation of signal transmission from cellular receptors, e.g., growth-factor receptors; entry of cells into mitosis; and the regulation of cytoskeleton function, e.g., actin bundling. Thus, the molecules of the present invention may be involved in: 1) the regulation of transmission of signals from cellular receptors, e.g., cell growth factor receptors; 2) the modulation of the entry of cells, e.g., precursor cells, into mitosis; 3) the modulation of cellular differentiation; 4) the modulation of cell death; and 5) the regulation of cytoskeleton function, e.g., actin bundling.

Inhibition or over stimulation of the activity of protein kinases involved in signaling pathways associated with cellular growth can lead to perturbed cellular growth, which can in turn lead to cellular growth related disorders. As used herein, a “cellular growth related disorder” includes a disorder, disease, or condition characterized by a deregulation, e.g., an upregulation or a downregulation, of cellular growth. Cellular growth deregulation may be due to a deregulation of cellular proliferation, cell cycle progression, cellular differentiation and/or cellular hypertrophy. Examples of cellular growth related disorders include cardiovascular disorders such as heart failure, hypertension, atrial fibrillation, dilated cardiomyopathy, idiopathic cardiomyopathy, or angina; proliferative disorders or differentiative disorders such as cancer, e.g., melanoma, prostate cancer, cervical cancer, breast cancer, colon cancer, or sarcoma.

As used herein, the term “protein kinase domain” includes an amino acid sequence of about 150 to 400 amino acid residues in length and having a bit score for the alignment of the sequence to the protein kinase domain (HMM) of at least 50. Preferably, a protein kinase domain includes at least about 200-400 amino acids, more preferably about 200-300 amino acid residues, or about 220-270 amino acids and has a bit score for the alignment of the sequence to the protein kinase domain (HMM) of at least 120 or greater. The protein kinase domain (HMM) has been assigned the PFAM Accession PF00069. An alignment of the protein kinase domain (amino acids 37 to 286 of SEQ ID NO:19) of human 2504 with a consensus amino acid sequence derived from a hidden Markov model yields a score of 229.1 (E=6.5e-65). The identified protein kinase domain of 2504 is depicted in SEQ ID NO:27. An alignment of the protein kinase domain (amino acids 44 to 276 of SEQ ID NO:22) of human 15977 with a consensus amino acid sequence derived from a hidden Markov model yields a score of 123.3 (E=4.3e-33). The identified protein kinase domain of 15977 is depicted in SEQ ID NO:29. An alignment of the protein kinase domain (amino acids 285 to 540 of SEQ ID NO:25) of human 14760 with a consensus amino acid sequence derived from a hidden Markov model yields a score of 251.1 (E=1.5e-71). The identified protein kinase domain of 2504 is depicted in SEQ ID NO:30.

In a preferred embodiment 2504, 15977, or 14760 polypeptide or protein has a “protein kinase domain” or a region which includes at least about 200-400 more preferably about 200-300 or 220-270 amino acid residues and has at least about 70% 80% 90% 95%, 99%, or 100% homology with a “protein kinase domain,” e.g., the protein kinase domain of human 2504, 15977, or 14760 (e.g., residues 37-286 of SEQ ID NO:19; residues 44-276 of SEQ ID NO:22, or residues 285-540 of SEQ ID NO:25).

A 2504, 15977, or 14760 molecule can further include a “serine/threonine kinase domain.”

As used herein, the term “serine/threonine kinase domain” includes an amino acid sequence of about 150 to 400 amino acid residues in length and having a bit score for the alignment of the sequence to the protein kinase domain (HMM) of at least 15. Preferably, a serine/threonine kinase domain includes at least about 200-400 amino acids, more preferably about 200-300 amino acid residues, or about 220-270 amino acids and has a bit score for the alignment of the sequence to the serine/threonine kinase domain (HMM) of at least 50 or greater. An alignment of the serine/threonine kinase domain (amino acids 24 to 286 of SEQ ID NO:19) of human 2504 with a consensus amino acid sequence derived from a hidden Markov model yields a score of 284.1 (E=1.8e-81). An alignment of the serine/threonine kinase domain (amino acids 44 to 329 of SEQ ID NO:22) of human 15977 with a consensus amino acid sequence derived from a hidden Markov model yields a score of 64.9 (E=1.8e-15). An alignment of the serine/threonine kinase domain (amino acids 285 to 540 of SEQ ID NO:25) of human 14760 with a consensus amino acid sequence derived from a hidden Markov model yields a score of 296.2 (E=4e-85). The identified serine/threonine kinase domains in 2504, 15977 and 14760 is depicted in SEQ ID NO:28.

In a preferred embodiment 2504, 15977, or 14760 polypeptide or protein has a “serine/threonine kinase domain” or a region which includes at least about 200-400 more preferably about 200-300 or 220-270 amino acid residues and has at least about 70% 80% 90% 95%, 99%, or 100% homology with a “serine/threonine kinase domain,” e.g., the serine/threonine kinase domain of human 2504, 15977, or 14760 (e.g., residues 24-286 of SEQ ID NO:19; residues 44-329 of SEQ ID NO:22, or residues 285-540 of SEQ ID NO:25).

To identify the presence of a “protein kinase” domain or a “serine/threonine kinase” domain in a 2504, 15977, or 14760 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference.

A 2504, 15977, or 14760 family member can include a protein kinase domain, e.g. a serine/threonine kinase domain.

As the 2504, 15977, or 14760 polypeptides of the invention may modulate 2504, 15977, or 14760-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 2504, 15977, or 14760-mediated or related disorders, as described below.

As used herein, a “2504, 15977, or 14760 activity”, “biological activity of 2504, 15977, or 14760” or “functional activity of 2504, 15977, or 14760”, refers to an activity exerted by a 2504, 15977, or 14760 protein, polypeptide or nucleic acid molecule on e.g., a 2504, 15977, or 14760-responsive cell or on a 2504, 15977, or 14760 substrate, e.g., a protein substrate, as determined in vivo or in vitro. In one embodiment, a 2504, 15977, or 14760 activity is a direct activity, such as an association with a 2504, 15977, or 14760 target molecule. A “target molecule” or “binding partner” is a molecule with which a 2504, 15977, or 14760 protein binds or interacts in nature, e.g., a protein containing one or more serine and or threonine residues. A 2504, 15977, or 14760 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 2504, 15977, or 14760 protein with a 2504, 15977, or 14760 receptor. For example, the 2504, 15977, or 14760 proteins of the present invention can have one or more of the following activities: 1) the regulation of transmission of signals from cellular receptors, e.g., cell growth factor receptors; 2) the modulation of the entry of cells, e.g., precursor cells, into mitosis; 3) the modulation of cellular differentiation; 4) the modulation of cell death; 5) the regulation of cytoskeleton function, e.g., actin bundling; or 6) the ability to phosphorylate a substrate.

Based on the above-described sequence similarities, the 2504, 15977, and 14760 molecules of the present invention are predicted to have similar biological activities as protein kinase family members. Thus, the 2504, 15977, and 14760 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, disorders associated with bone metabolism, immune disorders, hematopoietic disorders, cardiovascular disorders, liver disorders, viral diseases, pain or metabolic disorders.

In addition, the 2504, 15977, and 14760 molecules of the invention may modulate physiological and pathological processes in the cells or tissues where they are expressed. For example, Taq Man studies described herein show abundant expression of 2504, 15977, and 14760 mRNAs in neural tissues, including the brain cortex and hypothalamus. 15977 mRNA is also highly expressed in epithelial cells, astrocytes (glial cells), HUVEC cells, smooth muscle cells and fetal liver. 14760 mRNA is also abundantly expressed in the fetal liver, endothelial cells, fetal heart, fibroblasts, bone marrow glycophorin-positive cells, hepatocytes, cardiovascular cells, and skeletal muscle. Accordingly, these molecules can act as novel diagnostic targets and therapeutic agents of disorders involving the cells or tissues where they are expressed, e.g., neural (e.g., brain or astrocytic) disorders; cardiovascular and blood vessel (smooth muscle or endothelial cell) disorders; immune disorders (e.g., disorders involving glycophorin-positive cells); hepatic or liver disorders; skin disorders; skeletal disorders, among others.

Identification and Characterization of Human 2504, 15977, or 14760 cDNA and Genomic Sequence

The human 2504 sequence (SEQ ID NO:18), which is approximately 2297 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1503 nucleotides (nucleotides 154-1656 of SEQ ID NO:18; SEQ ID NO:20). The coding sequence encodes a 501 amino acid protein (SEQ ID NO:19).

The human 15977 sequence (SEQ ID NO:21), which is approximately 4417 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1377 nucleotides (nucleotides 337-1713 of SEQ ID NO:21; SEQ ID NO:23). The coding sequence encodes a 459 amino acid protein (SEQ ID NO:22).

The human 14760 sequence (SEQ ID NO:24), which is approximately 2046 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1788 nucleotides (nucleotides 119-1906 of SEQ ID NO:24; SEQ ID NO:26). The coding sequence encodes a 596 amino acid protein (SEQ ID NO:25).

Tissue Distribution of 2504, 15977, or 14760 mRNA

Endogenous human 2504, 15977, and 14760 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology.

To determine the level of 2504, 15977, and 14760 in various human tissues a primer/probe set was designed using Primer Express (Perkin-Elmer) software and primary cDNA sequence information. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. 2504, 15977, and 14760 mRNA levels were analyzed in a variety of samples of human tissues

Relative 2504 mRNA expression was determined by TaqMan assays on mRNA derived from the following tissues: monkey cortex; monkey dorsal root ganglion; monkey spinal cord; monkey sciatic nerve; monkey kidney; monkey hairy skin; monkey heart left ventricle; monkey gastro muscle; monkey liver; human brain; human spinal cord; human heart; human kidney; human liver; and human lung. The highest 2504 mRNA expression was observed in monkey cortex, human brain, and monkey and human spinal cords.

Relative 15977 mRNA expression was determined by TaqMan assays on mRNA derived from the following human tissues: (1) Aorta/normal; (2) Fetal heart/normal; (3) Heart normal; (4) Heart/congestive heart failure (CHF); (5) Vein/Normal; (6) Smooth muscle cells (SMC) (Aortic); (7) Spinal cord/Normal; (8) Brain cortex/Normal; (9) Brain hypothalamus/Normal; (10) Glial cells (Astrocytes); (11) Brain/Glioblastoma; (12) Breast/Normal; (13) Breast tumor/(invasive carcinoma (IDC); (14) Ovary/Normal; (15) Ovary/Tumor; (16) Pancreas; (17) Prostate/Normal; (18) Prostate/Tumor; (19) Colon/normal; (20) Colon/tumor; (21) Colon/IBD; (22) Kidney/normal; (23) Liver/normal; (24) Liver fibrosis; (25) Fetal Liver/normal; (26) Lung/normal; (27) Lung/tumor; (28) Lung/COPD; (29) Spleen/normal; (30) Tonsil/normal; (31) Lymph node/normal; (32) Thymus/normal; (33) Epithelial Cells (prostate); (34) Endothelial Cells (aortic); (35) Skeletal Muscle/Normal; (36) Fibroblasts (Dermal); (37) Skin/normal; (38) Adipose/Normal; (39) Osteoblasts (primary); (40) Osteoblasts (undifferentiated); (41) Osteoblasts (Diff); (42) Osteoclasts; (43) Aortic smooth muscle cells (SMC) Early; (44) Aortic SMC Late; (45) Shear human umbilical vein endothelial cells (HUVEC); and (46) Static HUVEC. Elevated 15977 mRNA expression was observed in epithelial cells, astrocytes (glial cells), normal brain (e.g., cortex and hypothalamus), HUVEC, and normal fetal liver.

Relative 14760 mRNA expression was determined by TaqMan assays on mRNA derived from the following human tissues: (1) Aorta/Normal; (2) Fetal Heart/Normal; (3) Heart/Normal; (4) Heart/CHF; (5) Vein/Normal; (6) SMC/aortic; (7) Nerve; (8) Spinal Cord/Normal; (9) Brain Cortex/Normal; (10) Brain hypothalamus; (11) Glial Cells (astrocytes); (12) Glioblastoma; (13) Breast/Normal; (14) Breast/IDC; (15) Ovary/Normal; (16) Ovary/Tumor; (17) Pancreas; (18) Prostate/Normal; (19) Prostate/tumor adenocarcinoma; (20) Colon/Normal; (21) Colon/Tumor; (22) Colon/IBD; (23) Kidney/Normal; (24) Liver/Normal; (25) Liver/Fibrosis; (26) Fetal Liver/Normal; (27) Lung/Normal; (28) COPD; (29) Spleen/Normal; (30) Tonsil/Normal; (31) Lymph Node/Normal; (32) Thymus/Normal; (33) Epithelial Cells; (34) Endothelial cells; (35) Skeletal Muscle/Normal; (36) Fibroblasts; (37) Skin/Normal; (38) Adipose/normal; (39) Osteoblast/Primary; (40) Osteoblast/undifferentiated; (41) Osteoblast/differentiated; and (42) Osteoclasts. Elevated 14760 mRNA expression was observed in normal brain (e.g., cortex and hypothalamus), and normal fetal liver and fetal heart.

Relative 14760 mRNA expression was determined by TaqMan assays on mRNA derived from the following tissues and cell lines: (1) Heart; (2) Lung; (3) Kidney; (4) Fetal Liver; (5) Spleen; (6) Granulocytes.; (7) NHDF mock; (8) NHLF mock; (9) NHLF TGF; (10) HepG2 Mock; (11) HepG2 TGF; (12) Pass Stell; (13) Liver Pool; (14) Control liver; (15) LF/NDR 191; (16) LF/NDR 193; (17) LF/NDR 079; (18) LN NDR 173; (19) Tonsil; (20) TH124 hr. MP39; (21) TH2 24 hr. MP39; (22) TH124 hr. MP21; (23) TH2 24 hr. MP21; (24) CD4; (25) CD8; (26) CD19; (27) CD3 MP42 rest; (28) CD14; (29) PBMC MOCK; (30) Bone marrow mononuclear cells (BM MNC); (31) CD34-positive cells (MPB CD34+); (32) Bone marrow glycophorin-positive cells (BM GPA+); (33) Cord Blood; (34) Erythroid; (35) Megakaryocytes; (36) Neutrophils (Neut) after 14 days in culture (d14); (37) CD14−/CD15+; (38) MBM CD11b; (39) HepG2; (40) HepG2.2.15; (41) MAI 01; (42) HL60; (43) K562; (44) Molt 4; (45) Hep3B Normoxia; and (46) Hep3B Hypoxia. Elevated 14760 mRNA expression was observed in pass stell, bone marrow glycophorin-positive cell lines, MOLT-4 cell lines and fetal liver.

Relative 14760 mRNA expression was determined using a cardiovascular organ panel by TaqMan assays on mRNA derived from the following cardiovascular tissues: normal atria; normal left ventricle; diseased right ventricle; diseased left ventricle; kidney; liver; and skeletal muscle. Elevated 14760 mRNA expression was observed in skeletal muscle and cardiovascular tissues.

Human 25501

The invention is based, at least in part, on the discovery of a novel transferase referred to herein as “25501”. The human 25501 sequence (SEQ ID NO:31), which is approximately 1971 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1512 nucleotides, including the termination codon (nucleotides indicated as coding of SEQ ID NO:31; SEQ ID NO:33). The coding sequence encodes a 503 amino acid protein (SEQ ID NO:32).

Human 25501 contains the following regions or other structural features (for general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420: a transfer domain (ProDom No. PD034341, SEQ ID NO:34) located at about amino acid residues 280 to 411 of SEQ ID NO:32; a recognition/binding domain located at about amino acid residues 30 to 250 of SEQ ID NO:32; six protein kinase C phosphorylation sites (Prosite PS00005) located at about amino acids 47 to 49, 126 to 128, 178 to 180, 181 to 183, 206 to 208, and 210 to 212 of SEQ ID NO:32; ten casein kinase II phosphorylation sites (Prosite PS00006) located at about amino acids 10 to 13, 41 to 44, 54 to 57, 126 to 129, 179 to 182, 222 to 225, 292 to 295, 357 to 360, 431 to 434, and 456 to 459 of SEQ ID NO:32; one cAMP/cGMP-dependent protein kinase phosphorylation site (Prosite PS00004) located at about amino acids 414 to 417 of SEQ ID NO:32; one tyrosine kinase phosphorylation site (Prosite PS00007) located at about amino acids 318 to 325 of SEQ ID NO:32; one amidation site (Prosite PS00009) located at about amino acids 377 to 380 of SEQ ID NO:32; and six N-myristoylation sites (Prosite PS00008) located at about amino acids 103 to 108, 281 to 286, 327 to 332, 337 to 342, 437 to 442, and 449 to 454 of SEQ ID NO:32.

The 25501 protein contains a significant number of structural characteristics in common with members of the transferase family, in particular, of methyltransferases. In general, transferases catalyze the transfer of one molecular group from a donor molecule to an acceptor molecule. Examples of such molecular groups include phosphate, amino, methyl, acetyl, acyl, phosphatidyl, phosphoribosyl, among other groups. The methyltransferase family is a large superfamily of enzymes that regulate biological processes by catalyzing the transfer of methyl groups to a wide variety of endogenous and exogenous compounds, including DNA, RNA, proteins, hormones, neurotransmitters, drugs, and xenobiotics (Weinshilboum et al. (1999) Annu. Rev. Pharmacol. Toxicol. 39:19-52).

Methylation of DNA can play an important role in the control of gene expression in mammalian cells. DNA methyltransferases are involved in DNA methylation and catalyze the transfer of a methyl group from S-adenosylmethionine to cytosine residues to form 5-methylcytosine, a modified base that is found mostly at CpG sites in the genome. The presence of methylated CpG islands in the promoter region of genes can suppress their expression. This process may be due to the presence of 5-methylcytosine, which apparently interferes with the binding of transcription factors or other DNA-binding proteins to block transcription. In different types of tumors, aberrant or accidental methylation of CpG islands in the promoter region has been observed for many cancer-related genes, resulting in the silencing of their expression. Such genes include tumor suppressor genes, genes that suppress metastasis and angiogenesis, and genes that repair DNA (Momparler and Bovenzi (2000) J. Cell Physiol. 183:145-54).

Methylation of proteins is a post-translational modification which can regulate the activity and subcellular localization of numerous proteins. Methylation of proteins can play an important role in protein repair and reversal of protein aging. Proteins undergo a variety of spontaneous degradation processes, including oxidation, glycation, deamidation, isomerization, and racemization. These non-enzymatic modifications can produce functionally damaged species that reflect the action of aging at the molecular level (Stadtman (1992) Science 257:1220-1224; Martin et al. (1996) Nat. Genet. 13:25-34). Methylation of these damaged proteins e.g., by protein L-isoaspartyl methyltransferase (Shimizu et al. (2000) Arch. Biochem. Biophys. 381:225-34) can play a part in the repair pathway. Protein methylation is also known to be important in cellular stress responses (Desrosiers and Tanguay (1988) J. Biol. Chem. 263:4686-4692). Moreover, protein methyltransferases have recently been demonstrated to be important in cellular signaling events, for example, in receptor-mediated and/or differentiation-dependent signaling (Lin et al. (1996) J. Biol. Chem. 271:15034-15044; Abramovich et al. (1997) EMBO J. 16:260-266).

Methylation is a process important for the catabolism of small molecules, such as thiol compounds and neurotransmitters. A deficiency in thiol compound detoxification by methylation is being investigated for its role in rheumatoid arthritis (Waring and Emery (1993) Baillieres Clin. Rheumatol. 6:337-50). Inhibition of dopamine methylation and inactivation by catechol-O-methyl transferase is a goal for therapy of Parkinson's disease (Goldstein and Lieberman (1992) Neurology 42(suppl):8-12).

As used herein, the term “transferase” includes a protein or polypeptide which is capable of catalyzing the transfer of a molecular group from a donor molecule to an acceptor molecule. In order to catalyze molecular group transfer, the transferases must recognize or bind the group's donor then catalyze the transfer of the group to an acceptor molecule. In the process, the transferase itself can become an intermediate acceptor molecule, e.g., the alkylation of an active site cysteine in O(6)-alkylguanine-DNA alkyltransferase (Daniels and Tainer (2000) Mutat. Res. 460:151-163). Members of a transferase family of proteins typically are cytoplasmic or nuclear proteins. Transferases, e.g. methyltransferases typically include conserved motifs, including at least one Prosite methyltransferase signature sequence, e.g. PS01261, PS00092, or PS01184. The 25501 molecules of the invention include regions homologous to these motifs.

A 25501 polypeptide can include a “transfer domain” or a region homologous with a “transfer domain”.

As used herein, the term “transfer domain” includes an amino acid sequence of about 50 to 250 amino acid residues in length and includes one, two, preferably three sequences homologous to the Prosite methylase or methyltransferase signature sequences PS01261, PS00092, and PS01184. Preferably, a transfer domain includes at least about 100 to 200 amino acids, more preferably about 120 to 150 amino acid residues, or about 130 to 140 amino acids and includes one, two, preferably three sequences homologous to Prosite methylase or methyltransferase signature sequences PS01261, PS00092, and PS01184. Preferably the Prosite sequences are arranged in the following order, first the PS01261, second the PS00092, third the PS01184 and are spaced about sixty amino acids or less from each other. Preferably a transfer domain catalyzes the transfer of a group, e.g. a methyl group from a donor to an acceptor molecule. The transfer domain of 25501 can be found at about amino acid residues 280 to 411 of SEQ ID NO:32.

A sequence similar to the Prosite sequence PS01261, the putative RNA methylase family UPF0020 signature, D-P-[LIVMF]-C-G-[ST]-G-x(3)-[LI]-E (SEQ ID NO:36) can be found in human 25501 at about amino acid residues 304 to 315 of SEQ ID NO:32, except an L replaces the [ST]. A sequence similar to the Prosite sequence PS00092, the N-6 adenine-specific DNA methylase signature, [LIVMAC]-[LIVFYWA]-x-[DN]-P—P—[FYW] (SEQ ID NO:37) can be found in human 25501 at about amino acid residues 371 to 377 of SEQ ID NO:32, except an L replaces the first P. A sequence similar to the Prosite sequence PS01184, the ubiE/COQ5 methyltransferase family signature 2, R—V-[LIVM]—K—[PV]-[GM]-G-x-[LIVMF]-x(2)-[LIVM]-E-x-S (SEQ ID NO:38) can be found in human 25501 at about amino acid residues 396 to 409 of SEQ ID NO:32, except an H replaces the K and the last three residues are L-S-E instead of E-x-S. In the above conserved signature sequences, and other motifs or signature sequences described herein, the standard IUPAC one-letter code for the amino acids is used. Each element in the pattern is separated by a dash (-); square brackets ([ ]) indicate the particular residues that are accepted at that position; x indicates that any residue is accepted at that position; and numbers in parentheses (( )) indicate the number of residues represented by the accompanying amino acid.

The transfer domain of the human 25501 protein is homologous, e.g., at least about 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or 41% identical to the ProDom family PD034341 (“VNG2242C Y71F9AL.1 MTH724 PH0338 AF1257 MJ0710 APE1835”) domain (ProDomain Release 2001.1). The ProDom PD034341 domain and can include one, two, preferably three Prosite methylase or methyltransferase signature sequences or sequences homologous to these sequences spaced sixty amino acids or less apart. A GAP alignment of the transfer domain (amino acids 280 to 411 of SEQ ID NO:32) of human 25501 with amino acid residues 1 to 133 of the 172 amino acid PD034341 domain consensus sequence (SEQ ID NO:34), derived from a BLAST search model results in 32% identity (as calculated from the blosum62 matrix).

In a preferred embodiment, a 25501 polypeptide or protein has a “transfer domain” or a region which includes at least about 100 to 200 more preferably about 120 to 150 or 130 to 140 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “transfer domain,” e.g., the transfer domain of human 25501 (e.g., residues 280 to 411 of SEQ ID NO:32).

Regions similar to the transfer domain are found in other proteins. For example, a transfer domain can be found in MGC:2454 (SEQ ID NO:35, accession number 13278783 in GenPept; corresponding to number BC004163 in GenBank). MGC:2454 is homologous to the 25501 protein in SEQ ID NO:32. An alignment of the 25501 protein with MGC:2454 results in about 94% overall sequence identity between the two sequences. Sequence identity of 100% can be found in regions beginning about amino acid 1 to 473 of MGC:2454 (SEQ ID NO:35) with amino acids about 31 to 503 of 25501, SEQ ID NO:32 (as calculated in matblas from the blosum62.iij matrix).

To make the determination that the “transfer” domain in a 25501 protein sequence or a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against a database of domains, e.g., the ProDom database (Corpet et al. (1999), Nucl. Acids Res. 27:263-267). The ProDom protein domain database consists of an automatic compilation of homologous domains. Current versions of ProDom are built using recursive PSI-BLAST searches (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402; Gouzy et al. (1999) Computers and Chemistry 23:333-340) of the SWISS-PROT 38 and TREMBL protein databases. The database automatically generates a consensus sequence for each domain. A BLAST search was performed against the database resulting in the PD034341 profile of the “transfer” domain in the amino acid sequence of human 25501 at about residues 280 to 411 of SEQ ID NO:32.

A 25501 molecule can further include a recognition/binding domain or regions homologous with a “recognition/binding domain.” As used herein, the “recognition/binding domain” includes an amino acid sequence of about 100 to 350 amino acid residues in length and whose secondary structure is characterized by a high alpha helical content. Table 14, below, illustrates the prediction of the likelihood of amino acid residues from this region of 25501 to belong to an element of secondary structure by two prediction methods.

As shown in Table 14, the prediction methods agree that the majority of residues in this region, in particular, residues 117 to 198 of SEQ ID NO:32, can form alpha helices. Proteins can use alpha helices to recognize and bind nucleic acid molecules. For example, the helix-turn-helix DNA binding domain is involved in a variety of protein-DNA interactions (Wintjens and Rooman (1996) J. Mol. Biol. 262:294-313), with variations in additional helices and helix arrangements distinguishing protein families from one another. Proteins can use alpha helices to determine the specificity of ligand interactions. For example, amino acid residues on helices in the ligand binding pocket of steroid receptors allow the discrimination between different steroid hormones (Ekena et al. (1998) J. Biol. Chem. 273:693-699).

In a preferred embodiment, a 25501 polypeptide or protein has a “recognition/binding domain” or a region which includes at least about 150 to 300 more preferably about 180 to 260 or 210 to 230 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “recognition/binding domain,” e.g., the recognition/binding domain of human 25501 (e.g., residues 30 to 250 of SEQ ID NO:32).

To identify the presence of a “recognition/binding” domain in a 25501 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be analyzed by a secondary structure prediction method that predicts the secondary structure of proteins based on the characteristics of each amino acid (Chou and Fasman (1974) Biochemistry 13:222-244 and Garnier et al. (1978) J. Mol. Biol. 120:97-120).

A 25501 family member can include at least one transfer domain. A 25501 family member also can include at least one recognition/binding domain. Furthermore, a 25501 family member can include at least one, two, three, four, five preferably six protein kinase C phosphorylation sites (Prosite PS00005); at least one, two, three, four, five, six, seven, eight, nine and preferably ten casein kinase II phosphorylation sites (Prosite PS00006); at least one tyrosine kinase phosphorylation site (Prosite PS00007); at least one cAMP/cGMP protein kinase phosphorylation sites (Prosite PS00004); at least one amidation site (Prosite PS00009); and at least one, two, three, four, five preferably six N-myristoylation sites (Prosite PS00008).

Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, e.g., the sequence from about amino acid 258 to 267, from about 353 to 363, and from about 100 to 108 of SEQ ID NO:32; all or part of a hydrophilic sequence, e.g., the sequence from about amino acid 121 to 132, from about 150 to 160, and from about 410 to 423 of SEQ ID NO:32; a sequence which includes a Cys, or a glycosylation site.

As the 25501 polypeptides of the invention can modulate 25501-mediated activities, they can be useful for developing novel diagnostic and therapeutic agents for transferase-associated or other 25501-associated disorders, as described below.

As used herein, a “transferase-associated activity” includes an activity which involves a transfer function, e.g. the transfer of a group, e.g. a methyl group from a donor molecule to an acceptor molecule. This function is implicated in a wide range of cell activities, including, but not limited to cell growth and cell processes, e.g., the regulation of cell proliferation, differentiation, migration, protein transport, gene expression, and/or intra- or intercellular signaling, and apoptosis. Members of the family can play a role in cancer, developmental syndromes, such as Fragile X and Rett (El-Osta and Wolf (2000) Gene Expr. 9:63-75), neurodegenerative disorders such as Alzheimer's disease (Shimizu et al. (2000) Arch. Biochem. Biophys. 381:225-34), and Parkinson's disease (Goldstein and Lieberman (1992) Neurology 42 (suppl4):8-12), and inflammatory disorders such as rheumatoid arthritis (Waring and Emery (1992) Baillieres Clin. Rheumatol. 6:337-50).

As used herein, a “25501 activity”, “biological activity of 25501” or “functional activity of 25501”, refers to an activity exerted by a 25501 protein, polypeptide or nucleic acid molecule on e.g., a 25501-responsive cell or on a 25501 substrate, e.g., a protein substrate, as determined in vivo or in vitro. In one embodiment, a 25501 activity is a direct activity, such as an association with a 25501 target molecule. A “target molecule” or “binding partner” is a molecule with which a 25501 protein binds or interacts in nature. In an exemplary embodiment, 25501 is a transferase, e.g., a methyltransferase, and thus has the ability to bind to, or interact with, a substrate or target molecule, e.g., a nucleic acid molecule (e.g. DNA or RNA), a small organic molecule (e.g., a hormone, a neurotransmitter or a coenzyme), or a protein; and/or the ability to transfer a group, e.g. a methyl group from a donor to an acceptor molecule, e.g. the substrate or target molecule.

A 25501 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 25501 protein with a 25501 receptor. Based on the above-described sequence structures and similarities to molecules of known function, the 25501 molecules of the present invention can have similar biological activities as transferase family members. For example, the 25501 proteins of the present invention can have one or more of the following activities: (1) the ability to interact with a 25501 substrate or target molecule (e.g., a non-25501 protein); (2) the ability to convert a 25501 substrate or target molecule to a product (e.g., transfer of a methyl group to or from the substrate or target molecule); (3) the ability to interact with and/or methyl transfer to a second non-25501 target molecule e.g., a nucleic acid molecule (e.g., DNA or RNA), a small organic molecule (e.g., a hormone, neurotransmitter or a coenzyme) or a protein; (4) the ability to regulate substrate or target molecule activity; (5) the ability to modulate intra- or intercellular signaling and/or gene transcription (e.g., either directly or indirectly); (6) the ability to modulate cellular targeting and/or transport of proteins; (7) the ability to modulate cellular proliferation, growth, or differentiation; (8) the ability to modulate cell migration and/or (9) the ability to modulate apoptosis.

The 25501 molecules of the invention can modulate the activities of cells in tissues where they are expressed. For example, 25501 mRNA is expressed in brain, in particular the astrocytes, which provide physical and biochemical support for neurons and interact with capillary endothelial cells to form the blood-brain barrier. 25501 mRNA also can be found in the ovary and prostate epithelium. 25501 mRNA also is expressed in tissues undergoing large amounts of growth, differentiation and angiogenesis such as fetal and neonatal kidney, fetal heart and fetal adrenal gland. 25501 mRNA also is expressed in cancerous tissue, especially malignant tumors, such as Wilm's tumor, lung tumor, colon tumor, metastases of colon tumor in the liver, metastases of prostate tumor in the liver, metastases of breast tumors in the lung and brain. Accordingly, the 25501 molecules of the invention can act as novel diagnostic targets or therapeutic agents for neurological disorders, ovarian disorders, prostate disorders, or proliferative and/or differentiative disorders or other transferase disorders.

Gene Expression Analysis of 25501 by TaqMan® Analysis

Human 25501 expression was measured by TaqMan® quantitative PCR (Perkin Elmer Applied Biosystems) in cDNA prepared from a variety of normal and diseased (e.g., cancerous) human tissues or cell lines.

The results indicate significant 25501 expression in brain, e.g. glial cells (e.g. a high level in astrocytes); a medium level in the ovary; in the prostate e.g. a medium level in prostate epithelium; in tissues undergoing large amounts of growth, differentiation and angiogenesis, e.g. medium levels in the fetus and neonate (e.g. fetal and neonatal kidney fetal heart and fetal adrenal gland); and in cancerous tissue, e.g. tumors (e.g. medium levels in lung tumor, colon tumor and metastases of colon tumor in the liver, and high levels in Wilm's tumor and metastases of prostate tumor in the liver).

Transcriptional Profiling

The expression profiles of samples of metastatic brain and lung tumors originating from human breast adenocarcinoma tumors were compared with the profiles samples from primary human breast adenocarcinoma tumors. Total RNA was isolated from the tissue samples. Reverse transcriptase was used to generate 33 P-dCTP-labeled cDNAs from the RNA. These experimental tissue cDNAs were hybridized to an array of molecules with known sequences. The nylon array contained 9600 elements, each with a PCR product from cDNA clones of the known genes. The hybridization levels from each tissue sample are measured and compared. Comparisons resulting in at least a 1.5-fold difference were judged as significant. The 25501 transcript was identified as being upregulated in the lung and brain metastatic tumors originating from human breast adenocarcinoma tumors.

Human 17903

The present invention is based, at least in part, on the discovery of a novel aminopeptidase referred to herein as “17903”. The present invention provides the human 17903 sequence (SEQ ID NO:39), which is approximately 3034 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 2178 nucleotides (nucleotides 18 to 2195 of SEQ ID NO:39; SEQ ID NO:41). The coding sequence encodes a 725 amino acid protein (SEQ ID NO:40).

The 17903 protein includes a Pfam Peptidase family M1 consensus domain, as well as Prodom consensus domains for aminopeptidases. For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420.

The 17903 protein contains a significant number of structural characteristics in common with members of the aminopeptidase M1 family of metallopeptidases. Aminopeptidases (APs) are a group of widely distributed exopeptidases that catalyze the hydrolysis of amino acid residues from the amino-terminus of polypeptides and proteins. The enzymes are found in plant and animal tissues, in eukaryotes and prokaryotes, and in secreted and soluble forms. Biological functions of aminopeptidases include protein maturation, terminal degradation of proteins, hormone level regulation, and cell-cycle control.

Aminopeptidases are implicated in a host of conditions and disorders including aging, cancers, inflammatory diseases, cataracts, cystic fibrosis and leukemias. In eukaryotes, APs are associated with removal of the initiator methionine. In prokaryotes the methionine is removed by methionine aminopeptidase subsequent to removal of the N-formyl group from the initiator N-formyl methionine, facilitating subsequent modifications such as N-acetylation and N-myristoylation. In E. coli AP-A (pepA), the xerB gene product is required for stabilization of unstable plasmid multimers.

APs are also involved in the metabolism of secreted regulatory molecules, such as hormones and neurotransmitters, and modulation of cell-cell interactions. In mammalian cells and tissues, the enzymes are apparently required for terminal stages of protein degradation, and EGF-induced cell-cycle control; and may have a role in protein turnover and selective elimination of obsolete or defective proteins. Furthermore, the enzymes are implicated in the supply of amino acids and energy during starvation and/or differentiation, and degradation of transported exogenous peptides to amino acids for nutrition. APs may also have a role in inflammation. Industrial uses of the enzymes include modification of amino termini in recombinantly expressed proteins. See A. Taylor (1993) TIBS 18: 1993:167-172.

Aminopeptidases have been identified in a wide variety of tissues and organisms, including zinc aminopeptidase and aminopeptidase M from rat kidney membrane; human aminopeptidase N from intestine; arginine aminopeptidase from liver; aminopeptidase N b from muscle; leukotriene-A4 hydrolase; leucine aminopeptidase (LAP) from bovine and hog lens and kidney; aminopeptidase A (xerB gene product) from E. coli ; yscl APE1/LAP4 and aminopeptidase A (pep4 gene product) from S. cerevisiae ; LAP from aeromonas ; dipeptidase from mouse ascites; methionine aminopeptidase from salmonella, E. coli, S. cerevisiae and hog liver; and D-amino acid aminopeptidase from ochrobactrum anthropi SCRC C1-38.

As used herein, the term “aminopeptidase” refers to a protein or polypeptide that is capable of catalyzing the cleavage of a polypeptide bond at the amino terminus of a polypeptide molecule through hydrolysis (i.e., possessing amino-terminal polypeptide hydrolytic activity or exopeptidase activity). As referred to herein, aminopeptidases preferably include a catalytic domain of about 150-350 amino acid residues in length, preferably 200-300 amino acid residues in length, or more preferably 220-280 amino acids in length. Based on the sequence similarities described above, the 17903 molecules of the present invention are predicted to have similar biological activities as aminopeptidase family members.

As the biological functions of aminopeptidases include protein maturation and protein degradation, they typically play a role in diverse cellular processes. In particular, aminopeptidases have been shown to have a role in tumor growth, metastasis, and angiogenesis; in inflammatory disorders including, but not limited to osteoarthritis and rheumatoid arthritis, multiple sclerosis, Crohn disease, psoriasis, periodontal disease, and asthma; in cataracts; in cystic fibrosis; in leukemias; and in aging.

A 17903 polypeptide can include an “aminopeptidase zinc-binding motif” or regions homologous with the “Peptidase M1 family of aminopeptidases”.

As used herein, the term “Peptidase M1 family of aminopeptidases domain” includes an amino acid sequence having a bit score for the alignment of the sequence to the Peptidase M1 family domain (HMM) of at least 8. Preferably, a peptidase M1 family of aminopeptidases domain includes at least about 150-350 amino acids, more preferably 200-300 amino acids, or about 220-280 amino acids and has a bit score for the alignment of the sequence to the aminopeptidase domain (HMM) of at least 16 or greater. The Peptidase M1 family (HMM) has been assigned the PFAM Accession PF01433. An alignment of the Peptidase M1 family of aminopeptidases domain of human 17903 (amino acids 195 to 445 of SEQ ID NO:40) with the consensus amino acid sequences derived from a hidden Markov model yields a bit score for the alignment of the sequence to the amino-peptidase domain (HMM) of 172 (E=4.3e-59). The identified consensus amino acid sequence for the Peptidase M1 family of aminopeptidases is depicted in SEQ ID NO:42.

In a preferred embodiment 17903 polypeptide or protein has a “peptidase M1 family of aminopeptidases domain” or a region which includes at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with the Peptidase M1 family of aminopeptidases (e.g., amino acid residues 195 to 445 of SEQ ID NO:40).

To identify the presence of a Peptidase M1 aminopeptidase region of homology in a 17903 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference.

As the 17903 polypeptides of the invention may modulate 17903-mediated activities, they may be useful for developing novel diagnostic and therapeutic agents for 17903-mediated or related disorders, as described below.

As used herein, a “17903 activity”, “biological activity of 17903” or “functional activity of 17903”, refers to an activity exerted by a 17903 protein, polypeptide or nucleic acid molecule on e.g., a 17903-responsive cell or on a 17903 polypeptide substrate, as determined in vivo or in vitro. In one embodiment, a 17903 activity is a direct activity, such as an association with a 17903 target molecule. A “target molecule” or “binding partner” or “ligand” or “substrate” is a molecule with which a 17903 protein binds or interacts in nature, e.g., a polypeptide that a 17903 protein cleaves. A 17903 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 17903 protein with a 17903 ligand. For example, the 17903 proteins of the present invention can have one or more of the following activities: 1) cleavage of a protein precursor to maturation; 2) catalysis of protein degradation; 3) regulation of hormone levels; 4) modulation of tumor cell growth and invasion; 5) modulation of angiogenesis; and 6) regulation of cell proliferation.

Polypeptides of the invention include fragments which include: all or a part of a hydrophobic sequence, e.g. residues from about 317 to 352 of SEQ ID NO:40; or all or part of a hydrophilic fragment, e.g. residues from about 676 to 704 of SEQ ID NO:40. Other fragments include a cysteine residue or an N-glycosylation site.

The expression profile for 17903 is depicted in Tables 15-29 below. As depicted in tables 15-29, 17903 is up-regulated in proliferating endothelial cells compared to arrested endothelial cells in 5 out of 5 independent experiments. 17903 is further up-regulated in some lung, breast, ovary, and brain tumors as compared to normal tissues. 17903 is expressed in hemangiomas and the expression levels in hemangiomas are 30-50 fold higher than the expression level in normal skin. In addition, 17903 is expressed in other angiogenic tissues such as Wilms tumors, uterine adenocarcinoma, neuroblastoma, fetal adrenal gland, and fetal kidney. Mouse 17903 is up-regulated in VEGF plugs as compared to parental plugs in the xenograft model. In the RIP-Taq mouse model, the expression of 17903 is up-regulated in tumor islets and the expression levels of 17903 correlate to the expression levels of VEGF at various stages of tumor development.

Expression of 17903 was measured in various clinical samples by in situ hybridization. 17903 was weakly expressed in one of two breast tumor epithelial cell samples, but not in either of two normal breast samples. Three of four primary colon tumor and metastases were positive for 17903 expression, while 17903 was not detected in the normal colon control. 17903 was expressed in five of seven samples of malignant epithelium of several histologically different lung tumor subtypes, but was not detected in the normal lung control sample. 17903 was expressed in both malignant ovary epithelium and normal stroma of the ovary.

The methods of the present invention are most relevant to those normal and diseased tissues where 17903 is expressed, including the tissues described above as well as those shown in Tables 15-29 below. The expression pattern of 17903 in human samples and mouse models suggest that 17903 plays a positive role in cellular proliferation (including endothelial proliferation), tumor angiogenesis, and/or tumorogenesis. Accordingly, inhibition of 17903 function may inhibit tumor angiogenesis and tumor growth.

Identification and Characterization of Human 17903 cDNAs

The human 17903 sequence (SEQ ID NO:39), which is approximately 3034 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 2175 nucleotides (nucleotides 18-2192 of SEQ ID NO:39; SEQ ID NO:41). The coding sequence encodes a 725 amino acid protein (SEQ ID NO:40).

Tissue Distribution of 17903 mRNA

The expression of 17903 was monitored in various tissues and cell types by quantitative PCR (TaqMan® brand quantitative PCR kit, Applied Biosystems) according to the kit manufacture's instructions. The results are shown below in Tables 15-29.

Human 3700

The invention is based, at least in part, on the discovery of a novel protein kinase, herein referred to as “3700”. The human 3700 cDNA sequence (SEQ ID NO:43), which is approximately 3353 nucleotide residues long including non-translated regions, contains a predicted methionine-initiated coding sequence of about 1884 nucleotide residues, excluding termination codon (i.e., nucleotide residues 157-2040 of SEQ ID NO:43; also shown in SEQ ID NO:45). The coding sequence encodes a 628 amino acid protein having the amino acid sequence SEQ ID NO:44.

Human 3700 contains the following regions or other structural features: a predicted pkinase domain (PF00069) at about amino acid residues 53-303 of SEQ ID NO:44, a protein kinases ATP-binding region signature sequence at residues 59 to 67 of SEQ ID NO:44, and a serine/threonine protein kinase active site signature sequence at residues 171 to 183 of SEQ ID NO:44. A transmembrane domain is predicted at about amino acid residues 234 to 250 of SEQ ID NO:44.

The human 3700 protein has predicted N-glycosylation sites (Pfam accession number PS00001) at about amino acid residues 121-124 and 576-579 of SEQ ID NO:44; predicted cAMP-/cGMP-dependent protein kinase phosphorylation sites (Pfam accession number PS00004>at about amino acid residues 290-293, 337-340, and 413-416 of SEQ ID NO:44; predicted protein kinase C phosphorylation sites (Pfam accession number PS00005) at about amino acid residues 30-32, 74-76, 82-84, 122-124, 142-144, 148-150, 289-291, 327-329, 339-341, 373-375, 377-379, and 616-618 of SEQ ID NO:44; predicted casein kinase II phosphorylation sites (Pfam accession number PS00006) located at about amino acid residues 15-18, 133-136, 148-151, 227-230, 293-296, 331-334, 377-380, 391-394, 461-464, 511-514, 523-526, 578-581, and 606-609 of SEQ ID NO:44; a predicted tyrosine kinase phosphorylation site at residues 453-460 of SEQ ID NO:44; predicted N-myristoylation sites (Pfam accession number PS00008) at about amino acid residues 320-325, 347-352, and 360-365 of SEQ ID NO:44; and a predicted cell attachment sequence at about amino acid residues 134-136 of SEQ ID NO:44.

Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, e.g., the sequence of about residues 234-250 of SEQ ID NO:44; all or part of a hydrophilic sequence, e.g., the sequence of residues 40-55 or 445-470 of SEQ ID NO:44; a sequence which includes a cysteine residue; or a glycosylation site.

For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997, Protein 28:405-420).

The 3700 protein contains a significant number of structural characteristics in common with members of the Protein Kinase family. Protein phosphorylation is influenced primarily by enzymes of two types, namely protein kinases (PKs) and protein phosphatases (PPs). PKs catalyze addition of a phosphate moiety to a protein amino acid residue (generally a serine, threonine, or tyrosine residue), and PPs catalyze removal of such moieties. The catalytic activities of PKs and PPs are, in turn, influenced by the state of the cell and the environment in which it finds itself. Phosphorylation of amino acid residues by a PK generally manifests itself in the form of faster cell growth, metabolism, or division, as greater motility, or in the form of higher gene transcription, although certain physiological processes are inhibited by protein phosphorylation. De-phosphorylation of amino acid residues by a PP, by contrast, generally manifests itself as slower (or halted) cell growth, division, or metabolism, as lower motility, or in the form of lower gene transcription. PK/PP-modulated protein phosphorylation is also involved in carcinogenesis.

Without being bound by any particular theory of operation, 3700 protein is believed to be a serine/threonine kinase.

A 3700 polypeptide can include a pkinase domain. As used herein, the term “pkinase domain” refers to a protein domain having an amino acid sequence of about 200-300 amino acid residues in length, preferably, at least about 225-300 amino acids, more preferably about 278 amino acid residues or about 251 amino acid residues and has a bit score for the alignment of the sequence to the pkinase domain (HMM) of at least 100 or greater, preferably 200 or greater, and more preferably 300 or greater. The pkinase domain has been assigned the PFAM accession PF00069.

In a preferred embodiment, 3700 polypeptide or protein has a pkinase domain or a region which includes at least about 200-300, more preferably about 225-300, 278, or 251 amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with a pkinase domain, e.g., the pkinase domain of human 3700 (e.g., residues 53-303 of SEQ ID NO:44).

To identify the presence of a pkinase domain profile in a 3700 receptor, the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for PF00069 and score of 100 is the default threshold score for determining a hit. For example, using ORFAnalyzer software, a pkinase domain profile was identified in the amino acid sequence of SEQ ID NO:44 (e.g., amino acids 53-303 of SEQ ID NO:44). Accordingly, a 3700 protein having at least about 60-70%, more preferably about 70-80%, or about 80-90% homology with the pkinase domain profile of human 3700 is within the scope of the invention.

In one embodiment, a 3700 protein includes at least one transmembrane domain. As used herein, the term “transmembrane domain” includes an amino acid sequence of about 5 amino acid residues in length that spans the plasma membrane. More preferably, a transmembrane domain includes about at least 10, 15, 20 or 22 amino acid residues and spans a membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zagotta W. N. et al. (1996, Annu. Rev. Neurosci. 19: 235-263), the contents of which are incorporated herein by reference. Amino acid residues 234 to about 250 of SEQ ID NO:44 comprise a transmembrane domain in a 3700 protein. In one embodiment, the amino-terminal domain of 3700 protein (i.e., about residues 1-233 of SEQ ID NO:44) is on the cytoplasmic side of a cellular membrane (e.g., the nuclear membrane or the cytoplasmic membrane) and the carboxyl-terminal domain (i.e., about residues 251-628 of SEQ ID NO:44) is on the non-cytoplasmic side of the same membrane. In another embodiment, the amino-terminal domain is oriented on the non-cytoplasmic side of the membrane and the carboxyl-terminal domain is oriented on the cytoplasmic side.

While not being bound by any particular theory of operation, 3700 protein is believed to be, in at least one embodiment, a nuclear membrane protein having its carboxyl-terminal domain oriented within the nuclear envelope. In this embodiment, 3700 protein is capable of transmitting signaling information from the cytoplasm to the nucleus, whereby, for example, gene transcription can be regulated.

In one embodiment of the invention, a 3700 polypeptide includes at least one pkinase domain. In another embodiment, the 3700 polypeptide includes at least one pkinase domain and at least one transmembrane domain. The 3700 molecules of the present invention can further include one or more of the N-glycosylation, cAMP-/cGMP-dependent protein kinase phosphorylation, protein kinase C phosphorylation, casein kinase II phosphorylation, tyrosine kinase phosphorylation, N-myristoylation, and cell attachment sites described herein, and preferably comprises most or all of them.

Because the 3700 polypeptides of the invention can modulate 3700-mediated activities, they can be used to develop novel diagnostic and therapeutic agents for 3700-mediated or related disorders, as described below.

As used herein, a “3700 activity,” “biological activity of 3700,” or “functional activity of 3700,” refers to an activity exerted by a 3700 protein, polypeptide or nucleic acid molecule on, for example, a 3700-responsive cell or on a 3700 substrate (e.g., a protein substrate) as determined in vivo or in vitro. In one embodiment, a 3700 activity is a direct activity, such as association with a 3700 target molecule. A “target molecule” or “binding partner” of a 3700 protein is a molecule (e.g., a protein or nucleic acid) with which the 3700 protein binds or interacts in nature. In an exemplary embodiment, such a target molecule is a 3700 receptor. A 3700 activity can also be an indirect activity, such as a cellular signaling activity mediated by interaction of the 3700 protein with a 3700 receptor.

The 3700 molecules of the present invention are predicted to have similar biological activities as PK family members. For example, the 3700 proteins of the present invention can have one or more of the following activities: (1) catalyzing formation of a covalent bond within or between an amino acid residue (e.g., a serine or threonine residue) and a phosphate moiety; (2) modulating cell signaling; (3) modulating cell growth; (4) modulating cell differentiation; (5) modulating tumorigenesis; (6) modulating entry of a cell into the cell cycle; (7) modulating progression of a cell through the cell cycle; (8) modulating mitogenesis; (9) modulating cell motility; (10) modulating a cell-to-cell interaction; (11) modulating cell metabolism; (12) modulating gene transcription; (13) modulating an immune response; (14) modulating angiogenesis; (15) modulating tissue (e.g., kidney or liver) repair or regeneration; (16) modulating establishment of atherosclerosis; (17) modulating progression of atherosclerosis; and (18) modulating signaling across the blood-brain barrier.

Thus, 3700 molecules described herein can act as novel diagnostic targets and therapeutic agents for prognosticating, diagnosing, preventing, inhibiting, alleviating, or curing PK-related disorders.

Other activities, as described below, include the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which 3700 molecules are expressed. Thus, the 3700 molecules can act as novel diagnostic targets and therapeutic agents for controlling disorders involving aberrant activities of these cells.

The 3700 molecules can also act as novel diagnostic targets and therapeutic agents for controlling cellular proliferative and/or differentiative disorders (e.g., hematopoietic neoplastic disorders, carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

Expression data included herein indicate that 3700 is highly expressed in tissues having endothelial or epithelial cell layers, such as in blood vessels, kidney, and pancreas. These data indicate that 3700 protein can be involved in a variety of disorders that afflict endothelial and epithelial tissues. Examples of such disorders include cardiovascular disorders such as atherosclerosis, arteriosclerosis, abnormal blood coagulation, and coronary artery disease.

3700 is expressed in aortic and coronary smooth muscle cells, indicating that 3700 can have a role in disorders that affect these tissues. Examples of these disorders include coronary artery disease and cardiac insufficiency. 3700 can also be involved in the response of aortic and coronary tissues to ischemic damage, such as that associated with cardiac infarction or thrombotic injury to coronary arteries.

Expression of 3700 is enhanced in the presence of inflammatory cytokines, indicating a role for 3700 in normal and aberrant inflammatory responses. 3700 can have a role in a variety of immune disorders in tissues in which it is expressed. By way of example, 3700 can have a role in prostatitis, pancreatitis, meningitis, severe allergic reactions, and in autoimmune disorders. Modulating the activity or expression of 3700 can affect the severity of the immune disorder.

Expression of 3700 increases with age in transgenic mice in which the apoE gene has been silenced. The apoE mouse is an accepted model of atherosclerosis, and genes that are upregulated in that model often have a role in establishment or progression of atherosclerosis. Inflammatory cytokines are also known to enhance expression of genes (e.g., those encoding VCAM and E-selectin) that are associated with establishment and progression of atherosclerosis. These observations indicate that 3700 is involved in atherosclerosis in humans, and the establishment and progression of atherosclerosis in humans can be modulated by modulating one or both of expression and activity of 3700. Expression of 3700 appears to be enhanced earlier than other known inflammatory effector molecules, indicating that inhibition of activity or expression of 3700 may have a more beneficial effect than therapeutic methods involving the other known inflammatory effector molecules.

The significant expression of 3700 in kidney tissues indicates a role for 3700 in the normal and aberrant functions of kidney tissues. Various kidney disorders can be associated with aberrant activity or expression of 3700. Examples of these kidney-related disorders in which 3700 can have a role include pancreatitis, endocrine and exocrine tumors of the pancreas, diabetes, pancreatic abscesses, pancreatic fibrocystic disease, and pancreatic cholera.

Expression of 3700 activity in astrocytes indicates that 3700 can have a significant role in modulating signaling between the blood and brain/central nervous system compartments. Ability of 3700 to contact molecules that are present in the bloodstream or in the cerebrospinal fluid and to modulate the phosphorylation state of a protein in response to such contact permits passage of a signal from one compartment to the other without the necessity for passage of a large molecule between the compartments. Regulation of 3700 expression by inflammatory cytokines indicates that 3700 protein can interact with relatively small peptide effectors which normally or aberrantly occur in blood or cerebrospinal fluid. Thus, modulation of 3700 activity or expression permits one to affect passage of signals between the blood and brain compartments.

Expression of 3700 in arterial tissue indicates that 3700 can have a role in formation of new blood vessels (angiogenesis), such as that associated with establishment or reestablishment of blood supply to a tumor or a wounded tissue. Higher levels of 3700 expression were detected in lung, colon, ovarian, and breast tumors than in the corresponding normal tissues. These observations indicate that 3700 can enhance establishment and increase of blood supply to tumors and other rapidly-growing tissues (e.g., traumatized arterial endothelium) and that modulation of 3700 activity, expression, or both, can limit establishment and increase of blood supply to such tissues.

3700 was more highly expressed in diseased liver tissue (e.g., liver tissue obtained from patients with fibrosed or HBV-infected livers) than in normal liver tissues. These observations indicate that 3700 can modulate liver tissue repair and that 3700 can also serve as an indicator of liver tissue damage. Increased expression of 3700 in damaged or diseased liver tissue indicates that such tissues are better able than non-damaged liver to react to the presence of inflammatory cytokines (e.g., inducing apoptosis of seriously damaged liver cells or increased attraction of cells which induce regeneration or repair of liver tissue) and that such tissues direct increased blood supply, relative to non-damaged liver tissues. These functions can be more generally applicable, meaning that increased expression of 3700 in cells of a non-liver tissue can enhance blood supply to the tissue and can enhance repair or regeneration of the tissue.

Modulation of 3700 activity, expression, or both can be used to inhibit, prevent, alleviate, or cure the disorders discussed herein. Furthermore, assessment of the level of 3700 activity, expression, or both, can be used to diagnose or prognosticate these disorders.

Without being bound by any particular theory of operation, it is believed that the ability of 3700 protein to phosphorylate proteins, combined with its transmembrane nature, indicates an ability of 3700 protein to transmit signals from the external environment of the cell to the interior of the cell. Protein phosphorylation (e.g., that associated with G-protein signaling) is known to be a method by which transcription of genes can be modulated in response to extracellular stimuli. 3700 protein can bind molecules (e.g., inflammatory cytokines such as tumor growth factor beta or endothelial growth factor) in the extracellular milieu, undergo a conformational or other change, and exhibit an intracellular protein kinase activity. The intracellularly phosphorylated protein can phosphorylate another protein or affect the conformation or protein-binding-state of a nucleic acid. Thus, directly or indirectly, 3700 can affect the likelihood or rate at which a gene is transcribed, thereby correlating occurrence of an intracellular gene product with the presence of an extracellular signaling molecule. In one embodiment, the membrane in which 3700 protein is embedded is the nuclear membrane, and 3700 protein catalyzes a change in the phosphorylation state of a nuclear membrane protein or an intranuclear protein in response to occurrence of a signaling molecule in the cytoplasm of the cell.

Identification and Characterization of Human 3700 cDNA

The human 3700 nucleotide sequence (SEQ ID NO:43), which is approximately 3353 nucleotides in length including non-translated regions, contains a predicted methionine-initiated coding sequence at about nucleotide residues 157-2040. The coding sequence encodes a 628 amino acid protein (SEQ ID NO:44).

Expression of the 3700 Gene

Tables 30-41 list the results of real time quantitative PCR (TAQMAN®) analyses of 3700 gene expression in selected cells and tissues. In the Tables, “M” means monkey.

Human 21529

The present invention is based, at least in part, on the identification of novel molecules, referred to herein as “21529”, also known as adenylate cyclase nucleic acid and polypeptide molecules, which play a key role in regulation of the cyclic AMP (cAMP) signal transduction pathway by virtue of their conversion of intracellular ATP into cAMP. In one embodiment, the adenylate cyclase molecules modulate the activity of one or more proteins involved in cellular metabolism associated with cell maintenance, growth, or differentiation, e.g., cardiac, epithelial, or neuronal cell maintenance, growth, or differentiation. In another embodiment, the adenylate cyclase molecules of the present invention are capable of modulating the phosphorylation state of one or more proteins involved in cellular metabolism associated with cell maintenance, growth, or differentiation, e.g., cardiac, epithelial, or neuronal cell maintenance, growth or differentiation, via their indirect effect on cAMP-dependent protein kinases, particularly protein kinase A, as described in, for example, Devlin (1997) Textbook of Biochemistry with Clinical Correlations (Wiley-Liss, Inc., New York, N.Y.). In addition, the receptors which trigger activity of the adenylate cyclases of the present invention are targets of drugs as described in Goodman and Gilman (1996), The Pharmacological Basis of Therapeutics (9 th ed.) Hartman & Limbard Editors, the contents of which are incorporated herein by reference. Particularly, the adenylate cyclase molecules of the invention may modulate phosphorylation activity in tissues in which the polypeptides are highly expressed, including but not limited to skeletal muscle, heart, cervix, vein, brain, pancreas, breast, fetal kidney, fetal liver, and fetal heart.

Furthermore, 21529 expression may be modulated in tissues in which the 21529 polypeptides are expressed including, but not limited to, skeletal muscle, heart, cervix, vein, brain, pancreas, breast, fetal kidney, fetal liver, and fetal heart, which provides a profile of expression in normal human tissues. In addition, upregulation is observed in breast carcinoma. Therefore, modulation is particularly relevant in this disorder. Further, 21529 downregulation is shown in both lung and colon carcinoma. Therefore, modulation is also relevant in these tissues. In colonic liver metastases, however, there is significant upregulation. Accordingly, modulation is important in these tissues. Furthermore, 21529 expression occurs in cardiovascular tissues, such as, but are not limited to, aorta, aorta with intimal proliferation (atheroplaques), coronary artery, internal mammary artery, heart, especially heart derived from patients with congestive heart failure and heart tissue derived from myopathic patients, ischemic heart, and saphenous vein, (the chief superficial vein found in the human leg). Finally, as further discussed herein, the 21529 gene is expressed in hypertrophic cardiac myocytes from diseased subjects. Accordingly, 21529 modulation is particularly relevant in disorders that include but are not limited to congestive heart failure, ischemia, hypertension, myocardial infarction, atherosclerosis, cardiomyopathy, and other diseases of the cardiovascular system as disclosed herein.

In a preferred embodiment, the adenylate cyclase molecules of the invention are used to modulate the cyclic AMP (cAMP) signal transduction pathway. Cyclic AMP is a second messenger produced in response to ligand-induced stimulation of certain G-protein-coupled receptors (GPCR). In the cAMP signal transduction pathway, binding of a ligand to a GPCR leads to the activation of adenylate cyclase, which then catalyzes the synthesis of cAMP. The newly synthesized cAMP can in turn activate a cAMP-dependent protein kinase, such as protein kinase A. The activated cAMP-dependent kinases can, through a series of intermediate steps, regulate transcription factors and stimulate expression of target genes, as well as phosphorylate other downstream target proteins that are involved in a host of metabolic pathways. In addition, activated cAMP-dependent protein kinases can phosphorylate a voltage-gated potassium channel protein and lead to the inability of the potassium channel to open during an action potential. The inability of the potassium channel to open results in a decrease in the outward flow of potassium, which normally repolarizes the membrane of a neuron, leading to prolonged membrane depolarization.

Cyclic AMP also influences cardiovascular physiology. For instance, cAMP activates protein kinase A (PKA). The activated subunits of PKA initiate a series of enzymatic reactions that ultimately activate multiple proteins that regulate both the rate and force of cardiac contraction. For instance, phosphorylation of the L-type calcium channel enhances calcium entry into cardiocytes leading to increased contractility. Upon phosphorylation of phospholamban, the inhibition exerted by the non phosphorylated form of phospholamban on the sarcoplasmic reticulum calcium pump is removed, and its rate of calcium uptake increased, thereby leading to a more rapid decrease of the cytosolic calcium concentration during diastole. Dissociation of the troponin C-calcium complex is also enhanced when troponin I is phosphorylated which leads to an accelerated relaxation rate. Such events result in the enhancement of cardiac output. This process rapidly reverses when agonist occupancy of the receptor ceases, i.e. the reuptake of norepinephrine into presynaptic stores. For a review, see for example, Yoshihiro et al. (1997) Circulation Research 80:297-304 and Castellano et al. (1997) Hypertension 29:715-722.

As the enzyme that catalyzes conversion of intracellular ATP to cAMP, adenylate cyclase plays a central role in the regulation of cellular cAMP concentrations. Disruption or modulation of adenylate cyclase activity affects intracellular concentrations of cAMP, which can in turn modulate the cAMP signal transduction pathway.

Many cardiovascular patho-physiological conditions result from modulations in the cAMP signaling pathway. Therefore, changes in concentration and function of receptors, G-proteins, and adenylate cyclase may thus constitute fundamental defects underlying certain cardiac diseases.

Alterations that accompany physiological changes in cardiovascular function include, for example, transformations of the myocardial structure and function such as a transition of the myosin heavy chain isoform (Imumo et al. (1987) J Clin Invest 79:970-977), accumulation of alpha-skeletal muscle actin mRNA (Schwartz et al. (1986) Circ Res 59:551-555) changes in troponin isoforms (Mayer et al. (1995) Curr Opin Cardiol 10:238-245) deterioration of Na+K+-ATPases (Charlemagne et al. (1986) J Biol Chem 261:185-189) and collagen remodeling of myocardium (Wever et al. (1988) Circ Res 62:757-763). Further changes in physiological cardiovascular function resulting from various forms of heart failure include alterations in arterial tone and reactivity and alterations in platelet function including aggregation, secretion, and clot formation and blood pressure elevation. (Marcil et al. (1996) Hypertension 28:83-90).

Adenylate cyclase has been implicated in many cardiovascular diseases. For example, adenylate cyclase activity and its responsiveness to various hormones is altered in hypertensive patients. Aberrant adenylate cyclase levels in hypertensive patients were restored toward normal following antihypertensive drug therapy (Marcil et al. (1996) Hypertension 28:83-90). In addition, studies of heart in human and animal models indicate adenylate cyclase has function in cardiomyopathy (Michael et al. (1995) Hypertension 25:962-970, Roth et al (1999) Circulation 99:3099-3099), ischemia (Sandhu et al. (1996) Circulation Research 78:137-147), myocardial infarction (Espinasse et al. (1999) Cardiovascular Research 42:87-98) and congestive heart failure (Kawahira et al. (1998) Circulation 98:262-267, Panza et al. (1995) Circulation 91:1732-1738). Additionally, studies have indicated that adenylate cyclase has function in clinical situations resulting in myocardial dysfunction such as cardiopulmonary bypass (Booth et al. (1998) Anesthesiology 89: 602-611). Decreased concentrations of adenylate cyclase also occur in chronic pacing-induced heart failure (Ishikawa et al. (1994) J Clin Invest 93:2224-9), whereas changes in activity of adenylate cyclase isoforms occur with activation of PKC (Kawabe et al. (1994) J Biol Chem 169: 16554-8), PKA (Chen et al. (1997) PNAS 94: 14100-4), aging and in pressure-overload failing right ventricles (Bristow et al. (1992) J Clin Invest 89:803-15).

As the enzyme that catalyzes conversion of intracellular ATP to cAMP, adenylate cyclase plays a central role in the regulation of cellular cAMP concentrations. Disruption or modulation of adenylate cyclase activity affects intracellular concentrations of cAMP, which can in turn modulate the cAMP signal transduction pathway. Modulation of this pathway can disrupt or alter cellular metabolism, growth, and differentiation, potentially leading to cellular growth related-disorders. As used herein, a “cellular growth-related disorder” includes a disorder, disease, or condition characterized by a deregulation, e.g., an upregulation or a downregulation, of cellular growth. Cellular growth deregulation may be due to a deregulation of cellular proliferation, cell cycle progression, cellular differentiation and/or cellular hypertrophy. Examples of cellular growth related disorders include cardiovascular disorders such as heart failure, hypertension, atrial fibrillation, dilated cardiomyopathy, idiopathic cardiomyopathy, or angina; proliferative disorders or differentiative disorders such as cancer, e.g., melanoma, prostate cancer, cervical cancer, breast cancer, colon cancer, or sarcoma. Disorders associated with the tissues in which 21529 is expressed are also encompassed, especially skeletal muscle, heart, aorta, cervix, vein, brain, pancreas, and fetal kidney. Other disorders include tumors of the breast, lung, and colon. Disorders that are particularly relevant with respect to expression of the adenylate cyclase are cardiovascular disorders. As described above, the 21529 adenylate cyclase is expressed in human cardiovascular tissues. Further, the 21529 gene is highly expressed in hypertrophic cardiac myocytes. Accordingly, disorders that are relevant include hypertension, atherosclerosis, ischemia, cardiomyopathy, congestive heart failure, myocardial infarction, and diseases of the cardiovascular system as disclosed herein.

The disclosed invention relates to methods and compositions for the modulation, diagnosis, and treatment of adenylate cyclase-associated or related disorders, particularly disorders resulting from aberrations in components of the cAMP signal transduction pathway, such as cAMP-dependent disorders, and disorders associated with cAMP-dependent protein kinases. Such disorders include, but are not limited to, disorders involving the skeletal muscle, heart, cervix, blood vessels, brain, pancreas, and cardiovascular system. Further relevant disorders include disorders involving the breast, and especially tumors of the breast.

Specifically, the present invention provides isolated nucleic acid molecules comprising nucleotide sequences encoding the 21529 adenylate cyclase polypeptide whose amino acid sequence is given in SEQ ID NO:47, or a variant or fragment of the polypeptide. A nucleotide sequence encoding an adenylate cyclase polypeptide of the invention, more particularly the polypeptide of SEQ ID NO:47, is set forth in SEQ ID NO:46 and 48.

A novel human gene, termed clone 21529 is provided. This sequence, and complements thereof, are referred to as “adenylate cyclase” sequences indicating that the gene sequences share sequence similarity to adenylate cyclase genes.

The novel 21529 adenylate cyclase gene encodes an approximately 3.52 Kb mRNA transcript having the corresponding cDNA set forth in SEQ ID NO:46. This transcript has a 3231 nucleotide open reading frame (nucleotides 247-3477 of SEQ ID NO:46; nucleotides 1-3231 of SEQ ID NO:48), which encodes a 1077 amino acid protein (SEQ ID NO:47). An analysis of the full-length 21529 polypeptide predicts that the N-terminal 50 amino acids may represent a region comprising a signal peptide. MEMSAT program analysis of the full-length 21529 polypeptide predicted transmembrane segments at amino acid residues (aa) 27-50, 61-79, 92-113, 120-136, 143-160, 174-190, 365-381, 408-424, 589-605, 612-631, 664-685, 713-736, 744-760, and 790-807 of SEQ ID NO:47. Transmembrane segments for the presumed mature peptide (aa 51-1077) were predicted at aa 11-29, 42-63, 70-86, 93-110, 124-140, 315-331, 358-374, 539-555, 562-581, 614-635, 663-686, 694-710, and 740-757 of SEQ ID NO:47. Prosite program analysis was used to predict various sites within the 21529 protein. N-glycosylation sites were predicted at aa 697-700, 704-707, 836-839, and 938-941 of SEQ ID NO:47, with the actual modified residue being the first amino acid. Protein kinase C phosphorylation sites were predicted at aa 6-8, 51-53, 202-204, 212-214, 218-220, 290-292, 526-528, 550-552, and 606-608 of SEQ ID NO:47, with the actual modified residue being the first amino acid. Casein kinase II phosphorylation sites were predicted at aa 51-54, 115-118, 202-205, 253-256, 290-293, 333-336, 359-362, 465-468, 495-498, 687-690, 878-881, 919-922, 941-944, 958-961, 968-971, and 1015-1018 of SEQ ID NO:47, with the actual modified residue being the first amino acid. Tyrosine kinase phosphorylation sites were predicted at aa 318-325, 437-444, 570-576, and 859-865 of SEQ ID NO:47, with the actual modified residue being the last amino acid. N-myristoylation sites were predicted at aa 35-40, 111-116, 137-142, 145-150, 184-189, 329-334, 345-350, 360-365, 368-373, 402-407, 412-417, 458-463, 654-659, 661-666, 936-941, 995-1000, 1008-1013, and 1055-1060 of SEQ ID NO:47, with the actual modified residue being the first amino acid. A prokaryotic membrane lipoprotein lipid attachment site was predicted at aa 745-755 of SEQ ID NO:47, and a leucine zipper pattern was predicted at aa 55-76 of SEQ ID NO:47. Guanylate cyclase signature sequences were predicted at aa 377-400 and 995-1018 of SEQ ID NO:47.

The 21529 adenylate cyclase protein possesses two adenylate/guanylate cyclase catalytic domains, from aa 264-448 and aa 864-1064 of SEQ ID NO:47, as predicted by HMMer, Version 2. Other domain matches predicted by HMMer included a copper/zinc superoxide dismutase domain, from aa 376-383 of SEQ ID NO:47, and a eubacterial secY protein domain, from aa 60-385 of SEQ ID NO:47.

The 21529 protein displays closest similarity to the rat adenylate cyclase IV (CYA4) (SP Accession Number P26770), approximately 86% identity over their 1075 amino acid overlap.

A plasmid containing the 21529 cDNA insert was deposited with the Patent Depository of the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va., on Apr. 6, 2000, and assigned Patent Deposit Number PTA-1661. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

The 21529 adenylate cyclase sequences of the invention are members of a family of molecules having conserved functional features. The term “family” when referring to the proteins and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having sufficient amino acid or nucleotide sequence identity as defined herein. Such family members can be naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of murine origin and a homolog of that protein of human origin, as well as a second, distinct protein of human origin and a murine homolog of that protein. Members of a family may also have common functional characteristics.

Another embodiment of the invention features isolated adenylate cyclase proteins and polypeptides having an adenylate cyclase protein activity. As used interchangeably herein, a “adenylate cyclase protein activity”, “biological activity of an adenylate cyclase protein”, or “functional activity of an adenylate cyclase protein” refers to an activity exerted by an adenylate cyclase protein, polypeptide, or nucleic acid molecule on an adenylate cyclase responsive cell as determined in vivo, or in vitro, according to standard assay techniques. An adenylate cyclase activity can be a direct activity, such as conversion of intracellular ATP to cAMP, or an indirect activity, such as a cellular activity mediated by generation of cAMP, such as any downstream cellular response associated with the cAMP signal transduction pathway. In a preferred embodiment, a 21529 adenylate cyclase activity includes at least one or more of the following activities: (1) modulating (stimulating and/or enhancing or inhibiting) cellular growth, differentiation, and/or function, particularly in cells in which the sequences are expressed, for example, cells of the skeletal muscle, heart, cervix, vein, brain, pancreas, fetal kidney, and breast tumors, and cardiovascular tissue; a protein kinase A cellular effect, such as release of hormones, glycogen metabolism, such as in liver, heart, and skeletal muscles; (2) modulating the cAMP signal transduction pathway; (3) modulating a target cell's cAMP concentration; (4) modulating cAMP-dependent protein kinase activity, such as protein kinase A; and (5) modulating the release of hormones, such as release of cortisol in the adrenal gland cells, thyroid hormones from the thyroid gland, testosterone from testicular Leydig cells, and melatonin from the pineal gland.

Isolation of 21529

Clone 21529 was isolated from a human spleen or heart cDNA library. The identified clone 21529 encodes a transcript of approximately 3.52 Kb (corresponding cDNA set forth in SEQ ID NO:46). The open reading frame (nucleotides 247-3477 of SEQ ID NO:46; nucleotides 1-3231 of SEQ ID NO:48) of this transcript encodes a predicted 1077 amino acid protein (SEQ ID NO:47). This novel gene is preliminarily mapped to human chromosome 14 using the mapping panel Genebridge 4 human RH.

A search of the nucleotide and protein databases revealed that 21529 encodes a polypeptide that shares similarity with several adenylate cyclases, the greatest similarity being seen with the rat adenylate cyclase type IV protein (SP Accession Number P26770). An alignment of the 21529 polypeptide with this rat protein, using the Clustal method with PAM250 residue weight table, demonstrates the overall close similarity between the two sequences and indicates that 21529 is the human ortholog of the rat adenylate cyclase type IV.

mRNA Expression of Clone 21529

Expression of the novel 21529 adenylate cyclase was measured by TaqMan® quantitative PCR (Perkin Elmer Applied Biosystems) in cDNA prepared from the following normal human tissues: thymus, skeletal muscle, liver, lung, thyroid, heart, ovary, aorta, placenta, cervix, lymph node, vein, brain, esophagus, pancreas, kidney, brain, prostate, liver, spleen, breast, colon, tonsil, small intestine, fetal kidney, fetal liver, fetal heart, and testis.

Probes were designed by PrimerExpress software (PE Biosystems) based on the 21529 sequence. The primers and probes for expression analysis of 21529 and β-2 microglobulin were as follows:

The 21529 sequence probe was labeled using FAM (6-carboxyfluorescein), and the β2-microglobulin reference probe was labeled with a different fluorescent dye, VIC. The differential labeling of the target adenylate cyclase sequence and internal reference gene thus enabled measurement in the same well. Forward and reverse primers and the probes for both β2-microglobulin and the target 21529 sequence were added to the TaqMan® Universal PCR Master Mix (PE Applied Biosystems). Although the final concentration of primer and probe could vary, each was internally consistent within a given experiment. A typical experiment contained 200 nM of forward and reverse primers plus 100 nM probe for β-2 microglobulin and 600 nM forward and reverse primers plus 200 nM probe for the target 21529 sequence. TaqMan matrix experiments were carried out on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). The thermal cycler conditions were as follows: hold for 2 min at 50° C. and 10 min at 95° C., followed by two-step PCR for 40 cycles of 95° C. for 15 sec followed by 60° C. for 1 min.

The following method was used to quantitatively calculate 21529 expression in the various tissues relative to β-2 microglobulin expression in the same tissue. The threshold cycle (Ct) value is defined as the cycle at which a statistically significant increase in fluorescence is detected. A lower Ct value is indicative of a higher mRNA concentration. The Ct value of the 21529 sequence is normalized by subtracting the Ct value of the β-2 microglobulin gene to obtain a Δ Ct value using the following formula: Δ Ct=Ct h21529 −Ct β-2 microglobulin . Expression is then calibrated against a cDNA sample showing a comparatively low level of expression of the 21529 sequence. The Δ Ct value for the calibrator sample is then subtracted from Δ Ct for each tissue sample according to the following formula: ΔΔ Ct= Δ Ct- sample − Δ Ct- calibrator . Relative expression is then calculated using the arithmetic formula given by 2 −ΔΔCt . Expression of the target 21529 sequence in each of the tissues tested was then analyzed as discussed in more detail below.

The mRNA for the putative adenylate cyclase 21529 is differentially expressed in all of the normal tissues tested. There was significant expression in pancreas, vein, brain, heart, and skeletal muscle; moderate expression in cervix, fetal kidney, fetal heart, liver, placenta, thyroid, ovary, breast, aorta, and brain; and lower expression in lymph node, esophagus, kidney, lung, spleen, testis, small intestine, fetal liver, colon, prostate, thymus, and tonsil. These data indicate this novel adenylate cyclase has a widely dispersed pattern of expression, a characteristic in common with the rat adenylate cyclase IV homolog.

TaqMan data obtained using an Oncology panel wherein normal breast, normal lung, normal colon and normal liver tissue samples were compared to breast tumor, lung tumor, colon tumor and liver tumor samples, respectively, demonstrated that 21529 was upregulated in breast and colon tumor samples compared to their respective normal tissue samples.

mRNA Expression of Clone 21529 in Human Cardiovascular Tissues

mRNA was hybridized as discussed above in the following cardiovascular tissues: aorta, aorta with intimal proliferation, coronary artery, mammary internal artery, heart, congestive heart failure heart samples, ischemic heart samples, myopathic heart samples, and saphenous vein. These were compared in terms of relative expression to the expression of the gene in skeletal muscle. Highest expression was observed in tissue from congestive heart failure patients and myopathic hearts. Significant expression was also observed in coronary artery and in the internal mammary artery. Further, significant expression was also observed in ischemic heart. Lower levels of expression were observed in the remainder of the tissues.

Further, in situ hybridization experiments were done against hypertrophic cardiac myocytes from diseased hearts. Results showed increased expression of the gene in the hypertrophic myocytes.

Human 26176

The present invention is based, at least in part, on the discovery of a novel calpain protease referred to herein as “26176”. The present invention provides isolated nucleic acid molecules comprising nucleotide sequences encoding the 26176 calpain protease polypeptide whose amino acid sequence is given in SEQ ID NO:50, or a variant or fragment of the polypeptide. A nucleotide sequence encoding the 26176 calpain protease polypeptides of the invention is set forth in SEQ ID NO:49. The sequences are members of the calpain family of thiol proteases, also referred to as the peptidase family C2.

Calpains refer to calcium-activated neutral proteinases, a superfamily of endopeptidases typically having cysteine-proteinase and calcium-binding characteristics. These proteinases cleave numerous substrate proteins in a limited manner, typically leading to modification of the function and/or activity rather than general degradation of the substrate.

Calpains are classified into two main groups, the typical or conventional calpains and the atypical calpains, based on their domain content and/or variation. The typical calpains are further subdivided into ubiquitous and tissue-specific calpains based on their predominate patterns of expression.

Two forms of ubiquitous calpains have been extensively characterized in vertebrates: the μ-calpains (calpain I, CAPN1) and the m-calpains (calpain II, CAPN2), which are activated in vitro by micro- and millimolar calcium concentrations, respectively. An intermediate μ/m calpain has been characterized in chicken.

The ubiquitous μ- and m-calpains are heterodimers, each having a distinct, but homologous, large 80 kDa subunit (referred to as μCL or mCL, respectively) and an identical small 30 kDa subunit (referred to as 30K or Cs). The large subunit has four domains, designated I-IV from the N-terminus to the C-terminus. The function of domain I is unclear. Domain II is the cysteine protease domain responsible for calpain protease activity. Domain III is homologous to a calmodulin-binding protein and is speculated to interact with the calcium-binding domains of the large (domain IV) and small subunits (domain VI), when calcium is bound, thereby freeing the protease domain for activity (Goll et al. (1992) BioEssays 14:549-556). Domain IV of the large subunit is a calmodulin-like calcium-binding domain containing four EF-hand calcium-binding motifs. Although structurally similar to calmodulin, domain IV is more similar to sorcin, ALG-2, and grancalcin. Sorcin is involved in the multi-drug resistance of cultured cell lines and was recently reported to associate with the cardiac ryanodine receptor. Grancalcin possibly plays a role in granule-membrane fusion and degranulation. ALG-2 is thought to be involved in apoptosis and is induced by tumor promoters. See Meyers et al. (1995) J. Biol. Chem. 270:26411-26418; Meyers et al. (1985) J. Cell Biol. 100:588-597; Vito et al. (1996) Science 271:521-525; Teahan et al. (1992) Biochem. J. 286:549-554; Boyhan et al. (1992) J. Biol. Chem. 267:2928-2933.

The small subunit of typical calpains contains two domains, which are designated V and VI from the N-terminus to the C-terminus. Domain V is an N-terminal glycine-clustering hydrophobic region. Domain VI, which is similar to domain IV of the large subunit, is also a calcium-binding domain containing six EF-hands, EF2-EF5 as in the large subunit, and EF1 and EF6. EF5 of domain VI does not bind calcium and is proposed to be involved in the heterodimeric binding of domains IV and VI during interaction between the large and small subunits.

Calpastatin is an endogenous inhibitor of most calpains, the tissue-specific calpain p94 being an exception. Calpastatin, which has five domains, is cleaved by calpain in the interdomain regions, generating inhibitory peptides. The inhibitory effect of calpastatin has been attributed to interactions with calpain domains II, III, IV, and VI. The reactive site of calpastatin shows no apparent homology to that of other protease inhibitors, and it contains the consensus sequence TIPPXYR (SEQ ID NO:52), which is essential for inhibition. See Kawasaki et al. (1989) J. Biochem. 106:274-281; Croall et al. (1994) Biochem. 33:13223-13230; Croall et al. (1991) Physiol. Rev. 71:813-847; Kawasaki et al. (1996) Mol. Membr. Biol. 13:217-224; Melloni et al. (1989) Trends Neurosci. 12:438-444; Sorimachi et al. (1997) J. Biochem. 328:721-732; and Johnson et al. (1997) BioEssays 19(11): 1011-1018.

Several typical tissue-specific calpains are known in vertebrates, including skeletal muscle p94 (nCL-1, calpain 3′, CAPN3), stomach nCL2 (CAPN4) and nCL 2′, and digestive tubule nCL4. While p94 contains EF hands, it does not require calcium for proteinase activity. p94 has a domain IV sequence similar to that of μCL and mCL, but it does not bind to a small 30 kDa subunit (Kinbara et al. (1997) Arch. Biochem. Biophys. 342:99-107). p94 contains unique insertion sequences called IS1 and IS2, which are found in domain II and between domains III and IV, respectively). IS2 contains a nuclear-localization-signal-like basic sequence (Arg-Pro-Xaa-Lys-Lys-Lys-Lys-Xaa-Lys-Pro (SEQ ID NO:53)). Connectin/titin binding is also attributed to IS2. p94 may change its localization in a cell-cycle dependent manner and may be involved in muscle differentiation by interacting with the MyoD family. In fact, a defect in the protease p94 is responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). See Sorimachi et al. (1995) J. Biol. Chem. 270:31158-31162; Sorimachi et al. (1993) J. Biol. Chem. 268:10593-10605; Gregoriou et al. (1994) Eur. J. Biochem. 223:455-464; and Belcastro et al. (1998) Mol. Cell. Biochem. 179 (1, 2):135-145.

Calpains have broad physiological and pathological roles related to the enzymes' diverse population of substrates. Calpain substrates include “PEST” proteins, which have high proline, glutamine, serine, and threonine contents; calpain and calpastatin; signal transduction proteins including protein kinase C, transcription factors c-Jun, c-Fos, and α-subunit of heterotrimeric G proteins; proteins involved in cell proliferation and cancer including P53 tumor suppressor, growth factor receptors (eg., epidermal growth factor receptor), c-Jun, c-Fos, and N-myc; proteins with established physiological roles in muscle including Ca ++ -ATPase, Band III, troponin, tropomyosin, and myosin light chain kinase; myotonin protein kinase; proteins with established physiological roles in the brain and the central nervous system including myelin proteins, myelin basic protein (MBP), axonal neurofilament protein (NFP), myelin protein MAG; cytosketetal and cell adhesion proteins including troponins, talin, neurofilaments, spectrin, microtubule associated protein MAP-2, tau, MAPIB, fodrin, desmin, α-actinin, vimentin, spectrin, integrin, cadherin, filamin, and N-CAM; enzymes including protein kinases A and C, and phospholipase C; and histones.

See Sorimachi et al. (1997) J. Biochem. 328:721-732; Johnson et al. (1997) BioEssays 19(11):1011-1018; Shields et al. (1999) J. Neuroscience Res. 55(5):533-541; and Belcastro et al. (1998) Mol. Cell. Biochem. 179 (1, 2):135-145.

Calpain is implicated in a wide variety of physiological processes including alteration of membrane morphology, long-term potentiation of memory, axonal regeneration, neurite extension, cell proliferation (division), gastric HCl secretion, embryonic development, secretory granule movement, cell differentiation and regulation, cytoskeletal and membrane changes during cell migration, cytoskeletal remodeling, sex determination, and alkaline adaptation in fungi. See Solary et al. (1998) Cell Biol. Toxicol. 14:121-132; Sorimachi et al. (1997) J. Biochem. 328:721-732; Johnson et al. (1997) BioEssays 19(11):1011-1018; Suzuki et al. (1998) FEBS Letters 433(1, 2):1-4; Franz et al. (1999) Mammalian Genome 10(3):318-321; Shields et al. (1999) J. Neuroscience Res. 55(5):533-541; Schnellmann et al. (1998) Renal Failure 20(5):679-686; Banik et al. (1998) Annals New York Acad. Sc. 844:131-137; Belcastro et al. (1998) Mol. Cell. Biochem. 179 (1, 2):135-145; and McIntosh et al. (1998) J. Neurotrauma 15(10):731-769.

Under pathological conditions, aberrant regulation and/or activity of calpain can be detrimental to cells and tissues. In this context, calpains are implicated in a wide variety of disease states including exercise-induced injury and repair; apoptosis including T cell receptor-induced apoptosis, HIV-infected cell apoptosis, ectoposide-treated cell apoptosis, nerve growth factor deprived neuronal apoptosis; ischemia, such as cerebral and myocardial ischemia; traumatic brain injury; Alzheimer's disease and other neurodegenerative diseases; demyelinating diseases including experimental allergic encephalomyelitis (EAE) and multiple sclerosis; LGMD2A muscular dystrophy; spinal cord injury (SCI); cancer; cataract formation; and renal cell death by diverse toxicants.

The disclosed invention relates to methods and compositions for the modulation, diagnosis, and treatment of calpain protease-mediated disorders. Such disorders include, but are not limited to, disorders associated with perturbed cellular growth and differentiation; exercise-induced injury and repair; apoptosis including T-cell receptor-induced apoptosis, HIV-infected cell apoptosis, ectoposide-treated cell apoptosis, nerve growth factor deprived neuronal apoptosis; ischemia; traumatic brain injury; Alzheimer's disease and other neurodegenerative diseases; demyelinating diseases including experimental allergic encephalomyelitis (EAE) and multiple sclerosis; LGMD2A muscular dystrophy; spinal cord injury (SCI); proliferative disorders or differentiative disorders such as cancer, e.g., melanoma, prostate cancer, cervical cancer, breast cancer, colon cancer, or sarcoma; and renal cell death associated with diverse toxicants.

The sequences of the invention find use in diagnosis of disorders involving an increase or decrease in protease expression relative to normal expression, such as a proliferative disorder, a differentiative disorder, or a developmental disorder. The sequences also find use in modulating protease-related responses. By “modulating” is intended the upregulating or downregulating of a response. That is, the compositions of the invention affect the targeted activity in either a positive or negative fashion.

One embodiment of the invention features protease nucleic acid molecules, preferably human protease molecules, which were identified based on a consensus motif or protein domain characteristic of the calpain family of thiol proteases. Specifically, a novel human gene, termed clone 26176, is provided. This sequence, and other nucleotide sequences encoding the 26176 protein or fragments and variants thereof, are referred to as “calpain protease sequences” indicating that the sequences share sequence similarity to other calpain protease genes.

The calpain protease gene designated clone 26176 was identified in a human T-cell cDNA library. Clone 26176 encodes an approximately 3.78 Kb mRNA transcript having the corresponding cDNA set forth in SEQ ID NO:49. This transcript has a 2439 nucleotide open reading frame (nucleotides 276-2714 of SEQ ID NO:49; nucleotides 1-2439 of SEQ ID NO:51), which encodes an 813 amino acid protein (SEQ ID NO:50). MEMSAT analysis of the full-length 26176 polypeptide predicts a transmembrane segment from amino acids (aa) 286-302 of SEQ ID NO:50. Prosite program analysis was used to predict various sites within the 26176 protein. An N-glycosylation site was predicted at aa 366-369 of SEQ ID NO:50 with the actual residue being the first residue. A cAMP- and cGMP-dependent protein kinase phosphorylation site was predicted at aa 759-762 of SEQ ID NO:50 with the actual phosphorylated residue being the last residue. Protein kinase C phosphorylation sites were predicted at aa 165-167, 215-217, 251-253, 281-283, 422-424, 594-596, 668-670, 689-691, and 710-712 of SEQ ID NO:50 with the actual phosphorylated residue being the first residue. Casein kinase II phosphorylation sites were predicted at aa 4-7, 48-51, 123-126, 205-208, 373-376, 393-396, 445-448, 490-493, 523-526, 551-554, 594-597, 657-660, 748-751, and 761-764 of SEQ ID NO:50 with the actual phosphorylated residue being the first residue. Tyrosine kinase phosphorylation sites were predicted at aa 20-26 and aa 320-326 of SEQ ID NO:50 with the actual phosphorylated residue being the last. N-myristoylation sites were predicted at aa 201-206, 390-395, 453-458, 630-635, and 698-703 of SEQ ID NO:50 with the actual modified residue being the first. An amidation site was predicted at aa 614-617 of SEQ ID NO:50. The calpain protease protein 26176 possesses a calpain family cysteine protease domain (domain II), from aa 231-537 of SEQ ID NO:50, and a calpain large subunit domain III, from aa 685-810 of SEQ ID NO:50, as predicted by HMMer, Version 2.

The protein displays the closest similarity to the human gene designated PalBH, (Accession Numbers GPU:gi [5102944] dbj [BAA78730] (AB028639).

The 26176 protein also displays similarity to the murine CAPN7 protein, approximately 93% identity and 95% overall similarity over a 768 amino acid overlap (amino acid residues 45-813 of the 26176 protein (SEQ ID NO:50)), indicating 26176 is the human ortholog of this murine protein.

A plasmid containing the 26176 cDNA insert was deposited with the Patent Depository of the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va., on Apr. 6, 2000, and assigned Patent Deposit Number PTA-1649. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. 112.

The calpain protease sequences of the invention are members of a protease family of molecules having conserved functional features. The term “family” when referring to the proteins and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having sufficient amino acid or nucleotide sequence identity as defined herein. Such family members can be naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of murine origin and an ortholog of that protein of human origin, as well as a second, distinct protein of human origin and a murine ortholog of that protein. Members of a family may also have common functional characteristics.

Preferred 26176 calpain protease polypeptides of the present invention have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:50. The term “sufficiently identical” is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 45%, 55%, or 65% identity, preferably 75% identity, more preferably 85%, 95%, or 98% identity are defined herein as sufficiently identical.

Another embodiment of the invention features isolated calpain protease proteins and polypeptides having a calpain protease protein activity. As used interchangeably herein, a “calpain protease protein activity”, “biological activity of a calpain protease protein”, or “functional activity of a calpain protease protein” refers to an activity exerted by a calpain protease protein, polypeptide, or nucleic acid molecule on a calpain-protease-responsive cell as determined in vivo, or in vitro, according to standard assay techniques. A calpain protease activity can be a direct activity, such as an association with or an enzymatic activity on a second protein, or an indirect activity, such as a cellular signaling activity mediated by interaction of the calpain protease protein with a second protein. In a preferred embodiment, a 26176 calpain protease activity includes at least one or more of the following activities: (1) modulating (stimulating and/or enhancing or inhibiting) cellular proliferation, differentiation, and/or function (e.g., in cells in which it is expressed, for example, cells within normal and carcinoma tissues, such as lung, liver, colon, and breast; brain and skeletal muscle cells, etc.); (2) modulating a calpain protease response; (3) modulating the entry of cells into mitosis; (4) modulating cellular differentiation; and (5) modulating cell death.

Isolation of 26176

Clone 26176 was isolated from a human T-cell cDNA library. The identified clone 26176 encodes a transcript of approximately 3.78 Kb (corresponding cDNA set forth in SEQ ID NO:49). The open reading frame (nucleotides 276-2714 of SEQ ID NO:49; nucleotides 1-2439 of SEQ ID NO:51) of this transcript encodes a predicted 813 amino acid protein (SEQ ID NO:50)

A search of the nucleotide and protein databases revealed that 26176 encodes a polypeptide that shares similarity with several calpain proteases, the greatest similarity being seen with the murine CAPN7 protein (EMB Accession Number AJ012475).

mRNA Expression of Clone 26176

Expression of the novel 26176 calpain protease was measured by TaqMan® quantitative PCR (Perkin Elmer Applied Biosystems) in cDNA prepared from the following human tissues: normal colon, colon carcinoma, normal liver, colon metastasis, normal lung, lung carcinoma, normal breast, and breast carcinoma.

Probes were designed by PrimerExpress software (PE Biosystems) based on the 26176 sequence. The primers and probes for expression analysis of 26176 and β-2 microglobulin were as follows:

The 26176 sequence probe was labeled using FAM (6-carboxyfluorescein), and the β2-microglobulin reference probe was labeled with a different fluorescent dye, VIC. The differential labeling of the target calpain protease sequence and internal reference gene thus enabled measurement in the same well. Forward and reverse primers and the probes for both β2-microglobulin and the target 26176 sequence were added to the TaqMan® Universal PCR Master Mix (PE Applied Biosystems). Although the final concentration of primer and probe could vary, each was internally consistent within a given experiment. A typical experiment contained 200 nM of forward and reverse primers plus 100 nM probe for β-2 microglobulin and 600 nM forward and reverse primers plus 200 nM probe for the target 26176 sequence. TaqMan matrix experiments were carried out on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). The thermal cycler conditions were as follows: hold for 2 min at 50° C. and 10 min at 95° C., followed by two-step PCR for 40 cycles of 95° C. for 15 sec followed by 60° C. for 1 min.

The following method was used to quantitatively calculate 26176 expression in the various tissues relative to β-2 microglobulin expression in the same tissue. The threshold cycle (Ct) value is defined as the cycle at which a statistically significant increase in fluorescence is detected. A lower Ct value is indicative of a higher mRNA concentration. The Ct value of the 26176 sequence is normalized by subtracting the Ct value of the β-2 microglobulin gene to obtain a Δ Ct value using the following formula: Δ Ct=Ct h26176 −Ct β-2 microglobulin. Expression is then calibrated against a cDNA sample showing a comparatively low level of expression of the 26176 sequence. The Δ Ct value for the calibrator sample is then subtracted from Δ Ct for each tissue sample according to the following formula: ΔΔ Ct= Δ Ct- sample − Δ Ct- calibrator . Relative expression is then calculated using the arithmetic formula given by 2 −ΔΔCt . Expression of the target 26176 sequence in each of the tissues tested was then analysed.

The mRNA for the putative calpain protease 26176 is expressed in a variety of tumors. There was significant upregulation in colon carcinoma and breast carcinoma. Accordingly, expression of the 26176 calpain protease is relevant to colon and breast carcinoma. In additional experiments, the gene was expressed in three out of four normal lung tissue samples but in 15 out of 16 lung carcinoma clinical samples. Accordingly, expression of the 26176 calpain protease is relevant to lung carcinoma as well. This is consistent with the hypothesis that proteases may function in carcinogenesis by inactivating or activating regulators of cell cycle, differentiation, apoptosis, or other processes affecting cancer development and/or progression. In view of the fact that the 26176 gene is up-regulated in colon carcinoma, the gene is useful for inhibiting tumor progression. Inhibition of expression of this 26176 protease can thus be used to decrease the progression of carcinogenesis.

In addition, Northern blot experiments showed expression of the 26176 calpain protease in bone, ovary, T-cell, spleen, and kidney tissue. Accordingly, the 26176 protease is relevant to disorders involving these tissues.

In addition, 26176 expression has been observed in heart, neuronal tissue, monocytes, and prostate. Accordingly, expression of the 26176 gene is relevant to disorders involving these tissues.

Finally, 26176 expression has been observed in parathyroid tumor and in thymus. Accordingly, detection of expression or modulation of expression of the 26176 gene in these tissues, and particularly in disorders involving these tissues, is relevant.

Human 26343

The present invention is based, at least in part, on the discovery of novel molecules, referred to herein “Oxidoreductase Protein”, “OP” or “26343” nucleic acid and protein molecules, which are novel members of a family of enzymes possessing oxidoreductase activity. These novel molecules are capable of oxidizing and/or reducing molecular groups by catalyzing the transfer of a hydride moiety and, thus, play a role in or function in a variety of cellular processes, e.g., proliferation, metabolism, differentiation, hormonal responses, and inter- or intra-cellular communication.

The oxidation and reduction of molecules is of critical importance in many cellular metabolic and catabolic pathways. “Redox” reactions play important roles in the production and breakdown of nearly all major metabolic intermediates, including amino acids, vitamins, energy molecules (e.g., glucose, sucrose, and their breakdown products), signal molecules (e.g., transcription factors and neurotransmitters), and nucleic acids. A large class of enzymes which facilitate some of these molecular alterations, termed oxidoreductases, have been identified. In the forward reaction, these enzymes catalyze the transfer of a hydride ion from the target substrate to the enzyme or a cofactor of the enzyme (e.g., NAD + , NADP + , FAD + ), thereby oxidizing the substrate. These enzymes may also participate in the reverse reaction, wherein a molecular group of the target molecule is reduced by the transfer of a hydride group from the enzyme. Members of the oxidoreductases family are found in nearly all organisms, from prokaryotes to Drosophila to humans. Both between species and within the same species, oxidoreductases vary widely; disparate family members are frequently classified by the cofactor used by the enzyme (e.g., NAD + , NADP + , FAD + ), or by the particular substrate(s) of the enzyme (see, for example, Cavener, D. R. (1992) J. Mol. Biol. 223:811-814).

Different oxidoreductases are specific for a wide array of biological and chemical substrates. For example, there exist oxidoreductases specific for steroids (Kass and Sampson (1998) Biochemistry 37:17990-800), neurotransmitters (Lamark et al. (1991) Mol. Microbiol. 5:1049-1064), energy metabolites (Krasney et al. (1990) Mol. Biol. Evol. 7:155-177; Frederick), alcohols (Ledeboer et al. (1985) Nucleic Acids Res. 13:3069-3082; Koutz et al. (1989) Yeast 5:167-177), lipids (Funk et al. (1992) Proc. Natl. Acad. Sci. USA 89:3962-3966), amino acid precursors and nucleotide precursors (Wright et al. (1993) Proc. Natl. Acad. Sci. USA 90:10690-10694). Accordingly, oxidoreductase activity contributes to the ability of the cell to grow and differentiate, to proliferate, and to communicate and interact with other cells. Therefore, a wide range of metabolic disorders and related pathogenic states relate to the oxidoreductases, both directly and indirectly (see, for example, Salazar et al. (1997) J. Biol. Chem. 272:26425-26433).

As used herein, the term “oxidoreductase” includes a molecule which is involved in the oxidation or reduction of a biochemical molecule (e.g., a metabolic precursor which contains a molecular group which can be oxidized or reduced) by catalyzing the transfer of a hydride ion to or from the biochemical molecule. Oxidoreductase molecules are involved in the metabolism and catabolism of biochemical molecules necessary for energy production or storage, for intra- or inter-cellular signaling, and for metabolism or catabolism of metabolically important biomolecules. Examples of oxidoreductases include glucose oxidases, methanol oxidases, choline dehydrogenases, glucose dehydrogenases, cholesterol oxidases, alcohol dehydrogenases, and cellobiose dehydrogenases.

The OP proteins of the present invention show homology to the choline dehydrogenase family of oxidoreductases. Choline dehydrogenase (CDH) is the first enzyme of the glycine betaine synthetic pathway. Betaine, an atypical amino acid that is non-proteinogenic yet important as an osmoprotectant, is synthesized by a two-step oxidation of choline. This reaction takes place in the mitochondrial matrix by the membrane bound CDH and betaine aldehyde dehydrogenase (Landfald and Strom (1986) J. Bacteriol. 165:849-55; Styrvold et al. (1986) J. Bacteriol. 165:856-63; Grossman and Hebert (1989) Am. J. Physiol. 256(1 Pt. 2):F107-12; Zhang et al. (1992) Biochim. Biophys. Acta. 1117:333-9). CDH is also coupled to the respiratory chain. Betaine is further important in mammalian organisms as a major methyl group donor and nitrogen source.

Methyl groups derived from betaine may be used for recycling homocysteine to methionine. It is known that some tumor cells have an increased need for methionine for survival. Methionine dependent tumor cells are unable to proliferate, and they arrest in the G2 phase of the cell cycle. For example, MCF-7 breast cancer cells grown in methyl-deficient media show inhibition of cell proliferation and induction of apoptosis. Fresh patient colon tumors have also been shown to be methionine dependent based on cell cycle analyses. Metastatic colon tumors have a higher methionine dependence than primary tumors. Other examples of methionine dependence in tumors have been seen in small cell lung cancer and gliomas.

Human 26343 is overexpressed in various tumors, e.g., colon tumors, as compared to normal tissues (see section below on expression levels). Human 26343 is further elevated in later stage tumors. Elevation of the levels of the 26343 molecules of the present invention in tumor cells may increase tumor survival by increasing the supply of methionine available to the tumor cells. Accordingly, inhibition of the 26343 molecules of the present invention may cause tumor cell growth arrest and/or apoptosis, making the 26343 molecules of the present invention useful for the treatment of cellular proliferation, growth, apoptosis, differentiation, and/or migration disorders.

The 26343 molecules of the present invention may also be useful for the treatment of disorders characterized by the aberrant or abnormal regulation of the levels of choline, betaine (e.g., a disorder associated with aberrant regulation of osmolarity by betaine), homocysteine (e.g., homocystinuria), and/or methionine in a subject.

The 26343 molecules of the present invention may still further be useful for the treatment of disorders affecting tissues in which 26343 protein is expressed, e.g., primary osteoblasts, pituitary, CaCO cells, keratinocytes, aortic endothelial cells, fetal kidney, fetal lung, mammary epithelium, fetal spleen, fetal liver, umbilical smooth muscle, RAII Burkitt Lymphoma cells, lung, prostate, K53 red blood cells, fetal dorsal spinal cord, insulinoma cells, normal breast and ovarian epithelia, retina, HMC-1 mast cells, ovarian ascites, d8 dendritic cells, megakaryocytes, human mobilized bone morrow, mammary carcinoma, melanoma cells, lymph, vein, U937/A70p B cells, A549con cells, WT LN Cap testosterone cells, esophagus, and other tissues and/or cell types described further below.

In an alternate embodiment, any and all of the above described disorders may simply be referred to as “OP associated or related disorders”.

For example, the family of OP proteins comprise at least one, and preferably three or more “transmembrane domains.” As used herein, the term “transmembrane domain” includes an amino acid sequence of about 15 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 10, 15, 20, 25, 30, 35, 40, 45 or more amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have a helical structure. In one embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acid residues of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zagotta W. N. et al. (1996) Annu. Rev. Neurosci. 19:235-63, the contents of which are incorporated herein by reference. Amino acid residues 41-57, 292-311, and 545-564 of the human 26343 polypeptide (SEQ ID NO:55) comprise transmembrane domains.

In another embodiment, an OP molecule of the present invention is identified based on the presence of an GMC oxidoreductase signature domain in the protein or corresponding nucleic acid molecule. As used herein, the term GMC oxidoreductase signature domain includes a protein domain having an amino acid sequence of about 375-650, more preferably about 450-600 amino acid residues, or most preferably about 500-550 amino acids and has a bit score for the alignment of the sequence to the GMC oxidoreductase signature domain (HMM) of at least about 100, 200, 300, 400, 500, 600, 700, 800, or more. Preferably, a GMC oxidoreductase signature domain includes at least about 526 amino acid residues and has a bit score for the alignment of the sequence to the GMC oxidoreductase signature domain (HMM) of about 767.7. The GMC oxidoreductase signature domain has been assigned the PFAM labels “GMC_oxred — 1” and “GMC_oxred — 2” under accession number PS00623 and PS00624, respectively (see the Pfam website, available online through Washington University in Saint Louis). GMC oxidoreductase signature domains are involved in oxidoreductase activity and are described in, for example, Cavener (1992) J. Mol. Biol. 223:811-814, the contents of which are incorporated herein by reference.

To identify the presence of a GMC oxidoreductase signature domain in an OP protein and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (see the Pfam website, available online through Washington University in Saint Louis). A search was performed against the HMM database resulting in the identification of a GMC oxidoreductase signature domain in the amino acid sequence of SEQ ID NO:55 (at about residues 41-567).

A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28:405-420, and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference.

Isolated OP proteins of the present invention, have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:55, or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:54 or 56. As used herein, the term “sufficiently identical” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains have at least 30%, 40%, or 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical. Furthermore, amino acid or nucleotide sequences which share at least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95% homology and share a common functional activity are defined herein as sufficiently identical.

As used interchangeably herein, a “OP activity”, “biological activity of OP,” or “functional activity of OP,” includes an activity exerted by an OP protein, polypeptide or nucleic acid molecule on an OP-responsive cell or tissue, or on an OP protein substrate, as determined in vivo, or in vitro, according to standard techniques. In one embodiment, an OP activity is a direct activity, such as an association with an OP-target molecule. As used herein, a “target molecule” or “binding partner” is a molecule with which an OP protein binds or interacts in nature, such that OP-mediated function is achieved. An OP target molecule can be a non-OP molecule or an OP accessory polypeptide or molecule of the present invention (e.g., NAD + , FAD + , or other cofactor). As used herein, an “accessory” peptide or molecule refers to a peptide or molecule whose presence is may be needed for the proper activity of a protein (e.g., a cofactor or a metal ion that is needed by an enzyme). In an exemplary embodiment, an OP target molecule is an OP ligand (e.g., choline and/or an acceptor molecule to be reduced or oxidized choline and/or an acceptor molecule to be reduced or oxidized). Alternatively, an OP activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the OP protein with an OP ligand. The biological activities of OP are described herein. For example, the OP proteins of the present invention can have one or more of the following activities: 1) modulation of metabolism and catabolism of biochemical molecules, e.g., molecules necessary for energy production or storage; 2) modulation of betaine synthesis from choline; 3) modulation of methionine synthesis from homocysteine; 4) modulation of intra- or inter-cellular signaling; 5) modulation of cellular proliferation and/or migration; and/or 6) modulation of hormonal responses.

Accordingly, another embodiment of the invention features isolated OP proteins and polypeptides having an OP activity. Other preferred proteins are OP proteins having one or more of the following domains: a transmembrane domain, a GMC oxidoreductase signature domain, and, preferably, an OP activity. Additional preferred OP proteins have at least one GMC oxidoreductase signature domain, and/or at least one transmembrane domain and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising a complement of the nucleotide sequence of SEQ ID NO:54 or 56.

Isolation of the Human 26343 or OP cDNA

The invention is based, at least in part, on the discovery of a 65.3 kD human gene encoding a novel protein, referred to herein as 26343 or OP. The entire sequence of the human clone Fbh26343 was determined and found to contain an open reading frame termed “human OP.” The 2343 nucleotide sequence encoding the human OP protein is set forth as SEQ ID NO:54. The protein encoded by this nucleic acid comprises about 594 amino acids and has the amino acid sequence set forth as SEQ ID NO:55. The coding region (open reading frame) of SEQ ID NO:54 is set forth as SEQ ID NO:56.

Analysis of the Human 26343 or OP Molecule

A search for domain consensus sequences was performed using the amino acid sequence of human 26343 or OP and a database of HMMs (the Pfam database, release 2.1) using the default parameters (described above). The search revealed a GMC oxidoreductase signature domain (Pfam label GMC_oxred; Pfam Accession Numbers PS00623 and PS00624) within SEQ ID NO:55 at residues 41-567.

A search was performed against the ProDom database resulting in the identification of a portion of the deduced amino acid sequence of human 26343 or OP (SEQ ID NO:55) which has a 39% identity to ProDom entry “FAD flavoprotein oxidoreductase precursor dehydrogenase lyase signal protein cellobiose isoform”) over residues 41 to 351 and 37% identical over residues 488-568. In addition, human 26343 or OP is 50% identical to ProDom entry “L-sorbosone dehydrogenase, FAD dependent” over residues 501-573 of SEQ ID NO:55. In addition, human 26343 or OP is 57% identical to ProDom entry “NADH:N-amido-scyllo-inosamine oxidoreductase” over residues 40-74 and 32% identical over residues 254 to 308 of SEQ ID NO:55.

A search was also performed against the Prosite database, and resulted in the identification of one possible glycosaminoglycan attachment site within the human OP protein at residues 308-311 of SEQ ID NO:55. In addition, protein kinase C phosphorylation sites were identified within the human 26343 or OP protein at residues 81-83, 85-87, 283-285, 494-496, 515-517, and 592-594 of SEQ ID NO:55. This search also identified casein kinase II phosphorylation sites at residues 37-40, 231-234, 415-418, 455-458, 494-497 of SEQ ID NO:55. A tyrosine phosphorylation site motif was also identified in the human 26343 or OP protein at residues 503-510 of SEQ ID NO:55. The search also identified the presence of N-myristoylation site motifs at residues 20-25, 47-52, 129-134, 296-301, 309-314, 329-334, 374-379, and 429-434 of SEQ ID NO:55. In addition, the search identified an amidation site at residues 234-237, and a GMC oxidoreductase signature sequence at amino acids 297-311 of SEQ ID NO:55.

An analysis of the possible cellular localization of the human 26343 or OP protein based on its amino acid sequence was performed using the methods and algorithms described in Nakai and Kanehisa (1992) Genomics 14:897-911, and available online through the PSORT server website. The results from this analysis predict that the human 26343 or OP protein is found in the mitochondria, in the cytoplasm, in the nucleus, and in peroxisome.

An analysis of putative post-translationally truncated variants indicated that the mature protein may have residue 16 of SEQ ID NO:55 (arginine) as the N-terminal residue.

Analysis of Human 26343 or OP Expression

The following describes the expression of human 26343 or OP mRNA in various tissues, tumors, cell lines, and disease models, as determined using the TaqMan™ procedure and in situ hybridization analysis.

For in situ analysis, various tissues, e.g., tissues obtained from liver or colon, were first frozen on dry ice.

As indicated by the data obtained from the TaqMan analysis, human 26343 was expressed highly in the following tissues: normal fetal heart, normal brain cortex, brain (hypothalamus), brain (glioblastoma), normal breast, breast tumor (IDC), prostate tumor, colon tumor, normal kidney, normal liver, fibrotic liver, normal fetal liver, and skeletal muscle. Human 26343 is also expressed in the following tissues: normal heart, heart (congestive heart failure), normal spinal cord, normal prostate, normal ovary, and lung (chronic obstructive pulmonary disease).

Human 26343 showed increased expression in 100% of the clinical colon tumor samples tested, compared with clinical normal colon tissue samples.

Human 26343 showed increased expression in 100% of the clinical liver metastasis samples tested, compared with clinical normal liver tissue samples.

Human 26343 showed increased expression in 57% of the clinical lung tumors tested, compared with clinical normal lung tissue samples.

Human 26343 showed expression in most Xenograft friendly cell lines, e.g., MCF-7, ZR75, T47D, DLD-1, SW 480, SW 620, HCT 116, Colo 205, NCIH 125, NCIH 322, NCIH 460, and A549. Colon tumor cell lines show increased 26343 expression in later stages as follows:

The results from the in situ hybridization analysis indicate that human 26343 is expressed in 100% of primary colon tumors tested and 100% of metastatic tumors tested, as compared to 0% in normal tissues tested.

The data also indicate that human 26343 is focally expressed in 20% of lung tumors tested, as compared to 0% of the corresponding normal tissues.

Cell Cycle Analysis

The following describes the results from studies designed to determine how the expression of human 26343 mRNA is regulated during the cell cycle.

Transcriptional profiling analysis showed that human OP expression was increased in aphidocholine synchronized MCF10a cells within the G0/G1 phase of the cell cycle.

Human 26343 also showed cell cycle regulated expression in aphidocholine synchronized HCT 116 colon carcinoma cells, with higher expression in the G2/M phase of the cell cycle.

Human 26343 also showed cell cycle regulated expression in aphidocholine synchronized A549 lung carcinoma cells.

Reintroduction of Smad4, a tumor suppressor gene in the TGFβ signaling pathway, into SW 480 cells (colon carcinoma cells that are deficient in the expression of Smad4) by transient transfection caused a decrease in the expression of human 26343 in these cells.

Human 26343 expression was upregulated in the RER− (replication error) cell lines Caco2 and SW 480, as compared to RER+ cell lines. RER− cell lines have increased difficulty in mismatch repair during DNA replication.

Increased expression of human 26343 in RER− cells and in Smad4 deficient cells indicates that increased human 26343 expression is associated with situations known to cause progression to later stage tumors, i.e., errors in TGFβ signaling and mismatch repair.

Measurement of Methionine Levels in Tumor Cells

The following describes the measurement of methionine levels in tumor cells, as may be determined using the methods of Tan, Y. et al. (1999) Clin. Cancer Res. 5:2157-2163, the contents of which are incorporated herein by reference.

Briefly, tumor methionine levels are determined using an HPLC machine (Hitachi L-5200A Intelligent pump; Hitachi, Ltd., Tokyo, Japan) after derivitization of serum amino acids with the fluoraldehyde reagent OPA as described in Tan, Y. et al. (1997) Anticancer Res. 17:3857-3860 and Lishko, V. K. et al. (1993) Anticancer Res. 13:1465-1468. Supernatants are prepared from tumor tissue after sonication for 30 seconds and subsequent centrifugation at 13,000 rpm for 10 minutes. Tumor supernatant samples (25 μl) are precipitated by acetonitrile (75 μl). Ten μl of supernatant are mixed with 5 μl of OPA. After 1 minute, 50 μl of 0.1 M sodium acetate (pH 7.0) are added, and a 20 μl sample is loaded on a reversed-phase Supelcosil LC-18-DB column (particle size: 5 μm, 25 cm×4.8 mm) at room temperature. The column is eluted with solution A (tetrahydrofuran:methanol:0.1 M sodium acetate (pH 7.2); 5:95:900) and solution B (methanol). A gradient from 20-60% of solution B, run a flow rate of 1.5 ml/min, resolves the amino acids. The eluate is read with a fluorescence spectrophotometer (Hitachi, F1000) at a wavelength of 350-450 nm. The limit of detection is ˜0.1 μM methionine.

Measurement of OP Choline Dehydrogenase Activity

The following describes the measurement of OP choline dehydrogenase activity in cells, as may be determined using the methods of Zhang, J. et al. (1992) Biochim. Biophys. Acta 1117:333-339, the contents of which are incorporated herein by reference.

The following methods are used to assay the choline dehydrogenase activity of the OP molecules of the invention. The methods are performed with purified OP molecules, or with mitochondrial preparations containing OP molecules, as described below.

Preparation of Mitochondria

A 12 gram wet weight tissue or cell sample (e.g., a normal tissue or cell sample, or a tumor sample) is homogenized in 108 ml 0.25 M sucrose at a temperature of not more than 4° C. and centrifuged at 700×g at 4° C. for 8 minutes. The supernatant is subsequently centrifuged at 17,000×g at 4° C. for 10 minutes. The resulting mitochondrial pellet is resuspended in 30 ml of 0.25 M sucrose and repeatedly treated as above at least three times. The purity of the mitochondria is confirmed by determining the activities of a mitochondrial marker, fumarase (Stenech, J. (1984) in Experimental Biochemistry (Stenech, J., ed.), pp. 400-401, Allyn and Bacon, Boston); a cytosolic marker, lactate dehydrogenase (Worthington Biochemicals, Freehold, N.J.); and a microsomal marker, glucose-6-phosphatase (Leloir, L. F. and Cardini, C. E. (1975) Methods Enzymol. 3:840-844). This preparation is kept frozen at −90° C. until used. The protein concentration of the mitochondria is determined by the method of Bradford ((1976) Anal. Biochem. 72:248-254).

Colorimetric OP Choline Dehydrogenase Assay

OP choline dehydrogenase activity may be measured by the PMS-DCIP colorimetric method, as described in Singer, T. P. (1974) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 22, pp. 133-169, John Wiley, New York; and Rendina, G. and Singer, T. P. (1959) J. Biol. Chem. 234:1605-1610.

Radioenzymatic Assay of OP Choline Dehydrogenase Activity

A mitochondrial preparation containing OP molecules, made using the methods described above, is incubated with [methyl- 14 C]choline (55 mCi/mmol; ICN Biomedicals, Irvine, Calif.) in reaction medium containing 40 mM Tris buffer (pH 7.6) or 40 mM glycine buffer (pH 8.5) for varying amounts of time at 37° C. The reaction is inactivated by adding 1/10 of the reaction volume in the form of 1.2 M HCl. Mixtures are extracted with one reaction volume of methanol and 2 volumes of chloroform. After briefly vortexing at room temperature, the phases are separated by low speed centrifugation and collected.

HPLC Purification of Choline, Betaine Aldehyde, and Betaine

50 μl of the methanol-water phase (see above) is mixed with 100 μl of methanol and then analyzed by HPLC (3×8C Pecosphere Cartridge, silica column (Perkin Elmer, Norwalk, Conn.)). The reaction products are eluted (at a flow rate of 1.5 ml/minute) with buffer A containing 800 ml acetonitrile, 68 ml ethanol, 5 ml of 3:2 (v/v) 1.0 M ammonium acetate-glacial acetic acid buffer, 127 ml water, and 10 ml 1.0 M potassium dihydrogen phosphate. The radioactivity of the eluent is determined using an on-line solid scintillant radiometric detector (Model BL 507A, Berthold, Nashua, N.H.). The efficiency of detection is determined using radiolabeled standards. After each sample is run, the column is washed for 5 minutes with buffer B containing the same components as buffer A in the following volumes (ml): 400:68:132:400:10. In a typical chromatogram, only the three peaks of interest are detected, i.e., the [methyl- 14 C]choline substrate, and the two oxidation products, betaine aldehyde and betaine (betaine aldehyde is an intermediate in the two-step oxidation process that produces betaine from choline). The combined radioactivity in these three peaks is taken as 100%. In order to determine the amount of each product formed, the following formula is used:

moles ⁢ ⁢ of ⁢ ⁢ product = DPM ⁢ ⁢ in ⁢ ⁢ peak total ⁢ ⁢ DPM moles ⁢ ⁢ choline ⁢ ⁢ substrate ⁢ ⁢ in ⁢ ⁢ incubation ⁢ ⁢ medium

Partial Purification of OP Choline Dehydrogenase

Mitochondria are centrifuged at 17,000×g at 4° C. to remove the original buffer. The mitochondrial pellet is resuspended in 1.2 M sucrose, 0.05 mM EDTA, and 40 mM ammonium acetate for 45 minutes at 25° C. The resulting preparation is centrifuged at 24,000×g at 4° C. for 10 minutes (Beckman, Ti 50.2 rotor). The resulting pellet is referred to interchangeably herein as an “aged mitochondrial pellet” or “aged mitochondria” (Lin, C. S. and Wu, R. D. (1986) J. Prot. Chem. 5:193-200).

The aged mitochondrial pellet is resuspended by gentle stirring in 60 mM glycine-NaOH buffer (pH 10) at 4° C. for 40 minutes. The resulting preparation is centrifuged at 24,000×g at 4° C. for 10 minutes. The supernatant is discarded after centrifugation.

The pellet is extracted with 0.2 mg digitonin per mg protein. Digitonin is dissolved in 0.25 M warm sucrose and sonicated with a probe sonicator for 1-2 minutes and then chilled and gently added drop-wise to the mitochondrial preparation over a 5 minutes period. After incubation for 25 minutes at 4° C., the preparation is centrifuged at 24,000×g at 4° C. for 10 minutes. The pellet is resuspended in 0.25 M NaCl (Lin and Wu (1986) supra).

The digitonin-extracted mitochondrial preparation in 0.25 M NaCl is sonicated at 4° C. for 5 minutes and subsequently centrifuged at 100,000×g at 4° C. for 30 minutes (Beckman, Ti 50.2 rotor). The pellet is resuspended in a buffer containing 0.12 M sucrose, 0.05 mM EDTA, 6.0 mM choline (Fisher, Springfield, N.J.; recrystallized in methanol), 0.03 M potassium phosphate, and 1.0 M NaCl. Lubrol WX (0.2 mg per mg protein; Serva, Feinbiochemica, Heidelberg, Germany) is added, the preparation is shaken for 10 minutes at 4° C. and subsequently centrifuged at 100,000×g for 30 minutes. The solubilized OP molecules are present in the supernatant (Lin and Wu (1986) supra).

Thin Layer Chromatography Separation of Choline Oxidation Product

Choline, betaine aldehyde, and betaine are purified by thin layer chromatography on silica gel plates (LK5D; Whatman Company) developed with a mixture containing chloroform, methanol, and 0.1 M HCl (65:30:4; v/v) and visualized by staining in iodine vapor.

Measurement of OP Choline Dehydrogenase Activity

In one experiment, 62.5 μg OP protein (mitochondrial preparation) is incubated with 0.572 μCi, 0.15 mM [methyl- 14 C]choline in Tris buffer (pH 7.6) at 37° C. The total reaction volume is 150 μl. In another experiment, 31.5 μg OP protein (mitochondrial preparation) is incubated with 0.27 μCi, 0.13 mM [methyl- 14 C]choline in Tris buffer (pH 8.5) at 37° C. The total reaction volume is 150 μl. In still another experiment, varying amounts of OP protein (mitochondrial preparation) are incubated with 0.92 μCi, 0.2 mM [methyl- 14 C]choline in Tris buffer (pH 7.6) for 10 minutes at 37° C. The total reaction volume is 150 μl.

In another experiment, the effect of electron acceptors and cyanide on OP activity is measured. 0.5 mg of OP protein (mitochondrial preparation) is incubated with 0.1 mM, 0.41 μCi [methyl- 14 C]choline in Kregs-Hanseleit buffer (pH 7.75), in the presence of 1 mM potassium cyanide (KCN), phenazine methosulfate (PMS) and dichloroindophenol (DCIP), or PMS, DCIP, and KCN. The total reaction volume is 0.5 ml. The reaction mixture is incubated at 37° C. for 20 minutes. In another experiment, 11 μg OP protein (solubilized preparation) is incubated with 20 nmol, 0.27 μCi [methyl- 14 C]choline at 37° C. in 0.01 M KH 2 PO 4 (pH 7.7) for 40 minutes in the presence or absence of 0.1 mM NAD + , or 1.0 mM PMS, or 0.1 mM PMS and 0.1 mM NAD + together. The total reaction volume is 195 μl.

In another experiment, the effect of changes in pH on OP activity is measured. 63 μg OP protein is incubated in 40 mM phosphate, 40 mM glycine, 40 mM Hepes, 40 mM boric acid, or 40 mM Tris buffer at different pH. The [methyl- 14 C]choline concentration is 0.8 μCi, 0.19 mM for Tris-HCl and phosphate buffer, and 0.25 μCi, 0.13 mM for other buffers. The reactions are carried out at 37° C. for 10 minutes.

Human 56638

The present invention is based, at least in part, on the identification of a novel neprilysin protease referred to herein as “56638”. The human 56638 sequence (SEQ ID NO:57), which is approximately 2953 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 2340 nucleotides, including the stop codon (SEQ ID NO:59). Although the ATG at position 1-3 of SEQ ID NO:59 is the preferred start site of translation, other embodiments are included wherein, e.g., the ATG at position 28-30 of SEQ ID NO:59 is the start site of translation. The coding sequence encodes an 779 amino acid protein (SEQ ID NO:58). The human 56638 protein of SEQ ID NO:58 is predicted to have a signal peptide at about amino acid 1-44 of SEQ ID NO:58.

Human 56638 sequence contains the following regions or other structural features: an M13 peptidase (neprilysin) domain (PF01431) from about amino acid 572 to 778 of SEQ ID NO:58, which includes the characteristic HEXXH zinc-binding active site of metallopeptidases (PS00142; SEQ ID NO:62) located at about amino acid 610 to 619 of SEQ ID NO:58.

The human 56638 sequence can additionally include: eight N-glycosylation sites (PS00001) located from about amino acid 156 to 159, from about amino acid 177 to 180, from about amino acid 207 to 210, from about amino acid 243 to 246, from about amino acid 350 to 353, from about amino acid 530 to 533, from about amino acid 638 to 641, and from about amino acid 657 to 660 of SEQ ID NO:58; one cAMP and cGMP-dependent protein kinase phosphorylation site (PS00004) from about amino acid 183 to 186 of SEQ ID NO:58; eleven protein kinase C phosphorylation sites (PS00005) from about amino acid 158 to 160, from about amino acid 244 to 246, from about amino acid 269 to 271, from about amino acid 361 to 363, from about amino acid 391 to 393, from about amino acid 412 to 414, from about amino acid 493 to 495, from about amino acid 503 to 505, from about amino acid 551 to 553, from about amino acid 726 to 728, and from about amino acid 735 to 737 of SEQ ID NO:58; eight casein kinase II phosphorylation sites (PS00006) from about amino acid 137 to 140, from about amino acid 158 to 161, from about amino acid 179 to 182, from about amino acid 429 to 432, from about amino acid 445 to 448, from about amino acid 482 to 485, from about amino acid 503 to 506, and from about amino acid 673 to 676 of SEQ ID NO:58; three tyrosine kinase phosphorylation sites (PS00007) from about amino acid 435 to 442, from about amino acid 520 to 526, and from about amino acid 645 to 653 of SEQ ID NO:58; nine N-myristoylation sites (PS00008) from about amino acid 9 to 14, from about amino acid 44 to 49, from about amino acid 78 to 83, from about amino acid 93 to 98, from about amino acid 547 to 552, from about amino acid 608 to 613, from about amino acid 683 to 688, from about amino acid 706 to 711, and from about amino acid 750 to 755 of SEQ ID NO:58; a prenyl group binding site (CAAX box) (PS00294) from about amino acid 776 to 779 of SEQ ID NO:58; and a signal peptide from about amino acid 1 to 44 of SEQ ID NO:58, resulting in a mature protein of 822 amino acids, from amino acid 45 to 779 of SEQ ID NO:58.

Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, e.g., the sequence of 560-570 of SEQ ID NO:58; all or part of a hydrophilic sequence, e.g., the sequence of 620-640 of SEQ ID NO:58; a sequence which includes a Cys or a glycosylation site.

For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420.

The 56638 protein contains a significant number of structural characteristics in common with members of the neprilysin family of metalloproteases.

The neprilysin family comprises a number of related enzymes that share high structural homology and a common catalytic mechanism that involves cleavage of a protein substrate by hydrolysis of an amide bond that depends upon the presence of a metal ion, e.g., zinc. Neprilysins are mammalian membrane metalloproteases which contain the active site consensus sequence VxxHExxH (SEQ ID NO:61; amino acids 610 to 619 of SEQ ID NO:58) found in other zinc metalloproteases. The histidines are two of the three Zn-coordinating ligands, and the glutamate plays a role in catalysis by polarizing a water molecule. The hydrolysis occurs through the formation of a pentacoordinated complex of the metal which includes the three Zn-coordinating amino acids of the peptidase, the oxygen of the scissile bond, and the water molecule that is initially bound to the Zn atom. For a review, see, Barrett (1995) Methods in Enzymol 248:263-283. In addition, neprilysin family members share other structural features. They can be highly glycosylated type II integral membrane proteins, and they can have a cluster of conserved cysteine residues following the transmembrane domain which are involved in stabilizing the active enzyme through the formation of sulfide bridges (Tanja et al (2000) Biochem Biophys Res Comm 271:565-570).

The human 56638 proteins of the present invention show significant homology to members of the neprilysin metallopeptidase family, and in particular, to the mouse NL1/SEP and the rat neprilysin II proteins (Ghaddar et al. (2000) Biochem J 347:419-429; Ikeda et al. (1999) J Biol Chem 274:32469-32477). Like mouse NL1/SEP and rat neprilysin II, 56638 is a secreted protein. 56638 has the characteristic VxxHExxH (SEQ ID NO:61) zinc-binding metallopeptidase consensus sequence (PS00142), located at about amino acid 610 to 619 of SEQ ID NO:58. Neprilysin family members include neprilysin, endothelin converting enzyme (ECE), Kell Blood group antigen, PEX, and X-converting enzyme (XCE), and soluble secreted endopeptidase (SSE). Examples of substrates of the neprilysin peptidase family include, but are not limited to, neuropeptides involved in pain control, e.g., enkephalin, somatostatin, and substance P; and vasoactive peptides that mediate inflammation and pain, e.g., neurotensin, atrial natriuretic peptide (ANP), neurokinin, tachykinin, bradykinin, and endothelin (Checler et al. (1983) J Neurochem 41:375; Matsas et al. (1983) Proc Natl Acad Sci USA 80:3111; Matsas et al. (1984) Biochem J 223:433; Stepehenson and Kenny (1987) Biochem J 241:237; Turner and Tanzawa (1997) FASEB J 11:355-364). Mouse NL1/Sep has been shown to cleave enkephalin in vivo. Enkephalin, a major substrate of neprilysin, is one of several naturally occurring morphine like substances released from nerve endings of the central nervous system and the adrenal medulla. It acts as an analgesic and sedative in the body and appears to affect mood and motivation. As neprilysin is responsible for the inactivation of enkephalin and other bioactive peptides involved in inflammation and pain, neprilysins are critical for the proper function of many physiological systems, including neurotransmission, pain control, inflammatory response, and vascular tone.

Other neprilysin family members include a marker of common acute lymphoblastic leukemia antigen present at the surface of B cells (Roques et al. (1993) Pharmacol Rev 45:87), and the Kell blood group antigen (Lee et al. (1999) Proc Natl Acad Sci USA 88:6353-6357). Kell antigens are highly immunogenic and may cause severe fetal anemia in sensitized mothers, erythroblastosis in newborn infants, and severe hemolytic reactions if mismatched blood is transfused.

A 56638 polypeptide can include a “neprilysin domain” or regions homologous with a “neprilysin domain.” A 56638 polypeptide can optionally further include a signal peptide; at least one, two, three, four, five, six, seven, preferably eight N-glycosylation sites; at least one cAMP and cGMP-dependent protein kinase phosphorylation site; at least one, two, three, four, five, six, seven, eight, nine, ten, preferably eleven, protein kinase C phosphorylation sites; at least one, two, three, four, five, six, seven, preferably eight, casein kinase II phosphorylation sites; at least one, two, preferably three, tyrosine kinase phosphorylation sites; at least one, two, three, four, five, six, seven, eight, preferably nine, N-myristoylation sites; at least one prenyl group binding site.

As used herein, the term “neprilysin domain” includes an amino acid sequence of about 50 to 350 amino acid residues in length, more preferably about 100 to 300 amino acid residues, or about 200 to 215 amino acids, and having a bit score for the alignment of the sequence to the neprilysin domain (HMM) of at least 100, preferably 150, more preferably 200, most preferably 250 or more. Preferably, the domain includes a zinc-binding active site of metallopeptidase domains (PS00142) located at about amino acid 610 to 619 of SEQ ID NO:58. The neprilysin domain (HMM) has been assigned the PFAM Accession Number PF01431. An alignment of the neprilysin domain (amino acids 572 to 778 of SEQ ID NO:58) of human 56638 with a consensus amino acid sequence derived from a hidden Markov model derived from PFAM (SEQ ID NO:60) yields a bit score for the alignment of 270.4 (E=2.4e-77).

In a preferred embodiment 56638 polypeptide or protein has a “neprilysin domain” or a region which includes at least about 50 to 350, more preferably about 100 to 300, or 200 to 215 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “neprilysin,” e.g., the neprilysin domain of human 56638 (e.g., residues 572 to 778 of SEQ ID NO:58).

To identify the presence of a “neprilysin” domain in a 56638 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth Enzymol 183:146-159; Gribskov et al. (1987) Proc Natl Acad Sci USA 84:4355-4358; Krogh et al. (1994) J Mol Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a “neprilysin” domain in the amino acid sequence of human 56638 at about residues 572 to 778 of SEQ ID NO:58. The identified neprilysin domain is depicted in SEQ ID NO:60.

A 56638 protein can further include a signal peptide, and is predicted to be a secreted protein. As used herein, a “signal peptide” or “signal sequence” refers to a peptide of about 20 to 60, preferably about 30 to 50, more preferably, about 44 amino acid residues in length which occurs at the N-terminus of secretory and integral membrane proteins and which contains a majority of hydrophobic amino acid residues. For example, a signal sequence contains at least about 20 to 60, preferably about 30 to 50, more preferably, 44 amino acid residues, and has at least about 40-70%, preferably about 50-65%, and more preferably about 55-60% hydrophobic amino acid residues (e.g., alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, or proline). Such a “signal sequence”, also referred to in the art as a “signal peptide,” serves to direct a protein containing such a sequence to a lipid bilayer. For example, in one embodiment, a 56638 protein contains a signal sequence of about amino acids 1 to 44 of SEQ ID NO:58. The “signal sequence” is cleaved during processing of the mature protein. The mature 56638 protein corresponds to amino acids 45 to 778 of SEQ ID NO:58.

As used herein, a “56638 activity,” “biological activity of 56638,” or “functional activity of 56638,” refers to an activity exerted by a 56638 protein, polypeptide or nucleic acid molecule on e.g., a 56638-responsive cell or on a 56638 substrate, e.g., a protein substrate, as determined in vivo or in vitro. In one embodiment, a 56638 activity is a direct activity, such as an association with a 56638 target molecule. A “target molecule” “substrate” or “binding partner” is a molecule with which a 56638 protein binds or interacts in nature. A 56638 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 56638 protein with a 56638 binding partner. In an exemplary embodiment, 56638 is an enzyme for an enkephalin substrate.

Based on the above-described sequence similarities and the tissue distribution described below, the 56638 molecules of the present invention are predicted to have similar biological activities as neprilysin metalloprotease family members. Thus, in accordance with the invention, a 56638 metalloprotease or subsequence or variant polypeptide may have one or more domains and, therefore, one or more activities or functions characteristic of a neprilysin metalloprotease family member, including, but not limited to, (1) the ability to modulate the activity of a bioactive peptide, (2) the ability to cleave a neprilysin substrate, e.g., enkephalin, (3) the ability to modulate pain or inflammation response, (4) the ability to modulate spermatid cell activity or infertility, or (5) the ability to modulate hematopoietic cell activity, e.g., erythroid cell activity or B cell activity. Thus, the 56638 molecules can act as novel diagnostic targets and therapeutic agents for controlling neprilysin associated disorders.

Neprilysin is involved in the inactivation of the opioid enkephalins in the brain, which induce analgesic responses. Inhibitors of neprilysin are thus able to potentiate the analgesic effects of exogenous enkephalins, as evaluated by analgesic tests on animals, e.g., the hot plate test, tail flick test, writhing test, paw pressure test, all electric stimulation test, tail withdrawal test, or formalin test (Roques et al. (1995) Methods in Enzymology 248:263-283). Thus, 56638 neprilysin or subsequence or variant having neprilysin activity is capable of cleaving one or more protein substrates, e.g., biologically active neuropeptides, e.g., enkephalin, substance P, or somatostatin, to modulate pain response.

Neprilysin family members are also involved in the inflammatory response. Besides, enkephalin, other neprilysin substrates include endothelin (a polypeptide produced by endothelial cells that stimulates contraction of the underlying smooth muscle of blood vessel walls), and vasoactive peptides that cause vasodilation and pain, e.g., neurotensin, atrial natriuretic peptide (ANP), neurokinin, tachykinin, bradykinin, and endothelin.

TaqMan analysis revealed that 56638 mRNA is expressed in human adrenal gland, brain, heart, kidney, liver, lung, mammary gland, placenta, prostate, salivary gland, muscle, small intestine, spleen, stomach, testes, thymus, trachea, uterus, spinal cord, skin, and dorsal root ganglion (DRG). The highest 56638 mRNA expression was observed in testes, trachea, brain, spinal cord and DRG.

As 56638 mRNA is highly expressed in human testis, it suggests a role for 56638 in, e.g., fertility or spermatid development. Human 56638 appears to be a human orthologue of mouse neprilyisn NL1/SEP and the rat neprilysin II proteins (Ghaddar et al. (2000) Biochem J 347:419-429; Tanja et al. (2000) Biochem Biophys Res Comm 271:565-570). Like 56638, mouse NL1/SEP and rat neprilysin II are highly expressed in testis, and are secreted proteins. The rat and mouse proteins have been localized to the seminiferous tubules and, specifically, to spermatids (Ibid). Testicular neprilysin enzymes may act to modulate enkephalins acting as intratesticular paracrine/autocrine factors. Thus, the 56638 molecules can act as novel diagnostic targets and therapeutic agents controlling sperm formation or other processes related to fertility, e.g., spermatogenesis or fertilization.

As 56638 mRNA is highly expressed in human trachea, it also suggests a role for 56638 in modulation of the activity of bioactive peptides in the trachea, bronchus, and lung. Thus, the 56638 molecules can act as novel diagnostic targets and therapeutic agents controlling respiratory disorders, e.g., chronic obstructive pulmonary disease, emphysema, amyloidosis, lung disease, lung cancer, sleep apnea, bronchitis, pneumonias, silicosis, pulmonary edema, interstitial restrictive lung diseases, pulmonary embolus, or pulmonary hypertension.

56638 mRNA is also highly and widely expressed in the central and peripheral nervous system. More specifically, high levels of 56638 mRNA expression were found in human brain, spinal cord and DRG. Taqman experiments in rat showed that 56638 is expressed in pituitary gland, spinal cord, brain, nerve, TRG, and DRG. In situ hybridization with a 56638 probe shows that 56638 is heterogeneously expressed in monkey CNS, including expression in cerebral cortex, spinal cord, brain stem nucleus and hypothalamus. Hence, 56638 is likely a neuropeptidase, e.g., a neuropeptidase involved in pain response.

Animal models of pain response include, but are not limited to, axotomy, the cutting or severing of an axon; chronic constriction injury (CCI), a model of neuropathic pain which involves ligation of the sciatic nerve in rodents, e.g., rats; or intraplantar Freund's adjuvant injection as a model of arthritic pain. Other animal models of pain response are described in, e.g., ILAR Journal (1999) Volume 40, Number 3 (entire issue). Taqman experiments on rodent models of pain response showed that the 56638 gene is up-regulated in DRG seven days after axotomy and seven days after CCI. In situ hybridization experiments in rat pain models show up-regulation of the 56638 gene one and seven days after axotomy and after complete Freund's adjuvant intraplantar injection. These levels go back to normal at later time points. No contralateral effects were observed. These experiments indicate a role for the 56638 molecule in pain response.

Therefore, neprilysin and 56638 associated disorders can detrimentally affect regulation and modulation of the pain response; and vasoconstriction, inflammatory response and pain therefrom. Examples of neprilysin associated disorders in which the 56638 molecules of the invention may be directly or indirectly involved include pain, pain syndromes, and inflammatory disorders, including inflammatory pain.

As the 56638 polypeptides of the invention may modulate 56638-mediated activities, they may be useful for developing novel diagnostic and therapeutic agents for 56638-mediated or related disorders. For example, the 56638 molecules can act as novel diagnostic targets and therapeutic agents controlling pain, pain disorders, and inflammatory disorders. For example, a 56638 inhibitor can be useful in the treatment of pain, as 56638 inhibition could increase the endogenous levels of enkephalins and thereby increase the associated analgesic response.

The 56638 molecules can also act as novel diagnostic targets and therapeutic agents controlling pain caused by other disorders, e.g., cancer, e.g., prostate cancer. For example, endothelin, which is inactivated by neprilysin, is associated with the excruciating, debilitating pain that comes when prostate cancer invades the bone (reviewed in Nelson and Carducci (2000) BJU Int 85 Suppl 2:45-8). In addition, a neprolysin family member can be a marker of common acute lymphoblastic leukemia antigen present at the surface of B cells (Roques et al. (1993) Pharmacol Rev 45:87). Accordingly, the 56638 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, or pain therefrom.

The 56638 molecules can also act as novel diagnostic targets and therapeutic agents for brain disorders.

In addition, a neprolysin family member can be a Kell blood group antigen (Lee et al. (1999) Proc Natl Acad Sci USA 88:6353-6357). Kell antigens are highly immunogenic and may cause severe fetal anemia in sensitized mothers, erythroblastosis in newborn infants, and severe hemolytic reactions if mismatched blood is transfused. Therefore, the 56638 molecules can also act as novel diagnostic targets and therapeutic agents controlling disorders related to hematopoietic cells, e.g., blood cell- (e.g., erythroid-) associated disorders, e.g., anemia, or erythroblastosis.

The 56638 nucleic acid and protein of the invention can be used to treat and/or diagnose a variety of immune disorders.

Identification and Characterization of Human 56638 cDNA

The human 56638 sequence (SEQ ID NO:57), which is approximately 2953 nucleotides long, including untranslated regions, contains a predicted methionine-initiated coding sequence of about 2340 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO:57; SEQ ID NO:59). The coding sequence encodes a 779 amino acid protein (SEQ ID NO:58).

Tissue Distribution of 56638 mRNA

Endogenous human 56638 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology.

To determine the level of 56638 in various human tissues a primer/probe set was designed using Primer Express (Perkin-Elmer) software and primary cDNA sequence information. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. 56638 mRNA levels were analyzed in a variety of samples of human tissues, and in rodent models of pain response.

Relative 56638 mRNA expression was determined using mRNA derived from human tissue samples, both normal, and tumor. The samples are derived from human adrenal gland, brain, heart, kidney, liver, lung, mammary gland, placenta, prostate, salivary gland, muscle, small intestine, spleen, stomach, testes, thymus, trachea, uterus, spinal cord, skin, and dorsal root ganglion (DRG). The highest 56638 mRNA expression was observed in spinal cord, DRG, small intestine, testes, and trachea.

TaqMan experiments in rat showed that 56638 is expressed in pituitary gland, spinal cord, brain, nerve, TRG and DRG. TaqMan experiments on rodent models of pain response showed that the 56638 gene is up-regulated in DRG 7 days after axotomy and in the CCI model of neuropathic pain (7 days). No regulation was observed in the model of inflammatory pain, and there was no regulation in rat spinal cord in any of the models analyzed.

In situ hybridization experiments with the human 56638 probe showed expression in monkey brain, a subpopulation of DRG neurons, in the epithelium of trachea, and small intestine, as well as skin. In situ hybridization in rat animal models show up-regulation of the 56638 gene one and seven days after axotomy and after CFA intraplantar injection. These levels go back to normal at later time points. No contralateral effects were observed.

Human 18610

The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as “t ransient r eceptor”, “TR-1” or “18610” nucleic acid and polypeptide molecules, which are novel members of the transient receptor potential channel family. Transient receptor potential channel family members are ion channels, e.g., calcium channels. These novel molecules are capable of, for example, modulating an ion-channel mediated activity (e.g., a calcium channel-mediated activity) in a cell, e.g., a neuronal, muscle (e.g., cardiac muscle), or liver cell.

Calcium signaling has been implicated in the regulation of a variety of cellular responses, such as growth and differentiation. There are two general methods by which intracellular concentrations of calcium ions may be increased: calcium ions may be freed from intracellular stores, transported by specific membrane channels in the storage organelle, or calcium ions may be brought into the cell from the extracellular milieu through the use of specific channels in the cellular membrane. In the situation in which the intracellular stores of calcium have been depleted, a specific type of calcium channel, termed a ‘capacitative calcium channel’ or a ‘store-operated calcium channel’ (SOC), is activated in the plasma membrane to import calcium ions from the extracellular environment to the cytosol (see Putney and McKay (1999) BioEssays 21:38-46). Calcium may also enter the cell via receptor-stimulated cation channels (see Hofmann et al. (2000) J. Mol. Med. 78:14-25).

Members of the capacitative calcium channel family include the calcium release-activated calcium current (CRAC) (Hoth and Penner (1992) Nature 355: 353-355), calcium release-activated non-selective cation current (CRANC) (Krause et al. (1996) J. Biol. Chem. 271: 32523-32528), and the transient receptor potential (TRP) proteins TRP1, TRP2, TRP4, and TRP5. Depletion of intracellular calcium stores activate these channels by a mechanism which is yet undefined, but which has been demonstrated to involve a diffusible factor using studies in which calcium stores were artificially depleted (e.g., by the introduction of chelators into the cell, by activating phospholipase C γ , or by inhibiting those enzymes responsible for pumping calcium ions into the stores or those enzymes responsible for maintaining resting intracellular calcium ion concentrations) (Putney, J. W. (1986) Cell Calcium 7:1-12; Putney, J. W. (1990) Cell Calcium 11:611-624).

Recently, it has been elucidated that three TRP family members, TRP3, TRP6, and a mouse homologue, TRP7, form a sub-family of receptors that are activated in a calcium store-depletion independent manner. TRP3 and TRP6 are activated by diacylglycerols in a membrane delimited manner (Hofmann et al. (1999) Nature 397:259-263). Similarly, murine TRP7 is activated via diacylglycerol stimulation by G q protein coupled receptors (Okada et al. (1999) J. Biol. Chem. 274:27359-27370).

The TRP channel family is one of the best characterized calcium channel protein families. These channels include transient receptor potential proteins and homologues thereof (to date, seven TRP homologues and splice variants have been identified in a variety of organisms), the vanilloid receptor subtype I (also known as the capsaicin receptor); the stretch-inhibitable non-selective cation channel (SIC); the olfactory, mechanosensitive channel; the insulin-like growth factor I-regulated calcium channel; the vitamin D-responsive apical, epithelial calcium channel (ECaC); and melastatin, and the polycystic kidney disease protein family (see, e.g., Montell and Rubin (1989) Neuron 2:1313-1323; Caterina et al. (1997) Nature 389: 816-824; Suzuki et al. (1999) J. Biol. Chem. 274: 6330-6335; Kiselyov et al. (1998) Nature 396: 478-482; Hoenderop et al. (1999) J. Biol. Chem. 274: 8375-8378; and Chen et al. (1999) Nature 401(6751): 383-386). Each of these molecules is 700 or more amino acids in length, and shares certain conserved structural features. Predominant among these structural features are six transmembrane domains, with an additional hydrophobic loop present between the fifth and sixth transmembrane domains. It is believed that this loop is integral to the activity of the pore of the channel formed upon membrane insertion (Hardie and Minke (1993) Trends Neurosci 16: 371-376). Although found in disparate tissues and organisms, members of the TRP channel protein family all serve to transduce signals by means of calcium entry into cells, particularly pain signals (see, e.g., McClesky and Gold (1999) Annu. Rev. Physiol. 61: 835-856; Harteneck, C. (2000) Trends Neurosci. 23(4):159), light signals (Hardie and Minke, supra), or olfactory signals (Colbert et al. (1997) J. Neurosci 17(21): 8259-8269). Thus, this family of molecules may play important roles in sensory signal transduction in general.

As used herein, an “ion channel” includes a protein or polypeptide which is involved in receiving, conducting, and transmitting signals in an electrically excitable cell, e.g., a neuronal or muscle cell. Ion channels include calcium channels, potassium channels, and sodium channels. As used herein, a “calcium channel” includes a protein or polypeptide which is involved in receiving, conducting, and transmitting calcium ion-based signals in an electrically excitable cell. Calcium channels are calcium ion selective, and can determine membrane excitability (the ability of, for example, a neuronal cell to respond to a stimulus and to convert it into a sensory impulse). Calcium channels can also influence the resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation. Calcium channels are typically expressed in electrically excitable cells, e.g., neuronal cells, and may form heteromultimeric structures (e.g., composed of more than one type of subunit). Calcium channels may also be found in non-excitable cells (e.g., adipose cells or liver cells), where they may play a role in, e.g., signal transduction. Calcium channels are described in, for example, Davila et al. (1999) Annals New York Academy of Sciences 868:102-17 and McEnery, M. W. et al. (1998) J. Bioenergetics and Biomembranes 30(4): 409-418, the contents of which are incorporated herein by reference. As the TR-1 molecules of the present invention are calcium channels modulating ion channel mediated activities (e.g., calcium channel mediated activities), they may be useful for developing novel diagnostic and therapeutic agents for ion channel associated disorders (e.g., calcium channel associated disorders).

As used herein, an “ion channel associated disorder” includes a disorder, disease or condition which is characterized by a misregulation of an ion channel mediated activity. For example, a “calcium channel associated disorder” includes a disorder, disease or condition which is characterized by a misregulation of a calcium channel mediated activity. Ion channel associated disorders, e.g., calcium channel associated disorders, include but are not limited to CNS disorders, pain disorders, cellular proliferation, growth, differentiation, or migration disorders.

As used herein, the term “pain signaling mechanisms” includes the cellular mechanisms involved in the development and regulation of pain, e.g., pain elicited by noxious chemical, mechanical, or thermal stimuli, in a subject, e.g., a mammal such as a human. In mammals, the initial detection of noxious chemical, mechanical, or thermal stimuli, a process referred to as “nociception”, occurs predominantly at the peripheral terminals of specialized, small diameter sensory neurons. These sensory neurons transmit the information to the central nervous system, evoking a perception of pain or discomfort and initiating appropriate protective reflexes. The TR-1 molecules of the present invention may be present on these sensory neurons and, thus, may be involved in detecting these noxious chemical, mechanical, or thermal stimuli and transducing this information into membrane depolarization events. Thus, the TR-1 molecules by participating in pain signaling mechanisms, may modulate pain elicitation and act as targets for developing novel diagnostic targets and therapeutic agents to control pain.

As used herein, a “cellular proliferation, growth, differentiation, or migration process” is a process by which a cell increases in number, size or content, by which a cell develops a specialized set of characteristics which differ from that of other cells, or by which a cell moves closer to or further from a particular location or stimulus. The TR-1 molecules of the present invention are involved in signal transduction mechanisms, which are known to be involved in cellular growth, differentiation, and migration processes. Thus, the TR-1 molecules may modulate cellular growth, differentiation, or migration, and may play a role in disorders characterized by aberrantly regulated growth, differentiation, or migration. Such disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletal dysplasia; neuronal deficiencies resulting from impaired neural induction and patterning; hepatic disorders; cardiovascular disorders; and hematopoietic and/or myeloproliferative disorders.

As used herein, an “ion channel mediated activity” includes an activity which involves an ion channel, e.g., an ion channel in a neuronal cell, a muscular cell, or a liver cell, associated with receiving, conducting, and transmitting signals, in, for example, the nervous system. Ion channel mediated activities (e.g., calcium channel mediated activities) include release of neurotransmitters or second messenger molecules (e.g., dopamine or norepinephrine), from cells, e.g., neuronal cells; modulation of resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation; participation in signal transduction pathways, and modulation of processes such as integration of sub-threshold synaptic responses and the conductance of back-propagating action potentials in, for example, neuronal cells (e.g., changes in those action potentials resulting in a morphological or differentiative response in the cell).

The family of TR-1 polypeptides comprise at least one “transmembrane domain” and preferably six transmembrane domains. As used herein, the term “transmembrane domain” includes an amino acid sequence of about 10-30 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 10, 15, 20, 25, or 30 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, alanines, valines, phenylalanines, prolines or methionines. Transmembrane domains are described in, for example, Zagotta W. N. et al, (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which are incorporated herein by reference. Amino acid residues 758-774, 856-876, 923-941, 957-974, 1000-1016, and 1071-1096 of the 18610 or TR-1 polypeptide comprise transmembrane domains. Accordingly, TR-1 polypeptides having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a transmembrane domain of human TR-1 are within the scope of the invention.

In another embodiment, a 18610 or TR-1 molecule of the present invention is identified based on the presence of at least one pore domain between the fifth and sixth transmembrane domains. As used herein, the term “pore domain” includes an overall hydrophobic amino acid sequence which is located between two transmembrane domains of a calcium channel protein, preferably transmembrane domains 5 and 6, and which is believed to be a major determinant of ion selectivity and channel activity in calcium channels. Pore domains are described in, for example Vannier et al. (1998) J. Biol. Chem. 273: 8675-8679 and Phillips, A. M. et al. (1992) Neuron 8, 631-642, the contents of which are incorporated herein by reference. TR-1 molecules having at least one pore domain are within the scope of the invention. A pore domain is found in the human TR-1 sequence (SEQ ID NO:64) at about residues 1036-1055.

In another embodiment, a TR-1 molecule of the present invention is identified based on the presence of at least one “transient receptor domain.” As used herein, the term “transient receptor domain” includes a protein domain having an amino acid sequence of about 40-175 amino acid residues which serves to transport ions. Preferably, a transient receptor domain includes at least about 48 amino acid residues. To identify the presence of a transient receptor domain in a TR-1 protein, and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein may be searched against a database of known protein domains (e.g., the HMM database). The transient receptor domain (HMM) has been assigned the PFAM Accession PF02164. A search was performed against the HMM database resulting in the identification of three transient receptor domains in the amino acid sequence of human 18610 (SEQ ID NO:64) at about residues 699-747, 849-1016, and 1079-1137 of SEQ ID NO:64.

A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28:405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference.

In a preferred embodiment, the TR-1 molecules of the invention include at least one transmembrane domain, preferably six transmembrane domains, at least one pore domain, and/or at least one transient receptor domain.

Isolated polypeptides of the present invention, preferably 18610 or TR-1 polypeptides, have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:64 or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:63 or 65. As used herein, the term “sufficiently identical” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology or identity across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical. Furthermore, amino acid or nucleotide sequences which share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology or identity and share a common functional activity are defined herein as sufficiently identical.

In a preferred embodiment, a TR-1 polypeptide includes at least one or more of the following domains: a transmembrane domain, and/or a pore domain, and/or a transient receptor domain, and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to the amino acid sequence of SEQ ID NO:64. In yet another preferred embodiment, a TR-1 polypeptide includes at least one or more of the following domains: a transmembrane domain, and/or a pore domain, and/or a transient receptor domain, and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:63 or SEQ ID NO:65. In another preferred embodiment, a TR-1 polypeptide includes at least one or more of the following domains: a transmembrane domain, and/or a pore domain, and/or a transient receptor domain, and has a 18610 or TR-1 activity.

As used interchangeably herein, a “TR-1 activity”, “biological activity of TR-1” or “functional activity of TR-1”, refers to an activity exerted by a TR-1 polypeptide or nucleic acid molecule on a TR-1 responsive cell or tissue, or on a TR-1 polypeptide substrate, as determined in vivo, or in vitro, according to standard techniques. In one embodiment, a TR-1 activity is a direct activity, such as an association with a TR-1-target molecule. As used herein, a “substrate,” “target molecule,” or “binding partner” is a molecule with which a TR-1 polypeptide binds or interacts in nature, such that TR-1-mediated function is achieved. A TR-1 target molecule can be a non-TR-1 molecule or a TR-1 polypeptide or polypeptide of the present invention. In an exemplary embodiment, a TR-1 target molecule is a TR-1 ligand, e.g., a calcium channel ligand such as calcium. Alternatively, a TR-1 activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the TR-1 polypeptide with a TR-1 ligand. The biological activities of TR-1 are described herein. For example, the TR-1 polypeptides of the present invention can have one or more of the following activities: (1) modulate membrane excitability, (2) influence the resting potential of membranes, (3) modulate wave forms and frequencies of action potentials, (4) modulate thresholds of excitation, (5) modulate neurite outgrowth and synaptogenesis, (6) modulate signal transduction, (7) participate in nociception, and (8) bind and transport calcium ions.

The nucleotide sequence of the isolated human TR-1 cDNA and the predicted amino acid sequence of the human TR-1 polypeptide are shown in SEQ ID NOs:63 and 64, respectively.

Isolation of the Human 18610 or TR-1 cDNA

The invention is based, at least in part, on the discovery of a human gene encoding a novel polypeptide, referred to herein as either human 18610 or TR-1. The entire sequence of the human clone Fbh18610 was determined and found to contain an open reading frame termed human “18610” or “TR-1.” The nucleotide sequence of the human 18610 gene, which is 7334 nucleotides in length, is set forth in the Sequence Listing as SEQ ID NO:63. The amino acid sequence of the human 18610 expression product is set forth in the Sequence Listing as SEQ ID NO:64. The 18610 polypeptide comprises about 1885 amino acids. The coding region (open reading frame) of SEQ ID NO:63 is set forth as SEQ ID NO:65.

Analysis of the Human 18610 or TR-1 Molecules

A search using the polypeptide sequence of SEQ ID NO:64 was performed against the HMM database in PFAM resulting in the identification of three potential transient receptor domains in the amino acid sequence of human TR-1 at about residues 699-747, 849-1016, and 1079-1137 of SEQ ID NO:64. A search also identified an ion transport protein domain in the amino acid sequence of human TR-1 (SEQ ID NO:64) at about amino acid residues 884-1096 and an AN1-like zinc finger domain at about residues 33-61 of SEQ ID NO:64.

The amino acid sequence of human TR-1 was analyzed using the program PSORT to predict the localization of the proteins within the cell. This program assesses the presence of different targeting and localization amino acid sequences within the query sequence. The results of the analyses show the likelihood of human 18610 or TR-1 (SEQ ID NO:64) being localized, for example, to the endoplasmic reticulum, the nucleus, and the plasma membrane.

A MEMSAT analysis of the polypeptide sequence of SEQ ID NO:64 was also performed, predicting eight potential transmembrane domains in the amino acid sequence of human 18610 or TR-1 (SEQ ID NO:64) at about residues 282-301, 507-524, 758-774, 856-876, 923-941, 957-974, 1000-1016, and 1127-1146 of SEQ ID NO:64. However, a structural, hydrophobicity, and antigenicity analysis resulted in the identification of six transmembrane domains (TM1-TM6) and one pore domain between transmembrane domains five and six. TM1 is at about residues 758-774 of SEQ ID NO:64, TM2 is at about residues 856-876 of SEQ ID NO:64, TM3 is at about residues 923-941 of SEQ ID NO:64, TM4 is at about residues 957-974 of SEQ ID NO:64, TM5 is at about residues 1000-1016 of SEQ ID NO:64, TM6 is at about residues 1071-1096 of SEQ ID NO:64, and the pore domain is at about residues 1036-1055 of the amino acid sequence set forth as SEQ ID NO:64.

Searches of the amino acid sequence of human 18610 were further performed against the Prosite database. These searches resulted in the identification in the amino acid sequence of human 18610 (SEQ ID NO:64) of a number of potential N-glycosylation sites at about residues 404-407, 550-553, 715-718, 805-808, 925-928, 1058-1061, 1485-1488, 1616-1619, 1794-1797, and 1870-1873 of SEQ ID NO:64, a number of potential cAMP and cGMP-dependent protein kinase phosphorylation sites at about residues 600-603, 754-757, 1493-1496, and 1521-1524 of SEQ ID NO:64, a number of potential kinase C phosphorylation sites at about residues 2-4, 12-14, 22-24, 103-105, 195-197, 318-320, 349-351, 523-525, 529-531, 547-549, 615-617, 697-699, 727-729, 836-838, 842-844, 1245-1247, 1410-1412, 1456-1458, 1491-1493, 1520-1522, 1547-1549, 1719-1721, 1871-1873, and 1880-1882 of SEQ ID NO:64, a number of potential casein kinase II phosphorylation sites at about residues 5-8, 12-15, 22-25, 87-90, 115-118, 299-302, 367-370, 406-409, 508-511, 593-596, 603-606, 675-678, 778-781, 795-798, 883-886, 1163-1166, 1191-1194, 1361-1364, 1413-1416, 1430-1433, 1524-1527, 1547-1550, 1576-1579, 1635-1638, 1652-1655, 1763-1766, 1779-1782, and 1871-1874 of SEQ ID NO:64, a number of potential tyrosine kinase phosphorylation sites at about residues 320-327, 1212-1220, and 1566-1574 of SEQ ID NO:64, a number of potential N-myristoylation sites at about residues 32-37, 99-104, 159-164, 174-179, 208-213, 317-322, 357-362, 402-407, 522-527, 940-945, 1293-1298, 1349-1354, 1385-1390, 1438-1443, 1556-1561, 1642-1647, 1734-1739, and 1790-1795 of SEQ ID NO:64, and an amidation site at about residues 597-600 of SEQ ID NO:64.

A search of the amino acid sequence of human 18610 (SEQ ID NO:64) was also performed against the ProDom database. The results of this search identified numerous matches against protein domains described as, for example, “receptor from F54D1.5 transient sequence,” “melastatin FIS chromosome receptor MTR1 transmembrane,” “melastatin receptor chromosome transmembrane transient potential related,” “melastatin FIS receptor MTR1 transmembrane chromosome,” “receptor channel potential transient NOMPC TRP2 2-beta 2-alpha,” “receptor transient potential-related,” “channel receptor calcium transient potential repeat vanilloid transmembrane ion transport,” “kinase serine/threonine-protein, ATP-binding transferase,” “kinase elongation serine/threonine-protein transferase factor-2 eukaryotic calcium/calmodulin-dependent repeat,” “kinase receptor-like,” and the like were identified.

Tissue Distribution of Human 18610 or TR-1 mRNA by PCR Analysis

The following describes the tissue distribution of human 18610 mRNA, as may be determined by Polymerase Chain Reaction (PCR) on cDNA libraries using oligonucleotide primers based on the human 18610 sequence. For in situ analysis, various tissues, e.g. tissues obtained from brain, are first frozen on dry ice.

Tissue Distribution of Human 18610 or TR-1 mRNA by TaqMan™ Analysis

This example describes the tissue distribution of human 18610 mRNA in a variety of cells and tissues, as determined using the TaqMan™ procedure.

A human tissue panel was tested revealing highest expression of human 18610 mRNA in the in Jurkat cells (T-cell leukemia cells) and K562 cells (chronic myeloid leukemia cells), indicating a role for 18610 in cellular proliferation, growth, differentiation, or migration disorders such as cancer.

Human 33217

The invention is based, at least in part, on the identification of a novel AMP binding enzyme, referred to herein as “33217”. The human 33217 sequence (see SEQ ID NO:66), which is approximately 2846 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 2058 nucleotides, including the termination codon (see SEQ ID NO:68). The coding sequence encodes a 685 amino acid protein (see SEQ ID NO:67).

Human 33217 contains the following regions or other structural features: an AMP-binding enzyme domain (PFAM Accession Number PF00501) located at about amino acid residues 144-585 of SEQ ID NO:67, which includes a predicted AMP-binding domain signature (PS00455) at about amino acids 295 to 306 of SEQ ID NO:67; two predicted N-glycosylation sites (PS00001) from about amino acids 359-362 and 608-611 of SEQ ID NO:67; one predicted glycosaminoglycan attachment site (PS00002) from about amino acids 56-59 of SEQ ID NO:67; one predicted cAMP/cGMP-dependent protein kinase phosphorylation site (PS00004) located at about amino acids 9-12 of SEQ ID NO:67; four predicted Protein Kinase C phosphorylation sites (PS00005) at about amino acids 101-103, 144-146, 207-209, and 646-648 of SEQ ID NO:67; seven predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino acids 58-61, 69-72, 144-147, 208-211, 552-555, 579-582, and 667-670 of SEQ ID NO:67; fourteen predicted N-myristylation sites (PS00008) from about amino acids 23-28, 29-34, 44-49, 163-168, 191-196, 199-204, 224-229, 303-308, 328-333, 370-375, 405-410, 453-458, 462-467, and 510-515 of SEQ ID NO:67; and one predicted amidation site (PS00009) from about amino acids 227-230 of SEQ ID NO:67.

Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, e.g., the sequence from about amino acid 30 to 40, from about 185 to 200, and from about 385 to 395 of SEQ ID NO:67; all or part of a hydrophilic sequence, e.g., the sequence of from about amino acid 85 to 100, from about 270 to 280, and from about 465 to 475 of SEQ ID NO:67.

For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420.

The 33217 protein contains a significant number of structural characteristics in common with members of the AMP-binding enzyme family.

Acetyl-Coenzyme A (Ac-CoA) is an activated form of acetate that is involved in lipid biosynthesis, energy metabolism, and other normal processes in human cells. Ac-CoA can be generated by catabolism of glucose (e.g., through operation of the Krebs cycle) or fatty acids.

Ac-CoA is a starting material used in biosynthesis of cholesterol, fatty acids, lipids, and biochemical products derived from these (e.g., sterol and other hormones). Ac-CoA is made by ligation of an adenylate moiety (derived by cleaving a pyrophosphonate moiety from ATP) with the acetyl carboxyl group, and then by substituting a CoA moiety in place of the adenylate moiety. Overall, the net reaction is: acetate+CoASH+ATP→Ac-CoA+AMP+PP i . This reaction is catalyzed by an enzyme designated acetyl-CoA synthetase (ACS; EC 6.2.1.1; sometimes designated acetate-CoA ligase, acetate thiokinase, or acetyl-activating enzyme).

ACS enzymes are involved in lipid synthesis and energy generation. A cytosolic form of human ACS has been cloned, and an in vitro enzymatic assay of ACS activity has been described (Luong et al. (2000) J. Biol. Chem. 275:26458-26466). In yeast and bacteria, expression of ACS can be induced or enhanced by one or more of a decrease in oxygen partial pressure, an increase in intracellular cAMP concentration, and increased carbon flux through acetate-associated metabolic pathways (Kratzer et al. (1997) Mol. Microbiol. 26:631-641; Hiesinger et al. (1997) FEBS Lett. 415:16-20; Kumari et al. (2000) J. Bacteriol. 182:4173-4179). ACS is also up-regulated in developing plant seeds (Ke et al. (2000) Plant Physiol. 123:497-508).

The AMP-binding enzyme family of proteins is characterized by a common domain, an “AMP-binding enzyme domain,” that permits the respective family members to act via and ATP-dependent covalent binding of AMP to their substrates.

A 33217 polypeptide can include a “AMP-binding enzyme domain” or regions homologous with a “AMP-binding enzyme domain.”

As used herein, the term “AMP-binding enzyme domain” includes an amino acid sequence of about 250 to 600 amino acid residues in length and having a bit score for the alignment of the sequence to the AMP-binding enzyme domain profile (Pfam HMM) of at least 100. Preferably, a AMP-binding enzyme domain includes at least about 350 to 500 amino acids, more preferably about 400 to 475 amino acid residues, or about 430 to 450 amino acids and has a bit score for the alignment of the sequence to the AMP-binding enzyme domain (HMM) of at least 130, 150, 190 or greater. The AMP-binding enzyme domain (HMM) has been assigned the PFAM Accession Number PF00501. Preferably, a 33217 polypeptide includes an AMP-binding domain signature having the consensus sequence [LIVMFY]-x(2)-[STG]-[STAG]-G-[ST]-[STEI]-[SG]-x-[PASLIVM]-[KR] (SEQ ID NO:70). Preferably, a 33217 polypeptide contains the AMP-binding domain signature located at amino acids 295-306 of SEQ ID NO:67.

In a preferred embodiment 33217 polypeptide or protein has a “AMP-binding enzyme domain” or a region which includes at least about 350 to 500 more preferably about 400 to 475 or 430 to 450 amino acid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “AMP-binding enzyme domain,” e.g., the AMP-binding enzyme domain of human 33217 (e.g., residues 144 to 585 of SEQ ID NO:67).

To identify the presence of a “AMP-binding enzyme” domain in a 33217 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence of the protein can be searched against the Pfam database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a “AMP-binding enzyme” domain in the amino acid sequence of human 33217 at about residues 144 to 585 of SEQ ID NO:67. The identified AMP-binding enzyme domain is depicted in SEQ ID NO:69.

Human 33217 is predicted to be an acetyl-CoA synthetase enzyme (i.e., an acetyl-CoA ligase). Amino acid residues 205-404 of SEQ ID NO:67 align with amino acid residues 1034-1633 of a Pseudomonas aeruginosa acetyl-CoA synthetase (GENBANK™ Accession number AAG06956) with 58% sequence identity (117/200). The BLAST score for this alignment is 642 (297.1 bits). In addition, amino acid residues 412-623 of SEQ ID NO:67 align with amino acid residues 1658-2293 of the Pseudomonas aeruginosa enzyme.

Amino acid residues 75-420 of SEQ ID NO:67 align with amino acid residues 617-1654 of a Tetrahymena pyriformis acetyl-CoA synthetase (GENBANK™ Accession number BAA86907) with 47% sequence identity (163/346). The BLAST score for this alignment is 864 (398.8 bits). In addition, amino acid residues 438-554 of SEQ ID NO:67 align with amino acid residues 1706-2056 of the Tetrahymena pyriformis enzyme, and amino acid residues 567-612 of SEQ ID NO:67 align with amino acid residues 2090-2227 of the Tetrahymena pyriformis enzyme.

A 33217 family member can include an AMP-binding enzyme domain and at least one AMP-binding domain signature. Furthermore, a 33217 family member can include at least one, preferably two predicted N-glycosylation sites (PS00001); at least one predicted glycosaminoglycan attachment site (PS00002); at least one predicted cAMP/cGMP-dependent protein kinase phosphorylation site (PS00004); at least one, two, three, and preferably four predicted protein kinase C phosphorylation sites (PS00005); at least one, two, three, four, five, six, and preferably seven predicted casein kinase II phosphorylation sites (PS00006); and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and preferably 14 predicted N-myristylation sites (PS00008); and at least one predicted amidation site (PS00009).

As the 33217 polypeptides of the invention may modulate 33217-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 33217-mediated or related disorders, as described below.

As used herein, a “33217 activity”, “biological activity of 33217” or “functional activity of 33217”, refers to an activity exerted by a 33217 protein, polypeptide or nucleic acid molecule. For example, a 33217 activity can be an activity exerted by 33217 in a physiological milieu on, e.g., a 33217-responsive cell or on a 33217 substrate, e.g., a protein substrate. A 33217 activity can be determined in vivo or in vitro. In one embodiment, a 33217 activity is a activity is a direct activity, such as acetyl-CoA ligase activity, e.g., acetyl-CoA synthetase activity (i.e., ligation of a CoA moiety with an acetate moiety coupled with removal of a pyrophosphate moiety from ATP; formation of acetyl-CoA from acetate and CoASH catalyzed by a 33217 protein, proceeding through formation of an acetyl-adenylate intermediate). A “target molecule” or “binding partner” is a molecule with which a 33217 protein binds or interacts in nature, e.g., an integral membrane protein. In an exemplary embodiment, 33217 is an enzyme that acts via an ATP-dependent binding of AMP to its substrate.

A 33217 activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the 33217 protein with a 33217 receptor. The features of the 33217 molecules of the present invention can provide similar biological activities as AMP-binding enzyme family members. For example, the 33217 proteins of the present invention can have one or more of the following activities: (1) acetyl-CoA ligase activity; (2) promotion of activation of acetate; (3) promotion of acetate utilization (4) enhancement of uptake of acetate into fatty acids and biochemical products made from fatty acids (e.g., lipids and hormones such as sterol hormones); (5) promoting angiogenesis; (6) enhancing or inducing expression of genes involved in angiogenesis; (7) enhancing tumor growth; (8) enhancing tumor cell survival; (9) inducing neo-angiogenesis; (10) inducing aberrant angiogenesis; (11) inducing tumorigenesis; (12) enhancing tumor cell metastasis; (13) enhancing tumor cell invasivity; or (14) agonizing or antagonizing one or more of (1)-(13).

The 33217 polypeptide is predicted to be a soluble protein that displays enzymatic activity. The 33217 polypeptide is likely to be localized in the cytosol of human cells, although it can also be localized within mitochondria. 33217 is expressed in several types of tumor cells and is expressed at a significantly lower level (or is not expressed) in the corresponding normal tissue. For example, 33217 is expressed in many tumor cells of glioblastomas (i.e., a type of brain tumor), but is expressed at a significantly lower level in normal brain cells. Similarly, 33217 is expressed in tumor cells of papillary serous ovarian tumors, but is expressed at a significantly lower level in normal ovarian cells. 33217 is also expressed in tumor cells of small cell lung tumors, but is expressed at a significantly lower level in normal lung cells and, apparently, in lung tumor cells of other types (e.g., non-small cell lung tumor cells).

Expression of 33217 correlates with expression of angiogenic factors, including VEGF, IL-8, Id3, and HIF-1a (as described below). Co-regulation of 33217 and known angiogenic factors is an indication that 33217 is among the proteins involved in promoting angiogenesis. Up-regulation of 33217 in tumor cells is an indication that this protein is involved in angiogenesis associated with tumor growth and survival. Involvement of other ACS enzymes in cell cycling, metabolic carbon flux, and seed development in non-human organisms suggests that 33217 has a role in shifting the metabolism of normal cells to adjust to altered growth conditions (e.g., hypoxia, metabolic changes associated with one or more of tumorigenesis, tumor growth, tumor invasion of surrounding tissues, and metastasis). Together, these observations indicate that 33217 has a role in survival, growth, invasiveness, and metastasis of tumor cells. Modulation (e.g., decrease or increase) of 33217 expression can therefore modulate these disease processes, indicating therapeutic, diagnostic, prognostic, and preventive utility for the nucleic acids, polypeptides, and other 33217-associated molecules described in this disclosure.

The 33217 enzymatic activity is predicted to include acetyl-CoA ligase activity, i.e., formation of acetyl-CoA thioesters, which can be used for lipid biosynthesis (and biosynthesis of biochemicals made from fatty acids and lipids, such as cholesterol and hormones like the sterol hormones) or oxidized and used as a cellular energy source. In particular, 33217 is predicted to display acetyl-CoA synthetase activity.

Based on the above-described sequence similarities and functional characterizations, the 33217 molecules of the present invention are predicted to have similar biological activities as AMP-binding enzyme family members. Thus, the 33217 molecules can act as novel diagnostic targets and therapeutic agents for fatty acid metabolism disorders and for cellular proliferative and/or differentiative disorders.

Disorders which may be treated or diagnosed by methods described herein include, but are not limited to, adrenoleukodystrophy, hypocholesterolemia, hypercholesterolemia, and disorders associated with an accumulation in the liver of fibrous tissue, such as that resulting from an imbalance between production and degradation of the extracellular matrix accompanied by the collapse and condensation of preexisting fibers. The methods described herein can be used to diagnose or treat hepatocellular necrosis or injury induced by a wide variety of agents including processes which disturb homeostasis, such as an inflammatory process, tissue damage resulting from toxic injury or altered hepatic blood flow, and infections (e.g., bacterial, viral and parasitic). For example, the methods can be used for the early detection of hepatic injury, such as portal hypertension or hepatic fibrosis. In addition, the methods can be employed to detect liver fibrosis attributed to inborn errors of metabolism, for example, fibrosis resulting from a storage disorder such as Gaucher's disease (lipid abnormalities) or a glycogen storage disease, A1-antitrypsin deficiency; a disorder mediating the accumulation (e.g., storage) of an exogenous substance, for example, hemochromatosis (iron-overload syndrome) and copper storage diseases (Wilson's disease), disorders resulting in the accumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) and peroxisomal disorders (e.g., Zellweger syndrome). Additionally, the methods described herein may be useful for the early detection and treatment of liver injury associated with the administration of various chemicals or drugs, such as for example, methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, or which represents a hepatic manifestation of a vascular disorder such as obstruction of either the intrahepatic or extrahepatic bile flow or an alteration in hepatic circulation resulting, for example, from chronic heart failure, veno-occlusive disease, portal vein thrombosis or Budd-Chiari syndrome.

Expression of 33217 was also detected in normal kidney, Wilm's tumor, uterine adenocarcinoma, fetal adrenal (very low), fetal kidney, fetal heart, normal heart, spinal cord, and lymphangioma tissues. Accordingly, 33217 nucleic acid sequences and fragments thereof, proteins encoded by these sequences and fragments thereof, as well as modulators of 33217 gene or protein activity can be useful in diagnosing or treating diseases that involve these tissues in which the 33217 is expressed.

Identification and Characterization of Human 33217 cDNA

The human 33217 sequence (SEQ ID NO:66) is approximately 2846 nucleotides long. The region between and inclusive of the initiation codon and the termination codon is a methionine-initiated coding sequence of about 2058 nucleotides, including the termination codon (nucleotides indicated as “coding” of SEQ ID NO:66; SEQ ID NO:68). The coding sequence encodes a 685 amino acid protein (SEQ ID NO:67).

Tissue Distribution of 33217 mRNA by TaqMan Analysis

Endogenous human 33217 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology.

To determine the level of 33217 in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of cell lines or human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues and cell lines shown in Tables 42, 43, and 44.

As shown in Tables 42 and 43, expression of 33217 correlates with expression of angiogenic factors, including VEGF, IL-8, Id3, and HIF-1a. Co-regulation of 33217 and angiogenic factors is an indication that 33217 participates in angiogenic processes.

As shown in Table 44, expression of 33217 is highly elevated in some lung tumor samples, as compared to normal lung tissue samples.

Human 21967

The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as Lysyl Oxidase Related-2 (“Lor-2”) molecules, “Lor-2” or “21967” nucleic acid and polypeptide molecules, which play a role in or function in a variety of cellular processes in the cardiovascular system, e.g., cardiac cell function. In another embodiment, the Lor-2 molecules of the present invention modulate the activity of one or more proteins involved in a cardiovascular disorder, e.g., congestive heart failure, ischemia, cardiac hypertrophy, ischemic-reperfusion injury.

As used herein, the term “cardiovascular disorder” includes a disease, disorder, or state involving the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. Examples of such disorders include hypertension, atherosclerosis, coronary artery spasm, coronary artery disease, valvular disease, arrhythmias, and cardiomyopathies.

As used herein, the term “congestive heart failure” includes a condition characterized by a diminished capacity of the heart to supply the oxygen demands of the body. Symptoms and signs of congestive heart failure include diminished blood flow to the various tissues of the body, accumulation of excess blood in the various organs, e.g., when the heart is unable to pump out the blood returned to it by the great veins, exertional dyspnea, fatigue, and/or peripheral edema, e.g., peripheral edema resulting from left ventricular dysfunction. Congestive heart failure may be acute or chronic. The manifestation of congestive heart failure usually occurs secondary to a variety of cardiac or systemic disorders that share a temporal or permanent loss of cardiac function. Examples of such disorders include hypertension, coronary artery disease, valvular disease, and cardiomyopathies, e.g., hypertrophic, dilative, or restrictive cardiomyopathies. Congestive heart failure is described in, for example, Cohn J. N. et al. (1998) American Family Physician 57:1901-04, the contents of which are incorporated herein by reference.

As used herein, the term “cardiac cellular processes” includes intra-cellular or inter-cellular processes involved in the functioning of the heart. Cellular processes involved in the nutrition and maintenance of the heart, the development of the heart, or the ability of the heart to pump blood to the rest of the body are intended to be covered by this term. Such processes include, for example, cardiac muscle contraction, distribution and transmission of electrical impulses, and cellular processes involved in the opening and closing of the cardiac valves. The term “cardiac cellular processes” further includes processes such as the transcription, translation and post-translational modification of proteins involved in the functioning of the heart, e.g., myofilament specific proteins, such as troponin I, troponin T, myosin light chain 1 (MLC1), and α-actinin.

Lysyl oxidase (“LOX”) is an extracellular copper enzyme that initiates the crosslinking of collagens and elastin by catalyzing oxidative deamination of the ε-amino group in certain lysine and hydroxylysine residues of collagens and lysine residues of elastin (Smith-Mungo and Kagan (1998) Matrix Biol. 16:387-398 and Kaman in Biology of Extracellular Matrix , ed. Mecham (1986) Academic Press pp. 321-389). Lysyl oxidase has been shown to be important in a variety of cellular and physiologic processes including biogenesis of connective tissue matrices and bone resorption. A deficiency in lysyl oxidase activity is found in two X-linked, recessively inherited connective tissue disorders, the type IX variant of the Ehlers-Danlos syndrome and the Menkes syndrome, and in the X-linked, recessively inherited mottled series of allelic mutant mice (all characterized by abnormalities in copper metabolism). (Byers et al. (1980) New Engl. J. Med. 303:61-65; Royce et al. (1980) Biochemistry J. 192:579-586; Kuivaniemi et al. (1982) J. Clin. Invest. 69:730-733; Kuivaniemi et al. (1985) Amer. J. Human. Genet. 37:798-808; Peltonen et al. (1983) Biochemistry 22:6156-6163; Rowe et al. (1977) J. Biol. Chem. 252:939-942; Starcher et al. (1977) Biochem. Biophys. Res. Commun. 78:706-712; Danks in The Metabolic Basis of Inherited Disease ”, eds. Stanbury et al. (1983), McGraw - Hill pp. 1251-1268). Increased lysyl oxidase activity has been associated with fibrotic disorders such as atherosclerosis, hypertension, and liver and pulmonary fibrosis. (Kagan, supra).

More recently there have been identified proteins having structural and/or functional similarities to lysyl oxidase. For example, a lysyl oxidase-like protein, referred to herein as “LOL”, was identified from a human skin fibroblast cDNA library that contains extensive homology to several coding domains within the human lysyl oxidase mRNA which is believed to be involved in collagen maturation. (Kenyon et al. (1993) J. Biol. Chem. 268:18435-18437 and Kim et al. (1995) J. Biol. Chem. 270:7176-7182). Recent cloning and analysis of the mouse LOL gene (Kim et al. (1999) J. Cell Biochem. 72:181-188) demonstrated that steady state levels of LOL mRNA and type III procollagen mRNA increased coincidentally early in the development of liver fibrosis. In contrast, steady state levels of lysyl oxidase mRNA increased throughout the onset of hepatic fibrosis and appeared in parallel with the increased steady state levels of pro-alpha (I) collagen mRNA, suggesting that the LOL protein is involved in the development of lysine-derived cross-links in collagenous substrates. Moreover, the substrate specificity of the LOL protein may be different to that of lysyl oxidase and this difference may be collagen-type specific.

Likewise, a protein referred to herein as lysyl-oxidase related protein (“Lor”) has been identified which inhibits many of the structural features of lysyl oxidase and is overexpressed in senescent fibroblasts and is believed to play a role in age-associated changes in extracellular proteins. (Saito et al. (1997) J. Biol. Chem. 272:8157-8160). Lor contains four domains referred to herein as scavenger receptor cysteine-rich domains (“SRCR domains”) which are believed to be involved in binding to other cell surface proteins or extracellular molecules. The SRCR domain joins a long list of other widely distributed cysteine-containing domains found in extracellular portions of membrane proteins and in secreted proteins (Doolittle (1985) Trends Biochem. Sci. 10:233-237; Krieger in Molecular Structures of Receptors , eds. Rossow et al. (1986) Horwood, Chichester, U.K. pp. 210-231). Examples include the EGF-like domain, immunoglobulin superfamily domains, the LDL receptor/complement. C9 domain, clotting factor Kringle domains, and fibronectin domains. These disulfide cross-linked domains appear to provide stable core structures that (i) are able to withstand the rigors of the extracellular environment; (ii) are well suited for a variety of biochemical tasks, often involving binding; and (iii) are readily juxtaposed to other types of domains to permit the construction of complex mosaic proteins. (Doolittle supra; Sudhof et al. (1985) Science 228:815-822).

Lysyl oxidases (“LOXs”) have been immunolocalized to the extracellular matrix regions of stroma surrounding early breast cancers (Decitre et al. (1998) Lab Invest 78:143-151), with decreased expression observed in the stroma surrounding invasive breast cancers (Peyrol et al. (1997) Am. J. Pathol. 150:497-507). A progressive loss of LOX expression has also been observed during prostrate cancer progression in mice (Ren et al. (1998) Cancer Res. 58:1285-1290). These observations suggest that lysyl oxidases may function as tumor suppressors.

It has further been shown that human Lor is highly expressed in all adherent tumor cell lines examined, but not in cell lines that grow in suspension (Saito et al., supra), suggesting that LOXs can increase the adhesion properties of tumor cells. Lor expression was demonstrated to be concomitant with upregulation of type I procollagen. As adhesion properties contribute to the ability of tumor cells to colonize new sites, a tumor-promoting role for LOXs is also probable.

One embodiment of the invention features Lor-2 nucleic acid molecules, preferably human Lor-2 molecules, which were identified from a cDNA library made from the heart of a patient with congestive heart failure (CHF). The Lor-2 nucleic acid and protein molecules of the invention are described in further detail in the following subsections.

In yet another embodiment, the isolated proteins of the present invention, preferably Lor-2 proteins, can be identified based on the presence at least one SRCR domain and/or a lysyl oxidase domain and/or and a signal sequence.

In a preferred embodiment, a Lor-2 family member includes at least 1, 2, 3, 4, or more scavenger receptor cysteine-rich (“SRCR”) domains. Scavenger receptors are proteins which have been implicated in the development of atherosclerosis and other macrophage-associated functions. For example, the type I mammalian macrophage scavenger receptors are membrane glycoproteins implicated in the pathologic deposition of cholesterol in arterial walls during atherogenesis (Freeman et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:8810-8814). Scavenger receptors are characterized by the presence of a cysteine-rich domain, which is proposed to be involved in binding of physiological ligands (e.g., cell-surface proteins). This cysteine rich domain is referred to herein and in the art as a scavenger receptor cysteine-rich (“SRCR”) domains. Intra- or intercellular binding of ligand to the SRCR domain is believed to play a role in signaling or adhesion

As defined herein, a SRCR domain includes a protein domain which is about 88-112 amino acid residues in length and has about 16-60% identity with a SRCR of type I human macrophage scavenger receptor (e.g., amino acid residues 353-450 of SEQ ID NO:80). In another embodiment, a SRCR is abuse 90-110, 92-108, 94-106, or 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, or 106 amino acid residues in length and has about 22-54%, 26-50%, 28-48%, or 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, or 47% identity with a SRCR of type I human macrophage scavenger receptor (e.g., amino acid residues 353-450 of SEQ ID NO:80). For example, a SRCR domain can be found in murine type I scavenger receptor (Accession No. 1709140) from about amino acid residues 360-457. SRCR domains also have been found in diverse secreted and other cell-surface proteins from humans (e.g., CD5 and complement factor I), mice (Ly-1), and sea urchins (speract receptor). Moreover, many proteins include more than one SRCR domain (e.g., Ly-1 includes 3 SRCR domains and the speract receptor includes 4 SRCR domains). Likewise, human Lor-2 includes 4 SRCR domains, as set forth below.

To identify the presence of an SRCR in a Lor-2 family member, the amino acid sequence of the protein family member can be searched against a database of HMMs (e.g., the Pfam database, release 3.3) e.g., using the default parameters. For example, the search can be performed using the hmmsf program (family specific) and threshold score of 15 for determining a hit. hmmsf is available as part of the HMMER package of search programs (HMMER 2.1.1, Dec. 1998) which is freely distributed by the Washington University school of medicine. In one embodiment, a hit to a SRCR HMM having a score of at least 30-40, preferably at least 50-60, more preferably at least 70-80, and more preferably at least 90 or more is determinative of the presence of a SRCR domain within a query protein. A search using the amino acid sequence of SEQ ID NO:72 was performed against the HMM database resulting in the identification of 4 SRCR domains in the amino acid sequence of SEQ ID NO:72. Accordingly, in one embodiment of the invention, a Lor-2 protein has an SRCR domain at about amino acids 51-145 of SEQ ID NO:72. (Score of 91.4 against the SRCR domain profile HMM Accession No. PF00530). In another embodiment, a Lor-2 protein has an SRCR domain at about amino acids 183-282 of SEQ ID NO:72. (Score of 35.8). In another embodiment, a Lor-2 protein has an SRCR domain at about amino acids 310-407 of SEQ ID NO:72. (Score of 128.9). In another embodiment, a Lor-2 protein has an SRCR domain at about amino acids 420-525 of SEQ ID NO:72. (Score of 55.2).

Lor-2 family members can further include at least one or more speract receptor repeated domain (“SRRD”) signatures. The speract receptor is a transmembrane glycoprotein of 500 amino acid residues (Dangott et al. (1989) PNAS U.S.A. 86:2128-2132) which consists of a large extracellular domain of 450 which contains four repeats of a ˜115 amino acids termed more speract receptor repeated domain or “SRRDs”. Multiple sequence alignment of the four repeats reveals at least 17 perfectly conserved residues (including six cysteines, six glycines, and three glutamates). A SRRD signature has been generated from an alignment of the four SRRDs and has the consensus sequence: G-x(5)-G-x(2)-E-x(6)-W-G-x(2)-C-x(3)-[FYW]-x(8)-C-x(3)-G, corresponding to SEQ ID NO:74. The SRRD signature is further described in PROSITE Document, Accession No. PDOC00348 and as PROSITE Accession No. PS00420. In one embodiment, a SRRD signature is included within a SRCR. For example, a SRRD can be found in a SRCR of the C-terminal section of the mammalian macrophage scavenger receptor type I (Freeman et al. (1990) PNAS U.S.A. 87:8810-8814). Likewise, a SRRD signature can be found within the SRCR domain of human Lor-2 from about amino acids 312-349 of SEQ ID NO:72.

The consensus sequences herein are described according to standard Prosite Signature designation (e.g., all amino acids are indicated according to their universal single letter designation; X designates any amino acid; X(n) designates any n amino acids, e.g., X (2) designates any 2 amino acids; [FYW] indicates any one of the amino acids appearing within the brackets, e.g., any one of F, Y, or W, in the alternative, any one of Phe, Tyr, or Trp; and {X} indicates any amino but the amino acid included within the brackets.)

Lor-2 family members can further include at least one domain characteristic of lysyl oxidase, referred to herein as a lysyl oxidase domain or “LOX domain”. Lysyl oxidase is an extracellular copper-dependent enzyme that catalyzes the oxidative deamination of peptidyl lysine residues in precursors of various collagens and elastins. The deaminated lysines are then able to form aldehyde cross-links. (Krebs et al. (1993) Biochem. Biophys. Acta. 1202:7-12). The amino acid sequence of lysyl oxidase includes a signal sequence (e.g., amino acids 1 to 21 of human lysyl oxidase set forth as SEQ ID NO:75, a pro-peptide region (e.g., amino acids 22 to 168 of SEQ ID NO:75), and a region corresponding to the active, processed protein (e.g., amino acids 169-417 of SEQ ID NO:75), which is responsible for the enzymatic function of the molecule. Lysyl oxidase can be further characterized by the presence of a copper-binding site (Krebs et al. (1993) Biochem. Biophys. Acta. 12-2:7-12) having four conserved histidine residues that presumably supply the nitrogen ligands for copper coordination, and a quinone cofactor binding site (Wang et al. (1996) Science 273:1078-1084) (e.g., his289, his292, his294, and his296 of SEQ ID NO:75), also referred to as a “copper talon”. The copper binding site of human Lor-2 can be found, for example, at about amino acids 286-296 of SEQ ID NO:75.

Accordingly, as used herein, the term “LOX domain” includes a protein domain which is about 245-275 amino acid residues in length, and has about 38-64% identity with the amino acid sequence of processed lysyl oxidase (e.g., amino acid residues 169-417 of SEQ ID NO:75). Preferably, a LOX domain is about 225-300, more preferably about 230-290 amino acid residues in length, and more preferably about 235-285, or 240-280 amino acid residues in length, and has about 34-65% identity, preferably about 42-62%, and more preferably about 46-56% or 50-52% identity with the amino acid sequence of processed lysyl oxidase (e.g., amino acid residues 169-417 of SEQ ID NO:75). For example, a LOX domain can be found in huLOL (SEQ ID NO:76) from about amino acids 310-574; in huLor (SEQ ID NO:77) from about amino acids 481-751; in mu Lor-2 (SEQ ID NO:78) from about amino acids 464-733; and in huLor-2 (SEQ ID NO:72) from about amino acids 463-732.

In another embodiment, a LOX domain is involved in a lysyl oxidase or lysyl oxidase-like function. Lysyl oxidase or lysyl oxidase-like functions include, for example, aminotransferase activity, peptidyl lysine oxidation, oxidative deamination of lysine, cross-linking of extracellular matrix components, copper binding, and/or copper metabolism. Lysyl oxidase or lysyl oxidase-like functions are described in detail, for example, in Kagan et al. in Catalytic Properties and structural components of lysyl oxidase , John Wiley & Sons (1995) pp. 100-121, the contents of which are incorporated herein by reference. In yet another embodiment, a LOX domain has at least one, preferably two, and more preferably three or four histidine residues corresponding to the conserved histidine residues of lysyl oxidase which are involved in copper binding. For example, a LOX domain of a human Lor-2 sequence set forth in SEQ ID NO:72 (e.g., amino acid residues 330-732 in SEQ ID NO:72) has four histidine residues (e.g., his604, his607, his609, and his611 of SEQ ID NO:72) which correspond to those of human lysyl oxidase set forth as SEQ ID NO:75.

A LOX domain in a protein can further be included within a lysyl oxidase-related region (“LOX-related region”). A LOX-related region within a protein (e.g., within a Lor-2 family member) includes a protein region which is about 380-580, preferably about 390-550, more preferably about 400, 420, 450 or 500 amino acid residues in length and has at least 30-35%, 40-45%, 50-55%, 60-65%, 70-75%, 80-85%, or 90-95% homology with, for example, the amino acid sequence of human LOX. To identify the presence of a LOX-related region in a Lor-2 family member, the amino acid sequence of the protein family member can be searched against the HMM database, as described previously. In one embodiment, a hit to a LOX HMM having a score of at least 100-110, preferably at least 120-130, more preferably at least 140-150, and more preferably at least 160 or more is determinative of the presence of a LOX-related region within a query protein. A search using the amino acid sequence of SEQ ID NO:72 was performed against the HMM database resulting a hit to a LOX HMM from about amino acids 330-732 of SEQ ID NO:72. (Score of 166.6 against the LOX domain profile HMM Accession No. PF01186). Similar LOX-related regions were identified in muLor-2 from about amino acids 318-733 of SEQ ID NO:78 (Score of 162.8), in huLOL from about amino acids 1-574 of SEQ ID NO:76 (Score of 382.2) and in huLor from about amino acids 358-751 of SEQ ID NO:77 (Score of 146.8). In yet another embodiment, a lysyl oxidase-related region has at least 40-45%, 50-55%, 60-65%, 70-75%, 80-85%, or 90-95% homology with the amino acid sequence of a LOX domain of a human Lor-2 sequence set forth in SEQ ID NO:72 (e.g., amino acid residues 330-732 in SEQ ID NO:72). The lysyl oxidase-related regions of huLOL, huLor, muLor-2 and huLor-2 are the amino acids corresponding to processed lysyl oxidase (e.g., amino acids 169-417 of SEQ ID NO:75).

Another embodiment of the invention features a protein of the invention, preferably a Lor-2 protein, which contains a signal sequence. As used herein, a “signal sequence” refers to a peptide containing about 25 amino acids which occurs at the N-terminus of secretory proteins and which contains a large number of hydrophobic amino acid residues. For example, a signal sequence contains at least about 17-33 amino acid residues, preferably about 20-30 amino acid residues, more preferably about 24-26 amino acid residues, and more preferably about 25 amino acid residues, and has at least about 35-65%, preferably about 38-50%, and more preferably about 40-45% hydrophobic amino acid residues (e.g., Valine, Leucine, Isoleucine or Phenylalanine). Such a “signal sequence”, also referred to in the art as a “signal peptide”, serves to direct a protein containing such a sequence to a lipid bilayer. For example, in one embodiment, a Lor-2 protein contains a signal sequence containing about amino acids 1-25 of SEQ ID NO:72.

In yet another embodiment, a protein of the invention, preferably a Lor-2 protein, encodes a mature protein. As used herein, the term “mature protein” refers to a protein of the invention, preferably a Lor-2 protein, from which the signal peptide has been cleaved. In an exemplary embodiment, a mature Lor-2 protein contains amino acid residues 26 to 753 of SEQ ID NO:72.

In yet another embodiment, Lor-2 family members include at least 1, 2, 3, 4, 5 or more N-glycosylation sites. Predicted N-glycosylation sites are found, for example, from about amino acid 111-114, 266-269, 390-393, 481-484, and 625-628 of SEQ ID NO:72.

Lor-2 family members can further include at least 1, 2, 3, 4, 5, 6, 7, 8, or more or more Protein kinase C (“PKC”) phosphorylation sites. Predicted PKC phosphorylation sites are found, for example, from about amino acid 97-99, 104-106, 221-223, 268-270, 352-354, 510-512, 564-566, and 649-651 of SEQ ID NO:72.

Lor-2 family members can further include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more Casein kinase II phosphorylation sites. Predicted casein kinase II phosphorylation sites are found, for example, from about amino acid 31-34, 68-71, 115-118, 120-123, 135-138, 330-333, 352-355, 377-380, 392-395, 411-414, 424-427, 493-496, 527-530, and 617-620 of SEQ ID NO:72.

Lor-2 family members can further include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more N-myristoylation sites. Predicted N-myristoylation sites are found, for example, from about amino acids 13-18, 116-121, 130-135, 273-278, 312-317, 359-364, 378-383, 403-408, 443-448, 451-456, 463-468, 470-475, 489-494, 506-511, 515-520, 521-526, 626-631, 661-666, and 746-751 of SEQ ID NO:72.

Lor-2 family members can further include at least one or more amidation sites. A predicted amidation site is found, for example, from amino acid 117-180 of SEQ ID NO:72. As used herein, the site(s) have a consensus sequence selected from: N-{P}-[ST]-{P}(SEQ ID NO:83), where N is a glycosylation site (see PROSITE document PS00001); [ST]-X—[RK] (SEQ ID NO:84), where S or T is a phosphorylation site (see PROSITE document PS00005); [ST]-X (2)-[DE] (SEQ ID NO:85), where S or T is a phosphorylation site (see PROSITE document PS00006); G-{EDRKHPFYW}-X (2)-[STAGCN]-{P}(SEQ ID NO:86), where G is an N-myristoylation site (see PROSITE Accession No. PS00008); and X-G-[RK]-[RK] (SEQ ID NO:87), where X is an amidation site (see PROSITE document PS00009). These sites are further described at the expasy website as PDOC00001, PDOC00005, PDOC00006, PDOC00008, and PS00009, respectively.

Isolated proteins of the present invention, preferably Lor-2 proteins, have an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:72 or are encoded by a nucleotide sequence which includes a nucleotide sequence sufficiently homologous to SEQ ID NO:71. As used herein, the term “sufficiently homologous” includes a first amino acid or nucleotide sequence which contains at least a minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains have at least 30%, 40% or 50% homology, preferably 55%, 60%, 65%, 70% or 75% homology, more preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently homologous. Furthermore, amino acid or nucleotide sequences which share at least 30%, 40% or 50% homology, preferably 55%, 60%, 65%, 70% or 75% homology, more preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology and share a common functional activity are defined herein as sufficiently homologous.

Accordingly, another embodiment of the invention features isolated Lor-2 proteins and polypeptides having a Lor-2 activity. Preferred proteins are Lor-2 proteins having at least a signal sequence, a LOX domain, and at least one SRRD signature. Other preferred proteins are Lor-2 proteins having at least two, three, or four SRRD signatures. Other preferred proteins are Lor-2 proteins having at least a signal sequence, a LOX domain, and a SRCR domain. Other preferred proteins are Lor-2 proteins having at least a signal sequence, a LOX domain, and at least two SCRC domains. Other preferred proteins are Lor-2 proteins having at least a signal sequence, a LOX domain, and at least three SCRC domains. Other preferred proteins are Lor-2 proteins having at least a signal sequence, a LOX domain, and at least four SCRC domains.

The nucleotide sequence of the isolated human Lor-2 cDNA and the predicted amino acid sequence of the human Lor-2 polypeptide are shown in SEQ ID NOs:71 and 72, respectively.

The human Lor-2 cDNA (set forth in SEQ ID NO:71), which is approximately 2920 nucleotides in length, encodes a protein having a molecular weight of approximately 83.166 kD (with signal sequence) and 80.404 kD (without signal sequence) and which is approximately 753 (with signal sequence) (SEQ ID NO:72) and 728 amino acid residues (without signal sequence) in length. An ˜3.0 kb Lor-2 message was found to be expressed most tissues tested but was most highly expressed in heart and placenta (at least heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas tissues were tested). High expression of Lor-2 was also observed in the G361 melanoma cell line and in the SW480 adenocarcinoma colon cell line (at least G361, SW480, HL60, Hela 53, K562, Molty, Raji, and A549 cell lines were tested).

In a preferred embodiment, Lor-2 proteins of the invention have an amino acid sequence of at least 600-900, preferably about 650-850, more preferably about 700-800, and even more preferably about 720-760, 728 or 753 amino acid residues in length.

As used interchangeably herein, a “Lor-2 activity”, “biological activity of Lor-2” or “functional activity of Lor-2”, includes an activity exerted by a Lor-2 protein, polypeptide or nucleic acid molecule as determined in vivo, in vitro, or in situ, according to standard techniques. In one embodiment, a Lor-2 activity is a direct activity, such as an association with a Lor-2-target molecule. As used herein, a “target molecule” is a molecule with which a Lor-2 protein binds or interacts in nature, such that Lor-2-mediated function is achieved. A Lor-2 target molecule can be a Lor-2 protein or polypeptide of the present invention or a non-Lor-2 molecule. For example, a Lor-2 target molecule can be a non-Lor-2 protein molecule. Alternatively, a Lor-2 activity is an indirect activity, such as an activity mediated by interaction of the Lor-2 protein with a Lor-2 target molecule such that the target molecule modulates a downstream cellular activity (e.g., interaction of a Lor-2 molecule with a Lor-2 target molecule can modulate the activity of that target molecule on a cardiac cell).

In a preferred embodiment, a Lor-2 activity is at least one or more of the following activities: (i) interaction of a Lor-2 protein with a Lor-2 target molecule; (ii) interaction of a Lor-2 protein with a Lor-2 target molecule, wherein the Lor-2 target is a ligand; (iii) interaction of a Lor-2 protein with a Lor-2 target molecule, wherein the Lor-2 target is an extracellular matrix component (e.g., collagen or elastin); and (iv) modification of a Lor-2 target molecule (e.g., posttranslational modification).

In yet another preferred embodiment, a Lor-2 activity is at least one or more of the following activities: (1) crosslinking an extracellular matrix component; (2) regulating bone resorption and/or metabolism; (3) regulating copper metabolism; (4) modulating maturation, stabilization and/or degradation of extracellular matrix components; (5) regulating cellular signaling; and (6) regulating cellular adhesion (e.g. adhesion of a tumor cell).

In another embodiment of the invention, a Lor-2 molecule or preferably, a Lor-2 modulator, is useful for regulating, preventing and/or treating at least one or more of the following diseases or disorders: (1) diseases or disorders involving impaired copper metabolism (e.g., type IX of the Ehlers-Danlos syndrome and the Menkes syndrome); (2) bone disorders (e.g., osteoporosis or osteoarthritis); (3) fibrotic disorders (e.g., atherosclerosis, tissue and/or organ fibrosis); (4) proliferative disorders (e.g., cancer, for example, prostate cancer, breast cancer, lung cancer and the like); (5) vascular disorders (e.g., ischemia, ischemic-reperfusion injury); and (6) cardiac trauma (e.g., iatrogenic, accidental).

In yet another embodiment of the invention, a Lor-2 molecule or preferably, a Lor-2 modulator, is useful for regulating, preventing and/or treating at least one or more of the following diseases or disorders: (1) cardiac hypertrophy and cardiomyopathy; (2) cardiac pathologies; (3) myocardial hypertrophy and cardiovascular lesions; (4) myocardial aneurysms; (5) atherosclerotic cardiovascular disease; (6) fibrotic disease; (7) osteoporosis; (8) metastasis/prostate cancer; (9) cellular senescence/tumor suppression; (10) tumor progression; (11) liver fibrosis; (12) wound healing; (13) hypertension; (14) diabetes; (15) arthritis; and (16) bone disease (e.g., osteoporosis or osteoarthritis).

In yet another embodiment, a Lor-2 modulator is useful for regulating (e.g., inhibiting) tumor progression. For example, Lor-2 may be secreted by a tumor cell facilitating adhesion (e.g., enhancing the adhesive properties) of the cell. Accordingly, Lor-2 modulators can be used to affect the adhesive properties of tumor cells (e.g., to surrounding tissues).

In yet another embodiment, a Lor-2 modulator, is useful for regulating or preventing immunosuppression by tumor cells. For example, Lor-2 may be secreted by a tumor cell, conferring on that cell a growth advantage (e.g., maintaining the growth, differentiation, and transformed phenotype of the tumor cell). In such a situation, secreted Lor-2 can inhibit cytoxicity (e.g., lymphocytotoxicity, for example, IL-2-induced lymphocytotoxicity). Accordingly, Lor-2 may function to suppress the generation and/or proliferation of lymphocytic cells (e.g., lymphocyte-activated killer cells).

Isolation of the Human 21967 or Lor-2 (i.e., Lysyl Oxidase Related-2) cDNA

The invention is based, at least in part, on the discovery of the human gene encoding 21967 or Lor-2. Human Lor-2 was isolated from a cDNA library which was prepared from tissue obtained from subjects suffering from congestive heart failure. Briefly, a cardiac tissue sample was obtained from a biopsy of a 42 year old woman suffering from congestive heart failure. mRNA was isolated from the cardiac tissue and a cDNA library was prepared therefrom using art-known methods (described in, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989). Using a program which identifies the presence of signal peptides (Nielsen, H. et al. (1997) Protein Engineering 10:1-6) a positive clone was isolated.

The sequence of the positive clone was determined and found to contain an open reading frame. The nucleotide sequence encoding the human 21967 or Lor-2 protein comprises about 2920 nucleic acids, and has the nucleotide sequence set forth as SEQ ID NO:71. The open reading frame of 21967 is disclosed in SEQ ID NO:73. The protein encoded by this nucleic acid comprises about 753 amino acids, and has the amino acid sequence set forth as SEQ ID NO:72.

Analysis of Human 21967 or Lor-2

A BLAST search (Altschul et al. (1990) J. Mol. Biol. 215:403) of the nucleotide and protein sequences of human Lor-2 revealed that Lor-2 is similar to the following protein molecules: a human lysyl oxidase-related protein (Accession No. U89942) having approximately 56.9% identity over amino acids 33-752 of Lor-2 (SEQ ID NO:72); and a second murine lysyl-oxidase related protein; (Accession No. AF053368) having approximately 92.6% identity over amino acids 1-753, e.g., over the entire length) of Lor-2 (SEQ ID NO:72). (Identities were calculated using the ALIGN algorithm of Huang and Miller (1991) Adv. Appl. Math. 12:373-381).

The Lor-2 protein is predicted to have a signal peptide from amino acid residues 1-25 of SEQ ID NO:72. Accordingly, a mature Lor-2 protein is predicted to include amino acid residues 26-753 of SEQ ID NO:72. Lor-2 is also predicted to have 5 N-glycosylation sites, 8 protein kinase phosphorylation (“PKC”) sites, 14 casein kinase II phosphorylation sites, 19 N-myristoylation sites, and 1 amidation site. Predicted N-glycosylation sites are found, for example, from about amino acid 111-114, 266-269, 390-393, 481-484, and 625-628 of SEQ ID NO:72. Predicted PKC phosphorylation sites are found, for example, from about amino acid 97-99, 104-106, 221-223, 268-270, 352-354, 510-512, 564-566, and 649-651 of SEQ ID NO:72. Predicted casein kinase II phosphorylation sites are found, for example, from about amino acid 31-34, 68-71, 115-118, 120-123, 135-138, 330-333, 352-355, 377-380, 392-395, 411-414, 424-427, 493-496, 527-530, and 617-620 of SEQ ID NO:72. Predicted N-myristoylation sites are found, for example, from about amino acids 13-18, 116-121, 130-135, 273-278, 312-317, 359-364, 378-383, 403-408, 443-448, 451-456, 463-468, 470-475, 489-494, 506-511, 515-520, 521-526, 626-631, 661-666, and 746-751 of SEQ ID NO:72. A predicted amidation site is found, for example, from amino acid 117-180 of SEQ ID NO:72.

Moreover, Lor-2 has a 4 scavenger receptor cysteine-rich domains from amino acid residues 51-145, 183-282, 310-407, and 420-525 of SEQ ID NO:72. The third scavenger receptor cysteine-rich domain includes a speract receptor repeated domain signature from amino acid residues 312-349 of SEQ ID NO:72. Lor-2 further has a lysyl oxidase domain from residues 330-732 of SEQ ID NO:72. Within the lysyl oxidase domain of Lor-2, there exists a fragment having significant homology to the lysyl oxidase putative copper-binding region, termed the “copper-binding talon”. A prosite consensus pattern describing the copper-binding talon is as follows: W-E-W—H—S—C—H-Q-H—Y—H (SEQ ID NO:79) (see also PROSITE documentation PDOC00716 and Krebs and Krawetz (1993) Biochem. Biophys. Acta 1202:7-12). Amino acid residues 601-701 of human Lor-2 (SEQ ID NO:72) have ˜73% identity with this consensus sequence (8/11 residues) including each of the four conserved histidines, three of which are believed to be copper ligands residing within an octahedral coordination complex of lysyl oxidase.

Analysis of primary and secondary protein structures of 21967 was performed as follows: alpha, beta turn and coil regions, Garnier-Robson algorithm (Garnier et al. (1978) J Mol Biol 120:97); alpha, beta, and turn regions, Chou-Fasman algorithm (Chou and Fasman (1978) Adv in Enzymol Mol 47:45-148); hydrophilicity and hydrophobicity plots, Kyte-Doolittle algorithm (Kyte and Doolittle (1982) J Mol Biol 157:105-132); alpha amphipathic and beta amphipathic regions, Eisenberg algorithm (Eisenberg et al. (1982) Nature 299:371-374); flexible regions, Karplus-Schulz algorithm (Karplus and Schulz (1985) Naturwissens - Chafen 72:212-213); antigenic index, Jameson-Wolf algorithm (Jameson and Wolf (1988) CABIOS 4:121-136); surface probability plot, Emini algorithm (Emini et al. (1985) J Virol 55:836-839).

Prediction of the Chromosomal Location of 21967 or Lor-2—Electronic Mapping

To predict the chromosomal location of Lor-2, the Lor-2 nucleotide sequence of SEQ ID NO:71 was used to query, using the BLASTN program (Altschul S. F. et al, (1990) J. Mol. Biol. 215: 403-410) with a word length of 12 and using the BLOSUM62 scoring matrix, a database of human nucleotide sequences originating from nucleotide molecules (e.g., EST sequences, STS sequences and the like) that have been mapped to the human genome. Nucleotide sequences which had been previously mapped to human chromosome 2 near the D2S145 marker (e.g., having Accession Nos. AA191602 and R55706) were found to have high sequence identity to portions of the Lor-2 nucleotide sequence (3′ UTR sequence) indicating that Lor-2 maps to the same chromosomal location. Moreover, it is predicted that allelic variants of Lor-2 will map the same chromosomal location and species orthologs of Lor-2 will map to loci syntenic with the human Lor-2 locus.

Confirmation and Analysis of the Chromosomal Location of 21967 or Lor-2—PCR Mapping

The hLor-2 gene was mapped to human chromosome 2 (i.e., 2p11-p13), which is syntenic to mouse chromosome 6, by PCR typing of the Genebridge (G4) radiation hybrid panel (Research Genetics, Inc., Huntsville, Ala.). Typing of the DNA and comparison to radiation hybrid map data at the Whitehead Institute Center for Genome Research (WICGR) tightly linked the hLor-2 gene to a region on human chromosome 2 between WI-5987 (13.9cR) and GCT1B4 (16.7cR).

The huLor-2 primers used in the PCR mapping studies were: forward—GCTTACCAAGAAACCCATGTCAGC (SEQ ID NO:81) and reverse—GGCAGTTAGTCAGGTGCTGC (SEQ ID NO:82). The radiation hybrid mapping studies were performed as follows: PCR reactions of radiation hybrid panels, GeneBridge 4 (Research Genetics, Inc., Huntsville, Ala.) were assembled in duplicate using an automated PCR assembly program on a TECAN Genesis. Each reaction consisted of: 5 μl DNA template (10 ng/μl), 1.5 μl 10×PCR buffer, 1.2 μl dNTPs (2.5 mM), 1.15 μl forward primer (6.6 μM) 1.15 μl reverse primer (6.6 μM0, and 5 μl 1:75 platinum Taq. The reactions were thermocycled on a Perkin-Elmer 9600 for 95° C. 10 minutes (for the platinum Taq), [95° C. 40 sec, 52° C. 40 sec, 72° C., 50 sec] 35X, 72° C., 5 minutes, 4° C. hold. Resulting PCR products were run out on a 2% agarose gel and visualized on a UV light box.

The positive hybrids for the Genebridge 4 panel were submitted to the Whitehead Genome Center for placement in relation to a framework map.

Human Lor-2 mapped in close proximity to known genes including actin, gamma 2, smooth muscle, enteric (“ACTG2”), nucleolysin TIA1, semaphorin W (“SEMAW”), dysferlin (“DYSF”), docking protein 1 (“DOK1), glutamine-fructose-6-phosphate transaminase 1 (“GFPT”), the KIAA0331 gene, deoxyguanosine kinase (“DGUOK”), the TSC501 gene, eukaryotic translation initiation factor 3, subunit 10 (“EIF3S1”), tachykinin receptor 1 (“TACR1”), tissue-type plasminogen activator (“PLAT”) and dual specificity phosphatase 11 (“DUSP11”). Nearby disease mutations and/or loci include Alstrom syndrome (“ALMS1”), an autosomal recessively inherited syndrome characterized by retinal degeneration, obesity, diabetes mellitus, neurogenous deafness, hepatic dysfunction, and in some cases, late onset cardiomyopathy (see e.g., Alstrom et al. (1959) Acta Psychiat. Neurol. Scand. 34 (suppl. 129):1-35; Alter and Moshang (1993) Am. J. Dis. Child. 147:97-99; Awazu et al. (1997) Am. J. Med. Genet. 69:13-16; Aynaci et al. (1995) ( Letter ) Clin. Genet. 48:164-166; Charles et al. (1990) J. Med. Genet. 27:590-592; Cohen and Kisch (1994) Israel J. Med. Sci. 30:234-236; Collin et al. (1997) Hum. Molec. Genet. 6:213-219; Collin et al. (1999) ( Letter ) Clin. Genet. 55:61-62; Connolly et al. (1991) Am. J. Med. Genet. 40:421-424; Goldstein and Fialkow (1973) Medicine 52:53-71; Macari et al. (1998) Hum. Genet. 103:658-661; Marshall et al. (1997) Am. J. Med. Genet. 73:150-161; Michaud et al. (1996) J. Pediat. 128:225-229; Millay et al. (1986) Am. J. Ophthal. 102:482-490; Rudiger et al. (1985) Hum. Genet. 69:76-78; Russell-Eggitt et al. (1998) Ophthalmology 105:1274-1280; Tremblay et al. (1993) Am. J. Ophthal. 115:657-665; Warren et al. (1987) Am. Heart J. 114:1522-1524 and Weinstein et al. (1969) New Eng. J. Med. 281:969-977), orofacial cleft 2 (“OFC2”) (see e.g., Carinci et al. (1995) ( Letter ) Am. J. Hum. Genet. 56:337-339; Pezzetti et al. (1998) Genomics 50:299-305 and Scapoli et al. (1997) Genomics 43:216-220) and Parkinsons disease 3 (see e.g., Di Rocco et al. (1996) Adv. Neurol. 69:3-11 and Gasser et al. (1998) Nature Genet. 18:262-265). Additional information regarding Alstrom syndrome, orofacial cleft 2 and Parkinson disease 3 can be found collected under Accession Nos. 203800, 602966 and 602404, respectively, in the Online Mendelian Inheritance in Man (“OMIM™”) database, the contents of which are incorporated herein by reference.

Moreover, the syntenic location on mouse chromosome 6 is near ovarian teratoma susceptibility 1 (“Ots-1”), dysruption of corticosterone in adrenal cortex cells (“Cor”), brain protein 1 (“Brp1”), lymphocyte antigen 36 (“Ly36”), major liver protein 1 (“Lvp1”), cerebellar deficient folia (“cdf”), motor neuron degeneration 2 (“mnd2”), truncate (“tc”) and faded (“fe”). Of particular interest are the Lor-2 neighbors Ots-1 and Cor, both of which a postulated to play a role in tumor susceptibility. The Ots-1 locus was identified by linkage analysis of female LT/Sv mice, a strain characterized by its abnormally high incidence of spontaneous ovarian teratomas, which are extremely rare for other mouse strains. Ots-1 was identified as the single major locus that increases the frequency of teratomas in a semidominant manner (Lee et al. (1997) Cancer Res. 57:590-593. Likewise, the cor locus was identified as being associated with a phenotype of the AJ mouse strain (a strain susceptible to many neoplasms and infectious agents, presumably due to a deficiency in the phophylactic activities of endogenous glucocorticoids (e.g., adrenalcortical corticosterone (“CS”)) (Thaete et al. (1990) Proc. Soc. Exp. Biol. Med. 194:97-102). Accordingly, at least two loci in the near vicinity of mouse Lor-2 on chromosome 6 are associated with tumor susceptibility. Additional information regarding the Ots-1 and Cor loci can be found collected under Accession Nos. MGI:85864 and MGI:58993, respectively, in the Mouse Genomics Informatics database, the contents of which are incorporated herein by reference. Likewise, information regarding the cdf locus, the mnd2 locus and the mouse Lor-2 gene (i.e., the mouse ortholog of human Lor-2) can be found collected under Accession Nos. MGI:86274, MGI:97039 and MGI:1337004, respectively.

Tissue Distribution of 21967 or Lor-2 mRNA

Standard molecular biology methods (Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) were used to construct cDNA libraries in plasmid vectors from multiple human tissues. Individual cDNA clones from each library were isolated and sequenced and their nucleotide sequences were input into a database. The Lor-2 nucleotide sequence of SEQ ID NO:71 was used to query the tissue-specific library cDNA clone nucleotide sequence database using the BLASTN program (Altschul S. F. et al, (1990) J. Mol. Biol. 215: 403-410) with a word length of 12 and using the BLOSUM62 scoring matrix. Nucleotide sequences identical to portions of the Lor-2 nucleotide sequence of SEQ ID NO:71 were found in cDNA libraries originating from human endothelial cells, lymph node, bone, heart, neuron, and testes. Lor-2 nucleic acid sequences, fragments thereof, proteins encoded by these sequences, and fragments thereof as well as modulators of Lor-2 gene or protein activity may be useful for diagnosing or treating diseases that involve the tissues in which the Lor-2 mRNA is expressed. Likewise, when a similar analysis was performed using the Lor-2 sequence of SEQ ID NO:71 to query publicly available nucleotide sequence databases (e.g., DBEST databases) using BLAST, sequences having high homology to the 3′ untranslated region of human Lor-2 were identified in a Soares placenta normalized library and in Soares testis, B-cell and lung normalized libraries.

Northern blot hybridization with RNA samples was next performed under standard conditions and washed under stringent conditions, i.e., 0.2×SSC at 65° C. A DNA probe was radioactively labeled with 32 P-dCTP using the Prime-It kit (Stratagene, La Jolla, Calif.) according to the instructions of the supplier. Filters containing various tissue and cell line mRNAs were probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.

On a human mRNA blot containing mRNA from heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas, Lor-2 transcript (˜3.0 kb) was detected in all tissues tested but was most strongly detected in heart and placenta. Moreover, Lor-2 mRNA was strongly expressed in the G361 melanoma cell line and in the SW480 adenocarcinoma colon cell lines (as compared to expression in the HL60, HeLa53, K562, Molty, Raji, and SW480 cell lines (SW480 cell line expressing a 2.4 kb transcript). Transcripts of 5 kb and 2 kb were also detected evidencing possible splice variants of Lor-2.

Testing of a larger panel of human tissues revealed the following expression levels. Expression levels were normalized to beta 2 expression.

Next, Lor-2 expression levels were measured in a variety of tissue and cell samples using the TaqMan™ procedure.

The highest expression was observed in osteoblasts, cervix, kidney and placenta on the normal human tissue panel tested.

Expression of 21967 or Lor-2 mRNA in Clinical Tumor Samples and in Xenograft Cell Lines

In this example, RT-PCR was used to detect the presence of Lor-2 mRNA in various tumor and metastatic tissue samples as compared to normal tissue samples. RT-PCR was also used to detect the presence of Lor-2 mRNA in various xenograft cell lines. In breast tissue, Lor-2 mRNA was detected in 0/1 normal tissue samples as compared to 3/4 tumor clinical samples after 30 cycles of PCR. In xenograft cell lines isolated from breast tissue, Lor-2 mRNA was detected in 1/1 normal and 3/3 xenograft cell lines (cell lines MCF7, ZR75 and T47D). In lung tissue, Lor-2 mRNA was detected in 0/2 normal tissue samples as compared to 2/8 tumor tissue samples. In xenograft cell lines isolated from lung tissue, Lor-2 mRNA was detected in 0/5 xenograft cell lines after 30 cycles of PCR. In a second experiment performed with lung tissue, Lor-2 mRNA was detected in 2/2 normal and 8/8 tumor tissue samples, as well as in 5/5 xenograft cell lines (cell lines A549, H69, H125, H322 and H460) after 35 cycles of PCR. In colon tissue, Lor-2 mRNA was detected in 2/2 normal, 5/5 tumor and 5/5 metastatic samples, as well as in 7/7 xenograft cell lines (cell lines HCT116, HCT15, HT29, SW620, SW480, DLD1 and KM12) after 35 cycles of PCR. In liver tissue, LOR-2 mRNA was detected in 2/2 normal samples after 35 cycles of PCR. These data reveal that there exists a correlation between tumors and Lor-2 expression, at least in breast and lung tissues.

To further investigate this finding, Lor-2 mRNA levels were measured by quantitative PCR using the TaqMan™ procedure as described above. The procedure was carried out on cDNA generated from various carcinoma samples and compared to normal counterpart tissue samples. In 5/7 breast carcinomas, a 2-86 fold upregulation of Lor-2 was observed as compared to 2/4 normal breast tissue samples. Likewise, in 4/7 lung carcinomas, a 2-17 fold upregulation was observed as compared to 3/4 normal lung tissue samples. The relative levels of Lor-2 mRNA detected in various normal, tumor and metastases samples are set forth in Table 47.

These data reveal a significant upregulation of Lor-2 mRNA in at least breast and lung carcinomas. Moreover, there was a significant upregulation of Lor-2 expression in metastatic as compared to normal liver samples. Given that the mRNA for Lor-2 is expressed in a variety of tumors, with significant upregulation in carcinoma samples in comparison to normal samples, it is believed that inhibition of Lor-2 activity may inhibit tumor progression by affecting the adhesive properties of the tumor cells to surrounding tissues.

Human 1983 (SLGP)

The present invention is based, at least in part, on the discovery of novel G-protein coupled receptor (GPCR) family members, referred to herein as SLGP protein and nucleic acid molecules. The human SLGP molecules are also referred to as “1983” molecules and the mouse SLGP molecules are also referred to as “12231 or “m1983” molecules. The present invention also provides methods and compositions for the diagnosis and treatment of cellular proliferation, growth, differentiation, or migration disorders (e.g., cancer, arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia).

The present invention is also based, at least in part, on the discovery that the novel SLGP molecules of the present invention are upregulated in in vitro proliferating and tube forming Human Dermal Microvascular Endothelial Cells (HMVEC) (see details below), are expressed in endothelial cells of glioblastomas as compared to normal brains (see details below), and are upregulated in VEGF-induced angiogenic xenograft plugs as compared to parental xenografts (see details below). Therefore, the SLGP molecules of the present invention modulate angiogenesis by endothelial cells (e.g., tumor endothelial cells). Accordingly, the SLGP molecules of the present invention are useful as targets for developing modulating agents to regulate a variety of cellular processes including angiogenesis (e.g., the proliferation, elongation, and migration of endothelial cells, such as endothelial cells in tumors). Angiogenesis is responsible for the formation of new vessels in tumor sites. The new vessels provide the oxygen and nutritional supply to tumors. Therefore, the SLGP modulators of the invention can modulate tumor formation and growth by modulating angiogenesis. For example, inhibition of the activity of an SLGP molecule can cause decreased angiogenesis, i.e., a decrease in cellular proliferation, elongation, and migration of endothelial cells and, thus, a decrease in the formation of new vessels, and a decrease in the supply of oxygen and nutrition to a tumor. Therefore, the SLGP modulators of the invention can be used to treat formation and growth of tumors, e.g., cancer, and other diseases characterized by excessive vessel formation such as arthritis and retinopathy. Additionally, increasing the activity of an SLGP molecule can cause increased angiogenesis and, therefore, increased vessel formation and can, thus, be used in treating diseases characterized by decreased vessel formation, e.g., tissue ischemia. Therefore, the SLGP molecules of the present invention are useful as targets and therapeutic agents for the modulation of diseases characterized by decreased angiogenesis, e.g., tissue ischemia, such as myocardial ischemia.

The SLGP protein is a GPCR that participates in signaling pathways within cells, e.g., signaling pathways involved in proliferation or differentiation. As used herein, a signaling pathway refers to the modulation (e.g., the stimulation or inhibition) of a cellular function/activity upon the binding of a ligand to the GPCR (SLGP protein). Examples of such functions include mobilization of intracellular molecules that participate in a signal transduction pathway, e.g., phosphatidylinositol 4,5-bisphosphate (PIP 2 ), inositol 1,4,5-triphosphate (IP 3 ) or adenylate cyclase; polarization of the plasma membrane; production or secretion of molecules; alteration in the structure of a cellular component; cell proliferation, e.g., synthesis of DNA and angiogenesis, e.g., proliferation, elongation, and migration of endothelial cells (e.g., tumor endothelial cells) to form new vessels (e.g., endothelial tubes); cell differentiation; and cell survival.

Regardless of the cellular activity modulated by SLGP, it is universal that as a GPCR, the SLGP protein interacts with a “G protein” to produce one or more secondary signals in a variety of intracellular signal transduction pathways, e.g., through phosphatidylinositol or cyclic AMP metabolism and turnover, in a cell. G proteins represent a family of heterotrimeric proteins composed of α, β and γ subunits, which bind guanine nucleotides. These proteins are usually linked to cell surface receptors, e.g., receptors containing seven transmembrane domains, such as the ligand receptors. Following ligand binding to the receptor, a conformational change is transmitted to the G protein, which causes the α-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the βγ-subunits. The GTP-bound form of the α-subunit typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cyclic AMP (e.g., by activation of adenylate cyclase), diacylglycerol or inositol phosphates. Greater than 20 different types of α-subunits are known in man, which associate with a smaller pool of β and γ subunits. Examples of mammalian G proteins include Gi, Go, Gq, Gs and Gt. G proteins are described extensively in Lodish H. et al. Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y., 1995), the contents of which are incorporated herein by reference.

As used herein, the phrase “phosphatidylinositol turnover and metabolism” includes the molecules involved in the turnover and metabolism of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) as well as to the activities of these molecules. PIP 2 is a phospholipid found in the cytosolic leaflet of the plasma membrane. Binding of a ligand to the SLGP activates, in some cells, the plasma-membrane enzyme phospholipase C that in turn can hydrolyze PIP 2 to produce 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP 3 ). Once formed IP 3 can diffuse to the endoplasmic reticulum surface where it can bind an IP 3 receptor, e.g., a calcium channel protein containing an IP 3 binding site. IP 3 binding can induce opening of the channel, allowing calcium ions to be released into the cytoplasm. IP 3 can also be phosphorylated by a specific kinase to form inositol 1,3,4,5-tetraphosphate (IP 4 ), a molecule which can cause calcium entry into the cytoplasm from the extracellular medium. IP 3 and IP 4 can subsequently be hydrolyzed very rapidly to the inactive products inositol 1,4-biphosphate (IP 2 ) and inositol 1,3,4-triphosphate, respectively. These inactive products can be recycled by the cell to synthesize PIP 2 . The other second messenger produced by the hydrolysis of PIP 2 , namely 1,2-diacylglycerol (DAG), remains in the cell membrane where it can serve to activate the enzyme protein kinase C. Protein kinase C is usually found soluble in the cytoplasm of the cell, but upon an increase in the intracellular calcium concentration, this enzyme can move to the plasma membrane where it can be activated by DAG. The activation of protein kinase C in different cells results in various cellular responses such as the phosphorylation of glycogen synthase, or the phosphorylation of various transcription factors, e.g., NF-kB. The language “phosphatidylinositol activity”, as used herein, includes an activity of PIP 2 or one of its metabolites.

Another signaling pathway in which the SLGP protein may participate is the cAMP turnover pathway. As used herein, “cyclic AMP turnover and metabolism” includes molecules involved in the turnover and metabolism of cyclic AMP (cAMP) as well as to the activities of these molecules. Cyclic AMP is a second messenger produced in response to ligand induced stimulation of certain G protein coupled receptors. In the ligand signaling pathway, binding of ligand to a ligand receptor can lead to the activation of the enzyme adenylate cyclase, which catalyzes the synthesis of cAMP. The newly synthesized cAMP can in turn activate a cAMP-dependent protein kinase.

The SLGP molecules of the present invention are involved in modulation of cellular proliferation, growth, differentiation, or migration processes. As used herein, a “cellular proliferation, growth, differentiation, or migration process” includes a process by which a cell e.g., an endothelial cell, increases in number, size, or content; by which a cell develops a specialized set of characteristics which differ from that of other cells; or by which a cell moves closer to or further from a particular location or stimulus (e.g., angiogenesis). As used herein, “cellular proliferation, growth, differentiation, or migration disorders” include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; and other diseases which are characterized by increased or deceased angiogenesis, including, but not limited to arthritis, retinal and optic disk neovascularization, and tissue ischemia, such as myocardial ischemia.

The activity of the SLGP proteins of the invention may also be implicated in cardiovascular disorders, congestive heart failure, or other cardiac cellular processes. As used herein, the term “cardiovascular disorder” includes a disease, disorder, or state involving the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. Examples of such disorders include hypertension, atherosclerosis, coronary artery spasm, coronary artery disease, valvular disease, arrhythmias, cardiomyopathies (e.g., dilated cardiomyopathy, idiopathic cardiomyopathy), arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrillation, long-QT syndrome, congestive heart failure, sinus node disfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, myocardial infarction, cardiac hypertrophy, and coronary artery spasm.

As used herein, the term “congestive heart failure” includes a condition characterized by a diminished capacity of the heart to supply the oxygen demands of the body. Symptoms and signs of congestive heart failure include diminished blood flow to the various tissues of the body, accumulation of excess blood in the various organs, e.g., when the heart is unable to pump out the blood returned to it by the great veins, exertional dyspnea, fatigue, and/or peripheral edema, e.g., peripheral edema resulting from left ventricular dysfunction. Congestive heart failure may be acute or chronic. The manifestation of congestive heart failure usually occurs secondary to a variety of cardiac or systemic disorders that share a temporal or permanent loss of cardiac function. Examples of such disorders include hypertension, coronary artery disease, valvular disease, and cardiomyopathies, e.g., hypertrophic, dilative, or restrictive cardiomyopathies. Congestive heart failure is described in, for example, Cohn J. N. et al. (1998) American Family Physician 57:1901-04, the contents of which are incorporated herein by reference.

As used herein, the term “cardiac cellular processes” includes intra-cellular or inter-cellular processes involved in the functioning of the heart. Cellular processes involved in the nutrition and maintenance of the heart, the development of the heart, or the ability of the heart to pump blood to the rest of the body are intended to be covered by this term. Such processes include, for example, cardiac muscle contraction, distribution and transmission of electrical impulses, and cellular processes involved in the opening and closing of the cardiac valves. The term “cardiac cellular processes” further includes processes such as the transcription, translation and post-translational modification of proteins involved in the functioning of the heart, e.g., myofilament specific proteins, such as troponin I, troponin T, myosin light chain 1 (MLC1), and α-actinin.

The novel SLGP molecules of the present invention comprise a family of molecules having certain conserved structural and functional features. The term “family” when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin. Members of a family may also have common functional characteristics.

For example, the family of G protein-coupled receptors (GPCRs), to which the SLGP proteins of the present invention bear significant homology, comprise an N-terminal domain, seven transmembrane domains (also referred to as membrane-spanning domains), six loop domains, and a C-terminal cytoplasmic domain (also referred to as a cytoplasmic tail). Members of the SLGP family also share certain conserved amino acid residues, some of which have been determined to be critical to receptor function and/or G protein signaling. For example, GPCRs usually contain the following features: a conserved asparagine residue in the first transmembrane domain; a cysteine residue in the second loop which is believed to form a disulfide bond with a conserved cysteine residue in the fourth loop; a conserved leucine and aspartate residue in the second transmembrane domain; an aspartate-arginine-tyrosine motif (DRY motif) at the interface of the third transmembrane domain and the third loop of which the arginine residue is almost invariant (members of the rhodopsin subfamily of GPCRs comprise a histidine-arginine-methionine motif (HRM motif) as compared to a DRY motif); a conserved tryptophan and proline residue in the fourth transmembrane domain; and conserved phenylalanine and leucine residues in the seventh transmembrane domain. Table 48 depicts an alignment of the transmembrane domain of 5 GPCRs. The conserved residues described herein are indicated by asterices.

The amino acid sequences of thrombin (Accession No. P25116), rhodopsin (Accession No. P08100), m1ACh (Accession No. P08482), IL-8A (Accession No. P25024), octopamine (Accession No. P22270), can be found as SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, respectively. Accordingly, GPCR-like proteins such as the SLGP proteins of the present invention contain a significant number of structural characteristics of the GPCR family. For instance, the SLGPs of the present invention contain conserved cysteines found in the first two loops (prior to the third and fifth transmembrane domains) of most GPCRs (cys490 and cys562 of SEQ ID NO:89). A highly conserved asparagine residue is present (asn125 in SEQ ID NO:89). SLGP proteins contains a highly conserved leucine (leu154 of SEQ ID NO:89). The two cysteine residues are believed to form a disulfide bond that stabilizes the functional protein structure. A highly conserved asparagine and arginine in the fourth transmembrane domain of the SLGP proteins is present (asp158 and arg218 of SEQ ID NO:89). Moreover, a highly conserved proline is present (pro307 of SEQ ID NO:89). Proline residues in the fourth, fifth, sixth, and seventh transmembrane domains are thought to introduce kinks in the alpha-helices and may be important in the formation of the ligand binding pocket. Moreover, a conserved tyrosine is present in the seventh transmembrane domain of SLGP-2 (tyr647 of SEQ ID NO:89).

In one embodiment, the SLGP proteins of the present invention contain at least one, two, three, four, five, six, or preferably, seven transmembrane domains. As used herein, the term “transmembrane domain” includes an amino acid sequence of about 15-40 amino acid residues in length, more preferably, about 15-30 amino acid residues in length, and most preferably about 18-25 amino acid residues in length, which spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an α-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zagotta W. N. et al, (1996) Annual Rev. Neuronsci. 19: 235-63, the contents of which are incorporated herein by reference. In a preferred embodiment, an SLGP protein of the present invention has more than one transmembrane domain, preferably 2, 3, 4, 5, 6, or 7 transmembrane domains. For example, transmembrane domains can be found at about amino acids 433-452, 465-481, 500-524, 533-553, 570-594, 619-635, and 642-666 of SEQ ID NO:89. In a particularly preferred embodiment, an SLGP protein of the present invention has 7 transmembrane domains.

In another embodiment, an SLGP is identified based on the presence of at least one Loop domain, also referred to herein as a ‘loop’. As defined herein, the term “loop” includes an amino acid sequence having a length of at least about 4, preferably about 5-10, preferably about 10-20, and more preferably about 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or 100-150 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide. Such loop regions may be located either extracellularly or in the cytoplasm. Accordingly, the N-terminal amino acid of a loop is adjacent to a C-terminal amino acid of a transmembrane domain in a naturally-occurring SLGP or SLGP-like molecule, and the C-terminal amino acid of a loop is adjacent to an N-terminal amino acid of a transmembrane domain in a naturally-occurring SLGP or SLGP-like molecule.

As used herein, a “cytoplasmic loop” includes an amino acid sequence located within a cell or within the cytoplasm of a cell. Also as used herein, an “extracellular loop” includes an amino acid sequence located outside of a cell, or extracellularly. For example, loop domains can be found at about amino acid residues 453-464, 482-499, 525-532, 554-569, 595-618, and 636-641 of SEQ ID NO:89.

In another embodiment of the invention, an SLGP is identified based on the presence of a “C-terminal domain”, also referred to herein as a C-terminal tail, in the sequence of the protein. As used herein, a “C-terminal domain” includes an amino acid sequence having a length of at least about 10, preferably about 10-25, more preferably about 25-50, more preferably about 50-75, even more preferably about 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, or 500-600 amino acid residues and is located within a cell or extracellularly. Accordingly, the N-terminal amino acid residue of a “C-terminal domain” is adjacent to a C-terminal amino acid residue of a transmembrane domain in a naturally-occurring SLGP or SLGP-like protein. For example, a C-terminal domain is found at about amino acid residues 667-690 of SEQ ID NO:89.

In another embodiment, an SLGP is identified based on the presence of an “N-terminal domain”, also referred to herein as an N-terminal loop in the amino acid sequence of the protein. As used herein, an “N-terminal domain” includes an amino acid sequence having about 1-500, preferably about 1-400, more preferably about 1-300, more preferably about 1-200, even more preferably about 1-100, and even more preferably about 1-50, 1-25, or 1-10 amino acid residues in length and is located outside of a cell orintracellularly. The C-terminal amino acid residue of a “N-terminal domain” is adjacent to an N-terminal amino acid residue of a transmembrane domain in a naturally-occurring SLGP or SLGP-like protein. For example, an N-terminal domain is found at about amino acid residues 1-432 of SEQ ID NO:89.

Accordingly in one embodiment of the invention, an SLGP includes at least one, preferably 6 or 7, transmembrane domains and and/or at least one loop. In another embodiment, the SLGP further includes an N-terminal domain and/or a C-terminal domain. In another embodiment, the SLGP can include six transmembrane domains, three cytoplasmic loops, and two extracellular loops, or can include six transmembrane domains, three extracellular loops, and 2 cytoplasmic loops. The former embodiment can further include an N-terminal domain. The latter embodiment can further include a C-terminal domain. In another embodiment, the SLGP can include seven transmembrane domains, three cytoplasmic loops, and three extracellular loops and can further include an N-terminal domain or a C-terminal domain.

In another embodiment, an SLGP is identified based on the presence of at least one “7 transmembrane receptor profile”, also referred to as a “Secretin family sequence profile”, in the protein or corresponding nucleic acid molecule. As used herein, the term “7 transmembrane receptor profile” includes an amino acid sequence having at least about 50-350, preferably about 100-300, more preferably about 150-275 amino acid residues, or at least about 200-258 amino acids in length and having a bit score for the alignment of the sequence to the 7tm — 1 family Hidden Markov Model (HMM) of at least 20, preferably 20-30, more preferably 30-40, more preferably 40-50, or 50-75 or greater. The 7tm — 1 family HMM has been assigned the PFAM Accession PF00001.

To identify the presence of a 7 transmembrane receptor profile in an SLGP, the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for PF00001 and a score of 15 is the default threshold score for determining a hit. For example, a search using the amino acid sequence of SEQ ID NO:89 was performed against the HMM database resulting in the identification of a 7 TM receptor profile in the amino acid sequence of SEQ ID NO:89. The results of the search are set forth below.

Score: 56.37  Seq: 421 678  Model: 75 348

*ksYYyvvYiIYTVGYSMSiaaLlvAMfIFcfFRrLHCtRNYIHMNMFms

+++Y+++  I  +G  +S++ L + +F F FF  +  TR +IH+N+

S

SLGP    421

IKDYNILTRITQLGIIISLICLAICIFTFWFFSEIQSTRTTIHKNLCCS  469

FILRaisWFIkDWvlyWmYsndeltwHCwMsivwCRivMfFMQYMMMtNY

L A  +F++    +N    +C I   +Y+ ++

+

SLGP    470 LFL-AELVFLVGINT---NTNKL----------

FCSIIAGLLHYFFLAAF  505

FWMLvEGvYLHTLIvMtFFsERqYFWWYylIGWGfPlVFitiWvItRcyY

WM +EG+ L+  +V   +  + +Y++G  +P+V ++  +  +

Y

SLGP    506

AWMCIEGIHLYLIVVGVIYNKGFLHKNFYIFGYLSPAVVVGFSAALGYRY  555

ENt..nCWDmNDnMwyWWIIrgPIMlsIvVNFFFFINIIRILMtKLRepq

+ T  CW++++N ++ W  +GP  L I+ N++ F  II+ + +

SLGP    556 YGTTKVCWLSTEN-

NFIWSFIGPACLIILGNLLAFGVIIYKVFRHTAGLK  604

MgEndMqqYWRlvKSTLlLIPLFGIHYMVFaWrPdNhwlwqIYMYFElsl

+    + +  L L+  +  +F  +   +++  y+ +

SLGP    605 PEVSCF--ENIRSCARGALALLLLGTTWIFGGLHVV-

HASVVTAYLFTVS  651

iSFQGFFVAiIYCFcNhEVQmEIRRrW*

SLGP    652 NAFQGMFIFLFLCVLSRKIQEEYYRLF  678

Accordingly, in one embodiment of the invention, an SLGP protein is a human SLGP protein having a 7 transmembrane receptor profile at about amino acids 421-678 of SEQ ID NO:89. Such a 7 transmembrane receptor profile has the amino acid sequence:

(SEQ ID NO:96)

Accordingly, SLGP proteins having at least 20-30%, 30-49%, 40-50%, 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with the 7 transmembrane receptor profile of human SLGP (e.g., SEQ ID NO:89) are within the scope of the invention.

In another embodiment, an SLGP is identified based on the presence of a “EGF-like domain” in the protein or corresponding nucleic acid molecule. As used herein, the term “EGF-like domain” includes a protein domain having an amino acid sequence of about 55-90, preferably about 60-85, more preferably about 65-80 amino acid residues, or about 70-79 amino acids and having a bit score for the alignment of the sequence to the EGF-like domain (HMM) of at least 6, preferably 7-10, more preferably 10-30, more preferably 30-50, even more preferably 50-75, 75-100, 100-200 or greater. The EGF-like domain HMM has been assigned the PFAM Accession PF00008. Preferably, one or more cysteine residues in the EGF-like domain are conserved among SLGP family members or other proteins containing EGF-like domains (i.e., located in the same or similar position as the cysteine residues in other SLGP family members or other proteins containing EGF-like domains). In a preferred embodiment, an “EGF-like domain” has the consensus sequence X(4)-C—X(0,48)-C—X(3,12)-C—X(1,70)-C—X(1,6)-C—X(2)-G-a-X(0,21)-G-X(2)-C—X, (where C=conserved cysteine involved in a disulfide bond, G=often conserved glycine, a=often conserved aromatic acid, X=any residue); corresponding to SEQ ID NO:97. In another preferred embodiment, an “EGF-like domain” has the consensus sequence C—X—C—X(5)-G-X(2)-C, the 3 C's are involved in disulfide bonds; corresponding to SEQ ID NO:98. In another preferred embodiment, an “EGF-like domain” has the consensus sequence C—X—C—X(2)-[GP]-[FYW]-X(4,8)—C, the three C's are involved in disulfide bonds; corresponding to SEQ ID NO:99.

To identify the presence of an EGF-like domain in an SLGP protein, make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for PF00008 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3)405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of an EGF-like domain in the amino acid sequence of SEQ ID NO:89. The results of the search, indicating that such a domain is found at residues 22 through 100 of SEQ ID NO:89, are set forth below:

Score: 6.16   Seq: 22 53   Model: 1 34

*CnpNPCmNgGtCvNtp.mYtCiCpeGYmyYtGrrC*

C+ +PC+ +++C+      C C +G   ++G

SLGP  22 CTKTPCLPNAKCEIRNGIEACYCNMG---FSGNGV  53

Score: 18.87   Seq: 62 100   Model: 1 34

*CnpN..PCmNgGtCvNtp.mYtCiCpeGYm.y.YtGrrC*

C ++  C +++ C+NT+ +Y+C C +G++ +  + R+

SLGP  62 CGNLTQSCGENANCTNTEGSYYCMCVPGFRSSSNQ 100

All amino acids are described using universal single letter abbreviations according to these motifs.

Such an EGF-like domain has the following amino acid sequence:

(SEQ ID NO:100)

Accordingly, SLGP proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with an EGF-like domain of human SLGP (e.g., SEQ ID NO:100) are within the scope of the invention.

In another embodiment, an SLGP is identified based on the presence of a “NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain” in the protein or corresponding nucleic acid molecule. As used herein, the term “NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain” includes a protein domain having an amino acid sequence of about 25-55, preferably about 30-50, more preferably about 35-45 amino acid residues, or about 40-43 amino acids and having a bit score for the alignment of the sequence to the NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain (HMM) of at least 6, preferably 7-10, more preferably 10-30, more preferably 30-50, even more preferably 50-75, 75-100, 100-200 or greater. The NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain HMM has been assigned the PFAM Accession PF00420.

To identify the presence of a NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain in an SLGP protein, make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for PF00420 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3)405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain in the amino acid sequence of SEQ ID NO:89. The results of the search, indicating that such a domain is found at residues 475 through 517 of SEQ ID NO:89, are set forth below.

Score: 6.77   Seq: 475 517   Model: 1 43

*MMMMthYHFiIMIaFmmGIMGIlMNRsHmMSMLMCLEmMMLSl*

++ + ++   +F+  I G+L +     ++ MC+E++ L L

SLGP     475 LVFLVGINTNTNKLFCSIIAGLLHYFFLAAFAWMCIE

GIHLYL   517

All amino acids are described using universal single letter abbreviations according to these motifs.

Such a NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain has the amino acid sequence:

(SEQ ID NO:101)

Accordingly, SLGP proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain of human SLGP (e.g., SEQ ID NO:101) are within the scope of the invention.

In another embodiment, an SLGP protein includes at least an EGF-like domain. In another embodiment, an SLGP protein includes at least an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain. In another embodiment, an SLGP protein includes at least a 7 transmembrane receptor profile. In another embodiment, an SLGP protein includes an EGF-like domain, and an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain. In another embodiment, an SLGP protein includes an EGF-like domain and a 7 transmembrane receptor profile. In another embodiment, an SLGP protein includes an EGF-like domain, and an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain, and a 7 transmembrane receptor profile.

In another embodiment, an SLGP protein includes an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain and a 7 transmembrane receptor profile. In another embodiment, an SLGP protein is human SLGP which includes an EGF-like domain having about amino acids 22-100 of SEQ ID NO:89. In another embodiment, an SLGP protein is human SLGP which includes an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain having about amino acids 475-517 of SEQ ID NO:89. In another embodiment, an SLGP protein is human SLGP which includes a 7 transmembrane receptor profile having about amino acids 421-678 of SEQ ID NO:89.

In yet another embodiment, an SLGP protein is human SLGP which includes a an EGF-like domain having about amino acids 22-100 of SEQ ID NO:89, an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain having about amino acids 475-517 of SEQ ID NO:89, and a 7 transmembrane receptor profile having about amino acids 421-678 of SEQ ID NO:89.

Preferred SLGP molecules of the present invention have an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:89 or SEQ ID NO:105. As used herein, the term “sufficiently homologous” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains have at least about 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains, are defined herein as sufficiently homologous. Furthermore, amino acid or nucleotide sequences which share at least 50%, preferably 60%, more preferably 70-80, or 90-95% homology and share a common functional activity are defined herein as sufficiently homologous.

As used interchangeably herein, an “SLGP activity”, “biological activity of SLGP” or “functional activity of SLGP”, refers to an activity exerted by an SLGP protein, polypeptide or nucleic acid molecule on an SLGP responsive cell as determined in vivo, or in vitro, according to standard techniques. In one embodiment, an SLGP activity is a direct activity, such as an association with a SLGP-target molecule. As used herein, a “target molecule” or “binding partner” is a molecule with which an SLGP protein binds or interacts in nature, such that SLGP-mediated function is achieved. An SLGP target molecule can be a non-SLGP molecule or an SLGP protein or polypeptide of the present invention. In an exemplary embodiment, an SLGP target molecule is an SLGP ligand. Alternatively, an SLGP activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the SLGP protein with an SLGP ligand.

In a preferred embodiment, an SLGP activity is at least one or more of the following activities: (i) interaction of an SLGP protein with soluble SLGP ligand (e.g., CD55); (ii) interaction of an SLGP protein with a membrane-bound non-SLGP protein; (iii) interaction of an SLGP protein with an intracellular protein (e.g., an intracellular enzyme or signal transduction molecule); (iv) indirect interaction of an SLGP protein with an intracellular protein (e.g., a downstream signal transduction molecule); and (v) modulation of cellular proliferation, growth, differentiation, or migration. In yet another preferred embodiment, an SLGP activity is at least one or more of the following activities: (1) modulation of cellular signal transduction, either in vitro or in vivo; (2) regulation of activation in a cell expressing an SLGP protein exposure to alpha-latrotoxin); (3) regulation of inflammation; or (4) modulation of angiogenesis (e.g., proliferation, elongation, and migration of endothelial cells (e.g. tumor endothelial cells), to form new vessels).

Accordingly, another embodiment of the invention features isolated SLGP proteins and polypeptides having an SLGP activity. Preferred SLGP proteins have at least one transmembrane domain and an SLGP activity. In a preferred embodiment, an SLGP protein has a 7 transmembrane receptor profile and an SLGP activity. In another preferred embodiment, an SLGP protein has an EGF-like domain and an SLGP activity. In another preferred embodiment, an SLGP protein has an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain and an SLGP activity. In still another preferred embodiment, an SLGP protein has a 7 transmembrane receptor profile, an EGF-like domain, and SLGP activity. In still another preferred embodiment, an SLGP protein has a 7 transmembrane receptor profile, an EGF-like domain, and an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain and an SLGP activity. In still another preferred embodiment, an SLGP protein has a 7 transmembrane receptor profile and an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain and an SLGP activity. In still another preferred embodiment, an SLGP protein has an EGF-like domain and an NADH-ubiquinone/plastoquinone oxidoreductase chain 4L domain and an SLGP activity. In still another preferred embodiment, an SLGP protein has a 7 transmembrane receptor profile, an EGF-like domain, an SLGP activity, and an amino acid sequence sufficiently homologous to an amino acid sequence of SEQ ID NO:89 or SEQ ID NO:105.

An alignment of the amino acid sequences of human SLGP (SEQ ID NO:89) and human CD 97 (Accession No. U76764, SEQ ID NO:102) generated utilizing the ALIGN program with the following parameter setting: PAM120, gap penalties: −12/−4 (Myers, E. and Miller, W. (1988) “Optimal Alignments in Linear Space” CABIOS 4:11-17) demonstrated a 27.9% identity between the two sequences.

An alignment of the nucleotide sequences of human SLGP (SEQ ID NO:88) and human CD 97 (Accession No. U76764, SEQ ID NO:103) generated utilizing the ALIGN program with the following parameter setting: PAM120, gap penalties: −12/−4 (Myers, E. and Miller, W. (1988) “Optimal Alignments in Linear Space” CABIOS 4:11-17) demonstrated a 41.8% identity between the two sequences.

The nucleotide sequence of the isolated human SLGP cDNA and the predicted amino acid sequence of the human SLGP polypeptide are shown in SEQ ID NOs:88 and 89, respectively.

The human SLGP cDNA, which is approximately 2987 nucleotides in length (SEQ ID NO:88), encodes a protein which is approximately 690 amino acid residues in length (SEQ ID NO:89).

The nucleotide sequence of the isolated mouse SLGP cDNA and the predicted amino acid sequence of the mouse SLGP polypeptide are shown in SEQ ID NOs:104 and 105, respectively.

The mouse SLGP cDNA, which is approximately 3952 nucleotides in length (SEQ ID NO: 104), encodes a protein which is approximately 689 amino acid residues in length (SEQ ID NO: 105).

Isolation of the Human and Mouse SLGP cDNAs

In order to identify novel secreted and/or membrane-bound proteins, a program termed ‘signal sequence trapping’ was utilized to analyze the sequences of several cDNAs of a cDNA library derived from bronchial epithelial cells which had been stimulated with the cytokine, TNFα. This analysis identified a human clone having an insert of approximately 3 kb containing a protein-encoding sequence of approximately 2987 nucleotides capable of encoding approximately 690 amino acids of SLGP (e.g., the starting methionine through residue 690 of, for example, SEQ ID NO:89).

The nucleotide sequence encoding the human SLGP protein is set forth as SEQ ID NO:88. The full length protein encoded by this nucleic acid is comprised of about 690 amino acids and has the amino acid sequence set forth as SEQ ID NO:89. The coding portion (open reading frame) of SEQ ID NO:88 is set forth as SEQ ID NO:90.

The nucleotide sequence encoding the mouse SLGP protein is set forth as SEQ ID NO:104. The full length protein encoded by this nucleic acid is comprised of about 689 amino acids and has the amino acid sequence set forth as SEQ ID NO:105. The coding portion (open reading frame) of SEQ ID NO:104 is set forth as SEQ ID NO:106.

Analysis of Human SLGP

A BLAST search (Altschul et al. (1990) J. Mol. Biol. 215:403) of the nucleotide sequence of human SLGP has revealed that SLGP is significantly similar to a protein identified as human CD 97 (Accession No. U76764; SEQ ID NO:102 and 103) and to a protein identified as rat latrophilin (Accession Nos. U78105, U72487).

The SLGP proteins of the present invention contain a significant number of structural characteristics of the GPCR family. For instance, the SLGPs of the present invention contain conserved cysteines found in the first 2 loops (prior to the third and fifth transmembrane domains) of most GPCRs (cys490 and cys562 of SEQ ID NO:89). A highly conserved asparagine residue is present (asn125 in SEQ ID NO:89). SLGP proteins contains a highly conserved leucine (leu154 of SEQ ID NO:89). The two cysteine residues are believed to form a disulfide bond that stabilizes the functional protein structure. A highly conserved asparagine and arginine in the fourth transmembrane domain of the SLGP proteins is present (asp158 and arg218 of SEQ ID NO:89). Moreover, a highly conserved proline is present (pro307 of SEQ ID NO:89). Proline residues in the fourth, fifth, sixth, and seventh transmembrane domains are thought to introduce kinks in the alpha-helices and may be important in the formation of the ligand binding pocket. Moreover, a conserved tyrosine is present in the seventh transmembrane domain of SLGP-2 (tyr647 of SEQ ID NO:89).

As such, the SLGP family of proteins, like the Secretin family of proteins, are referred to herein as G protein-coupled receptor-like proteins.

SLGP is predicted to contain the following sites: N-glycosylation site at residues 15-18, residues 21-24, residues 64-67, residues 74-77, residues 127-130, residues 177-180, residues 188-191, residues 249-252, residues 381-384, and at residues 395-398 of SEQ ID NO:89; Glycosaminoglycan attachment site at residues 49-52 of SEQ ID NO:89; cAMP- and cGMP-dependent protein kinase phosphorylation sites at residues 360-363 of SEQ ID NO:89; Protein kinase C phosphorylation sites at residues 135-137, residues 181-183, residues 233-235, residues 358-360, residues 363-365, residues 400-402, residues 457-459, residues 485-487, residues 558-560, and residues 667-669 of SEQ ID NO:89; Casein kinase II phosphorylation sites at residues 54-57, residues 68-71, residues 76-79, residues 94-97, residues 135-138, residues 150-153, residues 155-158, residues 161-164, residues 181-184, residues 190-193, residues 244-247, residues 310-313, residues 325-328, residues 346-349, and at residues 608-611 of SEQ ID NO:89; Tyrosine kinase phosphorylation site at residues 36-43, and residues 668-675 of SEQ ID NO:89; N-myristoylation sites at residues 38-43, residues 50-55, residues 80-85, residues 382-387, residues 388-393, residues 434-439, residues 480-485, residues 521-526, residues 584-589, and at residues 619-624 of SEQ ID NO:89; Aspartic acid and asparagine hydroxylation at residues 75-86 of SEQ ID NO:89, EF-hand calcium-binding domain at residues 153-165 of SEQ ID NO:89.

Tissue Distribution of SLGP mRNA by Northern Blot Hybridization

This Example describes the tissue distribution of SLGP mRNA, as determined by Northern blot hybridization.

Northern blot hybridizations with the various RNA samples were performed (Clontech Human Multi-tissue Northern I and a human normal and diseased heart tissue northern) under standard conditions and washed under stringent conditions. A 3.2 Kb and a 4.2 Kb mRNA transcript was detected in all tissues tested (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas), with the highest expression in heart. Specifically, the expression was found to be localized to endothelial cells in the heart. Additionally, these transcripts were found in both normal and diseased hearts.

Tissue Distribution Analysis of Human and Mouse SLGP cDNA

The following describes the tissue distribution of human and mouse SLGP cDNA, as determined using the TaqMan™ procedure.

The results from these analyses showed that Human SLGP is upregulated in tube forming Human Microvascular Endothelial Cells (HMVEC) and in proliferating HMVEC as compared to arresting HMVEC. Human SLGP is also upregulated in glioblastomas as compared to normal brain.

Additionally, mouse SLGP was shown to be upregulated in VEGF-induced angiogenic xenograft plugs as compared to parental plugs.

In Situ Hybridization Analysis of Human SLGP

The following describes the tissue distribution of human SLGP as determined using in situ hybridization analysis. For in situ analysis, tissues, e.g. brain and glioblastoma tissues, were first frozen on dry ice.

In situ hybridization results show that the human SLGP gene is expressed in endothelial cells of glioblastomas but not in endothelial cells of normal brains.

Analysis of Human and Mouse SLGP Expression

The following describes the expression of human and mouse SLGP as determined by transcriptional profiling experiments. Expression of human SLGP in proliferating HMVEC and arresting HMVEC was analyzed by transcriptional profiling. The results from this analysis demonstrate that human SLGP is up-regulated in proliferating HMVEC as compared to arresting HMVEC.

Expression of mouse SLGP in VEGF-induced angiogenic plugs and parental xenografts was also analyzed by transcriptional profiling. These results demonstrated that mouse SLGP expression is up-regulated in VEGF-induced angiogenic xenograft plugs as compared to parental xenografts.

Human 38555 and 593

The present invention is based, at least in part, on the discovery of human cDNA molecules which encode proteins which are herein designated 38555 (or 38555) and 593. The invention is also based on the discovery that the protein encoded by a previously described (but otherwise non-characterized) human brain cDNA clone is, or is functionally analogous to, a prostaglandin and thromboxane transmembrane transport protein. These three proteins are integral membrane proteins that facilitate transmembrane transport of charged organic compounds such as one or more of prostaglandins, thromboxanes, hexoses, disaccharides, hormones (e.g. insulin), peptides, neurotransmitters, cytokines, chemokines, and the like. The characteristics of each of these proteins and the cDNAs encoding them are now described separately.

Protein 38555

A cDNA encoding at least a portion of human 38555 protein was isolated from a library of human cDNA clones on the basis of homology to the amino terminal portion of the protein designated ‘human prostaglandin transporter’ (HPT) in the literature (U.S. Pat. No. 5,792,851; Lu et al. (1996) J. Clin. Invest. 98:1142-1149; Kanai et al. (1995) Science 268:866-869). Human protein 38555 is predicted by structural analysis to be a transmembrane transporter protein having twelve transmembrane domains.

The full length of the cDNA encoding human protein 38555 (SEQ ID NO:107) is 2563 nucleotide residues. The ORF of this cDNA, nucleotide residues 42 to 1970 of SEQ ID NO:107 (i.e. SEQ ID NO:109), encodes a 643-amino acid protein (SEQ ID NO:108) which exhibits amino acid sequence homology with HPT protein and other prostaglandin transporters. The human 38555 genomic sequence is shown as nucleotide residues 1-50,000 in SEQ ID NO:110 and nucleotide residues 50,124 in SEQ ID NO:118. The gene encoding human protein 38555 maps to human chromosome 15 at q26.1. A PAC clone including this region has been sequenced, and the sequence of that clone is listed in GenBank Accession number AC005319. It was not previously recognized that any protein, let alone protein 38555 was encoded within the portion of the genome encompassed by the PAC clone. The exon and intron structure of the genomic sequence is described in Tables 49 and 50. Table 49 lists the positions of exons in this sequence, and Table 50 lists intron positions and branch sites (bold residues in Table 50 indicate RNA splicing junctions.

In addition to full length human protein 38555, the invention includes fragments, derivatives, and variants of protein 38555, as described herein. These proteins, fragments, derivatives, and variants are collectively referred to herein as polypeptides of the invention or proteins of the invention.

The invention also includes nucleic acid molecules which encode a polypeptide of the invention. Such nucleic acids include, for example, a DNA molecule having the nucleotide sequence listed in SEQ ID NO:107 or some portion thereof, such as the portion which encodes human protein 38555, or a domain, fragment, derivative, or variant of protein 38555. These nucleic acids are collectively referred to as nucleic acids of the invention.

38555 proteins of the invention and nucleic acid molecules encoding them comprise a family of molecules having certain conserved structural and functional features, as indicated by the conservation of amino acid sequence between protein 38555 and HPT (SEQ ID NO:116), the human OatP sodium-independent organic anion transporter protein (GenBank Accession no. P46721; SEQ ID NO:117), human KIAA0880 protein (GenBank Accession no. 4240248; SEQ ID NO:115), and human protein 593 (as described herein, SEQ ID NO:113).

38555 proteins typically comprise a variety of potential post-translational modification sites (often within an extracellular domain), such as those described herein in Table 51, as predicted by computerized sequence analysis of human 38555 protein using amino acid sequence comparison software (comparing the amino acid sequence of protein 38555 with the information in the PROSITE database {rel. 12.2; February, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3}). In certain embodiments, a protein of the invention has at least 1, 2, 4, 6, 8, 10, 15, or 20 or more of the post-translational modification sites listed in Table 51.

Protein 38555 comprises domains which exhibit homology with known sugar (or other) transport domains and with Kazal domains. In one embodiment, the protein of the invention has at least one domain that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to one of these domains. Preferably, the protein of the invention has at least two domains, each of which is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to either the sugar (or other) transport domain or the Kazal domain of protein 38555.

Sugar (or other) transport domains occur in a variety of proteins involved in transmembrane transport of sugars and other metabolites. Other proteins which comprise such a domain include human glucose transporters GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, and GLUT7, Escherichia coli proteins AraE (arabinose-proton symporter), GalP (galactose-proton symporter), citrate-proton symport protein, KgtP (α-ketoglutarate permease), ProP (proline/betaine transporter), and XylE (xylose-proton symporter), Escherichia coli hypothetical proteins YabE, YdjE, and YhjE, Klebsiella pneumoniae citrate-proton symport protein, Zymomonas mobilis glucose facilitated diffusion protein, yeast high and low affinity glucose transport proteins (SNF3 and HXT1 through HXT14), yeast galactose transporter, yeast maltose permease, yeast myo-inositol transporter, yeast carboxylic acid transporter homolog JEN1, yeast hypothetical proteins YBR241c, YCR98c, and YFL040w, Klyveromyces lactis lactose permease, Neurospora crassa quinate transporter, Emericella nidulans quinate permease, Chlorella hexose carrier, Arabidopsis thaliana glucose transporter, spinach sucrose transporter, Leishmania donovani transporters D1 and D2, Leishmania enriettii probable transport protein LTP, Caenorhabditis elegans hypothetical protein ZK637.1, Haemophilus influenzae hypothetical proteins HI0281 and HI0418, and Bacillus subtilis hypothetical proteins YxbC and YxdF. Occurrence of a sugar (or other) transport domain in protein 38555 indicates that protein 38555 is involved in transmembrane transport of one or more compounds, most likely a compound having a molecular weight on the order of a hexose or greater (i.e. having a molecular weight greater than about 180). Examples of such compounds include prostaglandins, thromboxanes, hexoses, disaccharides, hormones (e.g. insulin), peptides, neurotransmitters, cytokines, chemokines, and the like. Protein 38555 thus mediates one or more of facilitated diffusion and symport or antiport (e.g. involving co-transport of a proton, a sodium ion, a potassium ion, or another physiological ion).

Kazal domains occur frequently in serine protease inhibitors. However, these domains also occur as extracellular domains in agrins, which are not thought to have roles as protease inhibitors. These domains are characterized by occurrence, preferably within an extracellular domain, of the consensus pattern

(SEQ ID NO:119) C-X (7 or 8) -C-X 6 -Y-X 3 -C-X (2 or 3) -C-

wherein standard single-letter amino acid residue codes are used, X being any amino acid residue, and subscripts referring to the number of residues. Agrins are involved in organization of neural synapses, including, for example, inter-neuronal synapses within the central nervous system (e.g. glutamatergic synapses) and neuromuscular junctions (Martin and Sanes (1997) Development 124:3909-3917; Lieth and Fallon (1993) J. Neurosci. 13:2509-2514). Agrins are also involved in organization of endothelial cells and astrocytes during formation and maintenance of the blood brain barrier. Thus, occurrence of a Kazal domain in protein 38555 indicates that this protein is involved in formation and maintenance of cell-to-cell interactions, and more particularly that the protein is involved in forming and maintaining neural synapses, including both neuron-to-neuron synapses and neuron-to-non-neural cell synapses (e.g. neuromotor and neuroendocrine synapses).

Human protein 38555 exhibits sequence similarity to HPT (GenBank Accession no. Q92959). An alignment of the amino acid sequences of human protein 38555 (SEQ ID NO:108) and HPT (SEQ ID NO:116) made using the ALIGN program of the GCG software package, pam120.mat scoring matrix, gap penalties −12/−4, demonstrates that the amino acid sequences of the proteins are 32.4% identical.

Protein 38555 is predicted by computerized amino acid sequence analysis (using the MEMSAT computer program) to be a twelve-transmembrane region integral membrane protein having transmembrane regions at approximately the following positions within SEQ ID NO:108: from about amino acid residue 8 to about residue 17; from about amino acid residue 29 to about residue 52; from about amino acid residue 59 to about residue 76; from about amino acid residue 129 to about residue 153; from about amino acid residue 164 to about residue 186; from about amino acid residue 215 to about residue 236;

from about amino acid residue 301 to about residue 324; from about amino acid residue 341 to about residue 361; from about amino acid residue 374 to about residue 392; from about amino acid residue 490 to about residue 513; from about amino acid residue 524 to about residue 548; and from about amino acid residue 575 to about residue 592.

Extracellular domains are predicted to include approximately amino acid residues 18 to 28, 77 to 128, 187 to 214, 325 to 340, 393 to 489, and 549 to 574 of SEQ ID NO:108. Intracellular domains are predicted to include approximately amino acid residues 1 to 7, 53 to 58, 154 to 163, 237 to 300, 362 to 373, 514 to 523, and 593 to 643 of SEQ ID NO:108.

Human protein 38555 can have additional amino acid residues at the amino terminal end of the sequence listed in SEQ ID NO:108 (i.e. the protein can have an additional portion at its amino terminus). For example, protein 38555 can have 1, 2, 4, 6, 10, 15, 20, 25, or 30 or more additional amino acid residues at the amino terminus indicated in SEQ ID NO:108.

As described elsewhere herein, relatively hydrophilic regions are generally located at or near the surface of a protein, and are more frequently effective immunogenic epitopes than are relatively hydrophobic regions. For example, the region of human protein 38555 from about amino acid residue 415 to about amino acid residue 430 appears to be located at or near the surface of the protein, while the region from about amino acid residue 440 to about amino acid residue 450 appears not to be located at or near the surface.

The predicted molecular weight of human protein 38555 is about 69.2 kilodaltons.

A monkey cDNA clone having significant homology with the human cDNA clone encoding protein 38555 was isolated from a monkey brain cDNA library, indicating that human protein 38555 is expressed in brain tissue, although it can, of course, be expressed in other tissues as well.

Biological Function of Human 38555 Proteins, Nucleic Acids Encoding them, and Modulators of These Molecules

Human 38555 proteins are involved in disorders which affect both tissues in which they are normally expressed and tissues in which they are normally not expressed. Based on the observation that 38555 protein is expressed in monkey brain and is therefore likely expressed in human brain tissue, human 38555 protein is involved in one or more biological processes which occur in brain and other neurological tissues. In particular, 38555 is involved in modulating growth, proliferation, survival, differentiation, and activity of cells including, but not limited to, central nervous system neurons, peripheral nervous system neurons, motor neurons, sensory neurons, and sympathetic and parasympathetic neural cells of the animal in which it is normally expressed. Protein 38555 is also involved in mediating interactions between cells, particularly between two neurons or between a neuron and a non-neuronal cell such as a muscle or endocrine cell. Thus, 38555 protein has a role in disorders which affect neuronal cells and cells which interact with neurons and their growth, proliferation, survival, differentiation, and activity.

Widespread expression of 38555 has been detected among human tissue types. Thus, the growth-, proliferation-, survival-, differentiation-, and activity-modulating activities of 38555 protein affect cells of many types. Thus, protein 38555 can affect cell-to-cell interactions in a wide variety of cell types.

The presence of the sugar (or other) transport domain in protein 38555 indicates that this protein is involved in transmembrane transport of one or more charged organic compounds such as prostaglandins, thromboxanes, neurotransmitters, hormones, small peptides, short polysaccharides (e.g. disaccharides), and the like. The proteins of the invention are therefore involved in one or more disorders relating to inappropriate uptake or release of such molecules (i.e. including inappropriate failure to take up or release such molecules). Protein 38555 is thus involved in one or more of a variety of cellular uptake and release disorders such as diabetes, nutritional disorders (e.g. vitamin deficiencies, and malnutrition), metabolic disorders (e.g. obesity, porphyrias, hyper- and hypolipoproteinemia, lipidoses, and water, electrolyte, mineral, and acid/base imbalances), and neural transmission disorders (e.g. inappropriate pain, dementia, multiple sclerosis, nerve root disorders, Alzheimer's disease, Parkinson's disease, depression, physical and psychological substance addiction, sexual dysfunction, schizophrenic disorders, delusional disorders, mood disorders, sleep disorders, and the like).

Occurrence of a Kazal domain in human protein 38555 further implicates this protein in neuronal development and transmission. The presence of this domain therefore indicates that 38555 protein is involved in disorders relating to inappropriate formation (i.e. including failure to form) and maintenance (i.e. including deterioration) of neuronal synapses, including both neuron-to-neuron synapses and neuron-to-non-neuronal cell synapses. Thus, in addition to the neural transmission disorders described above, protein 38555 is also implicated in disorders such as stroke, regeneration of chronically or traumatically damaged neuronal structures (including nerve, brain, and spinal cord), developmental neuronal disorders (e.g. spina bifida), neuronal cancers (e.g. gliomas, astrocytomas, ependymomas, pituitary adenomas, and the like), peripheral nerve deficit, cardiac insufficiency, and the like.

The observation that human protein 38555 shares sequence homology with proteins involved in transmembrane prostaglandin transport indicates that 38555 protein has activity identical or analogous to the activity of those proteins, i.e. that 38555 catalyzes or facilitates transmembrane transport of one or more prostaglandins, thromboxanes, other hormones or hormone-like molecules, or other charged organic compounds. Exemplary molecules which can be transported across cell membranes via protein 38555 include one or more charged organic compounds such as prostaglandins A 1 , A 2 , B 1 , B 2 , D 2 , E 1 , E 2 , F 1α , F 2α , G 2 , H 2 , I 2 , and J 2 and thromboxanes A 2 and B 2 . Uptake and release of prostaglandins and thromboxanes, for example, are known to be involved in a variety of physiological processes and disorders including glaucoma, ovum fertilization, sperm motility, pregnancy, labor, delivery, abortion, gastric protection, peptic ulcer formation, intestinal fluid secretion, liver protection, liver damage, liver fibrosis, pain stimulation, glomerular filtration, maintenance of body temperature, fever, airway resistance, asthma, chronic obstructive pulmonary disorder, modulation of blood pressure, hypertension, shock, modulation of inflammation, platelet aggregation, abnormal blood coagulation, atherosclerosis, arteriosclerosis, and coronary artery disease. Thus, polypeptides and nucleic acid molecules of the invention, and compounds which bind with or modulate one or more polypeptides and nucleic acid molecules of the invention can be used to prognosticate, diagnose, inhibit, or treat one or more of the disorders listed above or one or more disorders associated with the physiological processes listed above.

Protein 593

A cDNA encoding at least a portion of human 593 protein was identified by assembling isolated sequences derived from a library of human cDNA clones on the basis of homology with the nucleic acid sequence encoding human protein 38555. Human protein 593 is predicted by structural analysis to be a transmembrane transporter protein having twelve transmembrane domains.

The full length of the cDNA encoding human protein 593 (SEQ ID NO:111) is 2276 nucleotide residues. The ORF of this cDNA, nucleotide residues 1 to 1836 of SEQ ID NO:111 (SEQ ID NO:113), encodes a 612-amino acid protein (SEQ ID NO:112) which exhibits amino acid sequence homology with human protein 38555 and other prostaglandin transporters.

In addition to full length human protein 593, the invention includes fragments, derivatives, and variants of protein 593, as described herein. These proteins, fragments, derivatives, and variants are collectively referred to herein as polypeptides of the invention or proteins of the invention.

The invention also includes nucleic acid molecules which encode a polypeptide of the invention. Such nucleic acids include, for example, a DNA molecule having the nucleotide sequence listed in SEQ ID NO:111 or some portion thereof, such as the portion which encodes human protein 593, or a domain, fragment, derivative, or variant of protein 593. These nucleic acids are collectively referred to as nucleic acids of the invention.

Human 593 proteins of the invention and nucleic acid molecules encoding them comprise a family of molecules having certain conserved structural and functional features, as indicated by the close homology of human protein 593 (SEQ ID NO:112) to HPT (SEQ ID NO:117), the human OatP sodium-independent organic anion transporter protein (GenBank Accession no. P46721; SEQ ID NO:116), human KIAA0880 protein (GenBank Accession no. 4240248; SEQ ID NO:115), and human protein 38555 (as described herein, SEQ ID NO:108).

Human 593 proteins typically comprise a variety of potential post-translational modification sites (often within an extracellular domain), such as those described herein in Table 52, as predicted by computerized sequence analysis of human 593 protein using amino acid sequence comparison software (comparing the amino acid sequence of protein 593 with the information in the PROSITE database {rel. 12.2; February, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3}). In certain embodiments, a protein of the invention has at least 1, 2, 4, 6, 8, 10, 15, or 20 or more of the post-translational modification sites listed in Table 52.

Protein 593 comprises domains which exhibit homology with known sugar (or other) transport domains and with Kazal domains. In one embodiment, the protein of the invention has at least one domain that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to one of these domains. Preferably, the protein of the invention has at least two domains, each of which is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to either the sugar (or other) transport domain or the Kazal domain of protein 593.

Sugar (or other) transport domains occur in a variety of proteins involved in transmembrane transport of sugars and other metabolites. Other proteins which comprise such a domain include human glucose transporters GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, and GLUT7, Escherichia coli proteins AraE (arabinose-proton symporter), GalP (galactose-proton symporter), citrate-proton symport protein, KgtP (α-ketoglutarate permease), ProP (proline/betaine transporter), and XylE (xylose-proton symporter), Escherichia coli hypothetical proteins YabE, YdjE, and YhjE, Klebsiella pneumoniae citrate-proton symport protein, Zymomonas mobilis glucose facilitated diffusion protein, yeast high and low affinity glucose transport proteins (SNF3 and HXT1 through HXT14), yeast galactose transporter, yeast maltose permease, yeast myo-inositol transporter, yeast carboxylic acid transporter homolog JEN1, yeast hypothetical proteins YBR241c, YCR98c, and YFL040w, Klyveromyces lactis lactose permease, Neurospora crassa quinate transporter, Emericella nidulans quinate permease, Chlorella hexose carrier, Arabidopsis thaliana glucose transporter, spinach sucrose transporter, Leishmania donovani transporters D1 and D2, Leishmania enriettii probable transport protein LTP, Caenorhabditis elegans hypothetical protein ZK637.1, Haemophilus influenzae hypothetical proteins HI0281 and HI0418, and Bacillus subtilis hypothetical proteins YxbC and YxdF. Occurrence of a sugar (or other) transport domain in protein 593 indicates that protein 593 is involved in transmembrane transport of one or more compounds, most likely a compound having a molecular weight on the order of a hexose or greater (i.e. having a molecular weight greater than about 180). Examples of such compounds include prostaglandins, thromboxanes, hexoses, disaccharides, hormones (e.g. insulin), peptides, neurotransmitters, cytokines, chemokines, and the like. Protein 593 thus mediates one or more of facilitated diffusion and symport or antiport (e.g. involving co-transport of a proton, a sodium ion, a potassium ion, or another physiological ion). One, both, or neither of a glycosaminoglycan attached at the predicted glycosaminoglycan attachment site and a pyridoxal phosphate moiety attached at the predicted pyridoxal phosphate attachment site can, in conjunction with the amino acid sequence of protein 593, determine the specificity of the protein for transporting molecules across the membrane of a cell in which it is expressed.

Like human protein 38555, as described above, human protein 593 comprises a Kazal domain. Occurrence of a Kazal domain in protein 593 indicates that this protein is involved in formation and maintenance of cell-to-cell interactions, and more particularly that the protein is involved in forming and maintaining neural synapses, including both neuron-to-neuron synapses and neuron-to-non-neural cell synapses (e.g. neuromotor and neuroendocrine synapses).

Human protein 593 exhibits sequence similarity to HPT (GenBank Accession no. Q92959). Protein 593 is a twelve-transmembrane region integral membrane protein having transmembrane regions at approximately the following positions within SEQ ID NO:112: from about amino acid residue 1 to about residue 10; from about amino acid residue 33 to about residue 53; from about amino acid residue 62 to about residue 79; from about amino acid residue 118 to about residue 142; from about amino acid residue 153 to about residue 177; from about amino acid residue 200 to about residue 221; from about amino acid residue 262 to about residue 283; from about amino acid residue 314 to about residue 334; from about amino acid residue 347 to about residue 364; from about amino acid residue 469 to about residue 493; from about amino acid residue 509 to about residue 528; and from about amino acid residue 556 to about residue 579.

Extracellular domains are predicted to include approximately amino acid residues 11 to 32, 80 to 117, 178 to 199, 284 to 313, 365 to 468, and 529 to 555 of SEQ ID NO:112. Intracellular domains are predicted to include approximately amino acid residues 54 to 61, 143 to 152, 222 to 261, 335 to 346, 494 to 508, and 580 to 612 of SEQ ID NO:112.

Human protein 593 can have additional amino acid residues at the amino terminal end of the sequence listed in SEQ ID NO:112 (i.e. the protein can have an additional portion at its amino terminus). For example, protein 593 can have 1, 2, 4, 6, 10, 15, 20, 25, or 30 or more additional amino acid residues at the amino terminus indicated in SEQ ID NO:12.

As described elsewhere herein, relatively hydrophilic regions are generally located at or near the surface of a protein, and are more frequently effective immunogenic epitopes than are relatively hydrophobic regions. For example, the region of human protein 593 from about amino acid residue 240 to about amino acid residue 260 appears to be located at or near the surface of the protein, while the region from about amino acid residue 415 to about amino acid residue 430 appears not to be located at or near the surface.

The predicted molecular weight of human protein 593 is about 65.4 kilodaltons.

Biological Function of Human 593 Proteins, Nucleic Acids Encoding Them, and Modulators of These Molecules

Human 593 proteins are involved in disorders which affect both tissues in which they are normally expressed and tissues in which they are normally not expressed. Based on the observation that 593 protein exhibits amino acid sequence homology to human protein 38555, which is expressed in monkey brain and is therefore likely expressed in human brain tissue, human 593 protein is involved in one or more biological processes which occur in brain and other neurological tissues, although it can also be expressed in other tissues, and involved in disorders in those tissues as well. In particular, 593 is involved in modulating growth, proliferation, survival, differentiation, and activity of cells including, but not limited to, central nervous system neurons, peripheral nervous system neurons, motor neurons, sensory neurons, and sympathetic and parasympathetic neural cells of the animal in which it is normally expressed. Protein 593 is also involved in mediating interactions between cells, particularly between two neurons, or between a neuron and a non-neuronal cell such as a muscle or endocrine cell. Thus, 593 protein has a role in disorders which affect neuronal cells and cells which interact with neurons and their growth, proliferation, survival, differentiation, and activity.

Widespread expression of 593 has been detected among human tissue types. Thus, the growth-, proliferation-, survival-, differentiation-, and activity-modulating activities of 593 protein affect cells of many types. Thus, protein 593 can affect cell-to-cell interactions in a wide variety of cell types.

Protein 593 can also be expressed in other tissues which normally produce or are acted upon by prostaglandins and thromboxanes. Such tissues include, by way of example, blood tissues (e.g. blood platelets), epithelial tissues such as stomach, kidney, lung, uterus, vascular, and other epithelia, liver, ova, and spermatozoa. Protein 593 is thus involved in one or more disorders which affect these tissues, such as one or more of the tissues listed above in the discussion regarding protein 38555.

The presence of the sugar (or other) transport domain in protein 593 indicates that this protein is involved in transmembrane transport of one or more molecules such as neurotransmitters, prostaglandins, thromboxanes, hormones, small peptides, short polysaccharides (e.g. disaccharides), other charged organic compounds, and the like. The proteins of the invention are therefore involved in one or more disorders relating to inappropriate uptake or release of such molecules (i.e. including inappropriate failure to take up or release such molecules). Protein 593 is thus involved in one or more of a variety of cellular uptake and release disorders such as diabetes, nutritional disorders (e.g. vitamin deficiencies, and malnutrition), metabolic disorders (e.g. obesity, porphyrias, hyper- and hypolipoproteinemia, lipidoses, and water, electrolyte, mineral, and acid/base imbalances), and neural transmission disorders (e.g. inappropriate pain, dementia, multiple sclerosis, nerve root disorders, Alzheimer's disease, Parkinson's disease, depression, physical and psychological substance addiction, sexual dysfunction, schizophrenic disorders, delusional disorders, mood disorders, sleep disorders, and the like).

Occurrence of a Kazal domain in human protein 593 further implicates this protein in neuronal development and neuronal transmission processes. The presence of this domain therefore indicates that 593 protein is involved in disorders relating to inappropriate formation (i.e. including failure to form) and maintenance (i.e. including deterioration) of neuronal synapses, including both neuron-to-neuron synapses and neuron-to-non-neuronal cell synapses. Thus, in addition to the neural transmission disorders described above, protein 593 is also implicated in disorders such as stroke, regeneration of chronically or traumatically damaged neuronal structures (including nerve, brain, and spinal cord), developmental neuronal disorders (e.g. spina bifida), neuronal cancers (e.g. gliomas, astrocytomas, ependymomas, pituitary adenomas, and the like), peripheral nerve deficit, coronary insufficiency, angina, and the like.

The observation that human protein 593 shares sequence homology with proteins involved in transmembrane prostaglandin transport indicates that 593 protein has activity identical or analogous to the activity of those proteins, i.e. that 593 catalyzes or facilitates transmembrane transport of one or more prostaglandins, thromboxanes, other hormones or hormone-like molecules, or other charged organic compounds. Exemplary molecules which can be transported across cell membranes via protein 593 include charged organic compounds, such as one or more of prostaglandins A 1 , A 2 , B 1 , B 2 , D 2 , E 1 , E 2 , F 1α , F 2α , G 2 , H 2 , I 2 , and J 2 and thromboxanes A 2 and B 2 . Uptake and release of prostaglandins and thromboxanes, for example, are known to be involved in a variety of physiological processes and disorders including glaucoma, ovum fertilization, sperm motility, pregnancy, labor, delivery, abortion, gastric protection, peptic ulcer formation, intestinal fluid secretion, liver protection, liver damage, liver fibrosis, pain stimulation, glomerular filtration, maintenance of body temperature, fever, airway resistance, asthma, chronic obstructive pulmonary disorder, modulation of blood pressure, hypertension, shock, modulation of inflammation, platelet aggregation, abnormal blood coagulation, atherosclerosis, arteriosclerosis, and coronary artery disease. Thus, polypeptides and nucleic acid molecules of the invention, and compounds which bind with or modulate one or more polypeptides and nucleic acid molecules of the invention can be used to prognosticate, diagnose, inhibit, or treat one or more of the disorders listed above or one or more disorders associated with the physiological processes listed above.

Protein KIAA0880

A cDNA encoding at least a portion of human KIAA0880 protein was isolated by others from a human brain library of cDNA clones on the basis of the encoded protein being ‘large’ (Nagase et al. (1998) DNA Res. 5:355-364; GenBank submission assigned Accession no. AB020687, submitted Dec. 2, 1998). At the time this cDNA was isolated and submitted to GenBank, it was unknown by the isolators whether the encoded protein had any physiological relevance and, if it did, what that relevance might be. The present inventor has discovered that the protein encoded by the cDNA clone identified by Nagase et al. encodes a transmembrane transport protein that catalyzes transmembrane transport of charged organic compounds such as one or more prostaglandins. In view of this discovery, it is now possible to make use of protein KIAA0880 for the treatment of numerous disorders relating to aberrant transmembrane transport of prostaglandins and/or thromboxanes, and for other purposes.

The full length of the cDNA encoding human protein KIAA0880 (SEQ ID NO:114) is 4068 nucleotide residues and encodes a 709-amino acid protein (SEQ ID NO:115) which exhibits amino acid sequence homology with HPT and other prostaglandin transporters.

KIAA0880 proteins of the invention and nucleic acid molecules encoding them comprise a family of molecules having certain conserved structural and functional features, as indicated by its close homology to HPT (SEQ ID NO:116), the human OatP sodium-independent organic anion transporter protein (GenBank Accession no. P46721; SEQ ID NO:117), human 38555 protein (as described herein, SEQ ID NO:108), and human protein 593 (as described herein, SEQ ID NO:112).

KIAA0880 proteins typically comprise a variety of potential post-translational modification sites (often within an extracellular domain), such as those described herein in Table 53, as predicted by computerized sequence analysis of human KIAA0880 protein using amino acid sequence comparison software (comparing the amino acid sequence of protein KIAA0880 with the information in the PROSITE database {rel. 12.2; February, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3}). In certain embodiments, a protein of the invention has at least 1, 2, 4, 6, 8, or 10 or more of the post-translational modification sites listed in Table 53.

Protein KIAA0880 is predicted by computerized amino acid sequence analysis (using the MEMSAT computer program) to be a twelve-transmembrane region integral membrane protein having transmembrane regions at approximately the following positions within SEQ ID NO:115: from about amino acid residue 50 to about residue 69; from about amino acid residue 88 to about residue 108; from about amino acid residue 117 to about residue 134; from about amino acid residue 186 to about residue 206; from about amino acid residue 225 to about residue 249; from about amino acid residue 276 to about residue 297; from about amino acid residue 372 to about residue 394; from about amino acid residue 411 to about residue 432; from about amino acid residue 440 to about residue 463; from about amino acid residue 564 to about residue 587; from about amino acid residue 596 to about residue 612; and from about amino acid residue 651 to about residue 673

Extracellular domains are predicted to include approximately amino acid residues 70 to 87, 135 to 185, 250 to 275, 395 to 410, 464 to 563, and 613 to 650 of SEQ ID NO:115. Intracellular domains are predicted to include approximately amino acid residues 1 to 49, 109 to 116, 207 to 224, 298 to 371, 433 to 439, 588 to 595, and 674 to 709 of SEQ ID NO:115.

As described elsewhere herein, relatively hydrophilic regions are generally located at or near the surface of a protein, and are more frequently effective immunogenic epitopes than are relatively hydrophobic regions. For example, the region of human protein KIAA0880 from about amino acid residue 135 to about amino acid residue 155 appears to be located at or near the surface of the protein, while the region from about amino acid residue 160 to about amino acid residue 165 appears not to be located at or near the surface.

Human protein KIAA0880 exhibits sequence similarity to HPT (GenBank Accession no. Q92959; SEQ ID NO:117). An alignment between KIAA0880 (SEQ ID NO:115 and HPT (SEQ ID NO:117), made using the ALIGN program of the GCG software package, pam120.mat scoring matrix, gap penalties −12/−4, reveals that the amino acid sequences of the proteins are 39.5% identical.

The predicted molecular weight of human protein KIAA0880 is about 76.7 kilodaltons.

Biological Function of Human KIAA0880 Proteins, Nucleic Acids Encoding Them, and Modulators of These Molecules

Human KIAA0880 protein is involved in disorders which affect both tissues in which they are normally expressed and tissues in which they are normally not expressed. Based on the observation by others that KIAA0880 protein is expressed in human brain tissue and on the function of this protein as identified herein, human KIAA0880 protein is involved in one or more biological processes which occur in brain and other neurological tissues. In particular, KIAA0880 is involved in modulating growth, proliferation, survival, differentiation, and activity of cells including, but not limited to, central nervous system neurons, peripheral nervous system neurons, motor neurons, sensory neurons, and sympathetic and parasympathetic neural cells of the animal in which it is normally expressed. Protein KIAA0880 is also involved in mediating interactions between cells, particularly between two neurons, or between a neuron and a non-neuronal cell such as a muscle or endocrine cell. Thus, KIAA0880 protein has a role in disorders which affect neuronal cells and cells which interact with neurons and their growth, proliferation, survival, differentiation, and activity.

Widespread expression of KIAA0880 has been detected among human tissue types. Thus, the growth-, proliferation-, survival-, differentiation-, and activity-modulating activities of KIAA0880 protein affect cells of many types. Thus, protein KIAA0880 can affect cell-to-cell interactions in a wide variety of cell types.

Protein KIAA0880 is involved in transmembrane transport of one or more charged organic compounds such as prostaglandins, thromboxanes, and the like. Protein KIAA0880 mediates one or more of facilitated diffusion of the prostaglandin (or thromboxane or the like) and symport or antiport (e.g. involving co-transport of a proton, a sodium ion, a potassium ion, or another physiological ion).

Protein KIAA0880 is therefore involved in transmembrane transport of charged organic molecules such as one or more prostaglandins and thromboxanes in brain and other neural tissues in humans, and is thus involved in, and can be used to prognosticate, prevent, diagnose, or treat, one or more disorders related to inappropriate transmembrane transport (i.e. including inappropriate failure of transport) of prostaglandins, thromboxanes, and the like in neural tissues. Such disorders include, by way of example, neural transmission disorders (e.g. inappropriate pain, dementia, multiple sclerosis, nerve root disorders, Alzheimer's disease, Parkinson's disease, depression, physical and psychological substance addiction, sexual dysfunction, schizophrenic disorders, delusional disorders, mood disorders, sleep disorders, and the like) and disorders relating to inappropriate formation (i.e. including failure to form) and maintenance (i.e. including deterioration) of neuronal synapses, including both neuron-to-neuron synapses and neuron-to-non-neuronal cell synapses. Thus, in addition to the neural transmission disorders described above, protein KIAA0880 is also implicated in, and can be used to prognosticate, prevent, diagnose, or treat, one or more disorders such as stroke, regeneration of chronically or traumatically damaged neuronal structures (including nerve, brain, and spinal cord), developmental neuronal disorders (e.g. spina bifida), neuronal cancers (e.g. gliomas, astrocytomas, ependymomas, pituitary adenomas, and the like), peripheral nerve deficit, coronary insufficiency, angina, and the like. Exemplary molecules which can be transported across cell membranes via protein KIAA0880 include one or more charged organic compounds such as prostaglandins A 1 , A 2 , B 1 , B 2 , D 2 , E 1 , E 2 , F 1α , F 2α , G 2 , H 2 , I 2 , and J 2 and thromboxanes A 2 and B 2 . Uptake and release of prostaglandins and thromboxanes, for example, are known to be involved in a variety of physiological processes and disorders including glaucoma, ovum fertilization, sperm motility, pregnancy, labor, delivery, abortion, gastric protection, peptic ulcer formation, intestinal fluid secretion, liver protection, liver damage, liver fibrosis, pain stimulation, glomerular filtration, maintenance of body temperature, fever, airway resistance, asthma, chronic obstructive pulmonary disorder, modulation of blood pressure, hypertension, shock, modulation of inflammation, platelet aggregation, abnormal blood coagulation, atherosclerosis, arteriosclerosis, and coronary artery disease. Thus, polypeptides and nucleic acid molecules of the invention, and compounds which bind with or modulate one or more polypeptides and nucleic acid molecules of the invention can be used to prognosticate, diagnose, inhibit, or treat one or more of the disorders listed above or one or more disorders associated with the physiological processes listed above.

Biological Deposit

Clones encoding human 38555 and 593 proteins were deposited with ATCC on Jul. 22, 1999 in the form of a mixture of two plasmids, one (Ep65h2) encoding protein 38555, the other (Ep593) encoding protein 593. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

In order to check for the presence of Ep65h2 and Ep593 in the deposited mixture, an E. coli host strain (e.g. DH5α) is transformed using the mixture and plated and incubated on Luria broth plates containing 100 micrograms per milliliter ampicillin. About 10 to 20 transformants are selected and subjected to a standard plasmid minipreparation procedure. Each DNA is digested using restriction endonuclease EcoRI and the fragments are separated by, for example, agarose gel electrophoresis. Fragments are visualized (e.g. using ethidium bromide in the agarose gel). EcoRI digestion of Ep62h5 yields one band approximately 5.5 kB in size. EcoRI digestion of Ep62h5 yields two bands, one having a size of about 3.5 kB, and the other having a size of about 1.5 kB.

This deposit was made merely as a convenience to those of skill in the art. This deposit is not an admission that a deposit is required pursuant to 35 U.S.C. §112.

The 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein, fragments thereof, and derivatives and other variants of the sequence in SEQ ID NO:2, 6, 11, 19, 22, 25, 32, 40, 44, 47, 50, 55, 58, 64, 67, 72, 89, 105, 108 or 112 thereof are collectively referred to as “polypeptides or proteins of the invention” or “21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides or proteins”. Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as “nucleic acids of the invention” or “21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 nucleic acids.”

As used herein, the term “nucleic acid molecule” includes DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

The term “isolated or purified nucleic acid molecule” includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology (1989) John Wiley & Sons, N.Y., 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.

As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein, preferably a mammalian 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein, and can further include non-coding regulatory sequences, and introns.

An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one embodiment, the language “substantially free” means preparation of 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein (also referred to herein as a “contaminating protein”), or of chemical precursors or non-21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 chemicals. When the 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 (e.g., the sequence of SEQ ID NO:1, 3, 5, 7, 10, 12, 18, 20, 21, 23, 24, 26, 31, 33, 39, 41, 43, 45, 46, 48, 49, 51, 54, 56, 57, 59, 63, 65, 66, 68, 71, 73, 88, 90, 104, 106, 107, 109, 111 or 113) without abolishing or more preferably, without substantially altering a biological activity, whereas an “essential” amino acid residue results in such a change. For example, amino acid residues that are conserved among the polypeptides of the present invention, e.g., those present in the conserved domains, are predicted to be particularly unamenable to alteration.

A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:1, 3, 5, 7, 10, 12, 18, 20, 21, 23, 24, 26, 31, 33, 39, 41, 43, 45, 46, 48, 49, 51, 54, 56, 57, 59, 63, 65, 66, 68, 71, 73, 88, 90, 104, 106, 107, 109, 111 or 113, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

As used herein, a “biologically active portion” of a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein includes a fragment of a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein which participates in an interaction between a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 molecule and a non-21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 molecule. Biologically active portions of a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein, e.g., the amino acid sequence shown in SEQ ID NO:2, 6, 11, 19, 22, 25, 32, 40, 44, 47, 50, 55, 58, 64, 67, 72, 89, 105, 108 or 112, which include fewer amino acids than the full length 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein, and exhibit at least one activity of a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein. A biologically active portion of a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. Biologically active portions of a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein can be used as targets for developing agents which modulate a 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 mediated activity.

Calculations of homology or sequence identity (the terms “homology” and “identity” are used interchangeably herein) between sequences are performed as follows:

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453 algorithm which has been incorporated into the GAP program in the GCG software package using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers and Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Particular 21910, 56634, 55053, 2504, 15977, 14760, 25501, 17903, 3700, 21529, 26176, 26343, 56638, 18610, 33217, 21967, h1983, m1983, 38555 or 593 polypeptides of the present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:2, 6, 11, 19, 22, 25, 32, 40, 44, 47, 50, 55, 58, 64, 67, 72, 89, 105, 108 or 112. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2, 6, 11, 19, 22, 25, 32, 40, 44, 47, 50, 55, 58, 64, 67, 72, 89, 105, 108 or 112 are termed substantially identical.

In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1, 3, 5, 7, 10, 12, 18, 20, 21, 23, 24, 26, 31, 33, 39, 41, 43, 45, 46, 48, 49, 51, 54, 56, 57, 59, 63, 65, 66, 68, 71, 73, 88, 90, 104, 106, 107, 109, 111 or 113 are termed substantially identical.

“Misexpression or aberrant expression”, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

“Subject”, as used herein, can refer to a mammal, e.g., a human, or to an experimental or animal or disease model. The subject can also be a non-human animal, e.g., a horse, cow, goat, or other domestic animal.

A “purified preparation of cells”, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

As used herein, cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

As used herein, the term “cancer” (also used interchangeably with the terms, “hyperproliferative” and “neoplastic”) refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Cancerous disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, e.g., malignant tumor growth, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state, e.g., cell proliferation associated with wound repair. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “cancer” includes malignancies of the various organ systems, such as those affecting lung, breast, cervix, ovary, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term “carcinoma” also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

Examples of cellular proliferative and/or differentiative disorders of the lung include, but are not limited to, tumors such as bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, metastatic tumors, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders of the breast include, but are not limited to, proliferative breast disease including, e.g., epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disorders involving the colon include, but are not limited to, tumors of the colon, such as non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.

Examples of cancers or neoplastic conditions, in addition to the ones described above, include, but are not limited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi sarcoma.

Proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

As used herein, disorders of the breast include, but are not limited to, disorders of development; inflammations, including but not limited to, acute mastitis, periductal mastitis, periductal mastitis (recurrent subareolar abscess, squamous metaplasia of lactiferous ducts), mammary duct ectasia, fat necrosis, granulomatous mastitis, and pathologies associated with silicone breast implants; fibrocystic changes; proliferative breast disease including, but not limited to, epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors including, but not limited to, stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, no special type, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.

As used herein, disorders involving the colon include, but are not limited to, congenital anomalies, such as atresia and stenosis, Meckel diverticulum, congenital aganglionic megacolon-Hirschsprung disease; enterocolitis, such as diarrhea and dysentery, infectious enterocolitis, including viral gastroenteritis, bacterial enterocolitis, necrotizing enterocolitis, antibiotic-associated colitis (pseudomembranous colitis), and collagenous and lymphocytic colitis, miscellaneous intestinal inflammatory disorders, including parasites and protozoa, acquired immunodeficiency syndrome, transplantation, drug-induced intestinal injury, radiation enterocolitis, neutropenic colitis (typhlitis), and diversion colitis; idiopathic inflammatory bowel disease, such as Crohn disease and ulcerative colitis; tumors of the colon, such as non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.

As used herein, disorders involving the kidney (or renal disorders) include, but are not limited to, congenital anomalies including, but not limited to, cystic diseases of the kidney, that include but are not limited to, cystic renal dysplasia, autosomal dominant (adult) polycystic kidney disease, autosomal recessive (childhood) polycystic kidney disease, and cystic diseases of renal medulla, which include, but are not limited to, medullary sponge kidney, and nephronophthisis-uremic medullary cystic disease complex, acquired (dialysis-associated) cystic disease, such as simple cysts; glomerular diseases including pathologies of glomerular injury that include, but are not limited to, in situ immune complex deposition, that includes, but is not limited to, anti-GBM nephritis, Heymann nephritis, and antibodies against planted antigens, circulating immune complex nephritis, antibodies to glomerular cells, cell-mediated immunity in glomerulonephritis, activation of alternative complement pathway, epithelial cell injury, and pathologies involving mediators of glomerular injury including cellular and soluble mediators, acute glomerulonephritis, such as acute proliferative (poststreptococcal, postinfectious) glomerulonephritis, including but not limited to, poststreptococcal glomerulonephritis and nonstreptococcal acute glomerulonephritis, rapidly progressive (crescentic) glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis (membranous nephropathy), minimal change disease (lipoid nephrosis), focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis, IgA nephropathy (Berger disease), focal proliferative and necrotizing glomerulonephritis (focal glomerulonephritis), hereditary nephritis, including but not limited to, Alport syndrome and thin membrane disease (benign familial hematuria), chronic glomerulonephritis, glomerular lesions associated with systemic disease, including but not limited to, systemic lupus erythematosus, Henoch-Schönlein purpura, bacterial endocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary and immunotactoid glomerulonephritis, and other systemic disorders; diseases affecting tubules and interstitium, including acute tubular necrosis and tubulointerstitial nephritis, including but not limited to, pyelonephritis and urinary tract infection, acute pyelonephritis, chronic pyelonephritis and reflux nephropathy, and tubulointerstitial nephritis induced by drugs and toxins, including but not limited to, acute drug-induced interstitial nephritis, analgesic abuse nephropathy, nephropathy associated with nonsteroidal anti-inflammatory drugs, and other tubulointerstitial diseases including, but not limited to, urate nephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma; diseases of blood vessels including benign nephrosclerosis, malignant hypertension and accelerated nephrosclerosis, renal artery stenosis, and thrombotic microangiopathies including, but not limited to, classic (childhood) hemolytic-uremic syndrome, adult hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, and other vascular disorders including, but not limited to, atherosclerotic ischemic renal disease, atheroembolic renal disease, sickle cell disease nephropathy, diffuse cortical necrosis, and renal infarcts; urinary tract obstruction (obstructive uropathy); urolithiasis (renal calculi, stones); and tumors of the kidney including, but not limited to, benign tumors, such as renal papillary adenoma, renal fibroma or hamartoma (renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma, and malignant tumors, including renal cell carcinoma (hypernephroma, adenocarcinoma of kidney), which includes urothelial carcinomas of renal pelvis.

Examples of disorders of the lung include, but are not limited to, congenital anomalies; atelectasis; diseases of vascular origin, such as pulmonary congestion and edema, including hemodynamic pulmonary edema and edema caused by microvascular injury, adult respiratory distress syndrome (diffuse alveolar damage), pulmonary embolism, hemorrhage, and infarction, and pulmonary hypertension and vascular sclerosis; chronic obstructive pulmonary disease, such as emphysema, chronic bronchitis, bronchial asthma, and bronchiectasis; diffuse interstitial (infiltrative, restrictive) diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia (pulmonary infiltration with eosinophilia), Bronchiolitis obliterans -organizing pneumonia, diffuse pulmonary hemorrhage syndromes, including Goodpasture syndrome, idiopathic pulmonary hemosiderosis and other hemorrhagic syndromes, pulmonary involvement in collagen vascular disorders, and pulmonary alveolar proteinosis; complications of therapies, such as drug-induced lung disease, radiation-induced lung disease, and lung transplantation; tumors, such as bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pathologies of the pleura, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.

As used herein, disorders involving the pancreas include those of the exocrine pancreas such as congenital anomalies, including but not limited to, ectopic pancreas; pancreatitis, including but not limited to, acute pancreatitis; cysts, including but not limited to, pseudocysts; tumors, including but not limited to, cystic tumors and carcinoma of the pancreas; and disorders of the endocrine pancreas such as, diabetes mellitus; islet cell tumors, including but not limited to, insulinomas, gastrinomas, and other rare islet cell tumors.

As used herein, disorders involving the ovary include, for example, polycystic ovarian disease, Stein-leventhal syndrome, Pseudomyxoma peritonei and stromal hyperthecosis; ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometeriod tumors, clear cell adenocarcinoma, cystadenofibroma, brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecoma-fibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors.

Aberrant expression and/or activity of the molecules of the invention can mediate disorders associated with bone metabolism. “Bone metabolism” refers to direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which can ultimately affect the concentrations in serum of calcium and phosphate. This term also includes activities mediated by the molecules of the invention in bone cells, e.g. osteoclasts and osteoblasts, that can in turn result in bone formation and degeneration. For example, molecules of the invention can support different activities of bone resorbing osteoclasts such as the stimulation of differentiation of monocytes and mononuclear phagocytes into osteoclasts. Accordingly, molecules of the invention that modulate the production of bone cells can influence bone formation and degeneration, and thus can be used to treat bone disorders. Examples of such disorders include, but are not limited to, osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyroidism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced metabolism, medullary carcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoid antagonism, malabsorption syndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.

As used herein, “a prostate disorder” refers to an abnormal condition occurring in the male pelvic region characterized by, e.g., male sexual dysfunction and/or urinary symptoms. This disorder may be manifested in the form of genitourinary inflammation (e.g., inflammation of smooth muscle cells) as in several common diseases of the prostate including prostatitis, benign prostatic hyperplasia and cancer, e.g., adenocarcinoma or carcinoma, of the prostate.

Examples of immune, e.g., inflammatory, (e.g. respiratory inflammatory) disorders or diseases include, but are not limited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjögren's Syndrome, inflammatory bowel disease, e.g. Crohn's disease and ulcerative colitis, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, asthma, allergic asthma, chronic obstructive pulmonary disease, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, cases of transplantation, and allergy such as, atopic allergy.

As used herein, disorders involving the heart, or “cardiovascular disease” or a “cardiovascular disorder” includes a disease or disorder which affects the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. A cardiovascular disorder includes, but is not limited to disorders such as arteriosclerosis, atherosclerosis, cardiac hypertrophy, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, valvular disease, including but not limited to, valvular degeneration caused by calcification, rheumatic heart disease, endocarditis, or complications of artificial valves; atrial fibrillation, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, pericardial disease, including but not limited to, pericardial effusion and pericarditis; cardiomyopathies, e.g., dilated cardiomyopathy or idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, ischemic disease, arrhythmia, sudden cardiac death, and cardiovascular developmental disorders (e.g., arteriovenous malformations, arteriovenous fistulae, raynaud's syndrome, neurogenic thoracic outlet syndrome, causalgia/reflex sympathetic dystrophy, hemangioma, aneurysm, cavernous angioma, aortic valve stenosis, atrial septal defects, atrioventricular canal, coarctation of the aorta, ebsteins anomaly, hypoplastic left heart syndrome, interruption of the aortic arch, mitral valve prolapse, ductus arteriosus, patent foramen ovale, partial anomalous pulmonary venous return, pulmonary atresia with ventricular septal defect, pulmonary atresia without ventricular septal defect, persistance of the fetal circulation, pulmonary valve stenosis, single ventricle, total anomalous pulmonary venous return, transposition of the great vessels, tricuspid atresia, truncus arteriosus, ventricular septal defects). A cardiovascular disease or disorder also can include an endothelial cell disorder.

As used herein, disorders involving the brain include, but are not limited to, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states—global cerebral ischemia and focal cerebral ischemia—infarction from obstruction of local blood supply, intracranial hemorrhage, including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lacunar infarcts, slit hemorrhages, and hypertensive encephalopathy; infections, such as acute meningitis, including acute pyogenic (bacterial) meningitis and acute aseptic (viral) meningitis, acute focal suppurative infections, including brain abscess, subdural empyema, and extradural abscess, chronic bacterial meningoencephalitis, including tuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme disease), viral meningoencephalitis, including arthropod-borne (Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplex virus Type 2, Varicella-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency virus 1, including HIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy, AIDS-associated myopathy, peripheral neuropathy, and AIDS in children, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, fungal meningoencephalitis, other infectious diseases of the nervous system; transmissible spongiform encephalopathies (prion diseases); demyelinating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis, and other diseases with demyelination; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer disease and Pick disease, degenerative diseases of basal ganglia and brain stem, including Parkinsonism, idiopathic Parkinson disease (paralysis agitans), progressive supranuclear palsy, corticobasal degenration, multiple system atrophy, including striatonigral degenration, Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntington disease; spinocerebellar degenerations, including spinocerebellar ataxias, including Friedreich ataxia, and ataxia-telanglectasia, degenerative diseases affecting motor neurons, including amyotrophic lateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedy syndrome), and spinal muscular atrophy; inborn errors of metabolism, such as leukodystrophies, including Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, and Canavan disease, mitochondrial encephalomyopathies, including Leigh disease and other mitochondrial encephalomyopathies; toxic and acquired metabolic diseases, including vitamin deficiencies such as thiamine (vitamin B 1 ) deficiency and vitamin B 12 deficiency, neurologic sequelae of metabolic disturbances, including hypoglycemia, hyperglycemia, and hepatic encephatopathy, toxic disorders, including carbon monoxide, methanol, ethanol, and radiation, including combined methotrexate and radiation-induced injury; tumors, such as gliomas, including astrocytoma, including fibrillary (diffuse) astrocytoma and glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain stem glioma, oligodendroglioma, and ependymoma and related paraventricular mass lesions, neuronal tumors, poorly differentiated neoplasms, including medulloblastoma, other parenchymal tumors, including primary brain lymphoma, germ cell tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors, including schwannoma, neurofibroma, and malignant peripheral nerve sheath tumor (malignant schwannoma), and neurocutaneous syndromes (phakomatoses), including neurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindau disease.

As used herein, skeletal muscle disorders include, but are not limited to, muscular dystrophy (e.g., Duchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, and congenital muscular dystrophy), motor neuron diseases (e.g., amyotrophic lateral sclerosis, infantile progressive spinal muscular atrophy, intermediate spinal muscular atrophy, spinal bulbar muscular atrophy, and adult spinal muscular atrophy), myopathies (e.g., inflammatory myopathies (e.g., dermatomyositis and polymyositis), myotonia congenita, paramyotonia congenita, central core disease, nemaline myopathy, myotubular myopathy, and periodic paralysis), tumors such as rhabdomyosarcoma, and metabolic diseases of muscle (e.g., phosphorylase deficiency, acid maltase deficiency, phosphofructokinase deficiency, debrancher enzyme deficiency, mitochondrial myopathy, carnitine deficiency, carnitine palmityl transferase deficiency, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, lactate dehydrogenase deficiency, and myoadenylate deaminase deficiency).

As used herein, an “endothelial cell disorder” includes a disorder characterized by aberrant, unregulated, or unwanted endothelial cell activity, e.g., proliferation, migration, angiogenesis, or vascularization; or aberrant expression of cell surface adhesion molecules or genes associated with angiogenesis, e.g., TIE-2, FLT and FLK. Endothelial cell disorders include tumorigenesis, tumor metastasis, psoriasis, diabetic retinopathy, endometriosis, Grave's disease, ischemic disease (e.g., atherosclerosis), and chronic inflammatory diseases (e.g., rheumatoid arthritis).

Disorders involving the liver (hepatic disorders) include, but are not limited to, hepatic injury; jaundice and cholestasis, such as bilirubin and bile formation; hepatic failure and cirrhosis, such as cirrhosis, portal hypertension, including ascites, portosystemic shunts, and splenomegaly; infectious disorders, such as viral hepatitis, including hepatitis A-E infection and infection by other hepatitis viruses, clinicopathologic syndromes, such as the carrier state, asymptomatic infection, acute viral hepatitis, chronic viral hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and toxin-induced liver disease, such as alcoholic liver disease; inborn errors of metabolism and pediatric liver disease, such as hemochromatosis, Wilson disease, α 1 -antitrypsin deficiency, and neonatal hepatitis; primary bile acid malabsorption; intrahepatic biliary tract disease, such as secondary biliary cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and anomalies of the biliary tree; circulatory disorders, such as impaired blood flow into the liver, including hepatic artery compromise and portal vein obstruction and thrombosis, impaired blood flow through the liver, including passive congestion and centrilobular necrosis and peliosis hepatis, hepatic vein outflow obstruction, including hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive disease; hepatic disease associated with pregnancy, such as preeclampsia and eclampsia, acute fatty liver of pregnancy, and intrehepatic cholestasis of pregnancy; hepatic complications of organ or bone marrow transplantation, such as drug toxicity after bone marrow transplantation, graft-versus-host disease and liver rejection, and nonimmunologic damage to liver allografts; tumors and tumorous conditions, such as nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma of the liver and metastatic tumors.

Disorders which can be treated or diagnosed by methods described herein include, but are not limited to, disorders associated with an accumulation in the liver of fibrous tissue, such as that resulting from an imbalance between production and degradation of the extracellular matrix accompanied by the collapse and condensation of preexisting fibers. The methods described herein can be used to diagnose or treat hepatocellular necrosis or injury induced by a wide variety of agents including processes which disturb homeostasis, such as an inflammatory process, tissue damage resulting from toxic injury or altered hepatic blood flow, and infections (e.g., bacterial, viral and parasitic). For example, the methods can be used for the early detection of hepatic injury, such as portal hypertension or hepatic fibrosis. In addition, the methods can be employed to detect liver fibrosis attributed to inborn errors of metabolism, for example, fibrosis resulting from a storage disorder such as Gaucher's disease (lipid abnormalities) or a glycogen storage disease, A1-antitrypsin deficiency; a disorder mediating the accumulation (e.g., storage) of an exogenous substance, for example, hemochromatosis (iron-overload syndrome) and copper storage diseases (Wilson's disease), disorders resulting in the accumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) and peroxisomal disorders (e.g., Zellweger syndrome). Additionally, the methods described herein can be used for the early detection and treatment of liver injury associated with the administration of various chemicals or drugs, such as for example, methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, or which represents a hepatic manifestation of a vascular disorder such as obstruction of either the intrahepatic or extrahepatic bile flow or an alteration in hepatic circulation resulting, for example, from chronic heart failure, veno-occlusive disease, portal vein thrombosis or Budd-Chiari syndrome.

Additionally, the molecules of the invention can play an important role in the etiology of certain viral diseases, including but not limited to Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators of the activity of the molecules of the invention could be used to control viral diseases. The modulators can be used in the treatment and/or diagnosis of viral infected tissue or virus-associated tissue fibrosis, especially liver and liver fibrosis. Also, such modulators can be used in the treatment and/or diagnosis of virus-associated carcinoma, especially hepatocellular cancer.

Disorders related to reduced platelet number, thrombocytopenia, include idiopathic thrombocytopenic purpura, including acute idiopathic thrombocytopenic purpura, drug-induced thrombocytopenia, HIV-associated thrombocytopenia, and thrombotic microangiopathies: thrombotic thrombocytopenic purpura and hemolytic-uremic syndrome.

As used herein, neurological disorders include disorders of the central nervous system (CNS) and the peripheral nervous system, e.g., cognitive and neurodegenerative disorders, Examples of neurological disorders include, but are not limited to, autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, Korsakoff's psychosis, alcoholism, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder, as well as bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-1), and bipolar affective neurological disorders, e.g., migraine and obesity. Such neurological disorders include, for example, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states—global cerebral ischemia and focal cerebral ischemia—infarction from obstruction of local blood supply, intracranial hemorrhage, including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lacunar infarcts, slit hemorrhages, and hypertensive encephalopathy; infections, such as acute meningitis, including acute pyogenic (bacterial) meningitis and acute aseptic (viral) meningitis, acute focal suppurative infections, including brain abscess, subdural empyema, and extradural abscess, chronic bacterial meningoencephalitis, including tuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme disease), viral meningoencephalitis, including arthropod-borne (Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplex virus Type 2, Varicella-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency virus 1, including HIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy, AIDS-associated myopathy, peripheral neuropathy, and AIDS in children, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, fungal meningoencephalitis, other infectious diseases of the nervous system; transmissible spongiform encephalopathies (prion diseases); demyelinating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis, and other diseases with demyelination; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer's disease and Pick's disease, degenerative diseases of basal ganglia and brain stem, including Parkinsonism, idiopathic Parkinson's disease (paralysis agitans) and other Lewy diffuse body diseases, progressive supranuclear palsy, corticobasal degenration, multiple system atrophy, including striatonigral degenration, Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntington's disease, senile dementia, Gilles de la Tourette's syndrome, epilepsy, and Jakob-Creutzfieldt disease; spinocerebellar degenerations, including spinocerebellar ataxias, including Friedreich ataxia, and ataxia-telanglectasia, degenerative diseases affecting motor neurons, including amyotrophic lateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedy syndrome), and spinal muscular atrophy; inborn errors of metabolism, such as leukodystrophies, including Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, and Canavan disease, mitochondrial encephalomyopathies, including Leigh disease and other mitochondrial encephalomyopathies; toxic and acquired metabolic diseases, including vitamin deficiencies such as thiamine (vitamin B 1 ) deficiency and vitamin B 12 deficiency, neurologic sequelae of metabolic disturbances, including hypoglycemia, hyperglycemia, and hepatic encephatopathy, toxic disorders, including carbon monoxide, methanol, ethanol, and radiation, including combined methotrexate and radiation-induced injury; tumors, such as gliomas, including astrocytoma, including fibrillary (diffuse) astrocytoma and glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain stem glioma, oligodendroglioma, and ependymoma and related paraventricular mass lesions, neuronal tumors, poorly differentiated neoplasms, including medulloblastoma, other parenchymal tumors, including primary brain lymphoma, germ cell tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors, including schwannoma, neurofibroma, and malignant peripheral nerve sheath tumor (malignant schwannoma), and neurocutaneous syndromes (phakomatoses), including neurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindau disease. Further CNS-related disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incorporated herein by reference in its entirety.

As used herein, diseases of the skin (dermal disorders), include but are not limited to, disorders of pigmentation and melanocytes, including but not limited to, vitiligo, freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, and malignant melanoma; benign epithelial tumors, including but not limited to, seborrheic keratoses, acanthosis nigricans, fibroepithelial polyp, epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors; premalignant and malignant epidermal tumors, including but not limited to, actinic keratosis, squamous cell carcinoma, basal cell carcinoma, and merkel cell carcinoma; tumors of the dermis, including but not limited to, benign fibrous histiocytoma, dermatofibrosarcoma protuberans, xanthomas, and dermal vascular tumors; tumors of cellular immigrants to the skin, including but not limited to, histiocytosis X, mycosis fungoides (cutaneous T-cell lymphoma), and mastocytosis; disorders of epidermal maturation, including but not limited to, ichthyosis; acute inflammatory dermatoses, including but not limited to, urticaria, acute eczematous dermatitis, and erythema multiforme; chronic inflammatory dermatoses, including but not limited to, psoriasis, lichen planus, and lupus erythematosus; blistering (bullous) diseases, including but not limited to, pemphigus, bullous pemphigoid, dermatitis herpetiformis, and noninflammatory blistering diseases: epidermolysis bullosa and porphyria; disorders of epidermal appendages, including but not limited to, acne vulgaris; panniculitis, including but not limited to, erythema nodosum and erythema induratum; and infection and infestation, such as verrucae, molluscum contagiosum, impetigo, superficial fungal infections, and arthropod bites, stings, and infestations.

Additionally, molecules of the invention can play an important role in the regulation of metabolism or pain disorders. Diseases of metabolic imbalance include, but are not limited to, obesity, anorexia nervosa, cachexia, lipid disorders, and diabetes. Examples of pain disorders include, but are not limited to, pain response elicited during various forms of ti