Inhibitors of apoptosis

This application claims priority under 35USC120 to and is a division of U.S. Ser. No. 08/569,749, now U.S. Pat. No. 6,187,557, filed Dec. 8, 1995, which claims priority under 35USC120 to and is a continuation of U.S. Ser. No. 08/512,946, filed Aug. 8, 1995, now abandoned, both of which are incorporated herein by reference.

The field of this invention is human proteins involved in the inhibition of apoptosis, or programmed cell death.

Cellular apoptosis, or programmed cell death, may be initiated by a variety of different stimuli including viral infection, certain cell-culture conditions, cell-cell signaling, cytokines, etc. Elucidation of signal transduction pathways leading to apoptosis would provide valuable insight into a variety of pathogenic mechanisms. Accordingly, the ability to exogenously modulate the induction of apoptosis would yield therapeutic application for numerous clinical indications. In addition, components of such pathways would provide valuable targets for automated, cost-effective, high throughput drug screening and hence would have immediate application in domestic and international pharmaceutical and biotechnology drug development programs.

Relevant Literature

Rothe et al. (1994) Cell 78, 681-692, report the existence of tumor necrosis factor (TNF) receptor associated proteins which co-immunoprecipitate with a TNF receptor; see also Rothe, et al., pending U.S. patent application Ser. No: 08/446,915 now U.S Pat. No. 5,741,677. Roy, et al. (1995) Cell 80, 167-178 disclose the gene for a human neuronal apoptosis inhibitory protein. Birnbaum et al. (1994) J Virol 68, 2521-2528 disclose an inhibitor of apoptosis (iap) gene, Op-iap from the Orgyia pseudotsugata nuclear polyhedrosis virus (OpMNPV) with sequence similarity to two other viral genes: Cp-iap derived from Cydia pomonella granulosis virus (CpGV), and iap derived from the Autographa californica nuclear polyhedrosis virus (AcMNPV). Clem and Miller (1994), in Apoptosis II: The Molecular Basis of Apoptosis in Disease, pp 89-110, Cold Spring Harbor Laboratory Press, provide a recent review of apoptosis regulation by insect viruses.

The invention provides methods and compositions relating to novel human cellular inhibitor of apoptosis proteins (c-IAP). The subject proteins comprise a series of defined structural domain repeats and/or a RING finger domain; in particular, at least two of a first domain repeat comprising SEQUENCE ID NO: 5 or 6; a second domain repeat comprising SEQUENCE ID NO: 7 or 8; and a third domain repeat comprising SEQUENCE ID NO: 9 or 10; and/or a RING finger domain comprising SEQUENCE ID NO: 11 or 12, or a consensus sequences derived from these human genes. The proteins provide a c-IAP specific function, with preferred proteins being capable of modulating the induction of apoptosis; for example, by binding a human tumor necrosis factor receptor associated factor, TRAF. The compositions include nucleic acids which encode the subject c-IAP and hybridization probes and primers capable of hybridizing with the disclosed c-IAP genes.

The invention includes methods of using the subject compositions in therapy (e.g. gene therapy to enhance expression of a c-IAP gene), in diagnosis (e.g. genetic hybridization screens for c-IAP gene mutations, and in the biopharmaceutical industry (e.g. reagents for increasing yields of recombinant protein by enhancing host cell survival in culture, for screening chemical libraries for lead compounds for a pharmacological agent useful in the diagnosis or treatment of disease associated with apoptosis regulation, etc.).

The invention provides methods and compositions relating to novel cellular inhibitor of apoptosis proteins (c-IAPs). The nucleotide sequence of a natural cDNA encoding human c-IAP is shown as SEQUENCE ID NO:1 and the full conceptual translate is shown as SEQUENCE ID NO:2. The nucleotide sequence of another natural cDNA encoding human c-IAP2 is shown as SEQUENCE ID NO:3 and the full conceptual translate is shown as SEQUENCE ID NO:4. The human c-IAPs of the invention include incomplete translates of SEQUENCE ID NOS:1 and 3 or deletion mutants of SEQUENCE ID NOS: 2 and/or 4, which translates or deletions mutants have at least one of the human c-IAP specific activities described herein. In addition, the invention provides nonhuman mammalian homologs of the disclosed human c-IAPs. These homologs are encoded by natural cDNAs which are capable of specifically hybridizing with one or more of the disclosed human cDNAs under hybridization conditions describe below and are isolated using the methods and reagents described herein. For example, the amino acid sequence of a murine homolog of c-IAP1, and the sequence its cDNA are shown in SEQUENCE ID NOS: 14 and 13.

The subject proteins comprise a series of defined structural domain repeats and/or a RING finger domain shown to be necessary for human c-IAP specific function; generally including at least two of: a first domain repeat comprising SEQUENCE ID NO: 5, 6 or a consensus of 5 and 6, a second domain repeat comprising SEQUENCE ID NO: 7, 8 or a consensus of 7 and 8, and a third domain repeat comprising SEQUENCE ID NO: 9, 10 or a consensus of 9 and 10; and/or a RING finger domain comprising SEQUENCE ID NO: 11, 12 or a consensus of 11 and 12. Preferred domain repeat containing c-IAPs contain each of the three domain repeats. More preferred c-IAPs comprise the three domain repeats and the C-terminal RING finger. To secure or optimize the requisite function for the protein, the repeats are usually preceded (N-terminally) and separated by intervening regions of about 10 to about 100 residues, which regions preferably derive from those found in the natural c-IAP1 and c-IAP2 translates. Similarly, the RING finger domain of RING finger domain containing c-IAPs containing proteins is usually preceded by an N-terminal region of about 10 to 300 residues, usually 100 to 300 residues, which region preferably derives from those found in the natural c-IAP1 and c-IAP2 translates.

The proteins provide a human c-IAP1 or c-IAP2 (c-IAP1/2) specific activity or function which may be determined by convenient in vitro, cell-based, or in vivo assays. Preferred proteins are capable of modulating the induction of apoptosis. Such activity or function may be demonstrated in cell culture (e.g. cell transfections) or in animals (e.g. in vivo gene therapy, transgenics). c-IAP1/2 specific function can also be demonstrated by specific binding to a c-IAP1/2 specific binding target, including natural binding targets and nonnatural targets such as c-IAP1/2 specific antibodies. For example, c-IAPs comprising at least two of SEQUENCE ID NOS: 6, 7 and 8 are capable of specifically binding human tumor necrosis factor receptor associated factors 1 and 2 (TRAF1 and TRAF2) in simple in vitro binding assays. Finally, specific function can be assayed immunologically by the ability of the subject protein to elicit a c-IAP1/2 specific antibody in a rodent or rabbit. Generally, human c-IAP1/2-specificity of the binding agent is shown by binding equilibrium constants (usually at least about 10 7 M −1 , preferably at least about 10 8 M −1 , more preferably at least about 10 9 M −1) . A wide variety of cell-based and cell-free assays may be used to demonstrate human c-IAP1/2-specific binding; preferred are rapid in vitro, cell-free assays such as mediating or inhibiting human c-IAP1/2-protein (e.g. human c-IAP1-TRAF2) binding, immunoassays, etc.

The claimed human c-IAP proteins are isolated, partially pure or pure and are typically recombinantly produced. An “isolated” protein for example, is unaccompanied by at least some of the material with which it is associated in its natural state and constitutes at least about 2%, and preferably at least about 5% by weight of the total protein in a given sample; a partially pure protein constitutes at least about 10%, preferably at least about 30%, and more preferably at least about 60% by weight of the total protein in a given sample; and a pure protein constitutes at least about 70%, preferably at least about 90%, and more preferably at least about 95% by weight of the total protein in a given sample. A wide variety of molecular and biochemical methods are available for generating and expressing the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known in the art.

The invention provides human c-IAP1/2-specific binding agents including substrates, natural intracellular binding targets, etc. and methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, human c-IAP1/2-specific agents are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with improper utilization of a pathway involving human c-IAP1/2, e.g. apoptosis. Novel human c-IAP1/2-specific binding agents include human c-IAP1/2-specific antibodies and other natural intracellular binding agents identified with assays such as one- and two-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries, etc.

The invention also provides nucleic acids encoding the subject proteins, which nucleic acids may be part of human c-IAP1/2-expression vectors and may be incorporated into recombinant cells for expression and screening, transgenic animals for functional studies (e.g. the efficacy of candidate drugs for disease associated with c-IAP1/2 mediated signal transduction), etc., and nucleic acid hybridization probes and replication/amplification primers having a human c-IAP1/2 cDNA specific sequence contained in SEQUENCE ID NO: 1 or 3. Nucleic acids encoding human c-IAP1/2 are isolated from eukaryotic cells, preferably human cells, by screening cDNA libraries with probes or PCR primers derived from the disclosed human c-IAP1/2 cDNA.

In addition, the invention provides nucleic acids sharing sufficient sequence similarity with that of the disclosed human c-IAP1/2 cDNAs to effect hybridization thereto. Such human c-IAP1/2 cDNA homologs are capable of hybridizing to the human c-IAP1/2-encoding nucleic acid defined by SEQUENCE ID NO: 1 or 3 under stringency conditions characterized by a hybridization buffer comprising 30% formamide in 5×SSPE (0.18 M NaCl, 0.01 M NaPO 4 , pH7.7, 0.001 M EDTA) buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with the 0.2×SSPE. Preferred nucleic acids will hybridize in a hybridization buffer comprising 50% formamide in 5×SSPE buffer at a temperature of 42° C. and remain bound when subject to washing at 42° C. with 0.2×SSPE buffer at 42° C. Human c-IAP1/2 cDNA homologs can also be characterized by BLASTX (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410) probability scores. Using this nucleic acid sequence search program BLASTX, complete coding region human c-IAP1/2 cDNA homologs provide a Probability P(N) score of less than 1.0e-200. More preferred nucleic acids encode c-IAPs with at least about 50%, preferably at least about 60%, more preferably at least 70% pair-wise identity to at least one of SEQUENCE ID NOS: 2 and 4.

The subject nucleic acids are isolated, i.e. constitute at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction. The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, therapeutic nucleic acids, etc.; use in detecting the presence of human c-IAP1/2 genes and gene transcripts, in detecting or amplifying nucleic acids encoding additional human c-IAP1/2 homologs and structural analogs, and in gene therapy applications. When used as expression constructs, the nucleic acids are usually recombinant, meaning they comprise a sequence joined to a nucleotide other than that which it is joined to on a natural chromosome. The subject nucleic acids may be contained within vectors, cells or organisms.

In diagnosis, c-IAP1/2 hybridization probes find use in identifying wild-type and mutant c-IAP1/2 alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. In therapy, therapeutic c-IAP1/2 nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active c-IAP1/2. A wide variety of indications may be treated, either prophylactically or therapeutically with the subject compositions. For example, where cell-specific apoptosis or other limitation of cell growth is desired, e.g. neoproliferative disease, a reduction in c-IAP1/2 expression is effected by introducing into the targeted cell type c-IAP1/2 nucleic acids which reduce the functional expression of c-IAP1/2 gene products (e.g. nucleic acids capable of inhibiting translation of a c-IAP 1/2 protein). Conditions for treatment include restenosis, where vascular smooth muscle cells are involved, inflammatory disease states, where endothelial cells, inflammatory cells and glomerular cells are involved, myocardial infarction, where heart muscle cells are involved, glomerular nephritis, where kidney cells are involved, transplant rejection where endothelial cells are involved, infectious diseases such as HIV infection where certain immune cells and other infected cells are involved, or the like.

These c-IAP1/2 inhibitory nucleic acids are typically antisense: single-stranded sequences comprising complements of the disclosed c-IAP1/2 encoding nucleic acid. Antisense modulation of the expression of a given c-IAP1/2 protein may employ c-IAP1/2 antisense nucleic acids operably linked to gene regulatory sequences. Cell are transfected with a vector comprising a c-IAP1/2 sequence with a promoter sequence oriented such that transcription of the gene yields an antisense transcript capable of binding to endogenous c-IAP1/2 protein encoding mRNA. Transcription of the antisense nucleic acid may be constitutive or inducible and the vector may provide for stable extrachromosomal maintenance or integration. Alternatively, single-stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding a given c-IAP1/2 protein may be administered to the target cell, in or temporarily isolated from a host, at a concentration that results in a substantial reduction in expression of the targeted protein.

In other indications, e.g. certain hypersensitivities, atrophic diseases, etc., a reduction in apoptosis is desired. In these applications, an enhancement in c-IAP1/2 expression is effected by introducing into the targeted cell type c-IAP1/2 nucleic acids which increase the functional expression of c-IAP1/2 gene products. Conditions for treatment include multiple sclerosis, where certain neuronal cells are involved, inflammatory disease states such as rheumatoid arthritis, where bystander cells are involved, transplant rejection where graft cells are involved, infectious diseases such as HIV infection where certain uninfected host cells are involved, or the like. Such nucleic acids may be c-IAP1/2 expression vectors, vectors which upregulate the functional expression of an endogenous c-IAP1/2 allele, or replacement vectors for targeted correction of c-IAP1/2 mutant alleles.

Various techniques may be employed for introducing of the nucleic acids into viable cells. The techniques vary depending upon whether one is using the subject compositions in culture or in vivo in a host. Various techniques which have been found efficient include transfection with a retrovirus, viral coat protein-liposome mediated transfection, see Dzau et al., Trends in Biotech 11, 205-210 (1993). In some situations it is desirable to provide the nucleic acid source with an agent which targets the target cells, such as an antibody specific for a surface membrane protein on the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. In liposomes, the nucleic acid concentration in the lumen will generally be in the range of about 0.01 μM to 10 μM. For other techniques, the concentration and application rate is determined empirically, using conventional techniques to determine desired ranges.

Application of the subject therapeutics may be systemic or local, i.e. administered at the site of interest. Various techniques can be used for providing the subject compositions at the site of interest, such as injection, use of catheters, trocars, projectiles, pluronic gel, stents, sustained drug release polymers or other device which provides for internal access. Where an organ or tissue is accessible because of removal from the patient, such organ or tissue may be bathed in a medium containing the subject compositions, the subject compositions may be painted onto the organ, or may be applied in any convenient way. Systemic administration of the nucleic acid may be effected using naked DNA, lipofection, liposomes with tissue targeting (e.g. antibody).

The invention provides methods and compositions for enhancing the yield of many recombinantly produced proteins, such as tissue plasminogen activator (t-PA), by increasing maximum cell densities and survival time of host production cells in culture. Specifically, cultured cells are transfected with nucleic acids which effect the up-regulation of endogenous c-IAP or the expression of an exogenous c-IAP. For example, nucleic acids encoding functional c-IAP operably linked to a transcriptional promoter are used to over-express the exogenous c-IAP in the host cell (see, experimental section, below). Such transformed cells demonstrate enhanced survival ability at elevated cell densities and over extended culture periods.

The invention provides efficient methods of identifying pharmacological agents or lead compounds for agents active at the level of a human c-IAP 1/2 modulatable cellular function, particularly human c-IAP1/2 mediated signal transduction, especially in apoptosis. Generally, these screening methods involve assaying for compounds which modulate a human c-lAP1/2 interaction with a natural c-IAP1/2 binding target. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development. Target indications may include infection, genetic disease, cell growth and regulatory disfunction, such as neoplasia, inflammation, hypersensitivity, etc.

A wide variety of assays for binding agents are provided including protein-protein binding assays, immunoassays, cell based assays, etc. The human c-IAP1/2 compositions used the methods are usually added in an isolated, partially pure or pure form and are typically recombinantly produced. The human c-IAP1/2 may be part of a fusion product with another peptide or polypeptide, e.g. a polypeptide that is capable of providing or enhancing protein-protein binding, stability under assay conditions (e.g. a tag for detection or anchoring), etc. The assay mixtures comprise a natural intracellular human c-IAP1/2 binding target such as a TRAF. While native binding targets may be used, it is frequently preferred to use portions (e.g. peptides, nucleic acid fragments) thereof so long as the portion provides binding affinity and avidity to the subject human c-IAP1/2 conveniently measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which may be used to facilitate optimal binding and/or reduce non-specific or background interactions, etc. Also, reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.

The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the human c-IAP1/2 specifically binds the cellular binding target, portion or analog with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings. Incubations may be performed at any temperature which facilitates optimal binding, typically between 4 and 40° C., more commonly between 15° and 40° C. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening, and are typically between 0.1 and 10 hours, preferably less than 5 hours, more preferably less than 2 hours.

After incubation, the agent-influenced binding between the human c-IAP1/2 and one or more binding targets is detected by any convenient way. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. Separation may be effected by precipitation (e.g. TCA precipitation, immunoprecipitation, etc.), immobilization (e.g on a solid substrate), etc., followed by washing by, for examples, membrane filtration (e.g. Whatman's P-81 ion exchange paper, Polyfiltronic's hydrophobic GFC membrane, etc.), gel chromatography (e.g. gel filtration, affinity, etc.). In addition, one of the components usually comprises or is coupled to a label. A wide variety of labels may be employed—essentially any label that provides for detection of bound protein. The label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc. or indirect detection such as an epitope tag, an enzyme, etc. A variety of methods may be used to detect the label depending on the nature of the label and other assay components. For example, the label may be detected bound to the solid substrate or a portion of the bound complex containing the label may be separated from the solid substrate, and thereafter the label detected. Labels may be directly detected through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc. For example, in the case of radioactive labels, emissions may be detected directly, e.g. with particle counters or indirectly, e.g. with scintillation cocktails and counters.

The following experiments and examples are offered by way of illustration and not by way of limitation.

Experimental

The murine cellular inhibitor of apoptosis protein 1 (c-IAP1) was biochemically purified as a TNF-R2 associated protein using coimmunoprecipitation Rothe et al. (1994) supra. A large scale protein purification protocol provided material sufficient for peptide sequencing. Fully degenerate oligonucleotides corresponding to two of the isolated peptides were used to specifically amplify a 0.75 kb DNA fragment from mouse CT6 RNA by Reverse Transcription-PCR. This DNA fragment was used to isolate full-length cDNA clones from a mouse CT6 cDNA library by hybridization (50% formamide, 5×SSPE, 42° C.; filters washed at 42° C. with 0.2×SSPE, where 1×SSPE is 0.18 M NaCl, 0.01 M NaPO 4 , pH7.7, 0.001 M EDTA).

DNA sequence analysis predicted an open reading frame encoding a 612 amino acid protein that shows significant sequence similarity (36% amino acid identity) with the ‘inhibitor of apoptosis protein’ (IAP) from insect viruses (Clem, R. J. and Miller, L. K., 1994, supra) and the human ‘neuronal apoptosis inhibitory protein’ (NAIP) (23% amino acid identity), that is involved in spinal muscular atrophy (SMA) an inherited disease in humans (Roy et., 1995, supra). To obtain the human c-IAP1 gene, the originally amplified mouse DNA fragment was used as a probe to screen a HeLa cDNA library (30% formamide, 5×SSPE, 42° C.; filters washed at 42° C. with 0.2×SSPE). Sequence analysis of the isolated cDNA clones revealed that they correspond to two distinct genes, designated c-IAP1 and c-IAP2. The human c-IAP1 cDNA encodes a protein of 618 amino acids that is 84% identical to murine c-IAP1. The human c-IAP2 cDNA encodes a protein of 604 amino acids that shares a high degree of amino acid identity with both murine and human c-IAP1 (72% and 73%, respectively) and represents another member of the IAP superfamily.

Comparison of the amino acid sequence of members of the IAP superfamily reveals that they are comprised of at least three distinct domains. The N-terminal region of al IAP family members is comprised of ‘baculovirus IAP repeat’ (BIR) motifs (Birnbaum et al., 1994, supra). While the viral proteins contain two repeats, the mammalian homologs (c-IAP1, -2) possess three BIR motifs. Similarly, NAIP contains three BIR repeats. In addition to BIR motifs viral IAPs contain a C-terminal RING finger motif. This Zn-binding domain is also present in c-IAP1 and -2 but not in NAIP. Thus c-IAP1 and -2 define a distinct subfamily within the IAP superfamily that contain three BIR motifs and a RING finger motif. A RING finger domain is also present at the N-terminus of TRAF2 and has been shown to be involved in TRAF2 signal transduction. The RING finger motifs of c-IAP1 and -2 share significant sequence homology with the RING finger domains of viral IAPs but no homology with the TRAF2 RING finger domain besides the conserved cysteine and histidine residues. The region between the BIR domain and the RING finger domain of c-IAP1 and -2 is strongly conserved but does not reveal any significant homology to other members of the IAP family or any other proteins in the NCBI database.

A yeast two-hybrid system was used to determine how c-IAP1 and -2 interact with TNF-R2 and/or TRAFs. The following results were obtained indistinguishably for c-IAP1 and c-IAP2. Two-hybrid analysis revealed that c-IAP1 does not directly interact with TNF-R2. However, a direct interaction could be detected between c-IAP1 and TRAF2. The conserved TRAF domain of TRAF2 (amino acids 264-501) is sufficient to mediate this interaction. Consistently, c-IAP1 also interacted with TRAF1. Further analysis demonstrated that the coiled-coil region within the TRAF domain of TRAF2 (amino acids 251-358) is required for interaction with c-IAP1. In contrast, the C-terminal region of the TRAF domain (amino acids 359-501) that mediates the association of TNF-R2 with TRAF2 is dispensable for interaction of c-IAP1 with TRAF2. Thus c-IAP1 and TNF-R2 bind to non-overlapping docking sites within the TRAF domain of TRAF2. Consistently, c-IAP1 does not interact with TRAF3 (e.g. Cheng et al. (1995), supra), which does not contain a coiled-coil region with sequence similarity to TRAF2/TRAF1. Deletion mutagenesis of c-IAP1 indicated that the N-terminal half of the protein containing the three BIR motifs (amino acids 1-336 of c-IAP1 and 1-396 of c-IAP2) is sufficient for interaction with TRAF2 and TRAF1. Similarly, combinations of two of the three BIR motifs e.g. amino acid residues 46-99 and 204-249 of c-IAP1 and 29-82 and 189-234 of c-IAP2, separated by IAP1 derived intervening sequences of varying lengths are assayed for TRAF1 and TRAF2 binding. This indicates that BIR motifs represent a novel protein:protein interaction domain. The RING finger domain of c-IAP1/2 (amino acids 571-618 of c-IAP1 and 557-604 of c-IAP2) is not required for interaction with TRAFs, but rather mediates subsequent steps in the c-IAP1/2 signaling cascade. Similarly, a variety of c-IAP1 derived N-terminal leader sequences fused to the c-IAP1 RING finger domain are used to assay signal transduction mediation. In an analogous situation, the RING finger domain of TRAF2 has been demonstrated to be required for TRAF2-mediated activation of NF-κB.

A transfection based co-immunoprecipitation assay was used to investigate how c-IAP1 interacts with the complex of TNF-R2 and TRAFs. In this system c-IAP1 was N-terminally tagged with a FLAG epitope peptide and expressed in human embryonic 293 cells under the control of a constitutive CMV promotor (pRK vector). The c-IAP1 expression vector was transiently co-transfected into 293 cells with expression vectors for TNF-R2 and TRAFs. After 24-36 h, the cells were harvested and extracts immunoprecipitated with anti-TNF-R2 antibodies, followed by Western analysis with anti-FLAG antibodies. This assay demonstrated that while c-IAP1 associates directly with TRAF1 and TRAF2, its interaction with TNF-R2 is indirect and requires the hetetocomplex of TRAF1 and TRAF2. Thus, c-IAP1 is a component of the TNF-R2 (CD40)/TRAF signaling complex.

To determine the functional properties of c-IAP1 transient transfection assays were performed in human rhabdomyosarcoma KYM1 cells. The results indicate that overexpression of c-IAP1 but not of control vector, TRAF1 or TRAF2 protects KYM1 cells from TNF-induced programmed cell death (apoptosis). Hence, c-IAP1 regulates the cellular response to TNF by modulating TNF responsiveness, e.g. the initiation of an apoptotic or protective program. The transient transfection assay also finds use as a drug screening assay. In this application, candidate agents are screened as above for their ability to modulate the ability of c-IAP1 to downregulate apoptosis.

1. Protocol for human c-IAP1—TRAF2 binding assay.

A. Reagents:

Neutralite Avidin: 20 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM β-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors.

33 P human c-IAP1 10×stock: 10 −8 -10 −6 M unlabeled human c-IAP1 supplemented with 200,000-250,000 cpm of labeled human c-IAP1/21 (Beckman counter). Place in the 4° C. microfridge during screening.

Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB # 109894), 10 mg Aprotinin (BMB # 236624), 25 mg Benzamidine (Sigma # B-6506), 25 mg Leupeptin (BMB # 1017128), 10 mg APMSF (BMB # 917575), and 2 mM NaVo 3 (Sigma # S-6508) in 10 ml of PBS.

TRAF2: 10 −8 -10 −5 M biotinylated truncated TRAF2 (residues 264-501) in PBS.

B. Preparation of assay plates:

Coat with 120 μl of stock N-Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay:

Add 40 μl assay buffer/well.

Add 10 μl compound or extract.

Add 10 μl 33 P-human c-IAP1 (20,000-25,000 cpm/0.1-10 pmoles/well=10 −9 -10 −7 M final concentration).

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Add 40 μl biotinylated truncated TRAF2 (0.1-10 pmoles/40 ul in assay buffer)

Incubate 1 hour at room temperature.

Stop the reaction by washing 4 times with 200 μl PBS.

Add 150 μl scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding

b. Soluble (non-biotinylated truncated TRAF2) at 80% inhibition.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

TCTAAGTAGT ATCTTGGAAA TTCAGAGAGA TACTCATCCT ACCTGAATAT AAACTGAGAT 60

AAATCCAGTA AAGAAAGTGT AGTAAATTCT ACATAAGAGT CTATCATTGA TTTCTTTTGG 120

TGGTAAAAAT CTTAGTTCAT GTGAAGAAAT TTCATGTGAA TGTTTTAGCT ATCAAACAGC 180

ACTGTCACCT ACTCATGCAC AAAACTGCCT CCCAAAGACT TTTCCCAGGT CCCTCGTATC 240

AAAACATTAA GAGTATAATG GAAGATAGCA CGATCTTGTC AGATTGGACA AACAGCAACA 300

AACAAAAAAT GAAGTATGAC TTTTCCTGTG AACTCTACAG AATGTCTACA TATTCAACTT 360

TCCCCGCCGG GGTGCCTGTC TCAGAAAGGA GTCTTGCTCG TGCTGGTTTT TATTATACTG 420

GTGTGAATGA CAAGGTCAAA TGCTTCTGTT GTGGCCTGAT GCTGGATAAC TGGAAACTAG 480

GAGACAGTCC TATTCAAAAG CATAAACAGC TATATCCTAG CTGTAGCTTT ATTCAGAATC 540

TGGTTTCAGC TAGTCTGGGA TCCACCTCTA AGAATACGTC TCCAATGAGA AACAGTTTTG 600

CACATTCATT ATCTCCCACC TTGGAACATA GTAGCTTGTT CAGTGGTTCT TACTCCAGCC 660

TTTCTCCAAA CCCTCTTAAT TCTAGAGCAG TTGAAGACAT CTCTTCATCG AGGACTAACC 720

CCTACAGTTA TGCAATGAGT ACTGAAGAAG CCAGATTTCT TACCTACCAT ATGTGGCCAT 780

TAACTTTTTT GTCACCATCA GAATTGGCAA GAGCTGGTTT TTATTATATA GGACCTGGAG 840

ATAGGGTAGC CTGCTTTGCC TGTGGTGGGA AGCTCAGTAA CTGGGAACCA AAGGATGATG 900

CTATGTCAGA ACACCGGAGG CATTTTCCCA ACTGTCCATT TTTGGAAAAT TCTCTAGAAA 960

CTCTGAGGTT TAGCATTTCA AATCTGAGCA TGCAGACACA TGCAGCTCGA ATGAGAACAT 1020

TTATGTACTG GCCATCTAGT GTTCCAGTTC AGCCTGAGCA GCTTGCAAGT GCTGGTTTTT 1080

ATTATGTGGG TCGCAATGAT GATGTCAAAT GCTTTTGTTG TGATGGTGGC TTGAGGTGTT 1140

GGGAATCTGG AGATGATCCA TGGGTAGAAC ATGCCAAGTG GTTTCCAAGG TGTGAGTTCT 1200

TGATACGAAT GAAAGGCCAA GAGTTTGTTG ATGAGATTCA AGGTAGATAT CCTCATCTTC 1260

TTGAACAGCT GTTGTCAACT TCAGATACCA CTGGAGAAGA AAATGCTGAC CCACCAATTA 1320

TTCATTTTGG ACCTGGAGAA AGTTCTTCAG AAGATGCTGT CATGATGAAT ACACCTGTGG 1380

TTAAATCTGC CTTGGAAATG GGCTTTAATA GAGACCTGGT GAAACAAACA GTTCAAAGTA 1440

AAATCCTGAC AACTGGAGAG AACTATAAAA CAGTTAATGA TATTGTGTCA GCACTTCTAA 1500

ATGCTGAAGA TGAAAAAAGA GAGGAGGAGA AGGAAAAACA AGCTGAAGAA ATGGCATCAG 1560

ATGATTTGTC ATTAATTCGG AAGAACAGAA TGGCTCTCTT TCAACAATTG ACATGTGTGC 1620

TTCCTATCCT GGATAATCTT TTAAAGGCCA ATGTAATTAA TAAACAGGAA CATGATATTA 1680

TTAAACAAAA AACACAGATA CCTTTACAAG CGAGAGAACT GATTGATACC ATTTTGGTTA 1740

AAGGAAATGC TGCGGCCAAC ATCTTCAAAA ACTGTCTAAA AGAAATTGAC TCTACATTGT 1800

ATAAGAACTT ATTTGTGGAT AAGAATATGA AGTATATTCC AACAGAAGAT GTTTCAGGTC 1860

TGTCACTGGA AGAACAATTG AGGAGGTTGC AAGAAGAACG AACTTGTAAA GTGTGTATGG 1920

ACAAAGAAGT TTCTGTTGTA TTTATTCCTT GTGGTCATCT GGTAGTATGC CAGGAATGTG 1980

CCCCTTCTCT AAGAAAATGC CCTATTTGCA GGGGTATAAT CAAGGGTACT GTTCGTACAT 2040

TTCTCTCTTA AAGAAAAATA GTCTATATTT TAACCTGCAT AAAAAGGTCT TTAAAATATT 2100

GTTGAACACT TGAAGCCATC TAAAGTAAAA AGGGAATTAT GAGTTTTTCA ATTAGTAACA 2160

TTCATGTTCT AGTCTGCTTT GGTACTAATA ATCTTGTTTC TGAAAAGATG GTATCATATA 2220

TTTAATCTTA ATCTGTTTAT TTACAAGGGA AGATTTATGT TTGGTGAACT ATATTAGTAT 2280

GTATGTGTAC CTAAGGGAGT AGTGTCACTG CTTGTTATGC ATCATTTCAG GAGTTACTGG 2340

ATTTGTTGTT CTTTCAGAAA GCTTTGAATA CTAAATTATA GTGTAGAAAA GAACTGGAAA 2400

CCAGGAACTC TGGAGTTCAT CAGAGTTATG GTGCCGAATT GTCTTTGGTG CTTTTCACTT 2460

GTGTTTTAAA ATAAGGATTT TTCTCTTATT TCTCCCCCTA GTTTGTGAGA AACATCTCAA 2520

TAAAGTGCTT TAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2580

AAAAAAAAA 2589

618 amino acids

amino acid

single

linear

protein

2

Met His Lys Thr Ala Ser Gln Arg Leu Phe Pro Gly Pro Ser Tyr Gln

1 5 10 15

Asn Ile Lys Ser Ile Met Glu Asp Ser Thr Ile Leu Ser Asp Trp Thr

20 25 30

Asn Ser Asn Lys Gln Lys Met Lys Tyr Asp Phe Ser Cys Glu Leu Tyr

35 40 45

Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu

50 55 60

Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp Lys

65 70 75 80

Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp Lys Leu Gly

85 90 95

Asp Ser Pro Ile Gln Lys His Lys Gln Leu Tyr Pro Ser Cys Ser Phe

100 105 110

Ile Gln Asn Leu Val Ser Ala Ser Leu Gly Ser Thr Ser Lys Asn Thr

115 120 125

Ser Pro Met Arg Asn Ser Phe Ala His Ser Leu Ser Pro Thr Leu Glu

130 135 140

His Ser Ser Leu Phe Ser Gly Ser Tyr Ser Ser Leu Ser Pro Asn Pro

145 150 155 160

Leu Asn Ser Arg Ala Val Glu Asp Ile Ser Ser Ser Arg Thr Asn Pro

165 170 175

Tyr Ser Tyr Ala Met Ser Thr Glu Glu Ala Arg Phe Leu Thr Tyr His

180 185 190

Met Trp Pro Leu Thr Phe Leu Ser Pro Ser Glu Leu Ala Arg Ala Gly

195 200 205

Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala Cys Phe Ala Cys Gly

210 215 220

Gly Lys Leu Ser Asn Trp Glu Pro Lys Asp Asp Ala Met Ser Glu His

225 230 235 240

Arg Arg His Phe Pro Asn Cys Pro Phe Leu Glu Asn Ser Leu Glu Thr

245 250 255

Leu Arg Phe Ser Ile Ser Asn Leu Ser Met Gln Thr His Ala Ala Arg

260 265 270

Met Arg Thr Phe Met Tyr Trp Pro Ser Ser Val Pro Val Gln Pro Glu

275 280 285

Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Arg Asn Asp Asp Val

290 295 300

Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser Gly Asp

305 310 315 320

Asp Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg Cys Glu Phe Leu

325 330 335

Ile Arg Met Lys Gly Gln Glu Phe Val Asp Glu Ile Gln Gly Arg Tyr

340 345 350

Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser Asp Thr Thr Gly Glu

355 360 365

Glu Asn Ala Asp Pro Pro Ile Ile His Phe Gly Pro Gly Glu Ser Ser

370 375 380

Ser Glu Asp Ala Val Met Met Asn Thr Pro Val Val Lys Ser Ala Leu

385 390 395 400

Glu Met Gly Phe Asn Arg Asp Leu Val Lys Gln Thr Val Gln Ser Lys

405 410 415

Ile Leu Thr Thr Gly Glu Asn Tyr Lys Thr Val Asn Asp Ile Val Ser

420 425 430

Ala Leu Leu Asn Ala Glu Asp Glu Lys Arg Glu Glu Glu Lys Glu Lys

435 440 445

Gln Ala Glu Glu Met Ala Ser Asp Asp Leu Ser Leu Ile Arg Lys Asn

450 455 460

Arg Met Ala Leu Phe Gln Gln Leu Thr Cys Val Leu Pro Ile Leu Asp

465 470 475 480

Asn Leu Leu Lys Ala Asn Val Ile Asn Lys Gln Glu His Asp Ile Ile

485 490 495

Lys Gln Lys Thr Gln Ile Pro Leu Gln Ala Arg Glu Leu Ile Asp Thr

500 505 510

Ile Leu Val Lys Gly Asn Ala Ala Ala Asn Ile Phe Lys Asn Cys Leu

515 520 525

Lys Glu Ile Asp Ser Thr Leu Tyr Lys Asn Leu Phe Val Asp Lys Asn

530 535 540

Met Lys Tyr Ile Pro Thr Glu Asp Val Ser Gly Leu Ser Leu Glu Glu

545 550 555 560

Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys Lys Val Cys Met Asp

565 570 575

Lys Glu Val Ser Val Val Phe Ile Pro Cys Gly His Leu Val Val Cys

580 585 590

Gln Glu Cys Ala Pro Ser Leu Arg Lys Cys Pro Ile Cys Arg Gly Ile

595 600 605

Ile Lys Gly Thr Val Arg Thr Phe Leu Ser

610 615

2601 base pairs

nucleic acid

single

linear

cDNA

3

GGGCAGCAGG TTTACAAAGG AGGAAAACGA CTTCTTCTAG ATTTTTTTTT CAGTTTCTTC 60

TATAAATCAA AACTACCTCC CTAGAGAAAG GCTAGTCCCT TTTCTTCCCC ATTCATTTCA 120

TTATGAACAT AGTAGAAAAC AGCATATTCT TATCAAATTT GATGAAAAGC GCCAACACGT 180

TTGAACTGAA ATACGACTTG TCATGTGAAC TGTACCGAAT GTCTACGTAT TCCACTTTTC 240

CTGCTGGGGT CCCTGTCTCA GAAAGGAGTC TTGCTCGCGC TGGTTTCTAT TACACTGGTG 300

TGAATGACAA GGTCAAATGC TTCTGTTGTG GCCTGATGCT GGATAACTGG AAAAGAGGAG 360

ACAGTCCTAC TGAAAAGCAT AAAAAGTTGT ATCCTAGCTG CAGATTCGTT CAGAGTCTAA 420

ATTCCGTTAA CAACTTGGAA GCTACCTCTC AGCCTACTTT TCCTTCTTCA GTAACAAATT 480

CCACACACTC ATTACTTCCG GGTACAGAAA ACAGTGGATA TTTCCGTGGC TCTTATTCAA 540

ACTCTCCATC AAATCCTGTA AACTCCAGAG CAAATCAAGA TTTTTCTGCC TTGATGAGAA 600

GTTCCTACCA CTGTGCAATG AATAACGAAA ATGCCAGATT ACTTACTTTT CAGACATGGC 660

CATTGACTTT TCTGTCGCCA ACAGATCTGG CAAAAGCAGG CTTTTACTAC ATAGGACCTG 720

GAGACAGAGT GGCTTGCTTT GCCTGTGGTG GAAAATTGAG CAATTGGGAA CCGAAGGATA 780

ATGCTATGTC AGAACACCTG AGACATTTTC CCAAATGCCC ATTTATAGAA AATCAGCTTC 840

AAGACACTTC AAGATACACA GTTTCTAATC TGAGCATGCA GACACATGCA GCCCGCTTTA 900

AAACATTCTT TAACTGGCCC TCTAGTGTTC TAGTTAATCC TGAGCAGCTT GCAAGTGCGG 960

GTTTTTATTA TGTGGGTAAC AGTGATGATG TCAAATGCTT TTGCTGTGAT GGTGGACTCA 1020

GGTGTTGGGA ATCTGGAGAT GATCCATGGG TTCAACATGC CAAGTGGTTT CCAAGGTGTG 1080

AGTACTTGAT AAGAATTAAA GGACAGGAGT TCATCCGTCA AGTTCAAGCC AGTTACCCTC 1140

ATCTACTTGA ACAGCTGCTA TCCACATCAG ACAGCCCAGG AGATGAAAAT GCAGAGTCAT 1200

CAATTATCCA TTTTGAACCT GGAGAAGACC ATTCAGAAGA TGCAATCATG ATGAATACTC 1260

CTGTGATTAA TGCTGCCGTG GAAATGGGCT TTAGTAGAAG CCTGGTAAAA CAGACAGTTC 1320

AGAGAAAAAT CCTAGCAACT GGAGAGAATT ATAGACTAGT CAATGATCTT GTGTTAGACT 1380

TACTCAATGC AGAAGATGAA ATAAGGGAAG AGGAGAGAGA AAGAGCAACT GAGGAAAAAG 1440

AATCAAATGA TTTATTATTA ATCCGGAAGA ATAGAATGGC ACTTTTTCAA CATTTGACTT 1500

GTGTAATTCC AATCCTGGAT AGTCTACTAA CTGCCGGAAT TATTAATGAA CAAGAACATG 1560

ATGTTATTAA ACAGAAGACA CAGACGTCTT TACAAGCAAG AGAACTGATT GATACGATTT 1620

TAGTAAAAGG AAATATTGCA GCCACTGTAT TCAGAAACTC TCTGCAAGAA GCTGAAGCTG 1680

TGTTATATGA GCATTTATTT GTGCAACAGG ACATAAAATA TATTCCCACA GAAGATGTTT 1740

CAGATCTACC AGTGGAAGAA CAATTGCGGA GACTACAAGA AGAAAGAACA TGTAAAGTGT 1800

GTATGGACAA AGAAGTGTCC ATAGTGTTTA TTCCTTGTGG TCATCTAGTA GTATGCAAAG 1860

ATTGTGCTCC TTCTTTAAGA AAGTGTCCTA TTTGTAGGAG TACAATCAAG GGTACAGTTC 1920

GTACATTTCT TTCATGAAGA AGAACCAAAA CATCATCTAA ACTTTAGAAT TAATTTATTA 1980

AATGTATTAT AACTTTAACT TTCATCCTAA TTTGGTTTCC TTAAAATTTT TATTTATTTA 2040

CAACTCAACA AACATTGTTT TGTGTAACAT ATTTAATATA TGTATCTAAA CCATATGAAC 2100

ATATATTTTT TAGAAACTAA GAGAATGATA GGCTTTTGTT CTTATGAACG AAAAAGAGGT 2160

AGCACTACAA ACACAATATT CAATCAAAAT TTCAGCATTA TTGAAATTGT AAGTGAAGTA 2220

AAACTTAAGA TATTTGAGTT AACCTTTAAG AATTTTAAAT ATTTTGGCAT TGTACTAATA 2280

CCGGGAACAT GAAGCCAGGT GTGGTGGTAT GTGCCTGTAG TCCCAGGCTG AGGCAAGAGA 2340

ATTACTTGAG CCCAGGAGTT TGAATCCATC CTGGGCAGCA TACTGAGACC CTGCCTTTAA 2400

AAACAAACAG AACAAAAACA AAACACCAGG GACACATTTC TCTGTCTTTT TTGATCAGTG 2460

TCCTATACAT CGAAGGTGTG CATATATGTT GAATGACATT TTAGGGACAT GGTGTTTTTA 2520

TAAAGAATTC TGTGAGAAAA AATTTAATAA AACCCCCCAA ATTAAAAAAA AAAAAAAAAA 2580

AAAAAAAAAA AAAAAAAAAA A 2601

604 amino acids

amino acid

single

linear

protein

4

Met Asn Ile Val Glu Asn Ser Ile Phe Leu Ser Asn Leu Met Lys Ser

1 5 10 15

Ala Asn Thr Phe Glu Leu Lys Tyr Asp Leu Ser Cys Glu Leu Tyr Arg

20 25 30

Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu Arg

35 40 45

Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp Lys Val

50 55 60

Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp Lys Arg Gly Asp

65 70 75 80

Ser Pro Thr Glu Lys His Lys Lys Leu Tyr Pro Ser Cys Arg Phe Val

85 90 95

Gln Ser Leu Asn Ser Val Asn Asn Leu Glu Ala Thr Ser Gln Pro Thr

100 105 110

Phe Pro Ser Ser Val Thr Asn Ser Thr His Ser Leu Leu Pro Gly Thr

115 120 125

Glu Asn Ser Gly Tyr Phe Arg Gly Ser Tyr Ser Asn Ser Pro Ser Asn

130 135 140

Pro Val Asn Ser Arg Ala Asn Gln Asp Phe Ser Ala Leu Met Arg Ser

145 150 155 160

Ser Tyr His Cys Ala Met Asn Asn Glu Asn Ala Arg Leu Leu Thr Phe

165 170 175

Gln Thr Trp Pro Leu Thr Phe Leu Ser Pro Thr Asp Leu Ala Lys Ala

180 185 190

Gly Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala Cys Phe Ala Cys

195 200 205

Gly Gly Lys Leu Ser Asn Trp Glu Pro Lys Asp Asn Ala Met Ser Glu

210 215 220

His Leu Arg His Phe Pro Lys Cys Pro Phe Ile Glu Asn Gln Leu Gln

225 230 235 240

Asp Thr Ser Arg Tyr Thr Val Ser Asn Leu Ser Met Gln Thr His Ala

245 250 255

Ala Arg Phe Lys Thr Phe Phe Asn Trp Pro Ser Ser Val Leu Val Asn

260 265 270

Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Asn Ser Asp

275 280 285

Asp Val Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser

290 295 300

Gly Asp Asp Pro Trp Val Gln His Ala Lys Trp Phe Pro Arg Cys Glu

305 310 315 320

Tyr Leu Ile Arg Ile Lys Gly Gln Glu Phe Ile Arg Gln Val Gln Ala

325 330 335

Ser Tyr Pro His Leu Leu Glu Gln Leu Leu Ser Thr Ser Asp Ser Pro

340 345 350

Gly Asp Glu Asn Ala Glu Ser Ser Ile Ile His Phe Glu Pro Gly Glu

355 360 365

Asp His Ser Glu Asp Ala Ile Met Met Asn Thr Pro Val Ile Asn Ala

370 375 380

Ala Val Glu Met Gly Phe Ser Arg Ser Leu Val Lys Gln Thr Val Gln

385 390 395 400

Arg Lys Ile Leu Ala Thr Gly Glu Asn Tyr Arg Leu Val Asn Asp Leu

405 410 415

Val Leu Asp Leu Leu Asn Ala Glu Asp Glu Ile Arg Glu Glu Glu Arg

420 425 430

Glu Arg Ala Thr Glu Glu Lys Glu Ser Asn Asp Leu Leu Leu Ile Arg

435 440 445

Lys Asn Arg Met Ala Leu Phe Gln His Leu Thr Cys Val Ile Pro Ile

450 455 460

Leu Asp Ser Leu Leu Thr Ala Gly Ile Ile Asn Glu Gln Glu His Asp

465 470 475 480

Val Ile Lys Gln Lys Thr Gln Thr Ser Leu Gln Ala Arg Glu Leu Ile

485 490 495

Asp Thr Ile Leu Val Lys Gly Asn Ile Ala Ala Thr Val Phe Arg Asn

500 505 510

Ser Leu Gln Glu Ala Glu Ala Val Leu Tyr Glu His Leu Phe Val Gln

515 520 525

Gln Asp Ile Lys Tyr Ile Pro Thr Glu Asp Val Ser Asp Leu Pro Val

530 535 540

Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys Lys Val Cys

545 550 555 560

Met Asp Lys Glu Val Ser Ile Val Phe Ile Pro Cys Gly His Leu Val

565 570 575

Val Cys Lys Asp Cys Ala Pro Ser Leu Arg Lys Cys Pro Ile Cys Arg

580 585 590

Ser Thr Ile Lys Gly Thr Val Arg Thr Phe Leu Ser

595 600

55 amino acids

amino acid

single

linear

protein

5

Cys Glu Leu Tyr Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val

1 5 10 15

Pro Val Ser Glu Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly

20 25 30

Val Asn Asp Lys Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn

35 40 45

Trp Lys Leu Gly Asp Ser Pro

50 55

55 amino acids

amino acid

single

linear

protein

6

Cys Glu Leu Tyr Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val

1 5 10 15

Pro Val Ser Glu Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly

20 25 30

Val Asn Asp Lys Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn

35 40 45

Trp Lys Arg Gly Asp Ser Pro

50 55

46 amino acids

amino acid

single

linear

protein

7

Leu Ala Arg Ala Gly Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala

1 5 10 15

Cys Phe Ala Cys Gly Gly Lys Leu Ser Asn Trp Glu Pro Lys Asp Asp

20 25 30

Ala Met Ser Glu His Arg Arg His Phe Pro Asn Cys Pro Phe

35 40 45

46 amino acids

amino acid

single

linear

protein

8

Leu Ala Lys Ala Gly Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala

1 5 10 15

Cys Phe Ala Cys Gly Gly Lys Leu Ser Asn Trp Glu Pro Lys Asp Asn

20 25 30

Ala Met Ser Glu His Leu Arg His Phe Pro Lys Cys Pro Phe

35 40 45

48 amino acids

amino acid

single

linear

protein

9

Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Arg Asn Asp

1 5 10 15

Asp Val Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser

20 25 30

Gly Asp Asp Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg Cys Glu

35 40 45

48 amino acids

amino acid

single

linear

protein

10

Pro Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Asn Ser Asp

1 5 10 15

Asp Val Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser

20 25 30

Gly Asp Asp Pro Trp Val Gln His Ala Lys Trp Phe Pro Arg Cys Glu

35 40 45

52 amino acids

amino acid

single

linear

protein

11

Glu Glu Arg Thr Cys Lys Val Cys Met Asp Lys Glu Val Ser Val Val

1 5 10 15

Phe Ile Pro Cys Gly His Leu Val Val Cys Gln Glu Cys Ala Pro Ser

20 25 30

Leu Arg Lys Cys Pro Ile Cys Arg Gly Ile Ile Lys Gly Thr Val Arg

35 40 45

Thr Phe Leu Ser

50

52 amino acids

amino acid

single

linear

protein

12

Glu Glu Arg Thr Cys Lys Val Cys Met Asp Lys Glu Val Ser Ile Val

1 5 10 15

Phe Ile Pro Cys Gly His Leu Val Val Cys Lys Asp Cys Ala Pro Ser

20 25 30

Leu Arg Lys Cys Pro Ile Cys Arg Ser Thr Ile Lys Gly Thr Val Arg

35 40 45

Thr Phe Leu Ser

50

2862 base pairs

nucleic acid

single

linear

cDNA

13

TTCCTTTACA GTGAATACTG TAGTCTTAAT AGACCTGAGC TGACTGCTGC AGTTGATGTA 60

ACCCACTTTA GAGAATACTG TATGACATCT TCTCTAAGGA AAACCAGCTG CAGACTTCAC 120

TCAGTTCCTT TCATTTCATA GGAAAAGGAG TAGTTCAGAT GTCATGTTTA AGTCCTTATA 180

AGGGAAAAGA GCCTGAATAT ATGCCCTAGT ACCTAGGCTT CATAACTAGT AATAAGAAGT 240

TAGTTATGGG TAAATAGATC TCAGGTTACC CAGAAGAGTT CATGTGACCC CCAAAGAGTC 300

CTAACTAGTG TCTTGGCAAG TGAGACAGAT TTGTCCTGTG AGGGTGTCAA TTCACCAGTC 360

CAAGCAGAAG ACAATGAATC TATCCAGTCA GGTGTCTGTG GTGGAGATCT AGTGTCAAGT 420

GGTGAGAAAC TTCATCTGGA AGTTTAAGCG GTCAGAAATA CTATTACTAC TCATGGACAA 480

AACTGTCTCC CAGAGACTCG GCCAAGGTAC CTTACACCAA AAACTTAAAC GTATAATGGA 540

GAAGAGCACA ATCTTGTCAA ATTGGACAAA GGAGAGCGAA GAAAAAATGA AGTTTGACTT 600

TTCGTGTGAA CTCTACCGAA TGTCTACATA TTCAGCTTTT CCCAGGGGAG TTCCTGTCTC 660

AGAGAGGAGT CTGGCTCGTG CTGGCTTTTA TTATACAGGT GTGAATGACA AAGTCAAGTG 720

CTTCTGCTGT GGCCTGATGT TGGATAACTG GAAACAAGGG GACAGTCCTG TTGAAAAGCA 780

CAGACAGTTC TATCCCAGCT GCAGCTTTGT ACAGACTCTG CTTTCAGCCA GTCTGCAGTC 840

TCCATCTAAG AATATGTCTC CTGTGAAAAG TAGATTTGCA CATTCGTCAC CTCTGGAACG 900

AGGTGGCATT CACTCCAACC TGTGCTCTAG CCCTCTTAAT TCTAGAGCAG TGGAAGACTT 960

CTCATCAAGG ATGGATCCCT GCAGCTATGC CATGAGTACA GAAGAGGCCA GATTTCTTAC 1020

TTACAGTATG TGGCCTTTAA GTTTTCTGTC ACCAGCAGAG CTGGCCAGAG CTGGCTTCTA 1080

TTACATAGGG CCTGGAGACA GGGTGGCCTG TTTTGCCTGT GGTGGGAAAC TGAGCAACTG 1140

GGAACCAAAG GATGATGCTA TGTCAGAGCA CCGCAGACAT TTTCCCCACT GTCCATTTCT 1200

GGAAAATACT TCAGAAACAC AGAGGTTTAG TATATCAAAT CTAAGTATGC AGACACACTC 1260

TGCTCGATTG AGGACATTTC TGTACTGGCC ACCTAGTGTT CCTGTTCAGC CCGAGCAGCT 1320

TGCAAGTGCT GGATTCTATT ACGTGGATCG CAATGATGAT GTCAAGTGCT TTTGTTGTGA 1380

TGGTGGCTTG AGATGTTGGG AACCTGGAGA TGACCCCTGG ATAGAACACG CCAAATGGTT 1440

TCCAAGGTGT GAGTTCTTGA TACGGATGAA GGGTCAGGAG TTTGTTGATG AGATTCAAGC 1500

TAGATATCCT CATCTTCTTG AGCAGCTGTT GTCCACTTCA GACACCCCAG GAGAAGAAAA 1560

TGCTGACCCT ACAGAGACAG TGGTGCATTT TGGCCCTGGA GAAAGTTCGG AAGATGTCGT 1620

CATGATGAGC ACGCCTGTGG TTAAAGCAGC CTTGGAAATG GGCTTCAGTA GGAGCCTGGT 1680

GAGACAGACG GTTCAGCGGC AGATCCTGGC CACTGGTGAG AACTACAGGA CCGTCAATGA 1740

TATTGTCTCA GTACTTTTGA ATGCTGAAGA TGAGAGAAGA GAAGAGGAGA AGGAAAGACA 1800

GACTGAAGAG ATGGCATCAG GTGACTTATC ACTGATTCGG AAGAATAGAA TGGCCCTCTT 1860

TCAACAGTTG ACACATGTCC TTCCTATCCT GGATAATCTT CTTGAGGCCA GTGTAATTAC 1920

AAAACAGGAA CATGATATTA TTAGACAGAA AACACAGATA CCCTTACAAG CAAGAGAGCT 1980

TATTGACACC GTTTTAGTCA AGGGAAATGC TGCAGCCAAC ATCTTCAAAA ACTCTCTGAA 2040

GGAAATTGAC TCCACGTTAT ATGAAAACTT ATTTGTGGAA AAGAATATGA AGTATATTCC 2100

AACAGAAGAC GTTTCAGGCT TGTCATTGGA AGAGCAGTTG CGGAGATTAC AAGAAGAACG 2160

AACTTGCAAA GTGTGTATGG ACAGAGAGGT TTCTATTGTG TTCATTCCGT GTGGTCATCT 2220

AGTAGTCTGC CAGGAATGTG CCCCTTCTCT AAGGAAGTGC CCCATCTGCA GGGGGACAAT 2280

CAAGGGGACT GTGCGCACAT TTCTCTCATG AGTGAAGAAT GGTCTGAAAG TATTGTTGGA 2340

CATCAGAAGC TGTCAGAACA AAGAATGAAC TACTGATTTC AGCTCTTCAG CAGGACATTC 2400

TACTCTCTTT CAAGATTAGT AATCTTGCTT TATGAAGGGT AGCATTGTAT ATTTAAGCTT 2460

AGTCTGTTGC AAGGGAAGGT CTATGCTGTT GAGCTACAGG ACTGTGTCTG TTCCAGAGCA 2520

GGAGTTGGGA TGCTTGCTGT ATGTCCTTCA GGACTTCTTG GATTTGGAAT TTGTGAAAGC 2580

TTTGGATTCA GGTGATGTGG AGCTCAGAAA TCCTGAAACC AGTGGCTCTG GTACTCAGTA 2640

GTTAGGGTAC CCTGTGCTTC TTGGTGCTTT TCCTTTCTGG AAAATAAGGA TTTTTCTGCT 2700

ACTGGTAAAT ATTTTCTGTT TGTGAGAAAT ATATTAAAGT GTTTCTTTTA AAGGCGTGCA 2760

TCATTGTAGT GTGTGCAGGG ATGTATGCAG GCAAAACACT GTGTATATAA TAAATAAATC 2820

TTTTTAAAAA GTGAAAAAAA AAAAAAAAAA AAAAAAAAAA AA 2862

612 amino acids

amino acid

single

linear

protein

14

Met Asp Lys Thr Val Ser Gln Arg Leu Gly Gln Gly Thr Leu His Gln

1 5 10 15

Lys Leu Lys Arg Ile Met Glu Lys Ser Thr Ile Leu Ser Asn Trp Thr

20 25 30

Lys Glu Ser Glu Glu Lys Met Lys Phe Asp Phe Ser Cys Glu Leu Tyr

35 40 45

Arg Met Ser Thr Tyr Ser Ala Phe Pro Arg Gly Val Pro Val Ser Glu

50 55 60

Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp Lys

65 70 75 80

Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp Lys Gln Gly

85 90 95

Asp Ser Pro Val Glu Lys His Arg Gln Phe Tyr Pro Ser Cys Ser Phe

100 105 110

Val Gln Thr Leu Leu Ser Ala Ser Leu Gln Ser Pro Ser Lys Asn Met

115 120 125

Ser Pro Val Lys Ser Arg Phe Ala His Ser Ser Pro Leu Glu Arg Gly

130 135 140

Gly Ile His Ser Asn Leu Cys Ser Ser Pro Leu Asn Ser Arg Ala Val

145 150 155 160

Glu Asp Phe Ser Ser Arg Met Asp Pro Cys Ser Tyr Ala Met Ser Thr

165 170 175

Glu Glu Ala Arg Phe Leu Thr Tyr Ser Met Trp Pro Leu Ser Phe Leu

180 185 190

Ser Pro Ala Glu Leu Ala Arg Ala Gly Phe Tyr Tyr Ile Gly Pro Gly

195 200 205

Asp Arg Val Ala Cys Phe Ala Cys Gly Gly Lys Leu Ser Asn Trp Glu

210 215 220

Pro Lys Asp Asp Ala Met Ser Glu His Arg Arg His Phe Pro His Cys

225 230 235 240

Pro Phe Leu Glu Asn Thr Ser Glu Thr Gln Arg Phe Ser Ile Ser Asn

245 250 255

Leu Ser Met Gln Thr His Ser Ala Arg Leu Arg Thr Phe Leu Tyr Trp

260 265 270

Pro Pro Ser Val Pro Val Gln Pro Glu Gln Leu Ala Ser Ala Gly Phe

275 280 285

Tyr Tyr Val Asp Arg Asn Asp Asp Val Lys Cys Phe Cys Cys Asp Gly

290 295 300

Gly Leu Arg Cys Trp Glu Pro Gly Asp Asp Pro Trp Ile Glu His Ala

305 310 315 320

Lys Trp Phe Pro Arg Cys Glu Phe Leu Ile Arg Met Lys Gly Gln Glu

325 330 335

Phe Val Asp Glu Ile Gln Ala Arg Tyr Pro His Leu Leu Glu Gln Leu

340 345 350

Leu Ser Thr Ser Asp Thr Pro Gly Glu Glu Asn Ala Asp Pro Thr Glu

355 360 365

Thr Val Val His Phe Gly Pro Gly Glu Ser Ser Glu Asp Val Val Met

370 375 380

Met Ser Thr Pro Val Val Lys Ala Ala Leu Glu Met Gly Phe Ser Arg

385 390 395 400

Ser Leu Val Arg Gln Thr Val Gln Arg Gln Ile Leu Ala Thr Gly Glu

405 410 415

Asn Tyr Arg Thr Val Asn Asp Ile Val Ser Val Leu Leu Asn Ala Glu

420 425 430

Asp Glu Arg Arg Glu Glu Glu Lys Glu Arg Gln Thr Glu Glu Met Ala

435 440 445

Ser Gly Asp Leu Ser Leu Ile Arg Lys Asn Arg Met Ala Leu Phe Gln

450 455 460

Gln Leu Thr His Val Leu Pro Ile Leu Asp Asn Leu Leu Glu Ala Ser

465 470 475 480

Val Ile Thr Lys Gln Glu His Asp Ile Ile Arg Gln Lys Thr Gln Ile

485 490 495

Pro Leu Gln Ala Arg Glu Leu Ile Asp Thr Val Leu Val Lys Gly Asn

500 505 510

Ala Ala Ala Asn Ile Phe Lys Asn Ser Leu Lys Glu Ile Asp Ser Thr

515 520 525

Leu Tyr Glu Asn Leu Phe Val Glu Lys Asn Met Lys Tyr Ile Pro Thr

530 535 540

Glu Asp Val Ser Gly Leu Ser Leu Glu Glu Gln Leu Arg Arg Leu Gln

545 550 555 560

Glu Glu Arg Thr Cys Lys Val Cys Met Asp Arg Glu Val Ser Ile Val

565 570 575

Phe Ile Pro Cys Gly His Leu Val Val Cys Gln Glu Cys Ala Pro Ser

580 585 590

Leu Arg Lys Cys Pro Ile Cys Arg Gly Thr Ile Lys Gly Thr Val Arg

595 600 605

Thr Phe Leu Ser

610