AU774355B2

AU774355B2 - Novel bag proteins and nucleic acid molecules encoding them

Info

Publication number: AU774355B2
Application number: AU60383/99A
Authority: AU
Inventors: John C Reed; Shinichi Takayama
Original assignee: Sanford Burnham Prebys Medical Discovery Institute
Current assignee: Sanford Burnham Prebys Medical Discovery Institute
Priority date: 1998-09-09
Filing date: 1999-09-09
Publication date: 2004-06-24
Anticipated expiration: 2019-09-09
Also published as: EP1109824A4; WO2000014106A1; AU6038399A; CA2342027A1; WO2000014106A9; JP2002524068A; EP1109824A1

Description

WO 00/14106 PCT/US99/21053 NOVEL BAG PROTEINS AND NUCLEIC ACID MOLECULES ENCODING THEM STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under grant number CA-67329 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This invention relates generally to the fields of molecular biology and molecular medicine and more specifically to a novel family of proteins that can regulate protein folding. The functions of these proteins are potentially diverse, including promoting tumor cell growth and metastasis.

BACKGROUND INFORMATION The Hsc70/Hsp70-family of molecular chaperones participate in protein folding reactions, controlling protein bioactivity, degradation, complex assembly/disassembly, and translocation across membranes.

These proteins interact with hydrophobic regions within target proteins via a carboxyl (C)-terminal peptide binding domain, with substrate binding and release being controlled by the N-terminal ATP-binding domain of Hsc70/Hsp70.

Hsc70/Hsp70-assisted folding reactions are accomplished by repeated cycles of peptide binding, refolding, and release, WO 00/14106 PCTIUS99/21053 2 which are coupled to ATP hydrolysis by the ATP-binding domain (ATPase) of Hsc70/Hsp70 and by subsequent nucleotide exchange. The chaperone activity of mammalian Hsc70/Hsp70 is regulated by partner proteins that either modulate the peptide binding cycle or that target the actions of these chaperones to specific proteins and subcellular compartments. DnaJ-family proteins (Hdj-i/Hsp40; Hdj-2; Hdj-3) stimulate the ATPase activity of Hsc70/Hsp70, resulting in the ADP-bound state which binds tightly to peptide substrates. The Hip protein collaborates with Hsc70/Hsp70 and DnaJ homologues in stimulating ATP hydrolysis, and thus also stabilize Hsc70/Hsp70 complexes with substrate polypeptides, whereas the Hop protein may provide co-chaperone functions through interactions with the C-terminal peptide binding domain.

The Bcl-2 associated athanogene-1 (bag-1) is named from the Greek word athanos, which refers to anti-cell death. BAG-1 was previously referred to as Bcl-2-associated protein-i (BAP-1) in U.S. Patent No.

5,539,094 issued July 23, 1996, which is incorporated herein by reference. In this earlier patent, BAG-1 is described as a portion of the human BAG-1 protein, absent the N-terminal amino acids 1 to 85. In addition, a human protein essentially identical to human BAG-1 was described by Zeiner and Gehring, (Proc. Natl. Acad. Sci., USA 92:11465-11469 (1995)). Subsequent to the issuance of U.S.

Patent 5,539,094 the N-terminal amino acid sequence from 1 to 85 of human BAG-1 was reported.

BAG-1 and its longer isoforms BAG-1M (Rap46) and BAG-1L are recently described Hsc70/Hsp70-regulating proteins. BAG-1 competes with Hip for binding to the Hsc70/Hsp70 ATPase domain and promotes substrate release.

BAG-1. also reportedly stimulates Hsc70-mediated ATP 07/05 '04 FRI 13:45 FAX 61 7 3221 1245 GRIFFITH HACK 1008 3 hydrolysis by accelerating ADP/ATP exchange, analogous to the prokaryotic GrpE nucleotide exchange protein of the bacterial Hsc70 homologue, DnaK. Gene transfection studies indicate that BAG-1 proteins can influence a wide variety of cellular phenotypes through their interactions with Hs p 70 including increasing resistance to apoptosis, promoting cell proliferation, enhancing tumor cell migration and metastasis, and altering transcriptional activity of steroid hormone receptors.

Despite the notable progress in the art, there remains an unmet need for the further identification and isolation of additional homologous BAG protein species, and the nucleic acid molecules and/or nucleotide sequences that encode them. Such species would provide additional 15 means by which the identity and composition of the BAG domain, that is, the portion of the protein that is influencing or modulating protein folding, could be identified. In addition, such species would be useful for identifying agents that modulate apoptosis as candidates 20 for therapeutic agents, in particular, anticancer agents.

oo* The present invention satisfies these need, as well as providing substantial related advantages.

SuARRY OF THE INVENTION COMS ID No: SMBI-00739634 Received by IP Australia: Time 13:50 Date 2004-05-07 07/05 '04 FRI 13:45 FAX 61 7 3221 1245 GRIFFITH HACK 00oo9 3a According to one aspect of the present invention there is provided a substantially purified nucleic acid molecule having a nucleotide sequence corresponding to or complementary to a nucleotide sequence selected from the group consisting of (SEQ ID NO:1), (SEQ ID NO:3), (SEQ ID (SEQ ID NO:7), (SEQ ID NO;9), (SEQ ID NO:19), (SEQ ID NO:21) and (SEQ ID NO:23).

According to a further aspect of the present invention there is provided a substantially purified BAG family protein comprising of the amino acid sequence selected from the group consisting of (SEQ ID NO:4), (SEQ ID NO:6), (SEQ ID NO:8), (SEQ ID NO:10), (SEQ ID (SEQ ID N0:22) and (SEQ ID NO:24) or a fragment, a derivative or a mimetic thereof, wherein said protein, 15 fragment, derivative or mimetic has an ability to bind to •e According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a substantially purified BAG family protein 20 comprising of the amino acid sequence selected from the group consisting of (SEQ ID NO:2), (SEQ ID NO:4), (SEQ ID NO:6), (SEQ ID NO:8), (SEQ ID N0:10), (SEQ ID NO:20), (SEQ SID NO:22) and (SEQ ID NO:24), or a fragment, a derivative or a mimetic thereof, wherein said protein, fragment, derivative or mimetic has an ability to bind to Hsc70 and is useful for modulating tumor cell proliferation, cell migration and metastasis, and steroid hormone receptor function, and a pharmaceutically acceptable o*o* carrier According to a further aspect of the present invention there is provided a method of determining the risk of metastatic spread of cancer or prognosis of cancer patients by determining the level of expression of a BAGfamily protein or a fragment thereof, wherein said BAGfamily protein or fragment thereof comprises an amino acid sequence selected from the group consisting of (SEQ ID NO:4), (SEQ ID NO:6), (SEQ ID NO:8), (SEQ ID NO:10), (SEQ ID NO:20), (SEQ ID NO:22) and (SEQ ID NO:24), amino acid H:\MardR\Kepl\SpD ci\P4 Ri-pg-3at-7..doc 7/05/04 COMS ID No: SMBI-00739634 Received by IP Australia: Time 13:50 Date 2004-05-07 07/05 '04 FRI 13:45 FAX 61 7 3221 1245 GRIFFITH HACK 1010 3b sequence 157 to 204 of (SEQ ID NO:2), amino acid sequence 272 to 319 of (SEQ ID NO:2), amino acid sequence 378 to 457 of (SEQ ID NO:22), amino acid sequence 6 to 97 of (SEQ ID NO:24), amino acid sequence 180 to 257 of (SEQ ID NO:24), amino acid sequence 272 to 349 of (SEQ ID NO:24), and amino acid sequence 362 to 444 of (SEQ ID NO:24).

According to a further aspect of the present invention there is provided a substantially purified nucleic acid molecule having a nucleotide sequence corresponding to or complementary to at least contiguous nucleotides from a nucleotide sequence selected from the group consisting of (SEQ ID NO:3), (SEQ ID NO:7), (SEQ ID NO:9),(SEQ ID NO:21) and (SEQ ID NO:23).

According to a further aspect of the present 15 invention there is provided a substantially purified BAG family protein comprising of the amino acid sequence selected from the group consisting of (SEQ ID NO:2), (SEQ ID NO:4), (SEQ ID NO:6), (SEQ ID NO:8), (SEQ ID (SEQ ID NO:20), (SEQ ID NO:22) and (SEQ ID NO:24).

20 According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a substantially purified BAG family protein comprising of the amino acid sequence selected from the group consisting of (SEQ ID NO:2), (SEQ ID NO:4), (SEQ ID 25 NO:6), (SEQ ID NO:8), (SEQ ID NO:10), (SEQ ID NO:20), (SEQ ID NO:22) and (SEQ ID NO:24), and a pharmaceutically acceptable carrier.

H,\MaraR\Kep\SpCi\P4 l -pg3ath,7.5.04.doc 7/0D/G4i COMS ID No: SMBI-00739634 Received by IP Australia: Time 13:50 Date 2004-05-07 WO 00/14106 PCTIUS99/21053 4 Another aspect of the present invention provides an amino acid sequence present in the family of BAG-1 related proteins, that modulates Hsc70/Hsp70 chaperone activity, that is, the BAG domain.

Another aspect of the present invention provides novel polypeptide and nucleic acid compositions and methods useful in modulating Hsc70/Hsp70 chaperone activity.

Another aspect of the present invention is directed to methods for detecting agents that modulate the binding of the BAG family of proteins, such as BAG-1 (beginning at residue 116 of SEQ ID NO:2), and related proteins with the Hsc70/Hsp70 Family of proteins or with other proteins that may interact with the BAG-Family proteins.

Still another aspect of the present invention is directed to methods for detecting agents that induce the dissociation of a bound complex formed by the association of BAG-Family proteins with Hsc70/Hsp70 Family molecule chaperones or other proteins.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the full length cDNA sequence for human BAG-1 (SEQ ID NO:1) protein with the corresponding amino acid sequence (SEQ ID NO:2). Within the full length sequence are included the overlapping sub-sequences of BAG-1 (beginning at nucleotide 391), BAG-1M [beginning at nucleotide 260 of (SEQ ID and BAG-1L [beginning at nucleotide 46 of (SEQ ID NO:2)].

WO 00/14106 PCT/US9921053 Figures 2A and 2B combined shows the full length cDNA sequence (SEQ ID NO:3) aligned with the corresponding amino acid residues for human BAG-2 protein (SEQ ID NO:4) Figure 3 shows a cDNA sequence (SEQ ID aligned with the corresponding amino acid residues for human BAG-3 protein (SEQ ID NO:6).

Figure 4 shows the a cDNA sequence (SEQ ID NO:7) aligned with the corresponding amino acid residues for human BAG-4 protein (SEQ ID NO:8).

Figure 5 shows a cDNA sequence (SEQ ID NO:9) aligned with the corresponding amino acid residues for human BAG-5 protein (SEQ ID Figure 6A shows the full length cDNA sequence for C. elegans BAG-1 protein (SEQ ID NO:11).

Figure 6B shows the 210 amino acid sequence for C. elegans BAG-1 protein (SEQ ID NO:12) Figure 7A shows the full length cDNA sequence for C. elegans BAG-2 protein (SEQ ID NO:13) Figure 7B shows the 458 amino acid sequence for C. elegans BAG-2 protein (SEQ ID NO:14).

Figure 8A shows the full length cDNA sequence for S. pombe BAG-1A protein (SEQ ID Figure 8B shows the 195 amino acid sequence for S. pombe BAG-1A protein (SEQ ID NO:16).

WO 00/14106 PCT/US99/21053 6 Figure 9A shows the full length cDNA sequence for S. pombe BAG-1B protein (SEQ ID NO:17).

Figure 9B shows the 206 amino acid sequence for S. pombe BAG-1B protein (SEQ ID NO:18).

Figure 10 shows the topologies of the BAG-family proteins; human BAG proteins, BAG-1 (SEQ ID NO:2), BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6), BAG-4 (SEQ ID NO:8), (SEQ ID NO:10); S.pombe BAG-1A (SEQ ID NO:16)and BAG-1B (SEQ ID NO:18); and C. elegans BAG-1 (SEQ ID NO:12)and BAG-2 (SEQ ID NO:14). The relative positions of the BAG domains are shown in black, ubiquitinlike regions are represented in gray, WW domain are represented in strips. Nucleoplasmin-like nuclear localization sequence are also shown. The amino acid sequences of the BAG domain for human BAG-1 (SEQ ID NO:2), BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6), BAG-4 (SEQ ID NO:8), BAG-5 (SEQ ID NO:10), S.pombe BAG-1A (SEQ ID NO:16)and BAG-1B (SEQ ID NO:18), and C. elegans BAG-1 (SEQ ID NO:12)and BAG-2 (SEQ ID NO:14) are aligned demonstrating their homology. Black and gray shading represent identical and similar amino acids, respectively.

Figure 11 shows assays demonstrating the interaction of BAG-family proteins with Hsc70/ATPase. (A) Two-hybrid assays using yeast expressing the indicated fusion proteins. Blue color indicates a positive interaction, resulting in activation of the lacZ reporter gene. In vitro protein assays using GST-fusion proteins and 35 S-labeled in vitro translated proteins. (C) Co-immunoprecipitation assays using anti-Flag or IgGl control antibodies and lysates from 293T cells expressing Flag-tagged BAG-1 (beginning at residue 116 of SEQ ID WO 00/14106 PCTIUS99/21053 7 NO:2), BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6), Daxx, or Apaf-1.

Figure 12 shows surface plasmon resonance analysis of BAG-family protein interactions with Hsc70/ATPase. SDS-PAGE analysis of purified recombinant proteins. Representative SPR results of biosensor chips containing immobilized BAG proteins with and without maximally bound Figure 13 shows representative SPR results for biosensor chips containing immobilized BAG-1 (beginning at residue 116 at SEQ ID NO:2), BAG-1(AC), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) proteins. Hsc70/ATPase was flowed over the chips (arrow/left) until maximal binding was reached (response units), then flow was continued without Hsc70/ATPase (arrow/right). For BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6), Hsc70 was injected at 0.0175, 0.035, 0.07, 0.14, and 0.28 pM.

Figure 14 shows BAG-family protein modulation of chaperone activity. Protein refolding assay of chemically-denatured luciferase by Hsc70 plus DnaJ in the absence or presence of BAG and BAG-mutant proteins. (B) Concentration-dependent inhibition of protein refolding by BAG-family proteins [BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6)] but not by BAG-mutant (BAG-1 (C) Hsc70/Hsp40-mediated refolding of heat-denatured luciferase was assayed in the presence of (black bars) or absence of (striped bars) of 1.8 pM Hip, with (lanes 3-10) or without (lanes 1,2) various BAG-family proteins (1.8pM) as indicated (mean ±SE; A control (CNTL) is shown (lane 1) in which Hsc70 was replaced with an equivalent amount of

BSA.

WO 00/14106 PCT/US99/21053 8 Figure 15A shows an expanded cDNA sequence for human BAG-3 protein (SEQ ID NO:19).

Figure 15B shows the corresponding amino acid residues for the human BAG-3 protein (SEQ ID NO:20) of Figure Figure 15C shows the expanded cDNA sequence (SEQ ID NO:19) aligned with the corresponding amino acid residues for human BAG-3 protein of Figure 15A (SEQ ID Figure 16A shows an expanded cDNA sequence for human BAG-4 protein (SEQ ID NO:21) Figure 16B shows the corresponding amino acid residues for the human BAG-4 protein of Figure 16A (SEQ ID NO:22) Figure 16C shows the expanded cDNA sequence (SEQ ID NO:21) aligned with the corresponding amino acid residues for human BAG-4 protein of Figure 16A (SEQ ID NO:22).

Figure 17A shows an expanded cDNA sequence for human BAG-5 protein (SEQ ID NO:23) Figure 17B shows the corresponding amino acid residues for the human BAG-5 protein of Figure 17A (SEQ ID NO:24).

Figure 17C shows the expanded cDNA sequence (SEQ ID NO:23) aligned with the corresponding amino acid residues for human BAG-5 protein of Figure 17A (SEQ ID NO:24).

WO 00/14106 PCT/US99/21053 9 Figure 18 shows the topologies of the BAG-family proteins; human BAG proteins, BAG-1 (SEQ ID NO:2), BAG-2 (SEQ ID NO:4), expanded BAG-3 (SEQ ID NO:20), expanded BAG-4 (SEQ ID NO:22), expanded BAG-5 (SEQ ID NO:24); S.pombe BAG-1A (SEQ ID NO:16)and BAG-1B (SEQ ID NO:18); and C. elegans BAG-1 (SEQ ID NO:12)and BAG-2 (SEQ ID NO:14).

The relative positions of the BAG domains are shown in black, ubiquitin-like regions are represented in gray, WW domain are represented in strips. Nucleoplasmin-like nuclear localization sequence are also shown.

Definitions The term "apoptosis", as used herein, refers to the process of programmed cell death, although not all programmed cell deaths occur through apoptosis, as used herein, "apoptosis" and "programmed cell death" are used interchangeably.

The term "tumor cell proliferation", as used herein refers to the ability of tumor cells to grow and thus expand a tumor mass.

The term "cell migration", as used herein refers to the role cell motility plays in the invasion and potentially metastasis by tumor cells.

The term "metastasis", as used herein refers to the spread of a disease process from one part of the body to another, as in the appearance of neoplasms in parts of the body remote from the site of the primary tumor; results in dissemination of tumor cells by the lymphatics or blood vessels or by direct extension through serious cavitites or subarachnoid or other spaces.

WO 00/14106 PCT/US99/21053 The term "steroid hormone receptor function", as used herein refers to physiological, cellular and molecular functioning of receptors sites that bind with steroid hormones.

The term "substantially purified", as used herein, refers to nucleic acid.or amino acid.sequence that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and -most preferably 90% free from other components with which they are naturally associated.

"Nucleic acid molecule" as used herein refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single or double stranded, and represent the sense or antisense strand.

"Hybridization", as used herein, refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.

The terms "complementary" or "complementarity", as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T binds to the complementary sequence The term "homology", as used herein, refers to a degree of complementarity. There may be partial homology or complete homology identity). A partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid and is referred to using the functional term "substantially homologous." The inhibition of WO 00/14106 PCT/US9921053 11 hybridization of the completely complementary sequence to the target sequence may be examined using a hybridzation assay (Southern or northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding the hybridization) of a completely homologous sequence or probe to the target sequence under conditions of low stringency.

The term "antisense", as used herein, refers to nucleotide sequences which are commplementary to a specific DNA or RNA sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. Antisense molecules may be produced by any method, including synthesis by ligating the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a complementary strand. Once introduced into a cell, this transcribed strand combines with natural sequences produced by the cell to form duplexes. These duplexes then block either the further transcription or translation. In this manner, mutant phenotypes may be generated. The designation "negative" is sometimes used in reference to the antisense, and "positive" is sometimes used in reference to the sense strand.

"Amino acid sequence" as used herein refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited herein this term excludes an -amino acid sequence of a naturally occurring protein. "Amino acid sequence", "polypeptide" or "protein" are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

WO 00/14106 PCT[US99/21 053 12 The term "functional fragments" or "fragments", as used herein, with regard to a protein refers to portions of that protein that are capable of exhibiting or carrying out the activity exhibited by the protein as a whole. The portions may range in size from three amino acid residues to the entire amino acid sequence minus one amino acid.

For example, a protein "comprising at least a functional fragment of the amino acid sequence of SEQ ID NO:1", encompasses the full-length of the protein of SEQ ID NO:1 and portions thereof.

A "derivative" of a BAG protein, as used herein, refers to an amino acid sequence that is alterd by one or more amino acids. The derivative may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, substitution of an apolar amino acid with another apolar amino acid (such as replacement of leucine with isoleucine) The derivative may also have "nonconservative" changes, wherein a substituted amino acid has different but sufficiently similar structural or chemical properties that permits such a substitution without adversely effecting the desired biological activity, replacement of an amino acid with an uncharged polar R group with an amino acid with an apolar R group (such as replacement of glycine with tryptophan), or alternatively replacement of an amino acid with a charged R group with an amino acid with an uncharged Polar R group (such as replacement of lysine with asparagine).

WO 00/14106PC/S9215 PCTIUS99/21053 Amino Acids Apolar R Groups Amino Acid Radical Abbreviations 3-Letter 1-Letter alanine methyl ala A valine 2-propyl aal V leucine 2-methylpropyl leu L isoleucine 2-butyl ileI proline propyl* cyclized proP phenylalanine benzyl phe F trytophan 3-indolylmethl tyr methionine methylthioethyl met M Amino Acids Uncharged Polar R Groups Amino Acid fRadical Abbreviations 3-Letter j 1 -Letterd qlycine H gly G serine hydroxymethyl ser S threonine l-hydroxyethyl thr T cysteine thiolmethyl cy s C tyrosine 4-hydroxyphenylmethyl tyr Y asparagine aminocarbonylmethyl asn* N glutamine aminocarbonylethyl gln Q I Amino Acids Charged R Groups Amino Acid Radical Abbreviations I3-Letter 11-Letter aspartic acid carboxymethyl asp D glutamic acid carboxyethyl glu E lysine 4-aminobutyl lys K arginine 3-guanylpropyl arg R histidine 4-imidazoylmethyl__ his H WO 00/14106 PCTIUS99/21053 14 Similar minor modifications may also include amino acids deletions or insertions or both. Guidance in determining which amino acid residues may be modified as indicated above without abolishing the desired biological functionality may be determined using computer programs well known in the art, for example, DNASTAR software. In addition, the derivative may also result from chemical modifications to the encoded polypeptide, including but not limited to the following, replacement of hydrogen by an alkyl, acyl, or amino group; esterification of a carboxyl group with a suitable alkyl or aryl moiety; alkylation of a hydroxyl group to form an ether derivative. Further a derivative may also result from the substitution of a Lconfiguration amino acid with its corresponding Dconfiguration counterpart.

The term "mimetic", as used herein, refers to a molecule, the structure of which is developed from knowledge of the structure of a protein/polypeptide or portions thereof (such as BAG-1) and, as such, is able to effect some or all of the actions of BAG-1 protein.

"Peptide nucleic acid", as used herein, refers to a molecule which comprises an oligomer to which an amino acid residue, such as lysine, and an amino group have been added. These small molecules, also designated anti-gene agents, stop transcript elongation by binding to their complementary strand of nucleic acid (Nielsen, P.E. et al., Anticancer Drug Des. 8:53-63 (1993)) DETAILED DESCRIPTION OF THE INVENTION The present invention provides a family of BAG-1 related proteins from humans [BAG-1L (SEQ ID NO:2), BAG-IS beginning at residue 116 of SEQ ID NO:2, BAG-2 (SEQ ID WO 00/14106 PCTIUS99/21 053 NO:4), BAG-3 (SEQ ID NO:6) and (SEQ ID NO:20), BAG-4 (SEQ ID NO: 8) and (SEQ ID NO:22) and BAG-5 (SEQ ID NO:10) and (SEQ ID NO:24)], the invertebrate C.elegans [BAG-1 (SEQ ID NO:12), BAG-2 (SEQ ID NO:14)] and the fission yeast S.pombe [BAG-1A (SEQ ID NO:16), BAG-1B (SEQ ID NO:18)], specifically the full length amino acid sequences comprising human BAG-IL (SEQ ID NO:2), BAG-1 (beginning at residue 116 of SEQ ID NO:2), and BAG-2 (SEQ ID NO:4) C.

elegans BAG-1 (SEQ ID NO:12), and BAG-2 (SEQ ID NO:14), and S.pombe BAG-1A (SEQ ID NO:16) and BAG-1B (SEQ ID NO:18); and partial sequences comprising human BAG-3 (SEQ ID NO: 6) and (SEQ ID NO:20), BAG-4 (SEQ ID NO:8) and (SEQ ID NO:22), and BAG-5 (SEQ ID NO:10) and (SEQ ID NO:24) and functional fragments thereof. In particular, the invention provides the amino acid sequences comprising human BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6) and (SEQ ID NO:20), BAG-4 (SEQ ID NO:8) and (SEQ ID NO:22), and BAG-5 (SEQ ID NO:10) and (SEQ ID NO:24) proteins.

Another aspect of the present invention pro.vides the nucleic molecule and nucleotide sequences that encode the family of BAG-1 related proteins from humans [BAG-1 (SEQ ID NO:1), BAG-2 (SEQ ID NO:3), BAG-3 (SEQ ID NO:5) and (SEQ ID NO:19), BAG-4 (SEQ ID NO:7) and (SEQ ID NO:21) and (SEQ ID NO:9) and (SEQ ID NO:23)], the invertebrate C.elegans [BAG-1 (SEQ ID NO:11), BAG-2(SEQ ID NO:13)] and the fission yeast S.pombe [BAG-1A (SEQ ID NO:15), BAG-1B (SEQ ID NO:17)].

BAG-1L (SEQ ID NO:2) is a multifunctional protein that blocks apoptosis, promotes tumor cell metastasis, and contributes to factor-independent and p53-resistant cell growth. BAG-1L (SEQ ID NO:2) interacts with several types of proteins, including Bcl-2, some tyrosine kinase growth WO 00/14106 PCTIUS99/21053 16 factor receptors, steroid hormone receptors, and the p53induced cell cycle regulator Siah-1A.

BAG-1 is a regulator of Hsc70/Hsp70 family molecular chaperones. A carboxyl-terminal domain in this protein binds tightly to the ATPase domains of Hsc70 and 1 nM) (Zeiner.-M., Gebauer, and Gehring, U., EMBO J. 16: 5483-5490, (1997)). BAG-1 modulates the activity of these molecular chaperones, acting as an apparent functional antagonist of the Hsp70/Hsc70associated protein Hip (Hohfeld, J. and Jentsch, S., EMBO J. 16: 6209-6216, (1997); Takayama, Bimston, D.

Matsuzawa, Freeman, B. Aime-Sempe, Xie, Z., Morimoto, R. and Reed, J. EMBO J. 16: 4887-96, (1997); Zeiner, Gebauer, and Gehring, EMBO J.

16: 5483-5490, (1997)). In general, protein refolding is accomplished by Hsp70/Hsc70 through repeated cycles of target peptide binding and release, coupled to ATP hydrolysis (Ellis, Curr Biol. 7: R531-R533, (1997)).

BAG-1 appears to promote substrate release, whereas Hip stabilizes Hsp70/Hsc70 complex formation with target peptides (Hbhfeld, Minami, and Hartl, Cell.

83: 589-598, (1995)). Since each substrate interaction with Hsc70/Hsp70 is unique in terms of the optimal length of time the protein target should remain complexed with Hsc70/Hsp70 for achieving new conformations, the net effect of BAG-1 can be either enhancement or inhibition of the refolding reaction.

The 70kd heat shock family proteins (Hsp70/Hsc70) are essential to a variety of cellular processes and have been implicated in cancer, yet it is unclear how these proteins are regulated in vivo. A variety of co-chaperones have been identified which may target Hsp70/Hsc70 to different subcellular compartments or promote their WO 00/14106 PCT/US99/21053 17 interactions with specific protein or protein complexes.

BAG-1 appears to represent a novel Hsp70/Hsc70 regulator which differs functionally from all other mammalian cochaperones identified to date, such as members of the DnaJ-, Hip-, Hop-, and cyclophilin-families of proteins.

Another aspect of the present invention provides the amino acid sequence of a binding domain of about 40 to amino acids that bind the a Hsc70/Hsp70 ATPase domain.

The BAG domain is situated near the C-terminus, and the ubiquitin-like domains are situated near the N-terminus.

The BAG family of proteins of the present invention contain a common conserved C-terminal domain (the "BAG" domain) that facilitates binding to the ATPase domain of Hsp70/Hsc70. The carboxyl-terminal domain of BAG-1 binds to the ATPase domain of Hsc70/Hsp70 and regulates its chaperone function by acting as a ADP-ATP exchange factor.

Other domains of BAG-1 mediate interactions with proteins such as Bcl-2 and retinoic acid receptors (RARs), allowing BAG-1 to target Hsc70/Hsp70 to other proteins, presumably modulating their function by changing their conformations.

Human BAG-1 was previously shown to inhibit Hsc70/Hsp70 dependent refolding of. denatured protein substrates in vitro Takayama, et al., EMBO J 16, 4887- 96 (1997); M. Zeiner, M. Gebauer, U. Gehring, EMBO J. 16, 5483-5490 (1997); and J. H6hfeld, S. Jentsch, EMBO J. 16, 6209-6216 (1997)). In Example III, Part A the effects of recombinant human BAG-1, BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) were compared using in vitro protein refolding assays similar to those employed previously for assessing BAG-1. The study showed that addition of equimolar amounts of each of the recombinant proteins to Hsc70 resulted in significant inhibition of luciferase refolding, with BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) showing somewhat WO 00/14106 PCTIS99/21053 18 greater inhibitor activity than BAG-1 (Figure 4A). In a separate luciferase folding study BAG-1, BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) once again displayed inhibition of luciferase refolding, however in this study varying amounts of BAG-1, BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) were added relative to Hsc70 which resulting in concentration-dependent inhibition of Hsc70 chaperone activity, luciferase folding (Example III Part A).

Additional follow on studies using the same experimental protocols as the previous studies, as taught in Example IIA, have shown that BAG-4 (SEQ ID NO:22) also undergoes association with Yet another aspect of the present invention provides a nucleotide sequence having at least about nucleotides and, generally, about 25 nucleotides, preferably about 35 nucleotides, more preferably about nucleotides, and most preferably about 55 nucleotides that can hybridize or is complementary under relatively stringent conditions to a portion of the nucleic acid sequences shown in Figures 1-9 and Figures 15-17, in particular the BAG domain as shown in in Figure 1B, e.g., nucleotides 552-593 of human BAG-3, or nucleotides 167-221 of human BAG-4.

Yet another aspect of the present invention provides a compound of the formula, RN-R X R2X2R3X3R4XR5X5R6X6R XX8R9X9R l o

X

l o XR X -R wherein,

R

N is a group of 1 to 552 independently selected amino acids;

R

1 is a group of 3 independently selected amino acids; WO 00/14106 PCT/US99/21053 19

X

1 is an amino acid with a charged or uncharged R group, such as aspartic acid, glutamic acid, asparagine, or glutamine;

R

2 is a group of 7 independently selected amino acids;

X

2 is an amino acid with a charged R group, such as glutamic acid;

R

3 is a group of 5 independently selected amino acids;

X

3 is an amino acid with an apolar R group, such as leucine, methionine, or isoleucine;

R

4 is a group of 3 independently selected amino acids;

X

4 is an amino acid with charged R group, such as aspartic acid or glutamine acid;

R

5 is a single independently selected amino acid;

X

5 is an amino acid with apolar or uncharged R group, such as leucine, valine, methionine, alanine or threonine;

R

6 is a group of 15 independently selected amino acids;

X

6 is an amino acid with a charged or uncharged R group, such as arginine, lysine, glutamine or aspartic acid; R is a group of 2 independently selected amino acids;

X

7 is an amino acid with a charged R group, such as arginine;

X

8 is an amino acid with a charged R group, such as arginine or lysine;

R

9 is a group of 2 independently selected amino acids;

X

9 is an amino acid with an apolar R group, such as valine;

R

10 is a group of 3 independently selected amino acids; WO 00/14106 PCT/US99/21053 X1 is an amino acid with an uncharged R group, such as glutamine;

R

1 is a group of 2 independently selected amino acids; X is an amino acid with an apolar R group, such as leucine; and Rc is a group of 1 to 100 independently selected amino acids.

A nucleotide sequence of at least about nucleotides and, generally, about 25 nucleotides, preferably about 35 nucleotides, more preferably about nucleotides, and most preferably about 55 nucleotides can be useful, for example, as a primer for the polymerase chain reaction (PCR) or other similar reaction mediated by a polymerase such as a DNA or RNA polymerase (see PCR Protocols: A guide to methods and applications, ed. Innis et al. (Academic Press, Inc., 1990), which is incorporated herein by reference; see, for example, pages 40-41). In addition, such a nucleotide sequence of the invention can be useful as a probe in a hybridization reaction such as Southern or northern blot analysis or in a binding assay such as a gel shift assay.

A nucleotide sequence of the invention can be particularly useful as an antisense molecule, which can be DNA or RNA and can be targeted to all or a portion of the region or of the 5'-translated region of a bag-1 nucleic acid sequence in a cell. For example, an antisense molecule can be directed to at least a portion of the sequence shown as the BAG domain in Figure 1A, e.g., nucleotides 272-319 of human BAG-1L (SEQ ID NO:1), or nucleotides 79-147 of human BAG-5 (SEQ ID NO:9). Since the of a nucleic acid contains elements involved in the control of expression of an encoded protein, an antisense molecule directed to the 5'-region of a nucleic WO 00/14106 PCT/US99/21053 21 acid molecule can affect the levels of protein expressed in a cell.

A nucleotide sequence of the invention also can be useful as a probe to identify a genetic defect due a mutation of a gene encoding a BAG protein in a cell. Such a genetic defect can lead to aberrant expression of a BAG protein in the cell or to expression of an aberrant BAG protein, which does not properly associate with a Bcl-2related protein or Hsc70/Hsp70 protein in the cell. As a result, a genetic defect in a gene encoding, for example, human BAG-i can result in a pathology characterized by increased or decreased levels in protein folding.

Further a nucleotide compound or composition as taught in the present invention can be synthesized using routine methods or can be purchased from a commercial source. In addition, a population of such nucleotide sequences can be prepared by restriction endonuclease or mild DNAse digestion of a nucleic acid molecule that contains nucleotides as shown in the nucleotide sequences shown in Figures 1-9 and Figures 15-17 that encodes the amino acids sequences also shown in Figures 1-9 and Figures 15-17. Methods for preparing and using such nucleotide sequences, for example, as hybridization probes to screen a library for homologous nucleic acid molecules are well known in the art (see, for example, Sambrook et al., Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989); Ausubel et al., Current Protocols in Molecular Biology (Green Publ., NY 1989), each of which is incorporated herein by reference) A particular nucleotide sequence can be designed based, for example, on a comparison of the nucleic acid molecules encoding any one of the BAG family proteins, as shown in Figures 1-9 and Figures 15-17, with another in the family. Such a comparison allows, for example, the WO 00/14106 PCTIUS99/21053 22 preparation of a nucleotide sequence that will hybridize to a conserved region present in both nucleic acid molecules, thus providing a means to identify homologous nucleic acid molecules present in other cell types or other organisms.

In addition, such a comparison allows the preparation of a nucleotide sequence that will hybridize to a unique region of any of the BAG family nucleotide sequences, such as those corresponding to the BAG domain, thus allowing identification of other proteins sharing this motif. In this regard, it is recognized that, while the human BAG-3 proteins shown as Figures 3 and 20, and human proteins shown as Figures 5 and 24, are only partial sequences, a variant human BAG-3 or BAG-5 produced, for example, by alternative splicing can exist and can be identified using an appropriately designed nucleotide sequence of the invention as a probe. Such useful probes readily can be identified by inspection of the sequences shown in the disclosed Figures by a comparison of the encoding nucleotide sequences.

If desired, a nucleotide sequence of the invention can incorporate a detectable moiety such* as a radiolabel, a fluorochrome, a ferromagnetic substance, a luminescent tag or a detectable binding agent such as biotin. These and other detectable moieties and methods of incorporating such moieties into a nucleotide sequence are well known in the art and are commercially available. A population of labelled nucleotide sequences can be prepared, for example, by nick translation of a nucleic acid molecule of the invention (Sambrook et al., supra, 1989; Ausubel et al., supra, 1989).

One skilled in the art would know that a method involving hybridization of a nucleotide sequence of the invention can require that hybridization be performed under relatively stringent conditions such that nonspecific background hybridization is minimized. Such hybridization WO 00/14106 PCT[US9921053 23 conditions can be determined empirically or can be estimated based, for example, on the relative GC content of a sequence and the number of mismatches, if known, between the probe and the target sequence (see, for example, Sambrook et al., supra, 1989) The invention further provides antibodies specific for human BAG family protein. As used herein, the term "antibody" includes polyclonal and monoclonal antibodies, as well as polypeptide fragments of antibodies that retain a specific binding activity for human BAG-1 *of at least about 1 x l05 M 1. One skilled in the art would know that anti-BAG-i antibody fragments such as Fab, F(ab'), and Fv fragments can retain specific binding activity for human BAG-i (beginning at residue 116 of SEQ ID NO:2) and, thus, are included within the definition of an antibody.

In addition, the term "antibody" as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies and fragments that retain binding activity such as chimeric antibodies or humanized antibodies. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference.

One skilled in the art would know that purified BAG family protein, which can be prepared from natural sources or synthesized chemically or produced recombinantly, or portions of a BAG family protein, including a portion of human BAG family protein such as a synthetic peptide as described above, can be used as an immunogen. Such peptides useful for raising an antibody include, for example, peptide portions of the N-terminal amino acids or the BAG domain of any of the human BAG WO 00/14106 PCTfS99/21053 24 proteins (see Figure IB). A particularly advantageous use of such a protein is for the immunostaining, wherein the methods provides a process to contrast the immunostaining of BAG-family proteins in carcinoma cells with adjacent non-neoplastic prostatic epithelial and basal cells which are generally present in the same tissue sections. These results would be correlated with a Gleason grade to determine whether any of the BAG-family proteins tend to be expressed at higher or lower levels in histologically advanced tumors. From this process a determination can be made as to degree at which the disease is progressing in a given patient, a prognosis can be made.

Non-immunogenic fragments or synthetic peptides of BAG proteins can be made immunogenic by coupling the hapten to a carrier molecule such bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), as described in Example IV, below. In addition, various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art and described, for example, by Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1988), which is incorporated herein by reference.

EXAMPLES

The following examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof.

WO 00/14106 PCT/US99/21053 EXAMPLE I Isolation and Characterization of BAG-family cDNA Sequences This example describes methods for isolating and characterizing of BAG-family cDNA sequences from human, nematode and yeast.

A. Cloning of human BAG cDNA sequences Yeast two-hybrid library screening of a human Jurkat cell cDNA library was performed as described by Takayama et al., EMBO 16:4887-96 (1997); Matsuzawa et al., EMBO 17:2736-2747 (1998), which are incorporated herein by reference) using EGY48 strain yeast transformed with pGilda-Hsc70/ATPase (67-377 amino acids) and the lacZ reporter plasmid pSH18-34. Of the resulting ~5 x 106 transformants, 112 Leu' colonies were obtained after 1 week incubation at 30 0 C. Assay of P-galactosidase (3-gal) activity of these colonies resulted in 96 clones. Mating tests were then performed using RFY206 yeast strain transformed with pGilda, pGilda mBAG-1 (1-219), or pGilda Hsc70/ATPase. Of these, 66 displayed specific interactions with Hsc70/ATPase. The pJG4-5 cDNAs were recovered using KC8 E. coli strain which is auxotrophic for tryptophan (Trp). DNA sequencing revealed 3 partially overlapping human BAG-1, 4 identical and one overlapping cDNAs encoding BAG-2, and 2 partially overlapping BAG-3 clones.

Using the above described yeast two-hybrid screen with the ATPase domain of Hsc70 as "bait", several human cDNAs were cloned which encode portions of BAG-1 or of two other BAG-1-like proteins which are termed BAG-2 (SEQ ID NO 4) and BAG-3 (SEQ ID NO:6). The longest of the cDNAs for BAG-2 (SEQ ID NO:3) and BAG-3 (SEQ ID NO:5) contained open reading frames (ORFs) of 207 and 162 amino acids, respectively, followed by stop codons. All BAG-1 (SEQ ID WO 00/14106 PCTIUS99/21053 26 NO:1), BAG-2 (SEQ ID NO:3) and BAG-3 (SEQ ID NO:5) cDNAs obtained by two-hybrid library screening with contained a conserved domain of about 40-50 amino acids which are termed the "BAG" domain and are shown in Figure 10. These results demonstrate that a family of BAG-1related proteins all contain a conserved ~45 amino acid region near their C-terminus that binds Hsc70/Hsp70.

B. Identification of additional BAG-family proteins A search of the translated Genbank database using the bBLAST and FASTA search programs also identified human ESTs that provided sequences for further investigation of BAG-family proteins. The putative BAG-4 (SEQ ID NO:8) and (SEQ ID NO:10) proteins contain BAG-domains that share the greatest sequence similarity with the BAG-domain of BAG-3 (SEQ ID NO:6). These were designated BAG-4 (Accession number AA693697, N74588) and BAG-5 (Accession number AA456862, N34101). BAG-4 has 62% identity and ~81% similarity to BAG-3, and BAG-5 has 51% identity and similarity to BAG-3.

'Additional BAG-family orthologues or homologues were also identified using computer-based searches and resulted in BAG-family homologue in the nematode C. elegans and the fission yeast S. pombe. The C. elegans genome encodes two apparent BAG-family proteins, which are most similar in their overall sequences to the human BAG-1 (Afo39713, gi:2773211) (SEQ ID NO:12) and BAG-2 (SEQ ID NO:14) (Afo68719, gi:3168927). The S. pombe contains two BAG-family proteins that share the greatest overall sequence similarity with human BAG-1 (Alo23S54,gi/3133105 and Alo23634, gi/3150250). The human and C. elegans BAG-1 proteins as well as S. pombe BAG-1A all have ubiquitin-like domains near their N-termini (see Figure 10A) of unknown function.

WO 00/14106 PCTIUS99/21053 27 The overall predicted amino acid sequences of the C. elegans BAG-1 (SEQ ID NO:12) and S. pombe BAG-1A (SEQ ID NO:16) proteins are ~18% identical similar) and ~17% identical similar), respectively, to human BAG-1, implying origin from a common ancestral gene. The C.

elegans BAG-1 protein (SEQ ID NO:12), however, contains a to 7 amino acid insert in its BAG-domain as compared to the human, murine, and yeast BAG-1 homologues (see Figure and is more similar to BAG-2 (SEQ ID NO:4) in regard to its BAG-domain. C. elegans and human BAG-2 also may be derived from a common ancestor as the C-terminal 225 amino acid region which encompasses both the BAG domain and upstream region of both C. elegans and human BAG-2 share ~34% amino acid sequence identity and ~70% similarity. The human BAG-2 protein (SEQ ID NO:4), however, contains a 9 amino acid insert in its BAG-domain compared to it C.elegans counterpart (see Figure 10B). Evolutionary-tree prediction algorithms suggest that human and C. elegans BAG-2 represent a distinct branch of the BAG-family that is more evolutionarily distant from the other BAG-family proteins. None of the predicted BAG-family proteins contain recognizable regions analogous to those found in other Hsc70 regulatory proteins, such as the J-domains and G/F-domains of DnaJ family proteins and the Tetratricopeptide Repeat (TR) domains of Hip/Hop family proteins.

C. Yeast two-hybrid assay of BAG binding to The longest of the cDNAs obtained for the BAG-2 and BAG-3 proteins were expressed with N-terminal transactivation (TA) domains in yeast and tested by yeast two-hybrid assay for interactions with fusion proteins consisting of Hsp70/ATPase or a variety of unrelated proteins (Fas, Siah, Fadd) containing N-terminal LexA DNAbinding domains. TA-BAG-2 and TA-BAG-3 demonstrated WO 00/14106 PCT[S99/21053 28 positive interactions with LexA-Hsc70/ATPase, resulting in transactivation of a lacZ reporter gene that was under the control of LexA operators (Figure 11A). No interactions with LexA-Fas (cytosolic domain), LexA-Siah, LexA-Fadd, or LexA were detected (see Figure 11A) demonstrating that the BAG-2 and BAG-3 proteins interact specifically with Specific two-hybrid interactions between and either BAG-2 or BAG-3 were also observed when BAG-2 and BAG-3 were expressed as LexA DNA-binding domain fusion proteins and Hsc70/ATPase was fused with a TA domain (see Figure 11A; right panel). These results demonstrate that similarly to BAG-1, BAG-2 and BAG-3 specifically interact with In order to determine whether the BAG proteins are capable of forming heterodimers, coexpression of BAG-2 and BAG-3 in the yeast two-hybrid assay was also performed.

Coexpression of BAG-2 and BAG-3 failed to show interaction with BAG-1 or a deletion mutant of BAG-1 (AC) which is missing part of its C-terminal domain required for Hsp70/Hsc70 binding suggest that these proteins do not form heterdimers.

D. Isolation and characterization of the complete open reading frame sequences of BAG-2 and BAG-3 In order to deduce the complete ORFs of BAG-2 and BAG-3, a A-phage cDNA library was screened as follows, using hybridization probes derived from the two-hybrid screening. A human jurkat T-cell X-ZapII library cDNA library (Stratagene) was screened by hybridization using 3 2P-labeled purified insert DNA from the longest of the human BAG-2 (clone #11) and human BAG-3 (clone #28) cDNA clones. From about one million clones screened, 38 BAG-2 and 23 BAG-3 clones were identified, cloned, and their cDNA inserts recovered as pSKII plasmids using a helper phage method (Stratagene). DNA sequencing of A-phage derived WO 00/14106 PCTfUS99/21 053 29 human BAG-2 cDNA clones revealed an ORF encoding a predicted 211 amino acid protein, preceded by an in-frame stop codon. The longest human BAG-3 X-phage cDNA clone contains a continuous ORF of 682 amino acids followed by a stop codon, but without an identifiable start codon (see Figure Although BAG-1L (SEQ ID NO:2), BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6) all contain a homologous BAG domain near their C-terminus, the N-terminal regions of these proteins are dissimilar. Using a combination of search tools (Prosite Search: PP search, using the Prosite pattern database, BCM Search Launcher, Baylor College of Medicine, and Blocks Search), it was determined that the BAG-2 Nterminal region contains potential kinase phosphorylation sites but otherwise shares no apparent similarity with other proteins or known functional domains.

In contrast, the predicted N-terminal region BAG-3 contains a WW domain as shown in Figure 10A. WW domains have been identified in a wide variety of sighaling proteins, including a Yes kinase adaptor protein (YAP), the Na'-channel regulator Nedd4, formin-binding proteins, dystrophin, and the peptidyl prolyl cis-trans-isomerase Pin-1. These roughly 40 amino acid domains mediate protein interactions and bind the preferred peptide ligand sequence xPPxY (Sudol., TIBS, 21: 161-163, 1996, which is incorporated herein by reference).

WO 00/14106 PCTIUS99/21053 EXAMPLE II In vitro Association of BAG proteins and This example demonstrates that BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) bind Hsc70/ATPase in various in vitro assays.

A. Solution binding assay of BAG-2 and BAG-3 to Association of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) with Hsc70/ATPase was determine by an in vitro protein binding assay where Hsc70/ATPase or BAG-family proteins were expressed in bacteria as Glutathione S- Transferase (GST) fusion proteins. Purified cDNA sequences encoding residues 5 to 211 of human BAG-2 (clone #11) and the C-terminal 135 amino acids of human BAG-3 (clone #28) (see Figure 10A) were subcloned into the EcoRI/Xho I sites of pGEX4T-1 prokaryotic expression plasmid (Pharmacia; Piscataway, NJ). These plasmids as well as pGEX4T-1-BAG-1, pGEX-4T-1-BAG-1 and pGEX-4T-1-XL which have been described previously (Takayama et al.,supra (1997); Xie et al., Biochemistry, 37:6410-6418, (1998), which are incorporated herein by reference), were expressed in XL-1 blue strain E. Coli (Stratagene, Inc., La Jolla, CA) Briefly, a single colony was inoculated into 1L of LB media containing 50 Ag/ml ampicillin and grown at 37C overnight.

The culture was then diluted by half with fresh LB/ampicillin and cooled to room temperature for 1 hr, before inducing with 0.4mM IPTG for 6 h at 25 0

C.

Cells were recovered and incubated with 0.5 mg/ml lysozyme in 50 mM Tris (pH 150 mM NaC1, 1% 0.1% 2-mercaptoethanol, 5 mM EDTA, 1 mM PMSF and a mixture WO 00/14106 PCT/US99/21053 31 of other protease inhibitors obtained from Boehringer Mannheim (1697498) at room temperature for 0.5 h, followed by sonication. Cellular. debris were pelleted by centrifugation at 27,500g for 10 min and the resulting supernatants were incubated with 30 ml of glutathionine- Sepharose (Pharmacia) at 40C overnight. The resin was then washed with 20 mM Tris (pH 150 mM NaC1, 0.1% Tweenand 0.1% 2-mercaptoethanol until the OD 280nm reached <0.01. For removal of GST, the resin with immobilized GSTfusion protein was incubated with 10U of thrombin (Boehringer, Inc.) at 40C in 20 mM Tris (pH 150 mM NaC1, 0.1% Tween-20, 0.1% 2-Mercaptoethanol, and 2.5 mM CaC12 overnight. Released proteins were then purified on Mono Q (HR10/10, Pharmacia) by FPLC using a linear gradient of 0.5M NaCl at pH 8.0 and dialyzed into chaperone assay buffer.

The ability of BAG-2 (SEQ ID NO:4) or BAG-3 (SEQ ID NO:6) to bind Hsc70/ATPase in solution was then examined. GST control or GST-BAG proteins were immobilized on glutathione-Sepharose and tested for binding to labeled in vitro translated (IVT) proteins.

Immunoprecipitation and in vitro GST-protein binding assays were performed as described by Takayama et al., supra (1997), using pCI-Neo flag or pcDNA3-HA into which human Bag-2 (clone #11) or human BAG-3 (clone #28) had been subcloned for in vitro translation of labeled proteins or expression in 293T cells. As shown in Figure 11B, 3 'S-Hsc70/ATPase bound in vitro to GST-BAG-1, GST-BAG-2, and GST-BAG-3 but not to GST-BAG-1(AC) or several other control proteins. BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6) also exhibited little or no binding to themselves or to each other, demonstrating that these proteins do not strongly homo- or hetero-dimerize or oligomerize. It should be noted, however, that BAG-2 (SEQ WO 00/14106 PCT/US99/2105 32 ID NO:4) displayed weak interactions with itself in binding assays and produced a positive result in yeast two-hybrid experiments, demonstrating that it can have the ability to self-associate.

B. Binding of BAG proteins to Hsc70 in vivo The ability of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) proteins to interact in cells with Hsc70 was tested by expressing these proteins with N-terminal Flag epitope tags in 293T human epithelial cells using coimmunoprecipitation assays as described previously (Takayama et al., supra (1997)). cDNAs encoding the Xphage cloned regions of BAG-2 and BAG-3 were subcloned inframe into pcDNA3-Flag. Anti-Flag immune complexes prepared from 293T cells after transfection with plasmids encoding Flag-BAG-1, Flag-BAG-2, or Flag-BAG-3 were analyzed by SDS-PAGE/immunoblot assay. As shown in Figure antiserum specific to Hsc70 detected the presence of BAG proteins associated with Hsc70, whereas control immunecomplexes prepared with IgGi as well as anti-Flag immune complexes prepared from cells transfected with Flag-tagged control proteins, Daxx and Apaf-l, did not contain associated protein. These results further demonstrate that BAG-family proteins specifically bind to C. BIAcore assay of BAG protein binding to the ATPase domain of BAG-1 (beginning at residue 116 of SEQ ID NO:2) is known to bind tightly to the ATPase domain of (Stuart et al., J. Biol. Chem., In Press (1998)). BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) proteins were therefore, examined for their ability to bind to The affinity and binding kinetics of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) to Hsc70/ATPase was also compared to that of BAG-1 (beginning at residue 116 of WO 00/14106 PCTIS99/21053 33 SEQ ID NO:2) for Hsc70/ATPase, using a surface plasmon resonance technique (BIAcore) which has been described previously (Stuart et al., supra, (1998) which is incorporated herein by reference).

BAG-family proteins were produced in bacteria and purified to near homogeneity as shown in Figure 12A and described above in Example I. The purified BAG-1 (beginning at residue 116 of SEQ ID NO:2), -2 (SEQ ID NO:4), and -3 (SEQ ID NO:6) proteins were then immobilized on biosensor chips and tested for their interactions with in the soluble phase. Kinetic measurements were performed using a BIAcore-II instrument with CM5 sensor chip and Amine Coupling Kit (Pharmacia Biosensor AB, Sweden). Briefly, for immobilization of proteins, the sensor chip was equilibrated with HK buffer (10 mM Hepes (pH 150 mM KCL) at 5l/min, then activated by injecting 17 p1 of 0.2M N-ethyl-N'-(3-diethylaminopropyl)carbodiimide and 0.05M N-hydroxysuccinimide (NHS/EDC) followed by 35 ~l of the protein of interest, in 10 mM acetate, pH 3.5-4.5. Excess NHS-ester on the surface was deactivated with 17 Al 1M ethanolamine-HCL (pH8.5). After immobilization, 5jl of regeneration buffer (50 mM phosphate (pH 6.8) and 4M GuHC1) was injected. For binding assays, (Sigma, H8778) was dissolved in HK buffer, and injected at 10 Al/min across the prepared surface at various concentrations. The surface was regenerated after each injection with 5 Al of regeneration buffer. The rate constants Kass and Kdiss were generated with BIAevaluation softward 3.01 (Pharmacia Biosensor AB). Addition of to chips containing BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4) or BAG-3 (SEQ ID NO:6) resulted in concentration-dependent binding, as reflected by an increase in the Response Units (RU) measured at the chip surface (shown in Figure 3B). In contrast, failed to display interactions in BIAcore assays with a variety of control proteins as well as a mutant of BAG-1 WO 00/14106 PCTIS99/21 053 34 lacking a C-terminal portion of the BAG domain which is required for Hsc70-binding (Figure 3B) Furthermore, flowing of various control proteins such as GST, BSA and Bcl-XL over the BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) chips resulted in negligible interaction. These results further demonstrate the specificity with which BAG-family proteins interact with and bind to The rates of Hsc70 binding to BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6) proteins were similar, following pseudo first-order kinetics with estimated association rate constants (Ka) of 2.1, 2.1 and 2.4 x 105 M 1 sec respectively. After allowing binding of Hsc70 to immobilized BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) to reach plateau levels, the chaperone was removed from the flow solution and the dissociation rate was monitored. BAG-1 (beginning at residue 116 at SEQ ID NO:2) and BAG-2 (SEQ ID NO:4) exhibited similar dissociation rates, with relatively slow loss of Hsc70 from the chip surface, resulting in estimated dissociation rate constants (Kd) of 3.0 and 5.0 x 4 sec 1 respectively (see Figure 3B). In contrast, dissociated more rapidly from biosensor chips containing BAG-3 (see Figure 3B), yielding an estimated Kd of 1.7 x 10 3 sec From the kinetic data, the apparent affinities (KD =Kd/Ka) were calculated for binding of Hsc70 to BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6) and were estimated to equal about KD= 1.4nM, KD=2.4nM, and KD=7.4nM, respectively. These results demonstrate that the interactions of BAG-family proteins with Hsc70 occur with apparent affinities sufficient for physiological relevance.

WO 00/14106 PCTIUS99/21 053 EXAMPLE III BAG-family proteins inhibit Hsp70/Hsc70-dependent protein folding This example demonstrates that BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) proteins inhibit Hsp70/Hsc70dependent refolding of denatured proteins similarly to a BAG-1 (beginning at residue 116 of SEQ ID NO:2) protein.

The effects of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) protein on Hsp70/Hsc70-dependent protein refolding was determined using in vitro protein refolding assays similar to those described previously by Takayama et al., supra, 1998; Terada et al., J Cell Biol., 139:1089-1095, 1997, which are incorporated herein by reference. Briefly, luciferase (20AM) was denatured in 25 mM Hepes-KOH, pH 7.2, 50 mM potassium acetate, 5 mM DTT, 6M guanidine hydrochloride at ~25 0 C for 1 h. Denatured luciferase was diluted 1:40 into 25 mM Hepes-KOH, pH 7.2, 50 mM potassium acetate, 5 mM DTT. Hsc70 (1.8 AM), DnaJ (StressGen, Inc.) (0.9AM), and various purified recombinant proteins as indicated were added to refolding buffer (30 mM Hepes-KOH, pH 7.6, 120 mM potassium acetate, 3mM magnesium acetate, 2 mM DTT, 2.5 mM ATP) with 0.2 volume of diluted denatured luciferase to a final concentration of 0.1 AM. Luciferase activity was measured after 1.5 hr incubation at 35 0

C.

The combination of Hsc70 and DnaJ resulted in ATP-dependent refolding of chemically denatured firefly luciferase, with function of over half the denatured enzyme restored in a 90 minute reaction, as monitored by a chemiluminescence assay. In contrast, neither Hsc70 nor DnaJ alone were able to induce substantial refolding of denatured luciferase. Furthermore, little spontaneous WO 00/14106 PCT/US99/21053 36 restoration of luciferase activity was observed with control proteins, BSA, GST or Bcl-XL (see Figure 4A).

Addition of recombinant purified BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) to the above assays in amounts equimolar to Hsc70 (1.8 pM) resulted in striking inhibition of luciferase refolding. BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) displayed somewhat greater inhibitory activity than BAG-1 (beginning at residue 116 of SEQ ID NO:2) as shown in Figure 4A. In contrast, the BAG-1 (AC) protein, which fails to bind Hsc70 as well as several other control proteins, had no effect on luciferase refolding.

In an additional refolding assay, described previously by Minami et al., J Biol. Chem. 271:19617-24, 1996), purified Hsc70 and human DnaJ homolog Hdj-1 (Hsp were used with additional cofactors provided in reticulocyte lysates v:v) to produce a system capable of refolding denatured luciferase. Briefly, additional cofactors included, recombinant Luciferase (Promega: QuantiLum TM), that had been heat denatured at 42 0 C for min, 1.8 pM Hsc70 (Sigma; purified from bovine brain), 0.9 /M Hsp40, and various recombinant purified proteins.

Luciferase activity was measured (Promega luciferase assay kit) using a luminometer (EG&G Berthold, MicroLumat luminometer, Model #LB96P). All results were normalized relative to non-denatured luciferase that had been subjected to the same conditions. Control reactions lacking ATP, Hsc70, or Hsp40 resulted in negligible luciferase refolding.

Various amounts of purified BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6), relative to amounts of Hsc70 were used in the above-described protein refolding assay. Addition of BAG-family proteins resulted in a concentration-dependent WO 00/14106 PCT/US99/21 053 37 inhibition of Hsc70 chaperone activity. Furthermore, the BAG-2 (SEQ ID NO:4)and BAG-3 (SEQ ID NO:6) inhibition of chaperone activity was demonstrated to be as potent as that observed for BAG-1 (beginning at residue 116 of SEQ ID NO:2). In contrast, the BAG-1 (AC) mutant as well as other control proteins did not suppress refolding of denatured luciferase. These results indicate that BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) can inhibit Hsc70/Hsp70 dependent protein refolding activity to the same extent as BAG-1 (beginning at residue 116 of SEQ ID NO:2).

B. BAG competes with Hip for binding to It is known that BAG-1 competes with Hip for binding to Hsc70, with these proteins exerting opposite effects on Hsc70-mediated protein refolding (Hohfeld, J., and Jentsch, Embo 16:6209-6216, 1997, which is incorporated herein by reference). In order to determine whether BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) also compete with Hip for binding to Hsc70, refolding assays were performed as described above in the presence of Hip protein.

Hip was purified as His 6 -protein. The fusion protein was induced from pET28-Hip Prapapanich et al., Mol Cell Biol., 18:944-952, 1998, which is incorporated herein by reference) with 0.1 mM IPTG at 25 0 C for 6h in BL21 cells. Cells from 1L of culture were resuspended into ml of 50 mM Phosphate buffer (pH 150 mM NaC1, and 1% Tween-20 and then incubated with 0.5 mg/ml lysozyme at 25 0 C for 0.5h, followed by sonication. After centrifugation at 27,500g for 10 min, the resulting supernatant was mixed with 15 ml nickel resin (Qiagen, Inc.) at 4 0 C for 3 h with 25 mM imidazol. The resin was then washed with 50 mM phosphate buffer (pH 25 mM imidazol, 150 mM NaCl and 0.1% Tween-20 until the OD280nm WO 00/14106 PCT/US99/21053 38 reached a value of <0.01. His 6 -Hip protein was eluted with 250 mM imidazol in washing buffer (Qiagene, Inc.) and purified on Mono Q (HR10/10 Pharmacia) by FPLC using a linear gradient of 0.5M NaCI at pH 8.0, followed by dialysis in chaperone assay buffer.

In the refolding assay reactions, addition of purified Hip at equimolar concentrations relative to BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) (1.8 AM) completely negated the inhibitory effects of the BAG-family proteins on refolding of denatured luciferase (see Figure 4C). These results demonstrate that the suppression of Hsc70 chaperone activity by BAG-family proteins is reversible, and that Hip antagonizes the effects of not only BAG-1 (beginning at residue 116 of SEQ ID NO:2), but also of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6).

In summary, these results demonstrate that BAGfamily proteins all contain a conserved BAG domain near their C-terminus that binds Hsc70/Hsp70, and that human BAG-family proteins can bind with high affinity to the ATPase domain of Hsc70 and inhibit its chaperone activity through a Hip-repressable mechanism.

EXAMPLE IV EXPANDED NUCLEIC ACID AND AMINO ACID SEOUENCES FOR HUMAN BAG-3. BAG-4 AND Following the procedures disclosed herein, the nucleic acid and amino acids sequences to human BAG-3, BAG-4 and BAG-5 were further expanded. The expanded sequences for BAG-3, BAG-4 and BAG-5 are shown in Figures 15, 16 and 17, respectively, with their respective sequence identification numbers, "SEQ ID NO"s.

EDITORIAL NOTE APPLICATION NUMBER 60383/99 The following Sequence Listing pages 1 to 48 are part of the description. The claims pages follow on pages 39 to WO 00/14106 PCT/US99/21053 SEQUENCE LISTING <110> Reed, John C.

Takayama, Shinichi The Burnham Institute <120> Novel BAG Proteins and Nucleic Acid Molecules Encoding Them <130> FP-LJ 3646 <140> <141> <150> 09/150,489 <151> 1998-09-09 <160> 24 <170> PatentIn Ver. <210> 1 <211> 1291 <212> DNA <213> Homo sapiens <220> <221> <222>

CDS

(1080) <400> 1 acgccgcgct cagcttccat cgctgggcgg tcaacaagtg cgggc ctg get cag cgc 57 Leu Ala Gin Arg 1 ggg Gly ggg gcg cgg aga Gly Ala Arg Arg ccg Pro 10 cga ggc gac cgg Arg Gly Asp Arg gag Glu 15 cgg ctg ggt tcc Arg Leu Gly Ser cgg Arg ctg cgc gcc ctt Leu Arg Ala Leu cca ggc cgg gag Pro Gly Arg Glu ccg Pro cgc cag tcg gag Arg Gin Ser Glu ccc ccg Pro Pro gcc cag cgt Ala Gin Arg ggt Gly ccg cct ccc tct cgg cgt cca cct gcc cgg agt act Pro Pro Pro Ser Arg Arg Pro Pro Ala Arg Ser Thr gcc agc ggg cat gac cga ccc acc agg ggc gcc gcc gcc ggc get cgc WO 00/14 106 Ala Ser Gly PCT/US99/2 1053 His Asp Arg Pro Thr Arg Gly Ala Ala Ala Gly Ala Arg agg ccg Arg Pro cqg atg aag aag Arg Met Lys Lys acc cgg cgc cgc Thr Arg Arg Arg t cg Ser acc Thr cgg agc gag Arg Ser Glu gag Glu ttg acc cgg agc Leu Thr Arg Ser gag Glu 90 gag ttg acc ctg Glu Leu Thr Leu gag gaa gcg acc Glu Glu Ala Thr t gg Trp 100 agt gaa gag gcg Ser Glu Glu Ala acc Thr 105 cag agt gag gag Gin Ser Glu Giu gcg Al a 110 acc cag ggc gaa.

Thr Gin Gly Glu gag atg Glu Met 115 aat cgg agc Asn Arg Ser gag gtg acc Glu Val Thr 135 cag Gin 120 gag gtg acc cgg Glu Val Thr Arg gag gag tcg acc Glu Giu Ser Thr cgg agc gag Arg Ser Glu 130 gtg act gtc Val Thr Val 441 489 agg gag gaa atg Arg Glu Glu Met gcg Al a 140 gca gct ggg ctc Ala Ala Gly Leu a cc Thr 145 acc cac Thr His 150 agc aat gag aag Ser Asn Glu Lys cac His 155 gac ctt cat gtt Asp Leu His Val acc Thr 160 tcc cag cag ggc Ser Gin Gin Gly agc Ser 165 agt gaa cca gtt Ser Glu Pro Val gtc Val1 170 caa gac ctg gcc Gin Asp Leu Ala cag Gin 175 gtt gtt gaa gag Val Vai Glu Glu gtc Val1 180 ata ggg gtt cca Ile Gly Val Pro tct ttt cag aaa Ser Phe Gin Lys ct C Leu 190 ata ttt aag gga Ile Phe Lys Gly aaa tct Lys Ser 195 ctg aag gaa Leu Lys Giu atg Met 200 gaa aca ccg ttg Giu Thr Pro Leu t ca Ser 205 gca ctt gga ata Ala Leu Gly Ile caa gat ggt Gin Asp Gly 210 gaa gag gtt Glu Glu Val tgc cgg Cys Arg gaa cta Giu Leu 230 gtc Val1 215 atg tta att ggg Met Leu Ile Gly aaa Lys 220 aag aac agt cca Lys Asn Ser Pro cag Gin 225 729 777 aag aag ttg aaa Lys Lys Leu Lys cat His 235 ttg gag aag tct Leu Giu Lys Ser gtg Val1 240 gag aag ata gCt Glu Lys Ile Ala gac cag ctg gaa gag ttg aat aaa gag ctt act gga atc cag cag ggt WO 00/14106 WOOO/4106PCTIUS99/2 1053 Asp Gin 245 Leu Giu Giu Leu Asn Lys Giu Leu Thr Gly Ile Gin Gin Gly grin 255 260 ttt ctg ccc aag Phe Leu Pro Lys gat Asp 265 ttg caa gct gaa Leu Gin Ala Glu gct Ala 270 ctc tgc Leu

O

aaa ctt Lys Leu gat agg Asp Arg 275 aga gta aaa Arg Val Lys gac aca ctg Asp Thr Leu 295 gcc Ala 280 aca ata gag cag Thr Ile Glu Gin tt Phe 285 atg aag atc ttg Met Lys Ile Leu gag gag att Giu Giu Ile 290 ttg aaa agg Leu Lys Arg 921 969 atc ctg cca gaa Ile Leu Pro Giu aat Asn 300 ttc aaa gac agt Phe Lys Asp Ser aga Arg 305 aaa ggc Lys Gly 310 ttg gta aaa aag Leu Val Lys Lys cag gca ttc cta Gin Ala Phe Leu gcc Aia 320 gag tgt gac aca Giu Cys Asp Thr 1017 1065 gag cag aac atc Glu Gin Asn Ile tgc Cys 330 cag gag act gag Gin Giu Thr Giu cgg Arg 335 Gig cag tct aca Leu Gin Ser Thr aac Asn 340 ttt gcc ctg gcc gag tgaggtgtag cagaaaaagg ctgtgctgcc ctgaagaatg 1120 Phe Ala Leu Ala Giu 345 gcgccaccag ctctgccgtc tctggatcgg aatttacctg atttcttcag ggctgctggg 1180 ggcaactggc catttgccaa ttttcctact ctcacactgg ttctcaatga aaaatagtgt 1240 ctttgtgatt tgagtaaagc tcctattctg tttttcacaa aaaaaaaaaa a 1291 <210> 2 <211> 345 <212> PRT <213> Homo sapiens <400> 2 Leu Ala Gin Arg Gly Gly Ala Arg Arg Pro Arg Giy Asp Arg Giu Arg Leu Giy Ser Arg Leu Arg Ala Leu Arg Pro Gly Arg Giu Pro Arg Gin Arg Pro Pro Ser Giu Pro Pro Ala Gin Arg Giy Pro Pro Pro Ser WO 00/14106 WO 0014106PCT/US99/2 1053 Ala Arg Ala Gly Ser Thr Ala Ser Gly His Asp Arg Pro Thr Arq Gly Ala Ala 55 Ala Arg Arg Thr Pro 70 Leu Arg Met Lys Lys Thr Arg Leu Thr Arq Ser Glu Glu Se r Thr Arg Ser Gi u 90 Gin Glu Ala Thr Gly Glu Glu 115 Thr Arg Ser T rp 100 Met Giu Glu Ala Ser Glu Glu Arg Arg Ser Leu Ser Giu Ala Thr Gln 110 Glu Glu Ser Ala Gly Leu Asn Arg Ser Val Thr Arg Asp 125 Al a Giu Giu Val 130 Thr Val Thr 135 Se r Giu Glu Met Ala 140 Asp Thr Val Thr 145 Ser His 150 Ser Asn Giu Lys Leu His Val Thr 160 Gin Gin Gly Ser 165 Ile Glu Pro Val Val 170 Ser Asp Leu Ala Gin Val 175 Val Glu Giu Lys Gly Lys 195 Ile Gin Asp Va I Gly Val Pro Phe Gin Lys Ser Leu Lys Giu Met 200 Met Thr Pro Leu Ser 205 Lys Leu Ile Phe 190 Ala Leu Gly Asn Ser Pro Gly Cys Arg 210 Gin Glu Val1 215 Lys Leu Ile Gly Lys 220 Leu Glu Val Glu 225 Glu Leu 230 Gin Lys Leu Lys Giu Lys Ser Val 240 Lys Ile Ala Asp 245 Phe Leu Giu Glu Leu 250 Leu Lys Giu Leu Thr Giy 255 Ilie Gin Gin Lys Leu Asp 275 Leu Giu Glu 290 Gly 260 Arg Leu Pro Lys Asp 265 Thr Gin Ala Giu Arg Val Lys Ile Glu Gin Phe 285 Phe Ala Leu Cys 270 Met Lys Ile Lys Asp Ser Ile Asp Thr Leu 295 Leu Pro Glu Asn 300 WO 00/14106 WO 0014106PCTIUS99I2I 053 Arg Leu Lys Arg Lys Gly 305 310 Leu Val Lys Lys Val Gin Ala Phe Leu Ala 315 320 Giu Cys Asp Thr Val 325 Giu Gin Asn Ile Cys 330 Gin Glu Thr Glu Arg Leu 335 Gin Ser Thr Asn 340 Phe Ala Leu Ala Glu 345 <210> 3 <211> 1179 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (792) <400> 3 gcagccgcgg tgtcgcgaag tcctcccggg ttgcccccgc ggcgtcagag ggagggcggg cgccgcgttg gtgacggcga ccctqcagcc caaggagcgc tccactcgct gccgccggag 120 ggccggtgac ctcttggcta ccccgcgtcg gaggcttag atg gct cag gcg aag 174 Met Ala Gin Ala Lys ate aac gct aaa Ile Asn Ala Lys gcc Ala aae gag ggg cgc Asn Glu Gly Arg ttc Ph e 15 tqe cge tee tc Cys Arg Ser Ser tec atg Ser Met gct gac cgc Ala Asp Arg tcc Ser agc cgc ctg etg Ser Arg Leu Leu gag Gi u 30 agc Ctg gac cag Ser Leu Asp Gin ctg gag etc Leu Glu Leu caa gag aaa Gin Giu Lys 270 agg gtt Arg Val gaa ato Giu Ile agg cag Arg Gin gct ttg aga gaa Ala Leu Arg Glu gca Ala gca act gct gtt Ala Thr Ala Val gag Glu ctt ctg gaa atg Leu Leu Glu Met atc agt gac gga Ile Ser Asp Gly 75 atc Ile 60 cac agt atc caa His Ser Ile Gin aa t Asn age cag gac atg Ser Gin Asp Met gaa aga gaa gaa tta aat ctg act gca Glu Arg Giu Glu Leu Asn Leu Thr Ala 80 aac Asnr WO 00/14106 ogt ttg atg Arg Leu Met aga aac coo Arg Asn Pro gat gag gtg Asp Giu Vai 120 PCT/US99/2 1053 gga aga act ctc aco gtt gaa gtg tca gta gaa aca att Gly Arg Thr Leu Thr Val Glu Val Ser Val Giu Thr Ile cag Gin 95 aag Lys cag Gin 105 gtc Val1 caa gaa tcc Gin Glu Ser cta Leu 110 gat Asp cat goc aca His Aia Thr agg Arg 115 gc Al a 100 att att Ile Ile aag agt Lys Ser aat aag ttt Asn Lys Phe ctg Leu 125 goa Ala gat ttg gga Asp Leu Gly cat tta His Leu 135 cca gtt Pro Val atg tog cto tac Met Ser Leu Tyr gat cag aag ttt Asp Gin Lys Phe 155 aag aaa att. aag Lys Lys Ile Lys agt Ser 140 oaa Gin tgt toa tot Cys Ser Ser gag gtg Giu Val 145 ggo tgt Gly Cys too ata gta Ser Ile Val oca oat ggg Pro His Gly got ott. gaa Ala EU3 Giu 165 aga aat att Arg Asn Ile 510 558 606 654 702 750 150 ga t Asp cag Gin aga aga tta Arg Arg Leu 170 aag Lys gag act otg Giu Thr Leu 175 ott Leu

I

~ga got ;iy Ala gaa aac tot. gao Giu Asn Ser Asp 185 ggt too aaa aot Gly Ser Lys Thr goo ato aag Ala Ile Lys ota Leu 190 got Ala tta Leu ga a Glu otg oaa oaa aat Leu Gin Gin Asn tagtcttoaa ttttaattga tcagatqagg attttaatta oatatttcao gggoaaaaag tttagtttao acctaagago taactagtto aaaatattco totatotaga tattotgtgg catgaotgot tagaaattct atttacacaa tttgttaggt atcaagtato gattttttag atgaataoat.

ttttcctgtc ttactgg tacaoaa ataaoca ttoagtt attgaat agtttgt tggcatj gag oat tot aaa C Glu His Ser Lys C 195 ago aga tto a t_ Se -Ag Ph:~i~ S e x 2 1 0 Lggt gtaaaaatga ta Ott agttgacact ga .ttg tgaataaoaa aa tot tgtottgtao ta ggg gaaaaoaaac gt gaa tcacgcagtc ac ~aaatacta itagttgtt otagoaat ggatctag toagotag :ottgggca 852 912 972 1032 1092 1152 1179 WO 00/14106 <210> 4 <211> 211 <212> PRT <213> Homo sapiens PCT/US99/2 1053 <400> 4 Met 1 Arg Asp Ala Gin Ala Ser Ser Ser Gin Leu Glu Ile Asn Ala Lys Ala Asn Giu Gly Arg Phe Cys Ala Asp Arg Ser Ser 25 Ala Arg Leu Leu Leu Arg Val Val Giu Gin Giu 40 Leu Leu Arg Giu Al a His Giu Ser Leu Ala Thr Ala Ser Ile Gin Giu Lys Giu Leu Giu Met Asn Ser Ile Glu Gin Asp Met Arg Arg Ile Ser Asp Arg Giu Giu Leu Leu Thr Ala Leu Met Giy Arg Gin Leu Thr Val Giu Val Ser Vai Giu Ala Thr Arq 115 Giv Asn Ala Arg Asn Pro Gin 105 Val1 Gin Giu Ser Ile Asp Giu Val 120 Met Asn Lys Phe Leu 125 Al a Leu Lys His 110 Asp Asp Leu Cys Ser Ser Lys Ser His Ser Leu Tyr 130 Giu Val Ser 140 Gin Pro His Gly 145 Gly Pro 150 Asp Asp Gin Lys Phe 155 Lys Ser Ile Val Ile 160 Cys Ala Leu Gin Lys Lys Ile 170 Lys Arg Arg Leu Giu Thr 175 Leu Leu Arg His Ser Lys 195 Arg Phe Asn 210 As n 180 Gly Glu Asn Ser Asp 185 Thr Ala Ile Lys Leu Leu Giu 190 Ala Giu Ser Ala Gly Ser Lys 200 Leu Gin Gin As n 205 WO 00/14106 WO 0014106PCT[US99/2 1053 <210> <211> 2528 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (2031) <400> gcg gag oto cgc Ala Glu Leu Arg 1 atc Ile 5 caa ccc cgg gc Gin Pro Arg Ala qcg Ala 10 gcc aac ttc tct Ala Asn Phe Ser gga Ctg Gly Leu gao cag aag Asp Gin Lys ttc ccc tct Phe Pro Ser cta gcc ggc cag Leu Ala Gly Gin ota cot. coo ttt Leu Pro Pro Phe atc toc tc Ile Ser Ser cac ccc tot His Pro Ser ggc ago gag gag Giy Ser Giu Giu got.

Ala att too aga. oao Ile Ser Arg His otg gcc Leu Ala aog toa. ccc cog Thr Ser Pro Pro ct Pro 55 tta att cat aaa, Leu Ile His Lys ggt goc ogg ogo Gly Ala Arq Arg acg gcg gcg gc Thr Ala Ala Ala cgg Arg cg Arq ccc gga cac gto Pro Gly His Val ggc Gly ggo gga gag ggg Gly Giy Glu Gly Cco Pro 75 oca gag act cgg Pro Glu Thr Arg ogc Arg cog gag oca, gog Pro Giu Pro Ala cgc aco ogo gc Arg Thr Arg Ala oca gog Pro Ala ggc aga c00 Gly Arg Pro ccc ago atg ago Pro Ser Met Ser gc Ala 105 goc aco cac tog Ala Thr His Ser ccc atg atg Pro Met Met 110 coo gga tgg Pro Gly Trp cag gtg gog too ggo aac ggt Gin Val Ala Ser Gly Asn Gly 115 gao Asp 120 cgc gao cot ttg Arg Asp Pro Leu gag ato Glu Ile 130 aag ato gao cog Lys Ile Asp Pro cag Gin 135 aco ggc tgg coo Thr Gly Trp Pro ttc Phe 140 tto gtg gao cac Phe Val Asp His aao ago cgo aco act acg tgg aac gao cog ogo gtg coo tot gag ggc WO 00/14106 PCT/US99/21053 Ser Arg Thr Thr Thr Trp Asn Asp Pro Arg Val Pro Ser Giu Gly ccc aag gag Pro Lys Glu agg ctg ccg Arg Leu Pro cca ggo tao Pro Gly Tyr 195 act cca Thr-Pro 165 tcc tct qcc aat Ser Ser Ala Asn ggc cct Gly Pro 170 tcc cqg gag Ser Arg Glu qgc tct Gly Ser 175 528 cct Pro 180 got agg gaa ggo Ala Arq Glu Gly cac His 185 cct gtg tac cc Pro Val Tyr Pro cag ctc cga Gin Leu Arg 190 gag aac cgg Glu Asn Arg 576 624 att ccc att cct Ile Pro Ile Pro ctc cat gaa ggc Leu His Glu Gly got Ala 205 cag gtg Gin Val 210 cac cot ttc cat His Pro Phe His gtc Va1 215 tat ccc cag cct Tyr Pro Gin Pro ggg Gly 220 atg cag cga ttc Met Gin Arg Phe cga Arg 225 act gag gcg gca Thr Giu Ala Ala gcg got cct cag Ala Ala Pro Gin agg Arg 235 tcc cag tca cct Ser Gin Ser Pro ctg Leu 240 672 720 768 cgg ggc atg cca Arg Gly Met Pro gaa Glu 245 acc act cag cca Thr Thr Gin Pro gat Asp 250 aaa cag tgt gga Lys Gin Cys Gly cag gtg Gin Val 255 gca gcg gcg Ala Ala Ala tcc cag tct Ser Gin Ser 275 gca gcc cag ccc Ala Ala Gin Pro oca Pro 265 gcc tcc cac gga Ala Ser His Gly cct gag cgg Pro Giu Arg 270 tcc tog gc Ser Ser Ala 816 864 oca got gc tct Pro Ala Ala Ser gac Asp 280 tgo tca too tca Cys Ser Ser Ser too Ser 285 agc ctg Ser Leu 290 cot too too ggc Pro Ser Ser Gly ago ago ctg ggc Ser Ser Leu Gly cac cag otc ccg His Gin Leu Pro cgg Arg 305 ggg tao ato too Gly Tyr lie Ser ccg gtg ata cac Pro Val Ile His cag aac gtt aco Gin Asn Val Thr cgg Arg 320 912 960 1008 oca goa gc cag Pro Ala Ala Gin cCC Pro 325 too tto cac aaa Ser Phe His Lys gc Ala 330 cag aag acg cac Gin Lys Thr His tao oca Tyr Pro 335 gcg cag agg ggt gag tao cag aco cac cag cot gtg tao cac aag at 1 1056 WO 00/14106 Ala Gin Arg cag ggg gat Gin Gly Asp 355 PCTIUS99/21 053 Gly 340 Giu Tyr Gin Thr His 345 Gin Pro Val gac tgg gag ccc Asp Trp Giu Pro cgg Arg 360 ccc ctg cgg gcq Pro Leu Arg Ala Tyr His Lys Ile 350 gca. tcc ccg ttc Ala Ser Pro Phe 365 tca cca gcc agq Ser Pro Ala Arg 1104 agg tca Arg Ser 370 tct gtc cag ggt Ser Val Gin Giy gca Al a 375 tcg aqc cgg gag Ser Ser Arg Giu ggc Gly 380 agc acg cca ctc Ser Thr Pro Leu cac His 390 tcc ccc tcg ccc Ser Pro Ser Pro cqt gtg cac acc Arg Val His Thr 1152 1200 1248 gtc gac agg cct Vai Asp Arg Pro cag Gin 405 cag ccc atg acc Gin Pro Met Thr cat His 410 cqa gaa act gca Arg Giu Thr Ala cct gtt Pro Val 415 tcc cag cct Ser Gin Pro gaa. ctc cct Giu Leu Pro 435 gaa.

Giu 420 aac aaa cca. gaa Asn Lys Pro Giu agt Ser 425 aag cca ggc cca.

Lys Pro Gly Pro gtt gga. cca Val Gly Pro 430 aaa gag gtg Lys Giu Val 1296 1344 cct gga cac atc Pro Gly His Ile cca Pro 440 att caa gtg atc Ile Gin Val Ile cgc Arg 445 gat tct Asp Ser 450 aaa cct gtt tcc Lys Pro Val Ser cag Gin 455 aag ccc cca cct Lys Pro Pro Pro ccc Pro 460 tct gag aag gta Ser Giu Lys Val gag Giu 465 gtg aaa gtt ccc Val Lys Vai Pro cct Pro 470 gct cca gtt cct Ala Pro Val Pro cct cct ccc agc Pro Pro Pro Ser 1392 1440 1488 ggc cct tct gct Gly Pro Ser Ala gt c Val1 485 ccc tct tcc ccc Pro Ser Ser Pro aag Lys 490 agt gtg gct aca Ser Val Ala Thr gaa gag Giu Giu 495 agg gca gcc Arg Ala Ala gga gaa gcc Gly Giu Ala 515 ccc Pro 500 agc act gcc cct Ser Thr Ala Pro gca Al a 505 gaa gct aca cct Giu Ala Thr Pro cca aaa cca Pro Lys Pro 510 aaa gtg gaa.

Lys Val Giu 1536 1584 gag gct ccc cca Giu Ala Pro Pro aaa Lys 520 cat cca gga qtg His Pro Gly Val ct g Leu 525 gcc atc ctg gag aag qtg cag ggg ctg gag cag gct gta. gac aac ttt 13 1632 WO 00/14106 WO 0014106PCTIUS99/2 1053 Ala Ile 530 Leu Glu Lys Val Gin 535 Giy Leu Glu Gin Al a 540 Val Asp Asn Phe gaa Giu 545 ggc aag aag act Gly Lys Lys Thr aaa aag tac ctg Lys Lys Tyr Leu atc gaa gag tat Ile Glu Giu Tyr ttg Leu 560 1680 1728 acc aaa gag ctg Thr Lys Glu Leu ctg Leu 565 gcc ctg gat tca Ala Leu Asp Ser gtg Val1 570 gac ccc gag gga Asp Pro Glu Gly cga gcc Arg Ala 575 gat gtg cgt Asp Vai Arg ttg gaa aaa Leu Giu Lys 595 gcc agg aga gac Ala Arg Arg Asp ggt Gly 585 gtc: agg aag gtt Val Arg Lys Val cag acc atc Gin Thr Ile 590 caa gtc cag Gin Val Gin 1-776 1824 ctt gaa cag aaa Leu Giu Gin Lys gcc Al a 600 att gat gtc cca Ile Asp Vai Pro ggt Gly 605 gtc tat Vai Tyr 610 gaa ctc cag ccc Glu Leu Gin Pro aac ctt gaa gca Asn Leu Glu Ala gat Asp 620 cag cca. ctg cag Gin Pro Leu Gin gca Al a 625 atc atg gag atg Ile Met Glu Met ggt Gly 630 gcc gtg gca gca Ala Val Ala Ala gac Asp 635 aag ggc aag aaa Lys Gly Lys Lys aat Asn 640 1872 1920 1968 gct gga aat gca Ala Gly Asn Ala gat ccc cac aca Asp Pro His Thr gaa Glu 650 acc cag cag cca Thr Gin Gin Pro gaa gcc Glu Ala 655 aca gca gca Thr Ala Ala gcg Al a 660 act tca aac ccc Thr Ser Asn Pro agc Ser 665 agc: atg aca gac Ser Met Thr Asp acc cct ggt Thr Pro Gly 670 2016 aac cca gca gca ccg Asn Pro Ala Ala Pro 675 tagcctctgc cctgtaaaag tcagactcgg aaccgatgtg 2071 tgCtttaggg attttagttg catgcatttc agagacttta ggtcagttgg ttttgattag 2131 ctgcttggta tgcagtactt gggtgaggca aacactataa agggctaaaa gggaaaatga 2191 tgcttttctt caatattctt actcttgtac aattaangaa gttgcttgtt gtttgagaag 2251 tttaaccccg ttgcttgttc tgcagccctg tcnacttggg cacccccacc acctgttagc 2311 tgtggttgtg cactqtcttt tgtagctctg gactggaggg gtagatgggg agtc aattac 2371 WO 00/14106 WO 00/4 106PCTIUS99/2 1053 ccatcacata aatatqaaac atttatcaga aatgttgcca ttttaatgag atgattttct 2431 tcatctcata attaaaatac ctgactttag agagaqtaaa atgtgccagg agocatagga 2491 atatctgtat gttggatgac tttaatgcta catttth 2528 <210> 6 <211> 677 <212> PRT '<213> Homo sapiens <400> 6 Al a 1 Asp Phe Giu Leu Arg Gin Lys Phe Pro Ser Gly Gin Pro Arg Ala Al a 10 Leu Ala Asn Phe Ser Giy Leu Ala Gly Gin Leu 25 Ile Pro Pro Phe Ser Giu Giu Ser Arg His Ile Ser Ser His Pro Ser Arg Arg Arg Leu Ala Thr Ser Pro Pro Leu Pro Pro 55 Ile His Lys Gly Thr Gly His Val Pro Glu Thr Arg Gly Gly 70 Pro Giu Ser Met Gly Glu Gly Pro Ala Pro Ser Ala Ala Ala Ala Ala Arg Thr Arg Ala Pro Ala Gly Arg Pro Gin Val Ala 115 Glu Ile Lys 130 Asn Ser Ara Gin 100 Ser Thr His Ser 105 Arg Gly Asn Gly Asp 120 Thr Asp Pro Leu Pro 125 Phe Pro Met Met 110 Pro Gly.Trp Val Asp His Ile Asp Pro Thr Thr Thr 150 Thr Pro Ser Gly Trp Pro Phe 140 Val1 Asn Asp Pro 145 Pro Arg 155 Pro Pro Ser Giu Lys Giu Ser Ala Asn 165 Gly 170 Pro Ser Arg Giu Gly Ser 175 Leu Arg Arg Leu Pro Pro Ala Arg Giu Gly His Val Tyr Pro Gin WO 00/14106 WO 0014106PCT/US99/2 1053 180 Ile 185 Le u Pro Giy Tyr 195 Gin Val His Pro Ile Pro His Giu Giy Al a 205 Met 190 Giu Asn Arg Gin Arg Phe Pro Phe His 210 Arg Thr Val1 215 Ala Pro Gin Pro Gly 220 Ser Giu Ala Ala 225 Arg Ala 230 Thr Aia Pro Gin Arg 235 Lys Gin Ser Pro Leu 240 Val Giy Met Pro Giu 245 Ala Thr Gin Pro Asp 250 Al a Gin Cys Gly Ala Ala Ala Ser Gin Ser 275 Ser Leu Pro Ala Gln Pro Pro 265 Cys Ser His Giy Ala Ala Ser Asp 280 Ser Ser Ser Ser Ser 285 His Pro Glu Arg 270 Ser Ser Ala Gin Leu Pro Ser Ser Gly Ser Leu Gly 290 Arg Gly Ser 300 Gin Tyr Ile Ser 305 Pro Ile 310 Ser Vai Ile His Asn Val Thr Arg 320 Ala Ala Gin Pro 325 Glu Phe His Lys Al a 330 Gin Lys Thr His Tyr Pro 335 Ala Gin Arg Gin Gly Asp 355 Arg Ser Ser Tyr Gin Thr His 345 Pro Pro Vai Tyr Trp Glu Pro Arg 360 Ser Leu Arg Ala Ala 365 Ser His Lys Ile 350 Ser Pro Phe Pro Ala Arg Val Gin Gly Ser Arg Glu 370 Ser Ser Gly 380 Arg Thr Pro Leu 385 Val1 His 390 Gin Pro Ser Pro Val His Thr Asp Arg Pro Gln 405 As n Pro Met Thr His 410 Lys Giu Thr Ala Pro Val 415 Ser Gin Pro Giu Leu Pro Lys Pro Giu Ser 425 Ile Pro Gly Pro Val Gly Pro 430 Lys Glu Val Gly His Ile Pro 13 Gin Val Ile Arg WO 00/14106 435 Asp Ser Lys PCTIUS99/21 053 445 Ser Glu 465 Gly Lys Pro Val Ser Gin 455 Val Pro Pro Ala 470 Ala Val Pro Ser Pro Pro Pro Pro 460 Pro Giu Lys Val Pro Val Proi Pro Pro Ser Pro 480 Pro Ser Ser Pro Lys 490 Glu Val Ala Thr Giu Glu 495 Arg Ala Ala Gly Glu Ala 515 Ala Ile Leu 530 Glu Gly Lys 545 Thr Lys Glu Asp Val Arg Leu Glu Lys 595 Val Tyr Giu Pro 500 Glu Thr Ala Pro Ala 505 His Ala Thr Pro Ala Pro Pro Lys 520 Gly Pro Gly Val Leu 525 Val Pro Lys Pro 510 Lys Vai Giu Asp Asn Phe Glu Lys Val Gin 535 Lys Leu Glu Gin Al a 540 Ile Lys Thr Leu Leu 565 Gin Ala Asp 550 Ala Lys Tyr Leu Met 555 Asp Giu Glu Tyr Leu 560 Leu Asp Ser Val1 570 Val1 Pro Giu Gly Arg Ala 575 Arq Arg Asp 580 Leu Gi y 585 Ile Arq Lys Val Glu Gin Lys Asp Val Pro Gly 605 Gin Gin Thr Ile 590 Gin Val Gin Pro Leu Gin Leu Gin Pro 610 Ala Ile Se r 615 Al a Leu Giu Ala Asp 620 Lys Met Glu Met 625 Ala Gly 630 Asp Val Ala Ala Asp 635 Thr Gly Lys Lys Gly Asn Ala Giu 645 Pro His Thr Giu 650 Gin Gin Pro Thr Ala Ala Asn Pro Ala 675 Ala Thr 660 Ala Pro Ser Asn Pro Ser Ser Met Thr Asp Thr Pro Giy 665 670 WO 00/14106 <210> 7 <211> 1010 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (323)..(1009) <400> 7 aoqatatcot gtaagaocaa cgtatggtcc aacataocccc attatgcacc tggttatact catotggcaa cagcoaact aotgaagoao ooootottaa atgaccctgc ccattatc PCT/US99/2 1053 gaattgcaag ccaggccctg cagaccagtt ccagtctotc ggggcaggtt tt. atg gag gcoagagttt qaattottat acaaatggag gggoaaatac tgcctcatac tcaggggott actccacaga agttcoaagt aottaccgtt gttggatcta tooccagoag gaotgtoaag ocaggatatc ogoottcaca gaacotgga atg gta ato gta gtg tto cac aat 120 180 240 300 352 Met Glu Met Val Ile Val Val Phe His Asn cac ggc oga. otg His Gly Arg Leu tao Tyr ggt Gly gao cac aag aaa Asp His-Lys Lys gat Asp 20 tgg Trp gog tgg got tot Ala Trp Ala Ser oot ggt Pro Gly got. tat gga Ala Tyr Gly gca ooa 000 Ala Pro Pro ago agt ggc Ser Ser Gly atg Met ggo Gly ggo ogt tat Gly Arg Tyr 000 Pro 35 aot Thr oct toa toa Pro Ser Ser aat oto tac Asn Leu Tyr gaa. agt act Giu Ser Thr gcg 000 toa Ala Pro Ser oca tgg Cot Pro Trp Pro cag oag 000 Gin Gin Pro 400 448 496 544 592 640 tot 000 cag Ser Pro Gin toa Ser 65 tat Tyr cot. toa 000 Pro Ser Pro aag Lys ca o His gat tot toa tao Asp Ser Ser Tyr 000 Pro 80 agt Ser ago oaa toa Ser Gin Ser oca Pro oaa Gin too Ser ago atg aac Ser Met Asn tog ggg aoa Ser Gly Thr 105 ogq Arg gt~g Val1 aao ttt Cot Asn Phe Pro gto oat oag Val His Gin tao Tyr 100 WO 00/14106 aac aat gat Asn Asn Asp gag cot cag Giu Pro Gin 125 PCTIUS99/21 053 ga t Asp 110 tca gat cit tig Ser Asp Leu Leu gat Asp 115 tcc caa gtc cag Ser Gin Val Gin tat agi got Tyr Ser Ala 120 aac aat caa Asn Asn Gin otg tat ggt aat Leu Tyr Gly Asn gcc Al a 130 acc agt gac cat Thr Ser Asp His ccc Pro 135 gat caa Asp Gin 140 agt agc agt ct Ser Ser Ser Leu gaa gaa tgt gia Glu Giu Cys Val ct Pro 150 ica gat gaa agt Ser Asp Glu Ser cot cog agi att Pro Pro Ser Ile aaa ato ata oat Lys Ile Ile His gig Val1 165 otg gag aag gic Leu Glu Lys Val oag Gin 170 784 832 880 tat cii gaa caa Tyr Leu Glu Gin gaa Glu 175 gia gaa gaa tt Val Glu Giu Phe gga aaa aag aoa Gly Lys Lys Thr gao aaa Asp Lys 185 goa tao tgg Ala Tyr Trp gat tca gt Asp Ser Vai 205 cig gaa gaa aig Leu Giu Giu Met cia Leu 195 aco aag gaa ott Thr Lys Giu Leu ttg gaa ctg Leu Glu Leu 200 goc aga aaa Ala Arg Lys 928 976 gaa act ggg ggo Glu Thr Giy Gly cag Gin 210 gao tot gia cgg Asp Ser Val Arg cag Gin 215 gag got Glu Ala 220 git igi aag ati Val Cys Lys Ile cag Gin 225 goc ata tig gaa a Ala Ile Leu Glu 1010 <210> 8 <211> 229 <212> PRT <213> Homo sapiens <400> 8 Met Glu Met Val Ile 1 5 His Lys Lys Asp Ala Val Val Phe His His Gly Arg Leu Tyr Asp Trp Ala Ser Pro 25 Giy Ala Tyr Gly Met Gly Gly Gly Asn Leu Arg Tyr Pro Trp Pro Ser Ser Ala 40 Pro Ser Ala Pro Pro WO 00/14106 WO 0014106PCTIUS99/2 1053 Tyr Met Thr Glu Ser Thr Pro Trp Pro Ser Ser Gly Ser Pro Asp Ser Ser Tyr Gin Pro Pro Pro Ser Pro Pro Val Gin Gin Pro Tyr Ser Gin Ser Asp Gin Ser Met Asn Arg 90 His Asn Phe Pro Cys Ser Val His Gin Leu Leu Asp 115 Tyr 100 Giu Ser Ser Giy Thr 105 Vai Asn Asn Asp Asp Ser Asp 110 Leu Tyr Gly Ser Gin Vai Gin Tyr 120 Ser Aia Giu Pro Asn Aia 130 Thr Ser Asp His Pro 135 Asn Asn Gin Asp Gin 140 Ser Ser Ser Leu Pro 145 Giu Giu Cys Vai Ser Asp Giu Ser Thr 155 Pro Pro Ser Ile Lys 160 Lys Ile Ile His Vai 165 Leu Giu Lys Val Tyr Leu Giu Gin Giu Vai 175 Giu Giu Phe Giu Met Leu 195 Giy Lys Lys Thr Asp 185 Lys Ala Tyr Trp Leu Leu Giu 190 Glu Thr Giy Thr Lys Giu Leu Leu 200 Glu Leu Asp Ser Val1 205 Giy Gin 210 Asp Ser Val Arg Gin 215 Ala Arg Lys Glu Ala 220 Val Cys Lys Ile Gin 225 Aia Ile Leu Glu <210> <211> <212> <213> <220> <221> <222> 9 689

DNA

Homo sapiens

CDS

(482) <220> <221> unsure WO 00/14106 WO 0014106PCTIUS99/2 1053 <222> (105) <223> any amino acid <400> 9 ga gaa 'ata aaa aa t gaa ctt ctc caagca caa aac cct tct gaa ttg Gi Ile Lys Asn Giu Leu Leu Gin Ala Gin Asn Pro Ser Gin Len tac otg ago tc Tyr Leu Ser Ser aaa Lys aca gaa ttg cag Thr Glu Leu Gin ggt Gi y 25 tta att gga cag Leu Ile Giy Gin ttg gat Len Asp gag gta agt Glu Val Ser gca gtg atc Aia Val Ile gaa aaa aac ccc Gin Lys Asn Pro t gc Cys ato cgg gaa gc Ile Arg Glu Aia agg aga aga Arg Arg Arg ttg aag gag Len Lys Gin 143 191 gag gtg caa act Giu Val Gin Thr ctg Leu 55 ato aca tat att Ile Thr Tyr Ile gac Asp gcc ctt Ala Leu gag aaa aga aag Giu Lys Arg Lys ctg Leu 70 ttt gct tgt gag Phe Ala Cys .Giu gag Glu cac cca tco cat His Pro Ser His gcc gtc tgg aac Ala Val Trp Asn gt c Vai ctt gga aac ttg Len Gly Asn Len tot Ser 90 gag atc cag gga Giu Ile Gin Gly gaa Gin gtt ctt tca ttt Val Len Ser Phe gga aat cga ac Gly Asn Arg Thr gat Asp 105 aag aac tao atc Lys Asn Tyr Ile cgg ctg Arg Len 110 gaa gag otg Gin Giu Leu ctc Len 115 aco aag oag ctg Thr Lys Gin Leu cta Leu 120 gcc ctg gat gct Ala Leu Asp Ala gtt gat cog Val Asp Pro 125 gtg agg ott Vai Arg Leu 383 cag gga Gin Gly gog cag Ala Gin 145 gag aag tgt aag Giu Lys Cys Lys got Ala 135 goc agg aaa caa Ala Arg Lys Gin got.

Ala 140 aat att oto ago Asn Ile Leu Ser ctc gao ctg aaa Len Asp Len Lys tot Ser 155 gat gaa tgg gag Asp Giu Trp Gin tao Tyr 160 tgaaatacoa gagatctcao ttttgatact gttttgcact tcatatgtgc WO 00/14106 PCTIUJS99/2 1053 ttctatgtat agagagcttt cagttcattq atttatacgt gcatatttca gtctcagtat 592 ttatgattga agcaaattct attcaqtatc tgctgctttt gatqttqcaa qacaaatatc 652 attacagcac gttaactttt ccattcggat caaaaa-a 689 <210> <211> 160 <212> PRT <213> Homo sapiens <400> Glu Ile 1 Lys Asn Glu Leu Leu Gin Ala Gin Asn Pro Ser Giu Leu Tyr Leu Val Ser Ser Lys Ser Xaa Giu Thr Giu Leu Gin Gly 25 Ile Leu Ile Gly Gin Lys Asn Pro Val Ile Giu Cys 40 Ile Arg Giu Ala Arg Le u Leu Asp Giu Arq Arg Ala Lys Giu Ala Val Gin Thr Leu Glu Leu 55 Phe Thr Tyr Ile Asp His Lys Arg Lys Al a Leu 70 Leu Ala Cys Giu Glu Glu Pro Ser His Val Trp Asn Val Gly Giy Asn Leu Ile Gin Gly .Giu Val Leu Ser Phe Giu Leu Leu 115 Gly Giu Giu 130 Asn Arg Thr Asp 105 Al a Asn Tyr Ile Lys Gin Leu Leu 120 Al a Leu Asp Ala Val1 125 Val1 Arg Leu Giu 110 Asp Pro Gin Arg Leu Ala Lys Cys Lys Ala 135 Arq Lys Gin Al a 140 Gin Asn Ile Leu Ser Tyr Leu Asp Leu Lys Ser Asp Glu Trp Giu Tyr <210> 11 <211> 246 <212> DNA <213> Caenorhabditis elegans WO 00/14106 WO 00/4 106PCT[US99/2 1053 <400> 11 atgtctttcc qcctcttcgt tgaaatattt cactttcttt tccaqctttt tccccatctc gacctgcttt ggtttttcga gaaaaccacg ttccaaatca qcgacatctc tcaaattgag 120 atcataggct ttttgaagat tgctcaaatt atgcttctca tattgcatqa gcattttgaa 180 gcccgcgtca tcaaccaaag cattttttcc acccatcaca atgattttat cattttcttt 240 aaaatt 246 <210> 12 <211> 210 <212> PRT <213> Caenorhabditis elegans <400> 12 Met 1 Leu Leu Lys Val Asn Glu Giu Asn Arg Asp Arg Val1 5 Gin Ser Cys Ser Ser Gin Thr Thr Ile Asp Ile Gly Glu Asp Glu 25 Asn Ile Leu Thr Ile Ala Thr Lys Leu Leu Asp 40 Phe Asp Val Asp Val1 Asp Leu Giy Gin Giu Thr Met Asp Val Ser His Arg Giy Leu Ser Lys 55 Lys Leu Gin Giy Ala Ile Thr Leu Asn Gly Phe 70 Val Giu Asn Asp Lys 75 Phe Ile Val Met Gly Lys Asn Ala Leu Asn ASP Asp Ala Gly 90 Gin Lys Met Leu Met Gin Tyr Glu Lys Leu Arg Asp 115 Gin Val Glu Leu Ser Asn Leu 105 Arg Lys Ala Tyr Ala Asp Leu Giu 120 Leu Gly Phe Leu Giu 125 Lys Asp Leu Asn 110 Lys Pro Lys Tyr Phe Asn Met Gly Lys Giu Lys Lys 130 Giu Glu 145 Vali 140 Ala Giu Arg Giu Thr Leu Asp Giy Met Asn Ile Ile 155 160 WO 00/14106 WO 0014106PCTIUS99/21053 Thr Glu Thr Thr Pro 165 Giu Asn Gin Ala Lys 170 Arg Asn Arg Glu Lys Arg 175 Lys Thk Leu Leu Leu Arg 195 Asn Giy Ile Gin Thr Leu Leu Asn Gin Asn Asp Ala 185. 190 Arg Leu Gin Giu Tyr 200 Gin Ser Val Leu Giy Asp Ile Pro Giu 210 <210> 13 <211> 1377 <212> DNA <213> Gaenorhabditis elegans <220> <221> CDS <222> (1377) <400> 13 atg cca Met Pro 1 gtc gtg aac Val Val Asn 5 ata oca atc aaa Ile Pro Ile Lys ata Ile 10 ctt ggt cag aat Leu Gly Gin Asn caa tca Gin Ser cat agt cga His Ser Arg cca cca cag Pro Pro Gin aac too tcg tct Asn Ser Ser Ser tot Ser gtt gao aac gat Val Asp Asn Asp cga aat caa Arg Asn Gin tct cag caa Ser Gin Gin cag cca cot caa Gin Pro Pro Gin cog Pro 40 caa oca caa cag Gin Pro Gin Gin caa Gin caa tac Gin Tyr cag cag got oca Gin Gin Ala Pro aac Asn 55 gtg aat acc aat Val Asn Thr Asn atg Met cat oat tcc aac His His Ser Asn gga Gly ttc tca cot aac Phe Ser Pro Asn tto Phe oca tot ogt agt Pro Ser Arg Ser att cog gao ttt Ile Pro Asp Phe agt ttt tca tct Ser Phe Ser Ser ggg Gi y tto oca aac gat Phe Pro Asn Asp t ct Ser 90 gaa tgg tot tcg Glu Trp Ser Ser aat ttc Asn Phe WO 00/14 106 ccg tcg ttt Pro Ser Phe PCTIUS99/21 053 cca Pro 100 aat ttc cca agt Asn Phe Pro Ser ttc tca aat gga Phe Ser Asn Gly agt tct aat Ser Ser Asn 110 tog cca- aac Ser Pro Asn ttc ct gat ttt cca aga ttc Phe Pro Asp Phe Pro Arg Phe 115 gga Giy 120 aga gat gga gga Arg Asp Giy Gly cta Leu 125 384 432 cca ccg Pro Pro 130 atg caa gga tao Met Gin Gly Tyr agg Arg 135 aga agt oca aca Arg Ser Pro Thr cca Pro 140 aca tca act caa Thr Ser Thr Gin tct Ser 145 oca act tct aca Pro Thr Ser Thr aga cgc aao tct Arg Arg Asn Ser cag Gin 155 cag aat caa got Gin Asn Gin Ala cca caa tat tct Pro Gin Tyr Ser caa caa cca caa Gin Gin Pro Gin gct caa caa cgt Aia Gin Gin Arg cag aca Gin Thr 175 act ogt Thr Arg 480 528 576 act cct cog Thr Pro Pro t ca Ser 180 aca aaa got toa Thr Lys Ala Ser tot Ser 185 oga cca oca tot Arg Pro Pro Ser cgt Arg 190 gaa. oca aag Giu Pro Lys 195 gaa cot gag gta Giu Pro Giu Val gag aga. oca gca Giu Arg Pro Ala gt t ValI 205 att oca ttg Ile Pro Leu 624 oca tat Pro Tyr 210 gag aag aag gag Giu Lys Lys Glu aaa Lys 215 ooa otg gag aag Pro Leu Glu Lys aaa Lys 220 ggt agt ogt gat Gly Ser Arg Asp tot Ser 225 gga aag ggt gat Gly Lys Giy Asp gag Giu 230 aac ott gaa gag Asn Leu Giu Glu aac Asn 235 att goc aag ato Ile Ala Lys Ile 672 720 768 816 atc gga aag aat Ile Giy Lys Asn tgo gag tta tgt Cys Glu Leu Cys cog Pro 250 gaa. caa gaa. acg Giu Gin Giu Thr gao ggo Asp Giy 255 aag aga Lys Arg gao oca tot Asp Pro Ser gga aag aaa Giy Lys Lys 275 oca Pro 260 ota aco too oca Leu Thr Ser Pro aco gaa gga aag Thr Giu Gly Lys oca Pro 270 ctt oaa ogt aat Leu Gin Arg Asn caa Gin 280 agt gtt gtt gat Ser Vai Vai Asp ttc Phe 285 aat goc aag Asn Ala Lys 864 WO 00/14106 WO 0014106PCT/US99/2 1053 aca att Thr Ile 290 gtt act ttq gat Vai Thr Leu Asp att gaa tta caa Ile Giu Leu Gin gtt Val 300 gag cag ttg aga Giu Gin Leu Arq aaa Lys 305 aaa gct qct gaa Lys Aia Ala Giu ctc Leu 310 gaa atg gaa aaa Giu Met Gin Lys gag Gin 315 caa att ctt cqt Gin Ile Leu Arg 912 960 1008 cta gga gaa atc Leu Gly Gi Ile aqt Ser 325 gtt cat aac tgc Val His Asn Cys atg Met 330 ttc aaa ctg gaa Phe Lys Leu Glu gaa tgt Gin Cys 335 gat cgt Asp Arg gaa gag Giu Gin 340 att qaa gca atc Ile Giu Ala Ile act Thr 345 gac cga ttg aca Asp Arg Leu Thr aaa aga aca Lys Arg Thr 350 gaa cag aaa Gin Gin Lys 1056 aag aca gtt Lys Thr Val 355 caa gtt gtt gtc Gin Val Val Val act cca cga aat Thr Pro Arg Asn gaa Glu 365 1104 aaa gca Lys Ala 370 ctq gaa gat gca Leu Giu Asp Ala act Thr 375 ttg atg atc gat Leu Met Ile Asp gaa Gin 380 gtc gga gaa atg Val Gly Gin Met atg Met 385 cat tcg aat att His Ser Asn Ile gaa Gin 390 aag gct aag ctg Lys Ala Lys Len tgc Cys 395 cta caa acc tac Len Gin Thr Tyr 1152 1200 1248 1296 aac gcc tgt tcg Asn Ala Gys Ser tac Tyr 405 gaa gaa act gct Gin Glu Thr Ala gga Gi y 410 gcc acc tgc caa Ala Thr Cys Gin aac ttc Asn Phe 415 aic aag Ile Lys ttg aag atc Len Lys Ile ata Ile 420 att caq tgc gct Ile Gin Cys Ala gct Ala 425 gat gat cag aaa Asp Asp Gin Lys cgc Arg 430 cgt cgt Arg Arg aaa gca Lys Ala 450 ctg Len 435 gaa aat ctg atg Gin Asn Leu Met tct Ser 440 caa att gag aat Gin Ile Giu Asn gag aga acg Gin Arg Thr 1344 gat ttg atg gat Asp Len Met Asp caa agc gaa Gin Ser Gin 1377 <210> 14 <211> 458 <212> PRT WO 00/14106 <213> Caenorhabditis elegans PCTIUJS99/2 1053 <400> 14 Met 1 His Pro Val Val Ser Arg Ser Pro Gin Gin Asn As n Ile Pro Ile Lys Ile Leu Giy Gin Asn Gin Ser Ser Ser Ser Ser 25 Gin Val Asp Asn Asp Pro Pro Pro Gin Gin Tyr Gin Pro 40 Val Pro Gin Gin Gin His Arg Asn Gin Ser Gin Gin His Ser Asn Gin Aia Pro Giy Phe Asn Pro Asn Thr Asn Ser Pro Asn Ser Phe 70 Phe Ser Arg Ser Pro Asp Phe Pro Phe Ser Ser Giy Asn Pro Asn Asp Ser 90 Phe Trp Ser Ser Asn Phe Pro Ser Phe Phe Pro Asp 115 Pro Pro Met Pro 100 Phe Phe Pro Ser Giy 105 Arg Ser Asn Giy Pro Arg Phe Giy 120 Arg Asp Giy Gly Leu 125 Thr Ser Ser Asn 110 Ser Pro Asn Ser Thr Gin Gin Giy Tyr 130 Ser Pro Arg 135 Arg Ser Pro Thr Pro 140 Gin Thr Ser Thr Leu 150 Gin Arg Asn Ser Asn Gin Ala Pro 160 Gin Tyr Ser Gin 165 Thr Gin Pro Gin Gin Gin Arg Gin Thr 175 Thr Pro Pro Giu Pro Lys 195 Pro Tyr Giu 210 Ser 180 Giu Lys Aia Ser Ser 185 Glu Pro Pro Ser Pro Giu Val Pro 200 Pro Arg Pro Aia Arg Thr Arg 190 Ile Pro Leu Ser Arg Asp Lys Lys Giu Lys 215 Leu Giu Lys Lys 220 Ser Giy Lys Giy Asp Giu Asn Leu Giu Giu Asn Ile Ala Lys Ile Thr 230 240 WO 00/14106 WO 0014106PCTIUS99/21 053 Ile Giy Lys Asn As n 245 Cys Giu Leu Cys Pro 250 Giu Gin Giu Thr Asp Gly 255 Asp Pro Ser Gly Lys Lys 275 Pro 260 Leu Thr Ser Pro Ile 265 Thr Giu Gly Lys Pro Lys Arg 270 Asn Ala Lys Leu Gin Arg Asn Gin 280 Ser Vai Vai Asp Phe 285 Thr Ile 290 Vai Thr Leu Asp Lys 295 Ile Giu Leu Gin Val1 300 Glu Gin Leu Arg Lys 305 Lys Ala Aia Giu Leu 310 Giu Met Giu Lys Giu 315 Gin Ile Leu Arg Leu Gly Giu Ile Ser 325 Vai His Asn Cys Met 330 Phe Lys Leu Giu Giu Cys 335 Asp Arg Giu Lys Thr Vai 355 Ile Giu Ala Ile Thr 345 Asp Arg Leu Thr Lys Arg Thr 350 Giu Gin Lys Gin Val Val Val Thr Pro Arg Asn Giu 365 Lys Ala 370 Leu Giu Asp Ala Thr 375 Leu Met Ile Asp Giu 380 Val Giy Giu Met Met 385 His Ser Asn Ile Gi u 390 Lys Ala Lys Leu Leu Gin Thr Tyr Met 400 Asn Ala Cys Ser Tyr 405 Glu Giu Thr Ala Gly 410 Ala Thr Cys Gin Asn Phe 415 Leu Lys Ile Arg Arg Leu 435 Ile 420 Ile Gin Cys Ala Ala 425 Asp Asp Gin Lys Arg Ile Lys 430 Giu Arq Thr Glu Asn Leu Met Se r 440 Gin Ile Giu Asn Ala 445 Lys Aia 450 Asp Leu Met Asp Asp 455 Gin Ser Giu <210> <211> 588 <212> DNA (213> Schizosaccharomyces pombe WO 00/14106 WO 0014106PCT/US99/2 1053 <220> <221> CDS <222> (588) <400> 'at g Met 1 tca gaa aag Ser Giu Lys act Thr 5 ago aca gtt aca Ser Thr Val Thr ata Ile 10 cac tat gga aat His Tyr Gly Asn cag cga Gin Arg att gat Ile Asp ttt ccg gta Phe Pro Val goa Ala gto aat ota aat Val Asn Leu Asn acg tta agt gaa Thr Leu Ser Giu ctg Le u gat tta ott Asp Leu Leu gaa acg act gag Glu Thr Thr Giu tct gag aag aaa Ser Giu Lys Lys gto Val aag ott ttt Lys Leu Phe tao got Tyr Ala ggo aag ogt tta Gly Lys Arg Leu gao aaa aaa goo Asp Lys Lys Ala tta toa aaa ttg Leu Ser Lys Leu ggt Gly tta aaa aat oat Leu Lys Asn His agt Ser 70 aaa att ota tgt Lys Ile Leu Cys ata Ile 75 aga ooa oat aag Arg Pro His Lys oa a Gin 192 240 288 caa oga ggt too Gin Arg Gly Ser aag Lys gaa aaa gao aog Giu Lys Asp Thr gt t Vali 90 gag 000 got cog Giu Pro Ala Pro aaa gog Lys Ala gaa gog gag Giu Ala Giu aat Asn 100 cot gta ttt tog Pro Val Phe Ser att tot gga gaa Ile Ser Giy Glu ata aaa goo Ile Lys Ala 110 gao aat tao Asp Asn Tyr ato gat Ile Asp gta aat Vai Asn 130 cag Gin 115 tat gtt gao aaa Tyr Val Asp Lys gaa Giu 120 ctt too 000 atg Leu Ser Pro Met tao Tyr 125 aaa cog tog aac Lys Pro Ser Asn gat Asp 135 oca aag cag aaa Pro Lys Gin Lys aaa cag aaa cta Lys Gin Lys Leu atg Met 145 ata agt gaa ota Ile Ser Giu Leu ott Le u 150 tta caa cag ott tta aaa ttg gat gga Leu Gin Gin Leu Leu Lys Leu Asp Gly 155 gao gta otg ggc Asp Val Leu Gly ago Ser 165 gag aaa ttg ogt Giu Lys Leu Arg ttt Phe 170 gaa cgg aag oaa Giu Arg Lys Gin ott gtt Leu Val 17 WO 00/14106 WO 0014106PCTIUS99I2 1053 tct aag atc caa aaa atg ttg gat cac gtt gac caa aca agc caa gaa 576 Ser Lys Ile Gin Lys Met Leu Asp His Val Asp Gin Thr Ser Gin Glu 180 185 190 gtg gcc gca tag Val Ala Ala 195 <210> 16 <211> 195 <212> PRT <213> Schizosaccharomyces pombe <400> 16 Met Ser 1 31u Lys Thr 5 Ser Thr Val Thr Ile 10 His Tyr Gly Asn Gin Arg Phe Pro Val Asp Leu Leu Ala Val Asn Leu Asqn Giu 25 Thr Leu Ser Giu Leu Ile Asp Lys Leu Phe Glu Thr Thr Giu Ser Giu Lys Lys Tyr Ala Gly Lys Arg Leu Asp Lys Lys Ala Ser Leu Ser Lys Leu Arg Pro His Lys Gin Gly Leu Lys Asn His Ser Lys Ile Leu Cys Gin Arg Gly Ser Lys Giu Lys Asp Thr Val1 90 Glu Pro Ala Pro Lys Ala Giu Ala Glu Ile Asp Gin 115 Pro Val Phe Ser Arg 105 Ile Ser Gly Giu Ile Lys Ala 110 Asp Asn Tyr Tyr Val Asp Lys Giu 120 Leu Ser Pro Met Tyr 125 Val Asn 130 Lys Pro Ser Asn Asp 135 Pro Lys Gin Lys Asn 140 Lys Gin Lys Leu Met 145 Ile Ser Giu Leu Leu 150 Leu Gin Gin Leu Leu 155 Lys Leu Asp Giy Asp Val Leu Gly Ser 165 Giu Lys Leu Arg Phe 170 Glu Arg Lys Gin Leu Val 175 WO 00/14106 WO 0014106PCTJU 599/2 1053 Ser Lys Ile Gin Lys Met Leu Asp His Val Asp Gin Thr Ser Gin Giu 180 185 190 Val Ala Ala 195 <210> 17 <211> 621 <212> DNA <213> Schizosacoharomyces pombe <220> <221> CDS <222> <400> 17 atg Met 1 tct ttt ttt Ser Phe Phe acc cag ttg tgt tot atg gat aaa aaa tat tgg atc Thr 5 Gin Leu Gys Ser Asp Lys Lys Tyr Trp Ile tct cta got Ser Leu Ala aag aga got Lys Arg Ala gta ValI ttg toa gtt act Leu Ser Val Thr gtt Val1 25 ttg att agc qoa Leu Ile Ser Ala tta ttg aaa Leu Leu Lys act gaa acc gaa gat att gtc gtt gtt Thr Giu Thr Glu'Asp Ile Val Val Val 40 cat tao gat ggo His Tyr Asp Gly aat atg gtt tct Asn Met Val Ser gaa aag Giu Lys ttg aat ttt gtg Leu Asn Phe Val ttg Leu cga caa cca agg Arg Gin Pro Arg tac Tyr act agt ttt ott Thr Ser Phe Leu cgt Arg 70 cgc gtg tgc aac Arg Val Cys Asn ttt tca gta atg Phe Ser Val Met coo Pro 192 240 288 gac aaa gcg tct Asp Lys Ala Ser Ctc Leu aag tta aac ggg Lys Leu Asn Gly gtg Val1 90 aco cto aag gat Thr Leu Lys Asp ggt tca Gly Ser ott too gao Leu Ser Asp caa Gin 100 aat qtg oaa aat Asn Vai Gin Asn gga Giy 105 agt gaa tta gag Ser Giu Leu Giu cto gaa tta Leu Giu Leu 110 ccc aaa otg ago cog gca atg Pro Lys Leu Ser Pro Ala Met caa Gin caa att gaa gca Gin Ile Giu Ala tat ata gat gag Tyr Ile Asp Giu WO 00/14106 WO 0014106PCTIUJS99/21 053 115 125 ott caa Leu Gin 130 cag gat oto gtc Gin Asp Leu Val cct Pro 135 aaa att gaa gco Lys Ile Giu Ala tto Phe 140 tgo oaa tog tct Cys Gin Ser Ser 000 Pro 145 got tog goa oaa Ala Ser Ala Gin gat Asp 150 gtt oaa gat ttg Val Gin Asp Leu aoa cqo ott agt Thr Arg Leu Ser gaa Giu 160 432 480 528 aoa ttg ttg got Thr Leu Leu Ala atg ata aaa tta Met Ile Lys Leu gat Asp 170 got gtt aat gtt Ala Val Asn Val gaa gac Giu Asp 175 gao oca gaa Asp Pro Giu oaa tat ttg Gin Tyr Leu 195 got Al a 180 ogt ott aaa aga Arg Leu Lys Arg aaa Lys 185 gaa got att ogt Giu Ala Ile Arg tta tot caa Leu Ser Gin 190 agt aaa ota gat Ser Lys Leu Asp too Ser 200 aoo aag aat caa Thr Lys Asn Gin aao aaa tga Asn Lys 205 <210> 18 <211> 206 <212> PRT <213> Sohizosaooharomyoes pombe <400> 18 Met Ser Phe Phe Thr 1 5 Gin Leu Cys Ser Asp Lys Lys Tyr Trp Ile Ser Leu Ala Lys Arg Ala Val1 Leu Ser Val Thr Val1 25 Leu Ile Ser Ala Leu Leu Lys Tyr Asp Gly Thr Giu Thr Giu Asp 40 Ile Val Val Val His Giu Lys Leu Asn Phe Val Leu 55 Arg Gin Pro Arg Leu Asn Met Val Ser Tyr Thr Ser Phe Leu Arg Val Cys Asn Ala 75 Phe Ser Vai Met Pro Asp Lys Ala Ser Lys Leu Asn Gly Thr Leu Lys Asp Gly Ser Leu Ser Asp Gin Asn Vai Gin Asn Giy Ser Giu Leu Glu Leu Giu Leu WO 00/14106 WO 0014106PCT/US99/2 1053 100 Ser Pro Lys Leu 115 Leu Gin Gin Pro Ala Met Gin 120 Lys Ile Glu Ala Tyr 125 Asp Leu Val 130 Pro Ala Pro 135 Val1 Ile Glu Ala Ser Ala Gin 145 Thr Asp 150 Met Gin Asp Leu His 155 Ala Phe Cys 140 Thr Arg Val Asn 110 Ile Asp Giu Gin Ser Ser Leu Ser Glu 160 Val Giu Asp 175 Leu Ser Gin 190 Lys Leu Leu Ala Arg 165 Arg Ile Lys Leu Asp 170 Giu Asp Pro Giu Gin Tyr Leu 195 Ala 180 Ser Leu Lys Arg Ala Ile Arg Lys Leu Asp Ser 200 Lys Asn Gin Asn 205 <210> 19 211> 2534 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (2034) <400> 19 gcggagctcc gcatccaacc ccgggccgcg gccaacttct ctggactgga ctagccggcc agttgctacc tccctttatc tcctccttcc cctctggcag atttccagac acttccaccc ctctctggcc acgtcacccc cgcctttaat gcccgqcgcc ggcttcccgg acacgtcgqc ggcgqagagg ggcccacggc ocagagactc ggcgcccgga gccagcgccc cgcacccgcg ccccagcggg CCCagc atg agc gcc gcc acc cac tcq ccc atg atg cag gtq Met Ser Ala Ala Thr His Ser Pro Met Met Gin Val ccaqaagttt cgaggaggct tcataaaggt ggcggcccgg cagaccccaa gcg tcc Ala Ser 120 180 240 300 348 ggc aac ggt gac cgc qac cct ttg ccc ccc qga tgg gag ate aag atc Gly Asn Gly Asp Arg Asp Pro Leu Pro Pro Gly Trp Giu Ile Lys Ile 20 25 WO 00/14106 gao cog cag Asp Pro Gin act acg tgg Thr Thr Trp cca tcc tct Pro Ser Ser PCT/US99/21053 acc gqc tgg cc ttc tto gtg gao cac aao agc cgo aco Thr Gly Trp Pro Phe Phe Asp His Asn Ser Arg Thr aac Asn gac ccg cgc gtg Asp Pro Arg Val cCC Pro 55 tct gag ggo ccc Ser Glu Gly Pro aag gag act Lys Glu Thr otg ccg cct Leu Pro Pro 492 540 cc aat ggo cct Ala Asn Gly Pro too Ser 70 cgg gag ggo tct Arg Glu Gly Ser agg Arg got agg Ala Arg gaa ggo cac cct Glu Gly His Pro tao ccc cag otc Tyr Pro Gin Leu oga Arg cca ggo tao att Pro Gly Tyr Ile cCC Pro att oct gtg otc Ile Pro Val Leu oat His 100 gaa ggo got gag Glu Giy Ala Glu aac Asn 105 egg cag gtg cac Arg Gin Val His cct Pro 110 588 636 684 tto cat gto tat Phe His Val Tyr cCC Pro 115 cag cot ggg atg Gin Pro Gly Met cag Gin 120 oga tto oga act Arg Phe Arg Thr gag gcg Glu Ala 125 gca goa gcg Ala Ala Ala gaa aco act Glu Thr Thr 145 cct cag agg too Pro Gin Arg Ser cag Gin 135 toa cot otg cgg Ser Pro Leu Arg ggo atg oca Gly Met Pro 140 gcg gog gcg Ala Ala Ala 732 780 cag oca gat aaa Gin Pro Asp Lys tgt gga cag gtg Cys Gly Gin Val gca gc Ala Ala 160 cag ccc coa gc Gin Pro Pro Ala too Ser 165 cac gga cot gag His Gly Pro Glu cgg Arg 170 too cag tot oca Ser Gin Ser Pro got Ala 175 goo tot gao tgc Ala Ser Asp Cys too toa too too Ser Ser Ser Ser gc ago ctg cct Ala Ser Leu Pro too Ser 190 828 876 924 too ggc agg ago Ser Gly Arg Ser ago Ser 195 otg ggo agt cac Leu Gly Ser His cag Gin 200 otc cog ogg ggg Leu Pro Arg Gly tao ato Tyr Ile 205 too att ccg Ser Ile Pro gtg Val 210 ata cac gag cag Ile His Giu Gin aac Asn 215 gtt aco cgg oca Val Thr Arg Pro gca gc cag Ala Ala Gin 220 WO 00/14106 ccc tec ttc Pro Ser Phe 225 PCT/US99/21053 cac aaa gcc cag His Lys Ala Gin aag acg cac tac cca Lys Thr His Tyr Pro gcg Ala 235 cag agg ggt Gin Arg Gly 1020 gag tac Glu Tyr 240 cag acc cac cag Gin Thr His Gin cct Pro 245 gtg tac cac aag Val Tyr His Lys atc Ile 250 cag ggg gat gac Gin Gly Asp Asp tgg Trp 255 gag ccc cgg ccc Glu Pro Arg Pro ctg Leu 260 cgg gcg gca tec Arg Ala Ala Ser ccg Pro 265 ttc agg tca tct Phe Arg Ser Ser gtc Val 270 1068 1116 1164 1212 cag ggt gca tcg Gin Gly Ala Ser age Ser 275 cgg gag ggc tca Arg Glu Gly Ser cca Pro 280 gcc agg ago ago Ala Arg Ser Ser acg cca Thr Pro 285 agg cct Arg Pro ctc cac tcc Leu His Ser ccc Pro 290 tcg ccc atc cgt Ser Pro Ile Arg gtg Val 295 cac acc gtg gtc His Thr Val Val gac Asp 300 cag cag ccc Gin Gin Pro 305 atg acc cat cga Met Thr His Arg gaa Glu 310 act gca cct gtt Thr Ala Pro Val tcc Ser 315 cag cct gaa Gin Pro Glu 1260 aac aaa Asn Lys 320 cca gaa agt aag Pro Glu Ser Lys cca Pro 325 ggc cca gtt gga Gly Pro Val Gly cca Pro 330 gaa ctc cct cct Glu Leu Pro Pro gga Gly 335 cac ate cca att His Ile Pro Ile gtg atc cgc aaa Val Ile Arg Lys gtg gat tct aaa Val Asp Ser Lys cct Pro 350 1308 1356 1404 gtt tec cag aag Val Ser Gin Lys ccc Pro 355 cca cct ccc tct Pro Pro Pro Ser gag Glu 360 aag gta gag gtg Lys Val Glu Val aaa gtt Lys Val 365 ccc cct gct Pro Pro Ala gtc ccc tct Val Pro Ser 385 cca Pro 370 gtt cct tgt cct Val Pro Cys Pro cct Pro 375 ccc ago cct ggc Pro Ser Pro Gly cct tct get Pro Ser Ala 380 gca gcc ccc Ala Ala Pro 1452 1500 tec ccc aag agt Ser Pro Lys Ser get aca gaa gag Ala Thr Glu Glu agg Arg 395 agc act Ser Thr 400 gcc cct gca gaa Ala Pro Ala Glu get Ala 405 aca cct cca aaa Thr Pro Pro Lys cca Pro 410 gga gaa gcc gag Gly Glu Ala Glu 1548 WO 00/14106 WO 00/4 106PCTIUS99/2 1053 gct Ala 415 coo cca aaa cat Pro Pro DyHis cca Pro 420 gga gtg otg aaa Giy Val Leu Lys gaa gcc atc otg Giu Ala Ile Leu gag Giu 430 1596 1644 aag gtg cag ggg Lys Vai Gin Gly ctg Leu 435 gag cag gct gta Giu Gin Ala Val gao Asp 440 aac ttt gaa ggc Asn Phe Giu Giy aag aag Lys Lys 445 act gac aaa Thr Asp Lys ctg gcc ctg Leu Ala Leu 465 aag Lys 450 tao otg atg ato Tyr Leu Met Ile gaa Giu 455 gag tat ttg aco Glu Tyr Leu Thr aaa gag otg Lys Giu Leu 460 gtg ogt cag Vai Arg Gin 1692 1740 gat toa gtg gao Asp Ser Val Asp gag gga oga gc Giu Gly Arg Ala gat Asp 475 goc agg Ala Arg 480 aga gac ggt gtc Arg Asp Gly Val aag gtt cag aco Lys Vai Gin Thr at c Ile 490 ttg gaa aaa Ott Leu Glu Lys Leu gaa Glu 495 cag aaa gcc att Gin Lys Ala Ile gat Asp 500 qtc oca ggt caa Vai Pro Gly Gin cag gto tat Gin Val Tyr gaa oto 510 atg gag Met Giu 525 1788 1836 1884 cag ccc ago aao Gin Pro Ser Asn ctt Leu 515 gaa goa gat cag Glu Ala Asp Gin cca Pro 520 ctg cag goa ato Leu Gin Ala Ile atg ggt gc Met Gly Ala gtg Val1 530 goa gca gao aag Ala Ala Asp Lys ggc Gly 535 aag aaa aat got Lys Lys Asn Ala gga aat gca Gly Asn Ala 540 gca goa gcg Ala Ala Ala 1932 gaa gat Glu Asp act tca Thr Ser 560 ccc Pro 545 cac aca gaa aco His Thr Glu Thr cag cca gaa gc Gin Pro Glu Ala aca Thr 555 1980 2028 aac ccc ago ago Asn Pro Ser Ser atg Met 565 aca gac aco oct Thr Asp Thr Pro ggt Gly 570 aac cca goa gca Asn Pro Ala Ala cog Pro 575 tag cctotgccct gtaaaaatca gactcggaao cgatgtgtgc tttagggaat 2084 tttaagttgc atgcatttca gagaotttaa gtoagttggt ttttattagc tgcttggtat 2144 goagtaactt gggtggaggc aaaaoaotaa taaaagggct a aaaaggaaa atgatgottt 2204 WO 00/14106 tcttctatat ccgttgcttg gtgcactgtc ataaatatga ataattaaaa tatgttggat PCTIUS99/21 053 tcttactctg ttctqcagcc ttttqtagct aacatttatc tacctgactt gactttaatg tacaaataaa gaagttgctt qttgtttgag aaqtttaacc ctgtctactt gggcaccccc accacctgtt agctgtggtt ctggactgga ggggtagatg gggagtcaat tacccatcac agaaatgttg ccattttaat gaqatgattt tcttcatctc tagagagagt aaaatgtgcc aggagocata ggaatatctg ctacattttc 2264 2324 2384 2444 2504 2534 <210> <211> 575 <212> PRT <213> Homoa sapiens <400> Met 1 Gly Gin Ser Ala Ala Thr His Ser Pro Met Met Gin Val Ala Ser Gly Asn Asp Arg Asp Thr Gly Trp Pro Trp Leu Pro Pro Gly 25 Asp Giu Ile Lys Pro Phe Phe Ile Asp Pro Thr Thr Thr Thr Pro Ser His Asn Ser Trp Asn Asp Arg Glu Pro Arg Val Ser Ala Pro 55 Arg Giu Gly Pro Lys Le u Asn Gly Pro Giu Ser 70 Tyr Glu Gly Ser Arg Pro Pro Pro Ala Gly His Pro Val1 Glu Pro Gin Leu Arg 90 Arg Gly Tyr Ile Pro Ile Pro Val Leu Val Tyr Pro 115 Ala Ala Pro 130 Gly Ala Giu As n 105 Arg Gin Val His Pro Gly Met Gin 120 Se r Phe Arg Thr Glu 125 Met Pro Phe His 110 Ala Ala Ala Pro Giu Thr Gin Arg Ser Gin 135 Pro Leu Arg Gly 140 Thr Gin Pro Asp Lys Gin Cys Gly Gin Val Ala Ala Ala Ala Ala Ala WO 00/14106 PCT/US99/21053 Gin Pro Pro Ala Ser His 165 Gly Pro Glu Ser Gin Ser Pro Ala Ala 175 Ser Asp Cys Arg Ser Ser 195 Ser 180 Ser-Ser Ser Ser Ser 185 Ala Ser Leu Pro Ser Ser Gly 190 Ile Ser Ile Leu Gly Ser His Leu Pro Arg Gly Pro Val 210 Ile His Glu Gin Asn 215 Val Thr Arg Pro Ala 220 Ala Gin Pro Ser Phe 225 His Lys Ala Gin Thr His Tyr Pro Gin Arg Gly Glu Tyr 240 Gin Thr His Gin Val Tyr His Lys Gin Gly Asp Asp Trp Glu 255 Gin Gly Pro Arg Pro Ala Ser Ser 275 Leu 260 Arg Ala Ala Ser Pro 265 Phe Arg Ser Ser Arg Glu Gly Ser Pro 280 Ala Arg Ser Ser Thr 285 Pro Leu His Ser Pro 290 Ser Pro Ile Arg His Thr Val Val Asp 300 Arg Pro Gin Gin Pro 305 Met Thr His Arg Glu 310 Thr Ala Pro Val Gin Pro Glu Asn Pro Glu Ser Lys Pro 325 Gly Pro Val Gly Glu Leu Pro Pro Gly His 335 Ile Pro Ile Gin Lys Pro 355 Gin 340 Val Ile Arg Lys Glu 345 Val Asp Ser Lys Pro Val Ser 350 Val Pro Pro Pro Pro Pro Ser Lys Val Glu Val Lys 365 Ala Pro 370 Val Pro Cys Pro Pro Lys Ser Val 390 Pro 375 Pro Ser Pro Gly Pro 380 Ser Ala Val Pro Ser 385 Ser Ala Thr Glu Glu Ala Ala Pro Ser Thr 400 Ala Pro Ala Glu Ala Thr Pro Pro Lys Pro Gly Glu Ala Glu Ala Pro WO 00/14106 WO 0014106PCT/US99/2 1053 Pro Lys His Gin Gly Leu 435 Pro 420 Gly Val Leu Lys Val1 425 Glu Ala Ile Leu Glu Lys Val 430 Lys Thr Asp Giu Gin Ala Val Asn Phe Glu Gly Lys 445 Lys Lys 450 Tyr Leu Met Ile Giu 455 Glu Tyr Leu Thr Lys 460 Glu Leu Leu Ala Le u 465 Asp Ser Val Asp Pro 470 Giu Gly Arg Ala Val Arg Gin Ala Arg Asp Gly Val Lys Val Gin Thr Ile 490 Leu Giu Lys Leu Giu Gin 495 Lys Ala Ile Ser Asn Leu 515 Asp 500 Val Pro Gly Gin Val1 505 Gin Val Tyr Giu Leu Gin Pro 510 Glu Met Giy Giu Ala Asp Gin Leu Gin Ala Ile Met 525 Ala Val 530 Ala Aia Asp Lys Gly 535 Lys Lys Asn Ala Giy 540 Asn Ala Giu Asp Pro 545 His Thr Giu Thr Gin 550 Gin Pro Glu Ala Ala Ala Ala Thr Asn Pro Ser Ser Met 565 Thr Asp Thr Pro Gly 570 Asn Pro Ala Ala Pro 575 <210> <211> <212> <213> <220> <221> <222> 21 1966

DNA

Homno sapiens

CDS

(4 3) (1416) <400> 21 cggtggqagc ggggcgggaa gcgcttcagg gcagcggatc cc atq tcg gcc ctg Met Ser Ala Leu agg cgc tcg ggc tac 99c ccc aqt qac ggt ccg tcc tac ggc cqc tac WO 00/14106 WO 0014106PCT/US99/21053 Arq Arg Ser Gly Tyr Gly Pro Ser Asp Gly Pro Ser Tyr Gly Arg Tyr tac ggg oct ggq Tyr Gly Pro Gly qga gat gtg cog Gly Asp Val Pro gt a Val1 cac cca cct cca His Pro Pro Pro ccc ita Pro Leu 150 tat oct ctt Tyr Pro Leu cgc ggg ggc Arg Gly Gly cgc Arg cct gaa Oct ccc Pro Glu Pro Pro oct ccc att tcc Pro Pro Ile Ser tqg ogg gtg Trp Arg Val gqc gga gga Gly Gly Gly ggc cog gcg gag Gly Pro Ala Glu acc Thr 60 acc tgq ctg gga Thr Trp Leu Gly gaa Glu ggc gat Gly Asp ggo tao tat ccc Gly Tyr Tyr Pro tcg Se r 75 gga ggc gcc tgg Gly Gly Ala Trp cca Pro gag cct ggt cga Glu Pro Gly Arg gcc Al a gga gga agc cac Gly Gly Ser His cag Gln 90 gag cag cca cca Glu Gin Pro Pro Oct ago tao aat Pro Ser Tyr Asn tot Ser 100 294 342 390 aac tat tgg aat Asn Tyr Trp Asn tat cot gta aga Tyr Pro Val Arg 120 tot Ser 105 act gcg aga tot Thr Ala Arg Ser agg Arg 110 got cot tao oca Ala Pro Tyr Pro agt aca Ser Thr 115 oca gaa ttg caa Pro Glu Leu Gin ggc Gly 125 cag agt ttg aat Gin Ser Leu Asn tot tat aca Ser Tyr Thr 130 gca aat act Ala Asn Thr 438 aat gga Asn Gly goc toa Ala Ser 150 tat ggt oca aca Tyr Gly Pro Thr tao Tyr 140 coo oca ggc cot Pro Pro Gly Pro ggg Gi y 145 tao toa ggg got Tyr Ser Gly Ala tat gca cot ggt Tyr Ala Pro Gly act cag aco agt Thr Gin Thr Ser 486 534 582 630 tao Tyr 165 too aca gaa gtt Ser Thr Glu Val oca Pro 170 agt act tao ogt Ser Thr Tyr Arg tot ggc aac ago Ser Gly Asn Ser act oca gto tot Thr Pro Val Ser cgt Arg 185 tgg ato tat coo Trp Ile Tyr Pro cag Gin 190 cag gao tqt cag Gin Asp Cys Gin act gaa Thr Giu 195 gca coo cot ott agg ggg cag gtt oca gga tat ccg cot toa cag aac WO 00/14106 Ala Pro Pro cct gga atg Pro Gly Met 215 PCTIUS99/2 1053 Arg Giy Gin Val Pro 205 Gly Tyr Pro Pro Ser Gin Asn 210 aat cgt agt Asn Arg Ser aec ctg ccc cat Thr Leu Pro His tat Tyr 220 cct tat gga gat Pro Tyr Gly Asp ggt Gly 225 gtt cca Val Pro 230 caa tca gga ccg Gin Ser Gly Pro gta cga cca caa.

Val Arg Pro Gin gaa Giu 240 gat gcg tgg gct Asp Ala Trp Ala cct ggt gct tat Pro Gly Ala Tyr gga Gly 250 atg ggt ggc cgt Met Gly Gly Arg tat Tyr 255 ccc tgg cct tca Pro Trp Pro Ser gcg ccc tca gca.

Ala Pro Ser Ala ccc ggc aat ctc Pro Gly Asn Leu tac Tyr 270 atg act gaa agt Met Thr Glu Ser act tca Thr Ser 275 cca tgg cct Pro Trp Pro cag cag ccc Gin Gin Pro 295 aqc Ser 280 agt ggc tct ccc Ser Giy Ser Pro cag Gin 285 tca ccc cct Ser Pro Pro aag gat tct tca Lys Asp Ser Ser tac Tyr 300 ccc tat agc caa Pro Tyr Ser Gin tca ccc cca gtc Ser Pro Pro Val 290 tca gat caa. agc Ser Asp Gin Ser 305 tac gaa. tcc tcq Tyr Glu Ser Ser atg aac Met Asn 310 cgq cac aac ttt Arg His Asn Phe cct Pro 315 tgc agt gtc cat Cys Ser Val His cag Gin 320 ggg Gly 325 aca gtg atc aat Thr Val Ile Asn gaa Glu 330 gat tca gat ctt Asp Ser Asp Leu ttg Leu 335 gat tcc caa gtc Asp Ser Gin Val cag Gin 340 1014 1062 1110 tat aqt gct gag Tyr Ser Ala Glu cct Pro 345 cag ctg tat qqt Gin Leu Tyr Gly qcc acc agt gac Ala Thr Ser Asp cat ccc His Pro 355 aac aat caa Asn Asn Gin gat gaa agt Asp Giu Ser 375 gat Asp 360 caa agt agc agt Gin Ser Ser Ser ct t Le u 365 cct gaa gaa. tgt Pro Giu Giu Cys gta cct tca Val Pro Ser 370 gtg ctg gag Val Leu Glu 1158 1206 act cct ccg agt Thr Pro Pro Ser att Ile 380 aaa aaa atc ata Lys Lys Ile Ilie aag gtc cag tat ctt gaa caa gaa gta gaa gaa. ttt gta gga aaa aag 15 1254 WO 00/14106 Lys Val Gin 390 aca gac aaa Thr Asp Lys 405, ttg qaa otg Leu Glu Leu 900 aga aaa Ala Arg Lys PCTIUS99/2 1053 Lys Tyr Leu Glu Gin 395 ott Leu Glu Val Glu Glu Phe 400 ota Leu Val Gly Lys aoo aag gaa Thr Lys Glu gca tao Ala Tyr tgg T rp 410 gtt Val1 otg gaa gaa Leu'Glu Glu ott 1302 Le u 420 Gag 1350 gat Asp gag Glu 440 tca Ser 425 gct Ala gaa act ggg Glu Thr Gly ggo Gly 430 gac tot gta Asp Ser Val ogg Arg 435 aaa tta Lys Leu gtt tgt aag att cag goc ata otg gaa Val Cys Lys Ile Gin Ala Ile Leu Glu 445 450 tqa aaggatttag aaoaaagtgg aagoctgtta 1398 gaa aaa aaa gga tta Glu Lys Lys Gly Leu 455 otaaottgao caaagaacz tgacaagaag oaatacatt gaatggaaga atattttac ggaaaotatg gagttaocc totaoaagot gottattac aaootacoaq atgaaaotc tggatatctt gtcaoattt ttatagotta gaotttagc 1446

C

c t a c 9 t c a ttgattaggt oagottttcc oatgaagttg tattgooaag agoaggaggg atataatttg tgtaoattgt ttcttggact aaaaaaaaaa taattaooot tttgatttta ttttoagttt tagactcaot aaaoaoactt agaoaaaoag gactgotttc tctgttttgt aaaaaaaaaa ctttttgaaa tacttgaaaa toaqaogaat oottaaaaaa oaoaoaaoag gatgtgtttt aacatataot tttgttattt tgootgttga aotggcaaag gaatgtaata tttatggata gcttatoaga tttaaacato toatgtgtaa gcagtttaoa 1506 1566 1626 1686 1746 1806 1866 1926 1966 <210> 22 <211> 457 '<212> PRT <213> Homo sapiens <400> 22 Met Ser Ala Leu Arg Arg Ser Gly Tyr Gly Pro Ser Asp Gly Pro Ser 1 5 10 Tyr Gly Arg Tyr Tyr Gly Pro Gly Gly Gly Asp Val Pro Val His Pro 39 WOOO/14106 WO 0014106PCTIUS99/2 1053 Pro Pro Pro Pro Ser Trp Arg Tyr Pro Leu Giu Pro Pro Gin Thr Pro Pro Ile Trp Leu Gly Val Arg Gly Giu Gly Gly 55 Gly Pro Ala Giu Thr Gly Giy Giy Gly Giu Asp 70 Giy Tyr Tyr Pro Gly Aia Trp Pro Pro Gly Arg Gly Ser His Gin Thr Gin Pro Pro Tyr Pro Ser Tyr Asn Tyr Pro Ser 115 Asn Ser Tyr Ser 100 Thr Tyr Trp Asn Ser 105 Pro Ala Arg Ser Tyr Pro Vai Arg 120 Tyr Giu Leu Gin Gly 125 Pro Arg Ala Pro 110 Gin Ser Leu Pro Gly Pro Thr Asn Giy 130 Giv Ala Ala 135 Tyr Gly Pro Thr Tyr 140 T yr Asn Thr Ala Ser Gly Ala Tyr 155 Se r Ala Pro Gly Tyr 160 Gin Thr Ser Tyr 165 Thr Thr Giu Val Pro 170 T rp Thr Tyr Arg Ser Ser 175 Gly Asn Ser Cys Gin Thr 195 Pro Ser Gin Pro Val Ser Arg 185 Arg Ile Tyr Pro Ala Pro Pro Leu 200 Thr Gly Gin Val Gin Gin Asp 190 Gly Tyr Pro Tyr Gly Asp Asn Pro Gly Met 215 Gin Leu Pro His Tyr 220 Val Gi y 225 Asp Arg Ser Val Pro 230 Pro Ser Gly Pro Arg Pro Gin Ala Trp Ala Gly Ala Tyr Gly 250 Pro Gly Gly Arg Tyr Pro 255 Trp Pro Ser Giu Ser Thr Ser 260 Ser Pro Ser Ala Pro 265 Ser Gly Asn Leu Tyr Met Thr 270 Ser Pro Pro Pro Trp Pro Se r Gly Ser Pro Gin WO 00/14106 WO 0014106PCTIUS99/2 1053 Ser Pro 290 Pro Val Gin Gin Pro 295 Lys Asp Ser Ser Tyr 300 Pro Tyr Ser Gin Ser 305 Tyr Asp Gin Ser Met Giu Ser Ser Gly 325 Arg His Asn Phe Gys Ser Val His Thr Val Ile Asn Giu 330 Asp Ser Asp Leu Leu Asp 335 Ser Gin Vai Ser Asp His 355 Gin 340 Tyr Ser Aia Giu Pro 345 Gin Leu Tyr Giy Asn Aia Thr 350 Pro Giu Giu Pro Asn Asn Gin Asp 360 Gin Ser Ser Ser Leu 365 Cys Val 370 Pro Ser Asp Giu Ser 375 Thr Pro Pro Ser Ile 380 Lys Lys Ile Ile His 385 Val Leu Giu Lys Val 390 Gin Tyr Leu Giu Gin 395 Glu Vai Giu Glu Val Giy Lys Lys Thr 405 Asp Lys Aia Tyr Leu Leu Giu Giu Met Leu 415 Thr Lys Giu Ser Val Arg 435 Leu Giu Leu Asp Ser 425 Vai Giu Thr Giy Gly Gin Asp 430 Gin Ala Ile Gin Ala Arg Lys Glu 440 Ala Vai Cys Lys Leu Giu 450 Lys Leu Giu Lys Lys Gly Leu 455 <210> 23 <211> 4308 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1590) <400> 23 CCCCCCCCCC cccccccccc ccngaagacg cccggagcgg ctgctgcagc cagtagcggc WO 00/14106 WO 0014106PCT/US99/2 1053 coottcaccg gctgccccgc tcagacctag tcgggagggg tgcgaggcat gcagctgggg 120 gcccagotcc ggtgcogcao ccgtaaagg gctqatottc caoctogcca cctcagccac 180 gggacgccaa gaccgoatcc aattcagact tcttttggtg cttgtgaaac tgaacacaac 240 aaaagt atg gat atg gga aac caa cat cct tct att agt agg ctt cag Met Asp Met Gly Asn Gin His Pro Ser Ile Ser Arg Leu Gin 288 gaa Giu atc caa aag gaa Ile Gin Lys Giu gta Vai aaa agt gta gaa Lys Ser Val Giu caa gtt atc ggo Gin Val Ile Giy 336 384 agt ggt ctg tca Ser Giy Leu Ser gac aag aat tac Asp Lys Asn Tyr aag Lys aaa ctg gag agg Lys Leu Giu Arg att cta Ile Leu aca aaa cag Thr Lys Gin gat ait cag Asp Ilie Gin ott Leu ttt gaa ata gac Phe Giu Ile Asp tct Ser 55 gta gat act gaa Vai Asp Thr Giu gga aaa gga Giy Lys Giy gaa cgt ctt Giu Arg Leu 432 480 caa qct agg aag Gin Aia Arg Lys gca gca cag gag Ala Ala Gin Giu ctc aaa Leu Lys gag ttg gag cag Giu Leu Giu Gin gca aac cac cca Ala Asn His Pro cac His cgg att gaa ata Arg Ile Giu Ile cag Gin aac att ttt gag Asn Ile Phe Giu gaa Giu 100 goc cag too oto Aia Gin Ser Leu aga gag aaa att Arg Giu Lys Ile gtg Vali 110 528 576 624 oca ttt tat aat Pro Phe Tyr Asn gga Giy 115 ggc aac t go gta Giy Asn Cys Vai gat gag ttt gaa Asp Giu Phe Giu gaa ggc Giu Giy 125 ato caa gat Ile Gin Asp ato too ttg Ilie Ser Leu 145 ato Ile 130 att ctg agg ctg Ile Leu Arg Leu aca Thr 135 oat gtt aaa act His Val Lys Thr gga gga aaa Gly Gly Lys 140 ato tgt gcg Ile Cys Ala cgg aaa gca agg Arg Lys Ala Arg tat Tyr 150 cac act tta aco His Thr Leu Thr a aa Lys 155 gtg caa gag ata ato gaa gao tgc atg aaa aag oag cot too ctg cog Val Giln Gu Ile Ile Giu Asp Cys Met Lys Lys Gin Pro Ser Leu Pro WO 00/14106 WO 0014106PCT[US99/21 053 160 165 ott Leu 175 too gag qat gca Ser Giu Asp Ala cat His 180 cot tcc gtt gc Pro Ser Val Ala atc aac ttc gtq Ile Asn Phe Val atg Met 190 tgt gag gtg aac Cys Glu Val Asn gcc oga ggg gto Ala Arg Gly Val ctg Leu 200 att gca ott otg Ile Ala Leu Leu atg ggt Met Gly 205 gtg aac aac Val Asn Asn ctg ato got Leu Ile Ala 225 aat As n 210 gag aoo tgo agg Glu Thr Cys Arg oao His 215 gat Asp tta too tgt gtg Leu Ser Cys Val oto tog ggg Leu Ser Gly 220 aoa gaa ato Thr Glu Ile gao otg gat got Asp Leu Asp Ala ota Leu 230 gtg tgo ggo Val Cys Gly ogg Arg 235 aga aat Arg Asn 240 tat cgg agg gag Tyr Arg Arg Giu gta Val1 245 gta gaa gat ato Val Giu Asp Ile aao As n 250 aaa tta ttg aaa Lys Leu Leu Lys tat Tyr 255 otg gat ttg gaa Leu Asp Leu Giu gaa goa gao aca Giu Ala Asp Thr aaa goa ttt gao Lys Ala Phe Asp otg Leu 270 1008 1056 1104 aga oag aat oat Arg Gin Asn His too Ser 275 att tta aaa ata Ile Leu Lys Ile gaa Giu 280 aag gto oto aag Lys Val Leu Lys aga atg Arg Met 285 aga gaa ata Arg Giu Ile tao otg ago Tyr Leu Ser 305 aaa Lys .290 aat gaa ott oto Asn Giu Leu Leu caa Gin 295 goa oaa aao oot Ala Gin Asn Pro tot gaa ttg Ser Glu Leu 300 oag ttg gat Gin Leu Asp 1152 1200 too aaa aca gaa Ser Lys Thr Giu ttg Leu 310 cag ggt tta att Gin Gly Leu Ile gga Gly 315 gag gta Glu Val 320 agt ott gaa aaa Ser Leu Glu Lys aac Asn 325 000 tgo ato ogg Pro Cys Ile Arg gaa Giu 330 goo agg aga aga Ala Arg Arg Arg 1248 1296 goa Al a 335 gtg ato gag gtg Val Ile Giu Val oaa Gin 340 aot otg ato aca Thr Leu Ile Thr att gao ttg aag Ile Asp Leu Lys goc ott gag aaa aga aag otg ttt Ala Leu Giu Lys Arq Lys Leu Phe got tgt gag gag oao oca too cat Ala Cys Giu Giu His Pro Ser His 1344 WO 00/14106 WO 0014106PCTIUS99/2 1053 355 aac As n 360 ttg Le u aaa gcc gtc Lys Ala Val gtt ctt tca Vai Leu Ser 385 gaa gag ctg Gin Gin Len tgg Trp 370 ttt Phe gtc ott gga Val Leu Gly aao As n 375 aco Thr tot gag atc Ser Giu Ile 365 cag gqa gaa Gin Gly Giu 380 atc ogg ctg Ile Arg Leu gtt gat cog Vai Asp Pro qat gga aat Asp Giy Asn cga Arg 390 ct q Leu gat aag aac Asp Lys Asn tac Tyr 395 got Ala otc aoo aag Leu Thr Lys 400 cag gga Gin Giy cag Gin 405 aag Lys ota gcc ctg Leu Ala Leu gat Asp 410 oaa Gin gaa gag aag Gin Gin Lys got gcc agg Ala Ala Arg 415 aaa Lys 425 ,got gtg agg Ala Val Arq gog cag aat att ctc ago tat oto gao otg aaa tot gat Ala Gin Asn Ile Len Ser Tyr Leu Asp Len Lys Ser Asp 435 440 tao tga aataooagag atotoaottt tgataotgtt ttgcaottoa Tyr gaa tgg gac Gin Trp Gli 445 tatgtgctto 1392 1440 1488 1536 1584 1640 1700 1760 1820 1880 1940 2000 2060 2120 2180 2240 2300 2360 tatgt ataga tgattgaagc aoagoaogtt tttgtccttt taaatgggtt ttaoataata toaaaoaoaa tataaggcot agtotgttot ctaogtttta agccaaaaaa gagotttoag aaattotatt aaottttooa tttttgogtt gtgoaagoat otaggaaaat goagtacatg gattggtoot tttgtaaaoa gagaggaato cctttgagat ttoattgatt oagtatotgo ttoggatoat tttaatcaga taaaatgoag tatgagaaag ttotgtttca aoooagctta tttttagttg ttgtttttgt tgcattaaag tatacqtgoa tgottttgat tatotgtatg aaaoaaaat a gtotttoaga gggaaatttt ttatgo-toga aoggggtggg gaaaaacago gtgoaaoata agaagggata cttatagttt tatttcagto gt t gaaga o atgtggtgtg gaggoagot t atotagaaot tggttaaata tagaaggott gtttttttgt atotgoattt agaaaattat aaggaooago tccttagtga tcagtattta aaatatoatt gtttgtttgg ttgtagattt aggoataaoo agagtaaggt ttttttcaot ttgttoagao tococatoot gaaaaotaat aataataoot gtaaattcat tgtaagttgo ttttgtttgt aaaatctgag WO 00/14106 aaggatggga tacctgtagt taaatcagaa atggacagcc gtaacccaga ttagtaaatg aaggcttcct ctctgttgcc atgtgtattt aagtgccttq ttgcctggtc gatgctcatt gctaaaggag cagcttgtct aatgttctag tcccatacta ccacgttttg ttaaaatctt ccaggctgca gcgattctcc ggctaatttt aactcctgac qagccacggc tgggaaatca PCTIUS99/2 1053 acatttaaat tqgagqatga agtctgaatg ttgtcacacc gggaccaggc tcataactac ctgggtgcct tgaaagagcc ccataaatgc agaacatgtg atcctgttag gtgtaactct tagccttaaa ctcagtattt aatcgctgga ctgcaggtcc gccacagtag gaggaagagt gtgcagtggt tgcctcagcc tgtattttta ctcgtgatcc gcccagccca tggttacgct taagttaatg atactggaga tagcacataa tccccggtgc cttcctaggt acctgctcca gctgaacaac attaaagtca tttctgagqa ggtccgagtg agtacatctt gtgtagggag tacctaaaag cccaatcatg cggtgggqt c aactcctggc ttgtaggatt ttttattttt gccatctcag acctqagtag atagagttga gcccgcctcq qqaagagtt t tcaggcatat ggcctttaaa cgggttacca tggttctctt tgttttacaa tttctaggca ggaccaatca tgagctcatg gtcgtgcqtg tccggtacaa ttataacaga tggaaatcca atagtcactt atgacagaag aaaatccctt agagqqcagt aaccgcgggc gcttttctgt tatttatttt ctcactgcaa ctgqgagtac qatttcacca gccccccaaa ttaaattaga tcttccccag aaaaaaaaag atgtcaggtt Ctgttgtcca cgtgagggta gtcagctgtt gtgaaacctq tcatgggcat aagcatctct aatqatttcc ctcctccccc gggtaatatt taaacagctc catagccctt gctatgtctt cggtatttag tcaaggcag at cat aatt t tqagatggag cctccacctc aggcatgtgg tgatqgtcag gtqctgggat gctctgttta agtactactt gaaacactca atactaaaac aqqctgtaaa gacgctgtca aaccactcac ctcggaatta gtggtggttt cttctaaagg caaagttctg gggtcacctt ctctttcaga aaaqtagcta aacaaatctt tcctactaga gccgtgagct tcattggaat tagaatgctc tctctgttgc ccaggttcaa caccatgcct gctggtctcg taacgggtgt attataccac acattttaaa 2420 2480 2540 2600 2660 2720 2780 2840 2900 2960 3020 3080 3140 3200 3260 3320 3380 3440 3500 3560 3620 3680 3740 3800 WO 00/14106 tttcattttg gtagacgttt catcgtgtgt gtaattggtc ctagtttcaa ttgacaccgg actgcatgta gaaaagaat c attaaacgct PCTfUS99/21 053 taaagttaaa cagtcattct gagtacaggg atqgaaatga tctgttCtcq atttaqctct attcttaaac tqtactgcaa gtagtccatg tgtcagcatt tttcaaacaa tqctcagctc tcagattgac ttccttgtac tgtcggcctt tgggcttgtc gtaaaacct a ttcatgcc ccctttaaaa gtgtttgtgt ttccaccgtc cttgattgac cggattattc cgtggggagc acattgtatt ctccccaaaa gtgtccattg accttttgcc attttgaatt tgtcagqcat tactcctqca tgtttgtgtt gtatttttgt atgtgtggct ttctttgaaa aagctgtggg gttcacatgg ggctttgttt atgaaccctg aatatqagct gatctgtaat ttgggtctgc 3860 3920 3980 4040 4100 4160 4220 4280 4308 <210> 24 <211> 447 <212> PRT <213> Homo sapiens <400> 24 Met 1 Gin Leu Asp Met Gly Lys Giu Val Ser Asp Asp Gin His Pro Ser Ser Arg Leu Gin Glu Ile Ser Val Giu Gin 25 Lys Vai Ile Gly Gin Leu Phe Giu Lys Asn Tyr Ile Asp Ser 55 Lys Arq Aia Lys 40 Val1 Leu Giu Arg Ile Lys Phe Ser Giy Leu Thr Lys Giy Asp Ile Asp Thr Giu Gin Gin Gly Giu Ala Arg Aia Gin Glu Arg Leu Leu Gi u 70 Ala Lys Leu Giu Gin Asn His Pro Ile Giu Ile Gin Asn Ile Phe Giu Tyr Asn Giy Glu 100 Gly Gin Ser Leu Val1 105 Asp Glu Lys Ile Vai Pro Phe 110 Gly Ile Gin Asn Cys Val Thr 46 Glu Phe Giu Glu WO 00/14106 115 Asp Ile Ile PCTIUS99/2 1053 120 His 125 Gly Leu Arq Leu 130 Arg Thr 135 His Val Lys Thr Giy 140 Ile Lys Ile Ser Le u 145 Giu Lys Ala Arg Tyr 150 Gys Thr Leu Thr Cys Ala Val Gin 160 Ile Ile Glu Asp 165 Pro Met Lys Lys Gin 170 Ile Ser Leu Pro Leu Ser 175 Glu Asp Ala Val Asn Lys 195 Asn Asn Gin His 180 Ala Ser Val Ala Lys 185 Ile Asn Phe Val Arg Gly Val Leu 200 Leu Ala Len Leu Met 205 Ser Met Cys Glu 190 Gly Val Asn Gly Le Ile Thr Cys Arg 210 Ala Asp His 215 Asp Ser Cys Val Len 220 Thr Leu Asp Ala TLeu 230 Val1 Val Cys Gly Arg 235 Lys Giu Ile Arg Arg Arg Gin Giu Asp Ile Asn 250 Lys Leu Len Lys Tyr Len 255 Asp Len Giu Asn His Ser 275 Ile Lys Asn 290 Ser Ser Lys 305 Ser Leu Gin Ile Gin Val Giu Lys Arg 355 Glu 260 Ile Ala Asp Thr Thr 265 Lys Ala Phe Asp Len Lys Ile Gin 280 Ala Val Leu Lys Arg 285 Gin Leu Arg Gin 270 Met Arq Giu Leu Tyr Len Gin Leu Leu Gin 295 Gin Gin Asn Pro Ser 300 Gin Thr Giu Lys Asn 325 Gin Thr Gly Leu Ile Gly 315 Ala Len Asp Gin Val 320 Cys Ile Arq Gin 330 Ile Arq Arq Arg Ala Val 335 Len Ile Thr 340 Lys Tyr 345 Glu Asp Leu Lys Leu Phe Ala Cys 360 Gin His Pro Ser 365 Giy Glu Ala Len 350 His Lys Ala Gin Val Leu Val Trp Asn Val Leu Gly Asn Leu Ser Gin Ile Gin 47 WO 00/14106 370 PCTIUS99/21 053 Se r Phe Asp Gly Asn Arg 390 Leu Thr Lys Gin Leu 405 Glu Lys Cys Lys Ala 420 Ile Leu Ser Tyr Leu Thr Asp Lys Asn Tyr 395 Leu Ala Leu Asp Ala 410 Ala Arg Lys Gin Ala 425 Asp Leu Lys Ser Asp 440 380 Ile Arg Leu Glu Glu 400 Val-Asp Pro Gin Gly 415 Val Arg Leu Ala Gin 430 Glu Trp Glu Tyr 445 Gi u Asn 435

Claims

1. A substantially purified nucleic acid molecule having a nucleotide sequence corresponding to or complementary to a nucleotide sequence selected from the group consisting of (SEQ ID NO:1), (SEQ ID NO:3), (SEQ ID (SEQ ID NO:7), (SEQ ID NO:9), (SEQ ID NO:19), (SEQ ID NO:21) and (SEQ ID NO:23).

2. The nucleic acid of claim 1 having a nucleotide sequence corresponding to or complementary to a nucleotide sequence that encodes a functionally active BAG family protein selected from the group consisting of (SEQ ID NO:2), (SEQ ID NO:4), (SEQ ID NO;6), (SEQ ID NO:8), (SEQ ID NO:10), (SEQ ID NO:20), (SEQ ID N0:22) and (SEQ ID NO:24).

3. The nucleic acid of claim 2 selected from the group consisting of (SEQ ID NO:1), (SEQ ID NO:3), (SEQ ID 20 (SEQ ID NO:7), (SEQ ID NO:9), (SEQ ID NO;19), (SEQ ID NO:21) and (SEQ ID NO:23).

4. The nucleic acid of claim 2 complementary to a nucleotide sequence that encodes a functionally active BAG o o oo oo o ooeo *o oo o o *o g oooo ,ooo o *oo 25 protein selected from NO:2), (SEQ ID NO:4), ID NO:10), (SEQ ID NO: NO:24) 30 5. A substantially having the nucleotide

6. A substantially having the nucleotide

7- A substantially having the nucleotide the group consisting of (SEQ ID (SEQ ID NO:6), (SEQ ID NO:8), (SEQ (SEQ ID NO:22) and (SEQ ID purified nucleic acid molecule sequence of (SEQ ID NO:3). purified nucleic acid molecule sequence of (SEQ ID purified nucleic acid molecule sequence of (SEQ ID NO:7). n;\tarfl'\Kq\5p8CP41381~m-li~MJ15O 40 410914 COMS ID No: SMBI-00744173 Received by IP Australia: Time 16:08 Date 2004-05-11 11/05/2004 16:06 GRIFFITH HACK 4 00262837999 N0.802 0004

8. A substantially having the nucleotide

9. A substantially having the nucleotide A substantially having the nucleotide .0

11. A substantially having the nucleotide purified sequence purified sequence purified sequence purified sequence nucleic of (SEQ nucleic of (SEQ nucleic of (SEQ nucleic of (SEQ acid molecule ID NO:9). acid molecule ID NO:19). acid molecule ID NO:21). acid molecule ID NO:23). *0 *0 0 0 0 *go o*o ooooo

12. A substantially purified BAG family protein encoded by the nucleic acid molecule of claim 1.

13. A substantially purified BAG family protein comprising of the amino acid sequence selected from the group consisting of (SEQ ID NO:4), (SEQ ID NO:6), (SEQ ID 20 NO:8), (SEQ ID NO:10), (SEQ ID NO:20), (SEQ ID NO:22) and (SEQ ID NO;24) or a fragment, a derivative or a mimetic thereof, wherein said protein, fragment, derivative or mimetic has an ability to bind to 25 14. A substantially purified protein corresponding to the amino acid sequence of 164 to 211 of (SEQ ID NO:4).

15. A substantially purified protein corresponding to the amino acid sequence of 378 to 457 of (SEQ ID NO:22).

16. A substantially purified protein corresponding to the amino acid sequence of 6 to 97 of (SEQ ID NO:24).

17. A substantially purified protein corresponding to the amino acid sequence of 180 to 257 of (SEQ ID NO:24). r:\araI\Kep\Speol\413i1- aimms-1.&.o4.aoc 11/05/04 COMS ID No: SMBI-00744173 Received by IP Australia: Time 16:08 Date 2004-05-11 11/05/2004 16:06 GRIFFITH HACK 4 00262837999 N0.802 9005

18. A substantially purified protein corresponding to the amino acid sequence-of 272 to 349.of (SEQ ID NO:24).

19. A substantially purified protein corresponding to the amino acid sequence of 362 to 444 of (SEQ ID NO:24). A pharmaceutical composition comprising a nucleic acid molecule of claim 1 useful for modulating tumor cell proliferation, cell migration and metastasis, and steroid hormone receptor function.

21. A method of modulating tumor cell proliferation, cell migration and metastasis, and steroid hormone receptor function by administering a nucleic acid molecule of claim 1.

22. A pharmaceutical composition comprising a substantially purified BAG family protein comprising of the amino acid sequence selected from the group consisting 20 of (SEQ ID NO;2), (SEQ ID NO:4), (SEQ ID NO;6), (SEQ ID NO;8), (SEQ ID NO;10), (SEQ ID NO;20), (SEQ ID NO:22) and (SEQ ID NO:24), or a fragment, a derivative or a mimetic thereof, wherein said protein, fragment, derivative or mimetic has an ability to bind to Hsc70 and is useful 25 for modulating tumor cell proliferation, cell migration and metastasis, and steroid hormone receptor function and a pharmaceutically acceptable carrier.

23. A method of modulating tumor cell proliferation by administering a pharmaceutical composition of claim 21.

24. A method of modulating cell migration and metastasis S"by administering a pharmaceutical composition of claim 21. 9

25. A method of modulating steroid hormone receptor function by administering a pharmaceutical composition of claim 22. H:\MaraR\Kiep\Spqci\P4138L.-clai=-11.5s. .do n11/,'5/O COMS ID No: SMBI-00744173 Received by IP Australia: Time 16:08 Date 2004-05-11 11/05/2004 16:06 GRIFFITH HACK 4 00262837999 NO.802 P006 42

26. A substantially purified antibody that specifically binds to a BAG family protein of claim 13.

27. The antibody of claim 26, wherein said antibody is a monoclonal antibody.

28. A method for detecting the presence of a BAG family protein in a sample, comprising the steps of; a. obtaining the sample; b. adding to said an antibody of claim 26 under suitable conditions for the binding of said antibody with the BAG family protein, and c. detecting said bound BAG family protein.

29. A method for detecting the presence of a first nucleic acid molecule that encodes a BAG family protein in 20 a sample, comprising the steps of: 0 a. obtaining the sample; b. adding to said sample a second nucleic acid 25 molecule capable of hybridizing with said first nucleic acid molecule under suitable conditions for the binding of said second nucleic acid molecule with said first nucleic acid molecule; and c. detecting said hybridized first and second 30 nucleic acid molecules.

30. A method of determining the risk of metastatic spread of cancer or prognosis of cancer patients by •determining the level of expression of a BAG-family protein or a fragment thereof, wherein said BAG-family protein or fragment thereof comprises an amino acid sequence selected from the group consisting of (SEQ ID Nl\Ir<Ir\Ke.«;\Cpc\1413 ll-clim «-11.5.OI.doc 11W05/04 COMS ID No: SMBI-00744173 Received by IP Australia: Time 16:08 Date 2004-05-11 11/05/2004 16:06 GRIFFITH HACK 00262837999 N0.802 0007 43 NO:4), (SEQ ID NO:6), (SEQ ID NO:8), (SEQ ID NO;10), (SEQ ID NO:20), (SEQ ID NO:22) and (SEQ ID NO:24), amino acid sequence 157 to 204 of (SEQ ID NO:2), amino acid sequence 272 to 319 of (SEQ ID NQ:2), amino acid sequence 378 to 457 of (SEQ ID NO:22), amino acid sequence 6 to 97 of (SEQ ID NO:24), amino acid sequence 180 to 257 of (SEQ ID NO:24), amino acid sequence 272 to 349 of (SEQ ID NO:24), and amino acid sequence 362 to 444 of (SEQ ID NO:24).

31. The substantially purified nucleic acid molecule of claim 1, wherein said molecule has a nucleotide sequence corresponding to or complementary to (SEQ ID NO:1).

32. The substantially purified nucleic acid molecule of claim 1, wherein said molecule has a nucleotide sequence corresponding to or complementary to (SEQ ID NO:3).

33. The substantially purified nucleic acid molecule of claim 1, wherein said molecule has a nucleotide sequence S, 20 corresponding.to or complementary to (SEQ ID

34. The substantially purified nucleic acid molecule of claim 1, wherein said molecule has a nucleotide sequence corresponding to or complementary to (SEQ ID NO:7). eo

35. The substantially purified nucleic acid molecule of claim 1, wherein said molecule has a nucleotide sequence corresponding to or complementary to (SEQ ID NO:9). S 30 36. The substantially purified nucleic acid molecule of claim 1, wherein said molecule has a nucleotide sequence corresponding to or complementary to (SEQ ID NO:19).

37. The substantially purified nucleic acid molecule of claim 1, wherein said molecule has a nucleotide sequence i corresponding to or complementary to (SEQ ID NO;21)- H.\Mlt*\KoQp\Spaci\P43a-claisZ-11.S.4.dC 1i/05/04 COMS ID No: SMBI-00744173 Received by IP Australia: Time 16:08 Date 2004-05-11 11/05/2004 16:06 GRIFFITH HACK 4 00262837999 NO.802 008 44

38. The substantially purified nucleic acid molecule of claim 1, wherein said molecule has a nucleotide sequence corresponding to or complementary to (SEQ ID N0:23).

39. A substantially purified nucleic acid molecule having a nucleotide sequence corresponding to or complementary to at least 20 contiguous 'nucleotides from a nucleotide sequence selected from the group consisting of (SEQ ID NO:3), (SEQ ID NO:7), (SEQ ID NO;9),(SEQ ID NO:21) and (SEQ ID NO:23). The substantially purified nucleic acid molecule of claim 39, wherein said nucleotide sequence corresponds to or is complementary to at least 20 contiguous nucleotides of SEQ ID NO:3.

41. The substantially purified nucleic acid molecule of claim 39, wherein said nucleotide sequence corresponds to or is complementary to at least 20 contiguous nucleotides 20 of SEQ ID NO:7.

42. The substantially purified nucleic acid molecule of claim 39, wherein said nucleotide sequence corresponds to "or is complementary to at least 20 contiguous nucleotides 25 of SEQ ID NO:9.

43. The substantially purified nucleic acid molecule of claim 39, wherein said nucleotide sequence corresponds to or is complementary to at least 20 contiguous nucleotides 30 of SEQ ID NO:21.

44. The substantially purified nucleic acid molecule of claim 39, wherein said nucleotide sequence corresponds to or is complementary to at least 20 contiguous nucleotides of SEQ ID NO:23. H:\NarR\eP p\pcl\±4s1-claainBs-ll.5.04.doc 11/03/o COMS ID No: SMBI-00744173 Received by IP Australia: Time 16:08 Date 2004-05-11 11/05/2004 16:06 GRIFFITH HACK 00262837999 N0.802 9009 A substantially purified BAG family protein comprising of the amino acid sequence selected from the group consisting of (SEQ ID NO:2), (SEQ ID NO:4), (SEQ ID NO:6), (SEQ ID NO:8), (SEQ ID NO:10), (SEQ ID NO;20), (SEQ ID NO:22) and (SEQ ID NO:24).

46. A pharmaceutical composition comprising a substantially purified BAG family protein comprising of the amino acid sequence selected from the group consisting of (SEQ ID NO:2), (SEQ ID NO:4), (SEQ ID NO:6), (SEQ ID NO:8), (SEQ ID NO:10), (SEQ ID NO:20), (SEQ ID NO:22) and (SEQ ID NO:24) and a pharmaceutically acceptable carrier. C C C f go ooo'o o oo ooo oool H:\MaraR\lK[el\SpeCi\P41381-Cla8-ma-11.5.04.CO0 i1/05/04 COMS ID No: SMBI-00744173 Received by IP Australia: Time 16:08 Date 2004-05-11