Polypeptide that interacts with heat shock proteins Number:7,094,873 from the United States Patent and Trademark Office (PTO) owispatent

Title: Polypeptide that interacts with heat shock proteins

Abstract: An isolated polypeptide having negative regulating activity for a heat shock protein is provided. Also provided is an isolated nucleic acid encoding the polypeptide of the invention, methods for identifying inhibitors of the polypeptide and recombinant preparation of the polypeptide. Also provided are compositions such as inhibitor compositions.

Patent Number: 7,094,873 Issued on 08/22/2006 to Patterson,   et al.

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Inventors:	Patterson; Winston Campbell (Chapel Hill, NC), Ballinger; Carol A. (Santa Fe, TX)
Assignee:	Board of Regents, The University of Texas System (Austin, TX)
Appl. No.:	09/573,473
Filed:	May 17, 2000

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Current U.S. Class:	530/350 ; 536/23.5
Current International Class:	C07K 14/00 (20060101)
Field of Search:	536/23.5 530/350

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References Cited [Referenced By]

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U.S. Patent Documents

	4436815		March 1984		Hershberger et al.
	4440859		April 1984		Rutter et al.
	6043084		March 2000		Scanlan et al.
	6165767		December 2000		Lal et al.
	6218521		April 2001		Obata
	6338952		January 2002		Young

Foreign Patent Documents

	WO-9904265		Jan., 1999		WO

Other References

Yang, X, et al, 1998, Human BAG-1/RAP46 protein is generated as four isoforms by alternative initiation and overexpressed in cancer cells, Oncogene, vol. 17, No. 8, pp. 981-989. cited by examiner . Takayama, S. et al, 1995, Cloning and functional analysis of BAG-1: a novel Bcl-2 binding protein with anti-cell death activity. Cell, vol. 80, No. 2, pp. 279-284. cited by examiner . Froesch, BA, et al, 1998, BAG-1L protein enhances androgen receptor function, Journal of Biological Chemistry, vol. 273, No. 19, pp. 11660-11666. cited by examiner . Cato, ACB, et al, 2001, BAG-1 family of cochaperones in the modulation of nuclear receptor action, Journal of Steroid Biochemistry & Molecular Biology, vol. 78, No. 5, pp. 379-388. cited by examiner . Prapapanich, V, et al, 1996, Mutational analysis of the hsp-70-interacting protein Hip, Molecular and Cellular Biology, vol. 16, No. 11, pp. 6200-6207. cited by examiner . Jiang, J., et al, 2001, CHIP is a U-box-dependent E3 ubiquitin ligase, Journal of Biological Chemistry, vol. 276, No. 46, pp. 42938-42944. cited by examiner . Prapapanich, V, et al, 1998, Mutation of Hip's carboxyl-terminal region inhibits a transitional stage of progesterone receptor assembly, Molecular and Cellular Biology, vol. 18, No. 2, pp. 944-952. cited by exa- miner . Bork, P, 2000, Powers and pitfalls in sequence analysis: the 70% hurdle, Genome Research, vol. 10, pp. 398-400. cited by examiner . Burgess, WH, et al, 1990, Possible dissociation of the heparin-binding and mitogenic activities of heparin-binding (acidic fibroblast) growth factor-1, J Cell Biology, vol. 111, pp. 2129-2138. cited by examiner . Lazar, E, et al, 1988, Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities, Molecular and Cellular Biology, vol. 8, pp. 1247-1252. cited by examiner . Bowie, JU, et al, 1990, Deciphering the message in protein sequences: tolerance to amino acid substitutions, Science, vol. 247, pp. 1306-1310. cited by examiner . Scanlan, MJ, Direct Submission, Dec. 23, 1997, Database GenBank Accession No. AF039689, Homo sapiens antigen NY-CO-7 (NY-CO-7) mRNA, complete cds. cited by examiner . Gura T. Science Nov. 7, 1997; 278: 1040-1. cited by examiner . Bergers G, et al. Cur Opin Genetics Develop 2000; 10: 120-7. cited by exam- iner . Skolnick, J, et al. Trends Biotechnol Jan. 2000; 18: 34-9. cited by examin- er . De Plaen E, et al. Immunogenetics. 1994; 40: 360-9. cited by examiner . Tockman MS, et al. Cancer Res. 1992; 52 (Suppl.): 2711s-2718s. cited by examiner . Ward AM. Developmental Oncol. 1985; 21: 90-106. cited by examiner . Goebl M, et al. Trends Biochem Sci. May 1991;16 (5): 173-7. cited by exami- ner . Murata S, et al. EMBO Rep. Dec. 2001;2 (12): 1133-8. cited by examiner . Nickolay R, et al. J biol Chem. Jan. 23, 2004; 279 (4): 2673-8. cited by examiner . Murata S, et al. Int J Biochem Cell Biol. 2003; 35: 572-8. cited by examin- er . Kampringa et al. (Mol. Cell. Biol. Jul. 2003; 23 (14): 4948-4958). cited by examiner . Luque et al. (Biochemistry. Nov. 19, 2002; 41 (46): 13663-13671. cited by examiner . Vucic et al. (J. Biol. Chem. Dec. 18, 1998; 273 (51): 33915-33921). cited by examiner . Takada et al. (Mol. Endocrinol. 2000; 14 (5): 733-740). cited by examiner . Guo et al. (Proc. Natl. Acad. Sci. USA. Jun. 22, 2004; 101 ;(25): 9205-9210). cited by examiner . Ballinger et al., "p35 is a Novel Protein Highly Expressed in Striated Muscle that Interacts with Members of the Heat Shock Protein Family," Abstract 643, 71.sup.st Annual Meeting of the American Heart Association, Nov. 8-11, Dallas, Supplement to Circulation,98(17):125 (Oct. 27, 1998). cited by other . Ballinger et al., "Identification of CHIP, a Novel Striated Muscle-Restricted TPR-Containing Protein that Negatively Regulates Chaperone Functions," Abstract 370.7 and Poster, Annual Meeting of Professional Research Scientists and American Association of Anatomists, Experimental Biology '99, Apr. 17-21, Washington, D.C., The FASEB Journal, 13(4):A442, (Mar. 12, 1999). cited by other . Ballinger et al., "Identification of CHIP, a Novel Tetratricopeptide Repeat-Containing Protein That Interacts with Heat Shock Proteins and Negatively Regulates Chaperone Functions," Molecular and Cellular Biology, 19(6):4535-4545 (Jun. 1999; available on line May 17, 1999). cit- ed by other . Ballinger et al., "Ubiquitylation of HSC70 is Mediated by CHIP, a Novel Ubiquitylation Factor," Abstract and oral presentation, Cold Spring Harbor Meeting on Molecular Chaperones & Heat Shock Response, May 3-7, Cold Spring Harbor, N.Y. (May 4, 2000). cited by other . Barr et al., "7-Deaza-2'-Deoxyguanosine-5'-Triphosphate: Enhanced Resolution in M13 Dideoxy Sequencing," BioTechniques, 4(5):428-432 (1986). cited by other . Baumann et al., "Dexamethasone Regulates the Program of Secretory Glycoprotein Synthesis in Hepatoma Tissue Culture Cells," The Journal of Cellular Biology, 85:1-8 (1980). cited by other . Benaroudj et al., "The COOH-terminal Peptide Binding Domain Is Essential for Self-association of the Molecular Chaperone HSC70," The Journal of Biological Chemistry, 272(13):8744-8751 (1997). cited by other . "BLAST," National Institutes of Health [online] United States, [retrieved Oct. 23, 2000]. Retrived from the Internet. cited by other . Boice et al., "A Mutational Study of the Peptide-binding Domain of Hsc70 Guided by Secondary Structure Prediction," The Journal of Biological Chemistry, 272(40):24825-24831 (1997). cited by other . Braun et al., "The base of the proteasome regulatory particle exhibits chaperone-like activity," Nature Cell Biology, 1:221-226 (Aug. 1999). cit- ed by other . Broach, "[21] Construction of High Copy Yeast Vectors Using 2-.mu.m Circle Sequences," Methods in Enzymology: vol. 101 Recombinant DNA, pp. 307-325 (1983). cited by other . Carrello et al., "The Common Tetratricopeptide Repeat Acceptor Site for Steroid Receptor-associated Immunophilins and Hop Is Located in the Dimerization Domain of Hsp90," The Journal of Biological Chemistry, 274(5):2682-2689 (Jan. 29, 1999). cited by other . Chakrabarti et al., "Vaccina Virus Expression Vector: Coexpression of .beta.-Galactosidase Provides Visual Screening of Recombinant Virus Plaques," Molecular and Cellular Biology, 5(12):3403-3409 (1985). cited by other . Chappell et al., "The ATPase Core of a Clathrin Uncoating Protein," The Journal of Biological Chemistry, 262(2):746-751 (1987). cited by other . Chen et al., "Interactions of p60, a Mediator of Progesterone Receptor Assembly, with Heat Shock Proteins Hsp90 and Hsp70," Molecular Endocrinology, 10(6):682-693 (1996). cited by other . Clewell et al., "Supercoiled Circular DNA-Protein Complex in Escherichia coli: Purification and Induced Conversion to an Open Circular DNA Form," Proceedings of the National Academy of Sciences USA, 62:1159-1166 (1969). cited by other . Clewell, "Nature of Col E.sub.1 Plasmid Replication in Escherichia coli in the Presence of Chlorampenicol," Journal of Bacteriology, 110(2):667-676 (1972). cited by other . Cohen et al., "Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA," Proceedings of the National Academy of Sciences USA, 69(8):2110-2114 (1972). cited by other . Connell et al., "CHIP's Interaction with HSP90 Regulates Glucocorticoid Receptor Function and Degradation," Abstract and poster, Cold Spring Harbor Meeting on Molecular Chaperones & Heat Shock Response, May 3-7, Cold Spring Harbor, N.Y., 25 pages (May 4, 2000). cited by other . Connell et al., "The co-chaperone CHIP regulates protein triage decisions mediated by heat shock proteins," Nature Cell Biology, 3(1):93-96 (2001). cited by other . Das et al., "The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions," The EMBO Journal, 17(5):1192-1199 (1998). cited by other . De Boer et al., "The tac promoter: A functional hybrid derived from the trp and lac promoters," Proceedings of the National Academy of Sciences USA, 80:21-25 (1983). cited by other . Demand et al., "The Carboxy-Terminal Domain of Hsc70 Provides Binding Sites for a Distinct Set of Chaperone Cofactors," Molecular and Cellular Biology, 18(4):2023-2028 (1998). cited by other . Deuerling et al., "Trigger factor and DnaK cooperate in folding of newly synthesized proteins," Nature, 400:693-696 (Aug. 12, 1999). cited by othe- r . Dittmar et al., "Folding of the Glucocorticoid Receptor by the Reconstituted hsp90-based Chaperone Machinery," The Journal of Biological Chemistry, 272(20):13047-13054 (1997). cited by other . Dubiel et al., "Molecular cloning and expression of subunit 12: a non-MCP and non-ATPase subunit of the 26 S protease," FEBS Letters, 363:97-100 (1995). cited by other . Ellis, "The "Bio" in Biochemistry: Protein Folding Inside and Outside the Cell," Science, 272:1448-1449 (1996). cited by other . Fiers et al., "Complete nucleotide sequence of SV40 DNA," Nature, 273(5658):113-120 (1978). cited by other . Frydman et al., "Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones," Nature, 370:111-117 (1994). cit- ed by other . Frydman et al., "Principles of Chaperone-Assisted Protein Folding: Differences Between in Vitro and in Vivo Mechanisms," Science, 272:1497-1502 (1996). cited by other . "GenomeNet WWW server," Institute for Chemical Research, Kyoto University [online]. Kyoto, Japan, updated Oct. 14, 2000 [retrieved Oct. 23, 2000]. Retrieved from the Internet: <URL: http://www.genome.ad.jp/SIT/CLUSTALW.html>, 2 pages. cited by other . Goebl et al., "The TPR snap helix: a novel protein repeat motif from mitosis to transcription," Trends in Biochemical Sciences, 16(5):173-177 (1991). cited by other . Goeddel et al., "Synthesis of human fibroblast interferon by E. coli," Nucleic Acids Research, 8(18):4057-4074 1980). cited by other . Graham et a., "A New Technique for the Assay of Infectivity of Human Adenovirus 5 DNA," Virology, 52:456-467 (1973). cited by other . Gyuris et al., "Cdi1, a Human G1 and S Phase Protein Phosphatase That Associates with Cdk2," Cell, 75:791-803 (1993). cited by other . Harlow et al., Antibodies; a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Title page, publication page, and table of contents only, 9 pages (1988). cited by other . Hartl, "Molecular chaperones in cellular protein folding," Nature, 381:571-579 1996. cited by other . Hinnen et al., "Transformation of yeast," Proceedings of the National Academy of Sciences USA, 75(4):1929-1933 (1978). cited by other . Hirano et al., "Snap Helix with Knob and Hole: Essential Repeats in S. pombe Nuclear Protein nuc2.sup.+," Cell, 60:319-328 (1990). cited by othe- r . Hitzeman et al., "Isolation and Characterization of the Yeast 3-Phosphoglycerokinase Gene (PGK) by an Immunological Screening Technique," The Journal of Biological Chemistry, 255(24):12073-12080 (1980). cited by other . Hohfeld et al., "Hip, a Novel Cochaperone Involved in the Eukaryotic Hsc70/hsp40 Reaction Cycle," Cell, 83:589-598 (1995). cited by other . Hohfeld et al., "GrpE-like regulation of the Hsc70 chaperone by the anit-apoptotic protein BAG-1," The EMBO Journal, 16(20):6209-6216 (1997). cited by other . Hohfeld, "Regulation of the Heat Schock Cognate Hsc70 in the Mammalian Cell: The Characterization of the Anti-Apoptotic Protein BAG-1 Provides Novel Insights," Biological Chemistry, 379:269-274 (1998). cited by other . Holland et al., "The Primary Structures of Two Yeast Enolase Genes," The Journal of Biological Chemistry, 256(3):1385-1395 (1981). cited by other . Irmer et al., "Characterization of Functional Domains of the Eukaryotic Co-chaperone Hip," The Journal of Biological Chemistry, 272(4):2230-2235 (1997). cited by other . Johnson et al., "Characterization of a Novel 23-Kilodalton Protein of Unactive Progesterone Receptor Complexes," Molecular and Cellular Biology, 14(3):1956-1963 (1994). cited by other . Johnson et al., "A Proteolytic Pathway That Recognizes Ubiquitin as a Degradation Signal," The Journal of Biological Chemistry, 270(29):17442-17456 (1995). cited by other . Kay, "Structure-function relationships in the FK506-binding protein (FKBP) family of peptidylprolyl cis-trans isomerases," B iochemical Journal, 314:361-385 (1996). cited by other . Kieffer et al., "Cyclophilin-40, a Protein with Homology to the P59 Component of the Steroid Receptor Complex," The Journal of Biological Chemistry, 268(17):12303-12310 (1993). cited by other . Koegl et al., "A Novel Ubiquitination Factor, E4, Is Involved in Multiubiquitin Chain Assembly," Cell, 96:635-644 (Mar. 5, 1999). cited by other . Lamb et al., "Tetratrico peptide repeat interactions: to TPR or not to TPR?" Trends in Biochemical Sciences, 20:257-259 (1995). cited by other . Lee et al., "Selective Inhibitors of the Proteasome-dependent and Vacuolar Pathways of Protein Degradation in Saccharomyces cerevisiae," The Journal of Biological Chemistry, 271(44):27280-27284 (1996). cited by other . Liberek et al., "Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK," Proceedings of the National Academy of Sciences USA, 88:2874-2878 (1991). cited by other . Lu et al., "The Conserved Carboxyl Terminus and Zinc Finger-like Domain of the Co-chaperone Ydj1 Assist Hsp70 in Protein Folding," The Journal of Biological Chemistry, 273;(10):5970-5978 (1998). cited by other . Luckow et al., "High Level Expression of Nonfused Foreign Genes with Autographa californica Nuclear Polyhedrosis Virus Expression Vectors," Virology, 170:31-39 (1989). cited by other . Luders et al., "Cofactor-Induced Modulation of the Functional Specificity of the Molecular Chaperone Hsc70," Biological Chemistry, 379:1217-1226 (1998). cited by other . Mackett et al., "General Method for Production and Selection of Infectious Vaccinia Virus Recombinants Expressing Foreign Genes," Journal of Virology, 49(3):857-864 (1984). cited by other . Maheswaran et al., "Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70," Genes & Development, 12:1108-1120 (1998). cited by other . Mather, "Establishment and Characterization of Two Distinct Mouse Testicular Epithelial Cell Lines," Biology of Reproduction, 23:243-252 (1980). cited by other . Mather et al., "Culture of Testicular Cells in Hormone-Supplemented Serum-Free Medium," Annals of the New York Academy of Sciences, 383:44-68 (1982). cited by other . Maxam et al., "[57] Sequencing End-Labeled DNA with Base-Specific Chemical Cleavages," Methods in Enzymology, vol. 65, Nucleic Acids Part I, pp. 499-590 (1980). cited by other . Meacham et al., "The Hc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation," Nature Cell Biology, 3(1):100-105 (2001). cited by other . Messing et al., "A system for shotgun DNA sequencing," Nucleic Acids. Research, 9(2):309-321 (1981). cited by other . Minami et al., "Regulation of the Heat-shock Protein 70 Reaction Cycle by the Mammalian DnaJ Homolog, Hsp40," The Journal of Biological Chemistry, 271(32):19617-19624 1996). cited by other . Morimoto, "Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators," Gene & Development, 12:3788-3496 (1998). cited by other . Moss, "Vaccinia Virus Expression Vectors," Gene Transfer Vectors for Mammalian Cells, Miller et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Title page, publication page, table of contents, and pp. 10-14 (1987). cited by other . Nair et al., "Molecular Cloning of Human FKBP51 and Comparisons of Immunophilin Interactions with Hsp90 and Progesterone Receptor," Molecular and Cellular Biology, 17(2):594-603 (1997). cited by other . National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, GenBank Locus HUMHSP70D, Accession No. M11717 M15432, "Human heat shock protein (hsp 70) gene," [online]. Bethesda, MD [retrieved on Oct. 24, 2000]. Retrived from the Internet. cited by other . National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, GenBank Locus HSHSC70, Accession No. Y00371, "Human hsc 70 gene for 71 kd heat shock cognate protein," [online]. Bethesda, MD [retrieved on Oct. 24, 2000]. Retrived from the Internet. cited by other . National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, GenBank Locus CVU57443, Accession No. U57443, "Cloning vector pODB8, GAL4 DNA-binding domain vector, complete sequence," [online]. Bethesda, MD [retrieved on Jan. 25, 2001]. Retrieved from the Internet. cited by other . National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, GenBank Locus MMU20344, Accession No. U20344, "Mus musculus gut-enriched Kruppel-like factor GKLF mRNA," [online]. Bethesda, MD [retrieved on Nov. 3, 2000]. Retrieved from the Internet. cited by other . National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, GenBank Locus AF039689, Accession No. AF039689, "Homo sapiens antigen NY-CO-7 (NY-CO-7) mRNA," [online]. Bethesda, MD [retrieved on Oct. 24, 2000]. Retrieved from the Internet. cited by other . National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, GenBank Locus AF129084, Accession No. AF129084, "Drosophila melanogaster carboxy terminus of Hsp70-interacting protein (CHIP) mRNA," [online]. Bethesda, MD [retrieved on Oct. 24, 2000]. Retrieved from the Internet. cited by other . National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, GenBank Locus AF129085, Accession No. AF129085, "Homo sapiens carboxy terminus of Hsp70-interacting protein (CHIP) mRNA," [online]. Bethesda, MD [retrieved on Oct. 24, 2000]. Retrieved from the Internet. cited by other . National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, GenBank Locus AF129086, Accession No. AF129086, "Mus musculus carboxy terminus of Hsp7-interacting protein (Chip) mRNA," [online]. Bethesda, MD [retrieved on Oct. 24, 2000]. Retrieved from the Internet. cited by other . National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, GenBank Locus HS313D11, Accession No. Z92544, "Human DNa sequence from cosmid 313D11 from a contig on the short arm of chromosome 16," [online]. Bethesda, MD [retrieved on Oct. 24, 2000]. Retrieved from the Internet. cited by other . National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, GenBank Locus NM.sub.--004323, Accession No. NM.sub.--004323, "Homo sapiensBCL2-associated athanogene (BAG1), mRNA," [online]. Bethesda, MD [retrieved on Nov. 15, 2000]. Retrieved from the Internet. cited by other . Naylor et al., "Evidence for the Existence of Distinct Mammalian Cytosolic, Microsomal, and Two Mitochondrial GrpE-like Proteins, the Co-chaperones of Specific Hsp70 Members," The Journal of Biological Chemistry, 273(33):21169-21177 (1998). cited by other . Netzer et al., "Protein folding in the cytosol: chaperonin-dependent and -independent mechanisms," Trends in Biochemical Sciences, 23:68-73 (1998). cited by other . Owens-Grillo et al., "The Cyclosporin A-binding Immunophilin CyP-40 and the FK506-binding Immunophilin hsp56 Bind to a Common Site on hsp90 and Exist in Independent Cytosolic Heterocomplexes with the Untransformed Glucocorticoid Receptor," The Journal of Biological Chemistry, 270(35):20479-20484 (1995). cited by other . Owens-Grillo et al., "A Model of Protein Targeting Mediated by Immunophilins and Other Proteins That Bind to hsp90 via Tetratricopeptide Repeat Domains," The Journal of Biological Chemistry, 271(23):13468-13475 (1996). cited by other . Patterson et al., "Cloning and Functional Analysis of the Promoter for KDR/flk-1, a Receptor for Vascular Endothelial Growth Factor," The Journal of Biological Chemistry, 270(39):23111-23118 (1995). cited by oth- er . Patterson, Winston C., "Mechanisms of KDR-FLK-1 Gene Regulation," Grant Abstract, Grant No. 1K08HL03658-01 [online]. National Heart, Lung, and Blood Institute, National Institutes of Health, project dates Jul. 1, 1997-Jun. 30, 2002 [retrieved on Jan. 24, 2001]. Retrieved from the Internet. cited by other . Patterson, Winston C., "Mechanisms of KDR-FLK-1 Gene Regulation," Grant Abstract, Grant No. 5K08HL03658-02 [online]. National Heart, Lung, and Blood Institute, National Institutes of Health, project dates Jul. 1, 1997-Jun. 30, 2002 [retrieved on Jan. 24, 2001]. Retrieved from the Internet. cited by other . Patterson, Winston C., "Mechanisms of Neovascularization in Ahtersclerosis," Grant Abstract, Grant No. 1R03AG15234-01 [online]. National Institute on Aging, National Institutes of Health, project dates Sep. 30, 1997-Sep. 29, 1998 [retrieved on Jan. 24, 2001]. Retrieved from the Internet. cited by other . Patterson et al., Poster and abstract, "Flavopiridol Inhibits Vascular Smooth Muscle Cell Proliferation in Vitro and Neointimal Formation in Vivo After Carotid Injury in the Rat," Conifer, Excerpta Medica Medical Communications B.V. [online]. Abstract 407-4, American College of Cardiology meeting, New Orleans, LA, Mar. 7-10, 1999, available on-line Jan. 31, 1999[abstract retrieved on Feb. 16, 2001]. Retrieved from the Internet. cited by other . Patterson et al., Poster, Keystone Conference: Protein Folding, Degradation, and Molecular Chaperones, Apr. 10-16, Copper Mountain Resort, Copper Mountain, CO, 22 pages (1999). cited by other . Pratt et al., "Steroid Receptor Interactions with Heat Shock Protein and Immunophilin Chaperones," Endocrine Reviews, 18(3):306-360 1997). cited by other . Prodromou et al., "Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones," The EMBO Journal, 18(3):754-762 (Feb. 1, 1999). cited by other . Pukatzki et al., "A Novel Component Involved in Ubiquitination Is Required for Development of Dictyostelium discoideum," The Journal of Biological Chemistry, 273(37):24131-24138 (1998). cited by other . Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Title page, publication page, and table of contents only, 30 pages (1989). cited by other . Sanger et al., "DNA sequencing with chain-terminating inhibitors," Proceedings of the National Academy of Sciences US, 74(12):5463-5467 (1977). cited by other . Scanlan et al., "Characterization of Human Colon Cancer Antigens Recognized by Autologous Antibodies," International Journal of Cancer, 765):652-658 (1998). cited by other . Scherrer et al., "Structural and Functional Reconstitution of the Glucocorticoid Receptor-Hsp90 Complex," The Journal of Biological Chemistry, 265(3):21397-21400 1990). cited by other . Schneider et al., "Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90," Proceedings of the National Academy of Sciences USA, 93(25):14536-14541 (1996). cited by other . Schumacher et al., "ATP-dependent Chaperoning Activity of Reticulocyte Lysate," The Journal of Biological Chemistry, 269(13):9493-9499 (1994). cited by other . Segnitz et al., "The Function of Steroid Hormone Receptors Is Inhibited by the hsp90-specific Compound Geldanamycin," The Journal of. Biological Chemistry, 272(30):18694-18701 (1997). cited by other . Shields et al., "Two Potent Nuclear Localization Signals in the Gut-enriched Kruppel-like Factor Define a Subfamily of Closely Related Kruppel Proteins," The Journal of Biological Chemistry, 272(29):18504-18507 (1997). cited by other . Shimatake et al., "Purified .lamda. regulatory protein cII positively activates promoters for lysogenic development," Nature, 292(5819):128-132 (1981). cited by other . Sikorski et al., "A Repeating Amino Acid Motif in CDC23 Defines a Family of Proteins and a New Relationship among Genes Required for Mitosis and RNA Synthesis," Cell, 60:307-317 1990). cited by other . Smith et al., "Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector," Molecular and Cellular Biology, 3(12):2156-2165 (1983). cited by other . Smith et al., "Identification of a 60-Kilodalton Stress-Related Protein, p60, Which Interacts with hsp90 and hsp70," Molecular and Cellular Biology, 13:869-876 (1993). cited by other . Smith et al., "Molecular Chaperones: Biology and Prospects for Pharmacological Intervention," Pharmacological Reviews, 50(4):493-513 (1998). cited by other . Summers et al., "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures," Texas Agricultural Experiment Station Bulletin No. 1555, Department of Entomology, Texas Agricultural Experiment Station and Texas A&M University, Title page and pp. 1-56 (1987). cited by other . Takayama et al., "BAG-1 modulates the chaperone activity of Hsp70/Hsc70," The EMBO Journal, 16(16):4887-4896 (1997). cited by other . Teter et al., "Polypetide Flux through Bacterial Hsp70: DnaK Cooperates with Trigger Factor in Chaperoning Nascent Chains," Cell, 97:755-765 (Jun. 11, 1999). cited by other . Ungewickell et al., "Functional Interaction of the Auxilin J Domain with the Nucleotide- and Substrate-binding Modules of Hsc70," The Journal of Biological Chemistry, 272(31):19594-19600 (1997). cited by other . Urlaub et al., "Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity," Proceedings of the National Academy of Sciences USA, 77(7):4216-4220 (1980). cited by other . Whitesell et al., "Stable and Specific Binding of Heat Shock Protein 90 by Geldanamycin Disrupts Glucocorticoid Receptor Function in Intact Cells," Molecular Endocrinology, 10(6):705-712 (1996). cited by other . Wickner et al., "Posttranslational Quality Control: Folding, Refolding, and Degrading Proteins," Science, 286:1888-1893 (Dec. 3, 1999). cited by other . Wu et al., "Differential transcriptional regulation of the human thrombin receptor gene by the Sp family of transcription factors in human endothelial cells," Biochemical Journal, 330:1469-1474 (1998). cited by other . Wu et al., "The Human KDR/flk-1 Gene Contains a Functional Initiator Element That Is Bound and Transactivated by TFII-I,"0 The Journal of Biological Chemistry, 274(5):3207-3214 (Jan. 29, 1999). cited by other . Wu et al., "The human KDR/flk-1 gene contains a functional initiator element that is bound and trasnsactivated by TFII-I," Abstract 425.1, Annual Meeting of Professional Research Scientists and American Association of Anatomists, Experimental Biology '99, Apr. 17-21, Washington, D.C., The FASEB Journal, 13(4):A531 (Mar. 12, 1999). cited by other . Young et al., "Specific Binding of Tetratricopeptide Repeat Proteins to the C-terminal 12-kDa Domain of hsp90," The Journal of Biological Chemistry, 273(29):18007-18010 (1998). cited by other . Zeiner et al., "Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins," The EMBO Journal, 16(18):5483-5490 (1997). cited by other . Ballinger et al., "CHIP's Interaction with HSP90 Regulates Glucocorticoid Receptor Function and Degradation," Abstract and oral presentation, Cold Spring Harbor Meeting on Molecular Chaperones & Heat Shock Response, May 3-7, Cold Spring Harbor, N.Y. (May 4, 2000). cited by other . Benjamin et al., "Stress (Heat Shock) Proteins Milecular Chaperones in Cardiovascular Biology and Disease," Circulation Research Journal of the American Heart Association, Jul. 27, 1998; 83(2):117-132. cited by other . Chang et al., "Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase," Nature, 275(19):617-624 (1978). cited by other . Hu et al., "Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase," Proc. Natl. Acad. Sci. USA. 1996; 93:1060-1064. cited by other . Itakura et al., "Expression in Escherichia coli of a Chemically Synthesized Gene for the Hormone Somatostatin," Science, 198:1056-1063 (1977). cited by other . Netting, "Cells have molecule for protein triage," Science News Online, Jan. 27, 2001; 159(4). cited by other . Schute et al., "Disruption of the Raf-1-Hsp90 Molecular Complex Results in Destabilization of Raf-1 and Loss of Raf-1-Ras Association," The Journal of Biological Chemistry, 1995; 270(41):24585-24588. cited by other . Sharp et al., "Heat-Shock Protein Protection," Research News, 1999;22(3):97-99. cited by other . Slavotinek et al., "Unfolding the Role of Chaperones and Chaperonins in Human Disease," TRENDS in Genetics, Sep. 2001;17(19):528-535. cited by other . Smith et al., "Molecular chaperones: Biology and Prospects for Pharmacological Intervention," Pharmacological Reviews, 1998;50(4):493-513. cited by other . Xu et al., "Heat-shock protein hsp90 governs the activity of pp60.sup.v-src kinase," Proc. Natl. Acad. Sci. USA, Aug. 1993; 90:7074-7078. cited by other . Yenari et al., "The Neuroprotective Potential of Heat Shock Protein 70 (HSP70)," Molecular Medicine Today, Dec. 1999; 5:525-531. cited by other . Imai et al., "CHIP Is Associated with Parkin, a Gene Responsible for Familial Parkinson's Disease, and Enhances its Ubiquitin Ligase Activity," Molecular Cell, Jul. 2002;10: 55-67. cited by other . Taverna and Goldstein, "Why are Proteins So Robust To Site Mutations?" Journal of Mol. Biology, 2002;315:479-484. cited by other . Yan et al., "AtCHIP, a U-Box-Containing E3 Ubigquitin Ligase, Plays a Critical Role in Temperature Stress Tolerance in Arabidopsis," Plant Physiology, Jun. 2003;132:861-869. cited by other . Amalfitano and Parks, "Separating Fact from Ficton: Assessing he Potential of Modified Adenovirus Vectors for Use in Human Gene Therapy," Current Gene Therapy, 2002;2:111-133. cited by other . Houdebine, "Production of pharmaceutical proteins from transgenic animals," Journal of Biotechnology, 1994:34:269-287. cited by other . Pandha et al., "Oncological applications of gene therapy," Curr Opin Invest Drugs, 2000;1(1):122-34. cited by other . Patterson A.P., "Notification of a Serious Adverse Event," Memorandum Department of Health and Human Services, Jan. 14, 2003: 3 pgs. cited by other . Verma and Somia, "Gene therapy-promises, problems and prospects," Nature, Sep. 18, 1997;389:239-42. cited by other . "CHIP (C-terminus of Hsc70-Interacting Protein) A U-box-dependent E3 ubiquitin ligase," [online].Phoenix Pharmaceuticals, Inc. [retrieved on Jul. 19, 2005]. cited by other . "In Vitro Protein Expression Guide," Promega, 50 pgs, Apr. 2005. cited by other.

Primary Examiner: Rawlings; Stephen L.
Attorney, Agent or Firm: Mueting, Raasch & Gebhardt, P.A.

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Government Interests

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STATEMENT OF GOVERNMENT RIGHTS

The present invention was made with support from the National Institutes of Health under Grant Nos. HL03658 and AG15234. The government may have certain rights in this invention.

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Parent Case Text

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PRIORITY APPLICATION

This application claims benefit of U.S. Provisional Patent Application Ser. No. 60/134,433, filed May 17, 1999, which is incorporated herein by reference.

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Claims

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What is claimed is:

1. An isolated polypeptide comprising SEQ ID NO: 2.

2. The polypeptide of claim 1 wherein the polypeptide is a recombinant polypeptide.

3. A composition comprising the polypeptide of claim 1.

4. An isolated polypeptide comprising the amino acid sequence of amino acids 1 197 of SEQ ID NO:2, wherein the polypeptide binds to the carboxyl-terminal domain of the heat shock protein Hsc70.

5. The polypeptide of claim 4 having a molecular weight as determined by SDS polyacrylamide gel electrophoresis of about 30 kD to about 40 kD.

6. A composition comprising the polypeptide of claim 4.

7. An isolated polypeptide comprising SEQ ID NO:7.

8. A composition comprising the polypeptide of claim 7.

9. An isolated polypeptide encoded by a nucleic acid that hybridizes to the nucleic acid complement of SEQ ID NO:1 under hybridization conditions of 0.015 M NaCl/0.0015 M sodium citrate (SSC) and about 0.1% sodium dodecyl sulfate (SDS) at about 50.degree. C. to about 65.degree. C., wherein said polypeptide comprises at least the amino acid sequence of amino acid residues 1 197 of SEQ ID NO:2, and wherein said polypeptide binds with to the carboxyl-terminal domain of the heat shock protein Hsc70.

10. A composition comprising the polypeptide of claim 9.

11. An isolated polypeptide comprising the amino acid sequence of amino acids 1 197 of SEQ ID NO:7, wherein the polypeptide binds to the carboxyl-terminal domain of the heat shock protein Hsc70.

12. A composition comprising the polypeptide of claim 11.

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Description

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BACKGROUND

Multi-protein complexes, which are the product of protein--protein interactions, participate in a variety of cellular processes. Such exemplary cellular processes include, for example, cell signaling, gene regulation, protein assembly and degradation, and mechanical events such as sarcomere shortening. Conserved structural motifs in many proteins have evolved to facilitate the interaction of specific proteins in the assembly of multi-protein complexes. The tetratricopeptide repeat (TPR) domain is one such structural motif that was originally identified by sequence comparisons among yeast proteins (Hirano et al., Cell, 60:319 328 (1990), Sikorski et al., Cell, 60:307 317 (1990)). The TPR domain contains a 34-amino acid structural motif with a loose consensus that is present, usually as multiple tandem repeats, in proteins with many cellular functions, including mitosis, transcription, protein transport, and development (Lamb et al., Trends in Biochemical Sciences, 20:257 259 (1995)). Structural analysis of the TPR domain demonstrates that it forms two .alpha.-helical regions separated by a turn, such that opposed bulky and small side chains form a "knob and hole" structure (Hirano et al., Cell, 60:319 328 (1990)). It is thought that a hydrophobic surface of this particular TPR domain mediates protein--protein interactions between TPR- and non-TPR-containing proteins.

TPR-containing proteins are typically known to play a diverse and important role in cellular function. Several TPR-containing proteins are known to participate in interactions with the major members of the heat shock protein family, Hsp70, Hsc70, and Hsp90. It is believed that these TPR-containing proteins are necessary for appropriate regulation of protein folding and transport. The TPR domains of protein phosphatase 5, cyclophilin 40 (CyP-40), and FKBP52 are known to mediate binding of these proteins to Hsp90 and assist in trafficking of nuclear hormone receptors (J. E. Kay, Biochem. J., 314:361 385 (1996)). A different group of TPR-containing proteins are known to interact with Hsc70 and Hsp70. Hsc70-Interacting Protein (HIP), also known as p48, binds to the ATPase domain of Hsc70, stabilizes the ADP-bound conformation, and increases the affinity for substrate proteins (Hohfeld et al., Cell 83:589 598 (1995)). Hsc70 Hsp90-Organizing Protein (HOP), also known as p60 or Sti1, serves as a coupling factor that facilitates the cooperation between Hsc70 and Hsp90, although it does not directly assist in chaperoning functions (Schumacher et al., J. Biol. Chem., 269:9493 9499 (1994)). In contrast to HIP, HOP interacts with the carboxy-terminal domain of Hsc70 (Demand et al., Mol. Cell. Biol., 18:2023 2028 (1998)).

In addition to fHP, at least two other proteins are known to regulate the reaction cycle of mammalian Hsc70 and Hsp70. Hsp40 stimulates the ATPase activity of Hsc70 and thus promotes the conversion of ATP-bound, low substrate-affinity Hsc70 to ADP-bound, high substrate-affinity Hsc70 (J. Hohfeld, Biol. Chem., 379:269 274 (1998)). The reverse reaction cycle, which involves exchange of ATP for ADP and loss of substrate affinity, was recently shown to be facilitated by the anti-apoptotic protein BAG-1 (Zeiner et al., EMBO J., 16:5483 5490 (1997)). Whereas HIP inhibits this reverse reaction cycle and stabilizes the ADP-bound conformation, no cellular inhibitors of the forward reaction cycle of the binding of a heat shock protein to a substrate to form a protein--protein complex have yet been identified.

Thus, there is a need for further understanding cellular components that regulate and interact with heat shock proteins, and identifying such cellular components.

SUMMARY OF THE INVENTION

Described herein are the isolation and characterization of a polypeptide, particularly a tetratricopeptide repeat (TPR)-containing polypeptide that is an interactive partner with heat shock proteins. A preferred polypeptide is referred to herein as Carboxyl terminus of Hsc70-Interacting Protein or "CHIP," not only because of its ability to negatively regulate formation of heat shock protein-substrate complexes by interacting with the forward reaction cycle of heat shock proteins and other accessory chaperone cofactors, but also because the polypeptide interacts in an ATP-independent fashion with these heat shock proteins. The nucleic acid and deduced amino acid sequences of this polypeptide in humans, mice, and Drosophila are provided.

Accordingly, the present invention provides an isolated polypeptide that negatively regulates binding of a heat shock protein to a substrate. Preferably, the polypeptide negatively regulates the heat shock proteins Hsc70, Hsp70, and Hsp90. The polypeptide can be a recombinant polypeptide. The polypeptide preferably has a molecular weight of about 30 kD to about 40 kD as determined by SDS polyacrylamide gel electrophoresis.

The present invention also provides a preferred isolated polypeptide having an amino acid sequence having greater than about 40% sequence identity to that of at least one of SEQ ID NOs. 2, 7, and 8. A particularly preferred polypeptide contains at least amino acids 1 197 from any of the polypeptides of SEQ ID NOs. 2, 7, and 8 is capable of negatively regulating the binding of a heat shock protein to a substrate. The polypeptide can be recombinant.

In another preferred embodiment, the present invention further provides an isolated polypeptide having an amino acid sequence represented by at least one of SEQ ID NOs. 2, 7, and 8. Preferably, the polypeptide is capable of negatively regulating the binding of a heat shock protein to a substrate. The polypeptide can be recombinant.

In another preferred embodiment, the present invention also provides a polypeptide containing an amino acid sequence represented by at least one of SEQ ID NOs. 2, 7, and 8, wherein the polypeptide has a molecular weight as determined by SDS polyacrylamide gel electrophoresis of about 30 kD to about 40 kD.

Also provided is a preferred polypeptide that negatively regulates binding of a heat shock protein to a substrate wherein nucleic acid encoding the polypeptide hybridizes to the nucleic acid or the nucleic acid complement of at least one of SEQ ID NOs. 1, 9, 10, and 11 under hybridization conditions of 0.015 M NaCl/0.0015 M sodium citrate (SSC) and about 0.1% sodium dodecyl sulfate (SDS) at about 50.degree. C. to about 65.degree. C.

The present invention further provides a nucleic acid fragment capable of hybridizing to at least one of SEQ ID NOs. 1, 9, 10, and 11, or a complement of at least one of SEQ ID NOs. 1, 9, 10, and 11, under hybridization conditions of 0.015 M NaCl/0.0015 M sodium citrate (SSC) and about 0.1% sodium dodecyl sulfate (SDS) at about 50.degree. C. to about 65C. Preferably, the nucleic acid fragment encodes at least a portion of a polypeptide, which is preferably capable of negatively regulating binding of a heat shock protein to a substrate. The nucleic acid fragment may further be provided in a nucleic acid vector, such as an expression vector that is capable of producing at least a portion of a polypeptide.

In another embodiment, the present invention provides a host cell that includes the nucleic acid fragment described herein. The host cell can be prokaryotic or eukaryotic.

A particularly preferred embodiment of the present invention is an isolated nucleic acid fragment having the nucleic acid sequence represented by at least one of SEQ ID NOs. 1, 9, 10, and 11, or a complement thereof.

The present invention also provides a nucleic acid fragment having a nucleic acid sequence with at least about 60% nucleic acid identity to that of at least one of SEQ ID NOs. 1, 9, 10, and 11, and a complement of the nucleic acid fragment.

The present invention further provides a method for identifying an inhibitor of a polypeptide that has negative regulating activity for a heat shock protein, the method includes incubating the polypeptide with a compound under conditions that promote the negative regulating activity of the polypeptide when the compound is not present, and determining if the negative regulating activity of the polypeptide is reduced relative to the negative regulating activity of the polypeptide in the absence of the compound.

Also provided is a method of expressing a nucleic acid fragment that encodes a polypeptide, the presence of which is associated with negative regulation of a heat shock protein, the method includes expressing the nucleic acid fragment in a cultured host cell transformed with an expression vector containing the nucleic acid fragment operably linked to control sequences recognized by the host cell. A suitable host cell of the invention may be a prokaryotic or eukaryotic cell, preferably a prokaryotic cell, which can form a part of a gram negative or gram positive organism. Preferably, the host cell is an E. coli cell. Preferably, the nucleic acid fragment has a nucleic acid sequence represented by at least one of SEQ ID NOs. 1, 9, 10, and 11, or a complement thereof. The expressed polypeptide preferably contains an amino acid sequence having greater than about 40% amino acid sequence identity to that of at least one of SEQ ID NOs. 2, 7, and 8.

The present invention also provides a method for producing a recombinant polypeptide by: a) providing an expression vector that contains a nucleic acid fragment having a nucleic acid sequence with at least about 60% nucleic acid identity to that of at least one of SEQ ID NOs. 1, 9, 10, and 11, or a complement of the nucleic acid fragment, operably linked to control sequences recognized by a host cell; b) transforming the host cell with the expression vector; and c) culturing the transformed cell under conditions that allow expression of the recombinant polypeptide encoded by the nucleic acid fragment. A suitable host cell may be a prokaryotic or eukaryotic cell, preferably a prokaryotic cell, which can form a part of a gram negative or gram positive organism. Preferably, the host cell is an E. coli cell.

Further provided is a method for inhibiting a polypeptide that negatively regulates binding of a heat shock protein to a substrate in a mammal by administering to the mammal a composition containing an amount of an inhibitor to an isolated polypeptide having an amino acid sequence identity greater than about 40% to that of at least one of SEQ ID NOs. 2, 7, and 8. Preferably, the composition is therapeutically effective for a neoplastic disease, ischemic disease, or a disease characterized by inflammation.

The present invention also provides a method for inhibiting a nucleic acid that encodes a polypeptide that negatively regulates binding of a heat shock protein to a substrate in a mammal by administering to the mammal a composition containing an amount of an inhibitor to an isolated nucleic acid fragment having a nucleic acid sequence with at least about 60% nucleic acid identity to that of at least one of SEQ ID NOs. 1, 9, 10, and 11, or a complement of the nucleic acid fragment.

The present invention further provides an inhibitory composition having an amount of an inhibitor to an isolated polypeptide, which negatively regulates binding of a heat shock protein to a substrate, that is effective to immunize or treat a mammal for a neoplastic disease, an ischemic disease, or a disease characterized by inflammation. Preferably, the inhibitory composition is provided in an amount effective to provide a therapeutic effect to a mammal diagnosed with a neoplastic disease, an ischemic disease, or a disease characterized by inflammation. The composition may optionally contain a pharmaceutically acceptable carrier.

Definitions

"Polypeptide" as used herein refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like.

As used herein, the term "isolated" means that a polypeptide is either removed from its natural environment or synthetically derived. Preferably, the polypeptide is purified, i.e., essentially free from any other polypeptides and associated cellular products or other impurities.

The term "recombinant nucleic acid," or "recombinant polynucleotide" as used herein, refers to a nucleic acid of genomic, cDNA, semisynthetic, or synthetic origin which by virtue of its origin or manipulation is not associated with all or a portion of a nucleic acid with which it is associated in nature, is linked to a nucleic acid other than that to which it is linked in nature, or does not occur in nature.

The term "recombinant polypeptide" refers to a polypeptide that is not necessarily translated from a designated nucleic acid. The recombinant polypeptide may be generated in any manner, including for example, chemical synthesis, or expression of a recombinant expression system. A recombinant polypeptide may include one or more analogs of amino acid residues or unnatural amino acid residues in its sequence. Methods of inserting analogs of amino acid residues into a sequence are known in the art. The recombinant polypeptide further includes a "fusion polypeptide." As used herein, a "fusion" polypeptide is a product of a first nucleic acid, for example a polypeptide according to the present invention, and a second nucleic acid, for example a glutathione S-transferase sequence (GST), operably linked at either the carboxyl terminus or amino terminus of the first sequence. Expression of this fusion polypeptide results in a single or continuous polypeptide when expressed and isolated from a host cell. The product of this expression can enhance properties relating to, for example, purification, isolation, targeting, and increased immunogenicity.

The term "operably linked" is defined to mean that at least one nucleic acid or nucleic acid fragment is placed in a functional relationship with at least one other nucleic acid or nucleic acid fragment. For example, a nucleic acid fragment for a presequence or secretory leader can be operably linked to nucleic acid fragment that encodes a polypeptide or a fusion polypeptide according to the present invention. A promoter or enhancer is "operably linked" to a nucleic acid fragment, e.g., a coding region, if it affects the transcription of the sequence or if it is positioned so as to facilitate translation. Generally, "operably linked" means that nucleic acids being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors, linkers, or primers are used in accord with conventional practice.

A "coding region," also referred to as an "open reading frame" (ORF), is a nucleic acid which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding region are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding region can include, but is not limited to mRNA, cDNA, and recombinant nucleic acid.

"CHIP" refers to a preferred polypeptide according to the present invention that possesses "negative regulating activity" with respect to the binding of a heat shock protein to a substrate to form a heat shock protein-substrate complex, e.g., a protein--protein complex. Although not intending to be limited to a particular mechanism or theory, it is believed this "negative regulating activity" may occur by reducing or inhibiting heat shock protein ATPase activity, inhibiting or reducing binding of one or more accessory chaperone cofactors to a heat shock protein, by the direct binding or complexing of the heat shock protein with a CHIP polypeptide, and/or by the targeting of substrates for degradation. ATPase activity may be reduced or inhibited by the polypeptide's ability to interfere with the forward reaction cycle which includes the hydrolysis of ATP to ADP and inorganic phosphate and the subsequent binding of a heat shock protein to a substrate to form a protein--protein complex.

A "nucleic acid fragment" as used herein refers to a linear polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA and RNA. A nucleic acid fragment may include both coding regions and noncoding regions that can be obtained directly from a natural source (e.g., a microorganism), or can be prepared with the aid of recombinant techniques (including chemical synthetic techniques, as defined herein). A nucleic acid molecule may be equivalent to this nucleic acid fragment or it can include this fragment in addition to one or more other nucleotides. For example, the nucleic acid molecule of the invention can be a vector, such as an expression system or cloning vector.

"Recombinant host cells," "host cells," "cells," "cell lines," "cell cultures," and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells that can be, or have been used as recipients for a nucleic acid vector or other transfer nucleic acid, and include the progeny of the original cell which has been transformed. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total nucleic acid complement as the original parent, due to natural, accidental or deliberate mutation.

An "active analog" or "active fragment" of a polypeptide of the invention is one that is characterized by negative regulating activity as described herein. Active analogs and active fragments are described in greater detail herein.

"Amino acid identity" or "percentage amino acid identity," refers to a comparison of the amino acid residues of two polypeptides. The polypeptides to be compared are preferably aligned such that the amino acid residues are aligned to maximize the number of amino acids that the polypeptides have in common along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to maximize the number of shared amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. The percentage amino acid identity is the number of amino acids that the two sequences have in common within the alignment, divided by the number of amino acids in the amino acid sequence of interest, e.g., at least one of SEQ ID NOs. 2, 7, and 8 multiplied by 100. Preferably, the polypeptide has greater than a 40% amino acid identity, and more preferably at least about 50% amino acid identity to at least one of SEQ ID NOs. 2, 7, and 8. Amino acid identity may be determined, for example, using the sequence alignment program CLUSTAL W available on the World Wide Web at genome.ad.jp/SIT/CLUSTALW.html, and percent amino acid identity can be determined by BLAST 2 SEQUENCE'S at National Center for Biotechnology Information (NCBI) website: on the World Wide Web at .ncbi.nlm.nih.gov.

"Amino acid similarity" or "percentage amino acid similarity," refers to a comparison of the amino acid residues of two polypeptides wherein conservative amino acid residue substitutions are permitted, i.e., amino acid residues that share the same charge and the same polarity are considered "similar." Potential conservative amino acid residue substitutions are described in greater detail below. Preferably, a candidate polypeptide has at least about 60% amino acid similarity, and more preferably at least about 70% amino acid similarity. Amino acid similarity may be determined as described above in reference to determination of amino acid identity.

"Nucleic acid identity" or "percentage nucleic acid identity" refers to a comparison of the nucleic acids of two nucleic acid fragments as described herein. A "high degree" of nucleic acid sequence identity refers to a nucleic acid fragment that typically has at least about 60% nucleic acid sequence identity, and preferably at least about 70% nucleic acid sequence identity. Sequence identity may be determined, for example, using sequence techniques such as GCG FastA (Genetics Computer Group, Madison, Wis.), MacVector.TM. 4.5 (Kodak/IBI software package) or other suitable sequencing programs or methods known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Human CHIP (hCHIP) cDNA: Nucleic acid (upper line) (SEQ ID NO: 1) and deduced amino acid sequence (lower line) (SEQ ID NO: 2) of hCHIP. Residues containing the three tetratricopeptide repeat (TPR) domains (TPR1, amino acid residues 26 59), (TPR2, amino acid residues 60 93), and (TPR3, amino acid residues 94 127) are singly underlined. A region rich in highly charged residues (amino acid residues 143 190) is doubly underlined. A sequence similar to ubiquitin-proteasome-related proteins (amino acid residues 219 290) is represented by a dashed underline. Potential nuclear localization signals (amino acid residues 143 146 and amino acid residues 221 225) are in boldface.

FIG. 2. Comparison of TPR motifs: hCHIP TPR1 (amino acid residues 26 59 of SEQ ID NO: 2), hCHIP TPR2 (amino acid residues 60 93 of SEQ ID NO: 2), and hCHIP TPR3 (amino acid residues 94 127 of SEQ ID NO: 2) with the three TPR motifs of human HIP (hHIP), hHIP TPR1 (SEQ ID NO: 3), hHIP TPR2 (SEQ ID NO: 16), and hHIP TPR3 (SEQ ID NO: 17), the three TPR motifs of human protein phosphatase 5 (hPP5), hPP5 TPR1 (SEQ ID NO: 4), hPP5 TPR2 (SEQ ID NO: 18), and hPP5 TPR3 (SEQ ID NO: 19), and the three TPR motifs of human cyclophilin-40 (hCYP), hCYP TPR1 (SEQ ID NO: 5), hCYP TPR2 (SEQ ID NO: 20), hCYP TPR3 (SEQ ID NO: 13), illustrates a consensus sequence (SEQ ID NO: 6) for this class of TPR domains.

FIGS. 3A 3B (Hereinafter referred to as FIG. 3). Comparison of the human, mouse, and Drosophila CHIP amino acid sequences: The deduced amino acid sequences derived from human CHIP (SEQ ID NO: 2), mouse CHIP (SEQ ID NO: 7), and Drosophila CHIP (SEQ ID NO: 8), Open Reading Frames (ORFs) were aligned with GENEWORKS 2.5.1. (IntelliGenetics, Campbell, Calif.) using the default parameters. A consensus sequence (SEQ ID NO: 12) is also shown Similar or identical amino acid residues are boxed.

FIG. 4. The TPR domain of hCHIP and relative binding to Hsp70 and Hsc70: Diagram showing the location of the three TPR domains (amino acid residues 26 127), charged residues (amino acid residues 143 190), and proteasome-linked protein UFD2-like domains (amino acid residues 218 293) within the hCHIP coding region (SEQ ID NO: 2). The diagram also shows the constructs of five GST-hCHIP fusion proteins that contain one or more of these domains. GST construct 1 (amino acid residues 198 303) having an apparent molecular weight of about 40.2 kD, GST construct 2 (amino acid residues 1 142) having an apparent molecular weight of about 44.4 kD, GST construct 3 (amino acid residues 143 303) having an apparent molecular weight of about 46.6 kD, GST construct 4 (amino acid residues 1 197) having an apparent molecular weight of about 50.7 kD, and GST construct 5 (amino acid residues 1 303) having an apparent molecular weight of about 62.8 kD.

FIG. 5. Effects of hCHIP on ATPase activities and nucleotide binding of heat shock proteins: The ATPase activities of Hsc70 (black bars) and Hsp70 (white bars) were measured over 20 minutes in the presence or absence of hCHIP, Hsp40 and/or Hsp90 as indicated. Data were normalized to the ATPase activity of Hsc70 alone. * (or .dagger.) indicates a significant difference (p<0.05) between the ATPase activity of Hsc70 alone (or Hsp70 alone) and Hsc70+Hsp40 (or Hsp70+Hsp40). ** (or .dagger..dagger.) indicates a significant difference (p<0.05) between the ATPase activity of Hsc70+Hsp40 (or Hsp70+Hsp40) and Hsc70+Hsp40+CHIP (or Hsp70+Hsp40+CHIP). Each condition was repeated for a total of six replicates.

FIG. 6. Effects of CHIP on chaperone functions of heat shock proteins:

The aggregation of rhodanese was measured at 340 nm over 5 minutes in the absence (.diamond-solid.) or presence of hCHIP (.largecircle.), Hsp70+Hsp40 (.quadrature.), or CHIP+Hsp70+Hsp40 (.tangle-solidup.). The measured optical densities were normalized to the zero reading for each individual well of a microtiter plate and the increase in absorbance plotted as a percent of the total increase of rhodanese alone. Each condition was repeated for a total of eight replicates, and points represent the mean.+-.SEM.

FIG. 7. Luciferase activity was measured as an indication of refolding after thermal denaturation. The refolding reactions were performed with Hsc70 (.quadrature.) Hsp40 (.largecircle.), or both (.sup..DELTA.) in the absence (open symbols) and presence (closed symbols) of CHIP (+CHIP alone). Luciferase activity was measured at various intervals between 0 120 minutes and the activity for each reaction was normalized to luciferase in refolding buffer alone. Each condition was repeated for a total of 12 replicates, and points represent the mean.+-.SEM.

FIG. 8. Model of the eukaryotic reaction cycle in the presence of hCHIP, Hsp40, HIP and BAG-1: The forward reaction cycle, in which ATP is hydrolyzed to ADP and inorganic phosphate (Pi) is released, is enhanced by Hsp40. The biochemical data suggests that hCHIP blocks this forward reaction cycle. HIP stabilizes the ADP-bound, high substrate affinity conformation of Hsc70, and thus enhances chaperone activity. Conversely, BAG-1 accelerates nucleotide exchange, promoting substrate release and the formation of