WO2000014106A9 - Proteines bag et molecules d'acide nucleique les codant - Google Patents

Proteines bag et molecules d'acide nucleique les codant

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Publication number
WO2000014106A9
WO2000014106A9 PCT/US1999/021053 US9921053W WO0014106A9 WO 2000014106 A9 WO2000014106 A9 WO 2000014106A9 US 9921053 W US9921053 W US 9921053W WO 0014106 A9 WO0014106 A9 WO 0014106A9
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WIPO (PCT)
Prior art keywords
seq
bag
group
amino acid
nucleic acid
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PCT/US1999/021053
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English (en)
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WO2000014106A1 (fr
Inventor
John C Reed
Shinichi Takayama
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Burnham Inst
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Application filed by Burnham Inst filed Critical Burnham Inst
Priority to CA002342027A priority Critical patent/CA2342027A1/fr
Priority to EP99968670A priority patent/EP1109824A4/fr
Priority to AU60383/99A priority patent/AU774355B2/en
Priority to JP2000568863A priority patent/JP2002524068A/ja
Publication of WO2000014106A1 publication Critical patent/WO2000014106A1/fr
Publication of WO2000014106A9 publication Critical patent/WO2000014106A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system

Definitions

  • 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.
  • 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, 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-1/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.
  • Bcl-2 associated athanogene-1 (bag-1) is named from the Greek word a thano ⁇ , which refers to anti-cell death.
  • BAG-1 was previously referred to as Bcl-2-associated protein-1 (BAP-1) in U.S. Patent No. 5,539,094 issued July 23, 1996, which is incorporated herein by reference.
  • BAG-1 is described as a portion of the human BAG-1 protein, absent the N-terminal amino acids 1 to 85.
  • a human protein essentially identical to human BAG-1 was described by Zeiner and Gehring, ( Proc . Na tl . 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-IL 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 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 Hsc70/Hsp70, including increasing resistance to apoptosis, promoting cell proliferation, enhancing tumor cell migration and metastasis, and altering transcriptional activity of steroid hormone receptors.
  • BAG protein species 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 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 for therapeutic agents, in particular, anticancer agents. The present invention satisfies these need, as well as providing substantial related advantages .
  • the present invention provides a family of BAG-1 related proteins from humans [BAG-IL (SEQ ID NO:2), BAG-1
  • BAG-3 SEQ ID NO: 6
  • BAG-4 SEQ ID NO:20
  • BAG-lA (SEQ ID NO: 16), BAG-IB (SEQ ID NO:18)] and the nucleic acid molecules that encode them.
  • 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 N0:2), 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 .
  • Figure 1 shows the full length cDNA sequence for human BAG-1 (SEQ ID NO:l) 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-IM [beginning at nucleotide 260 of (SEQ ID NO:2)], and BAG-IL [beginning at nucleotide 46 of (SEQ ID NO:2)] .
  • 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 NO: 5) 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 NO:10).
  • 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. el egans BAG-1 protein (SEQ ID NO:12).
  • Figure 7A shows the full length cDNA sequence for C. el egans 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-lA protein (SEQ ID NO:15).
  • Figure 8B shows the 195 amino acid sequence for S . pombe BAG-lA protein (SEQ ID NO: 16) .
  • Figure 9A shows the full length cDNA sequence for S . pombe BAG-IB protein (SEQ ID NO:17).
  • Figure 9B shows the 206 amino acid sequence for S . pombe BAG-IB protein (SEQ ID NO:18).
  • FIG. 10 shows the topologies of the BAG-family proteins; human BAG proteins, BAG-1 (SEQ ID NO:2), BAG-2
  • BAG-5 (SEQ ID NO: 10); S . pombe BAG-lA (SEQ ID NO: 16) and
  • BAG-IB (SEQ ID NO: 18); and C. elegans BAG-1 (SEQ ID NO:12)and BAG-2 (SEQ ID NO:14).
  • A 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.
  • B 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-lA SEQ ID NO: 16
  • BAG-IB SEQ ID NO: 18
  • C. elegans BAG-1 SEQ ID NO: 12
  • BAG-2 SEQ ID NO: 14
  • 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.
  • B In vi tro protein assays using GST-fusion proteins and 35 S-labeled in vitro translated proteins.
  • Figure 12 shows surface plasmon resonance analysis of BAG-family protein interactions with Hsc70/ATPase.
  • A SDS-PAGE analysis of purified recombinant proteins.
  • B Representative SPR results of biosensor chips containing immobilized BAG proteins with and without maximally bound Hsc70/ATPase .
  • Figure 13 shows representative SPR results for biosensor chips containing immobilized BAG-1 (beginning at residue 116 at SEQ ID NO:2), BAG-1 ( ⁇ C) , 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).
  • 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 ⁇ M.
  • Figure 14 shows BAG-family protein modulation of Hsc70 chaperone activity.
  • A 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 Hsc70-mediated 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) .
  • Figure 15B shows the corresponding amino acid residues for the human BAG-3 protein (SEQ ID NO:20) of Figure 15A.
  • 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 NO:20) .
  • 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:
  • 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) .
  • Figure 18 shows the topologies of the BAG-family proteins; human BAG proteins, BAG-1 (SEQ ID NO:2), BAG-2
  • BAG-4 (SEQ ID NO:22), expanded BAG-5 (SEQ ID NO:24); S . pombe BAG-lA (SEQ ID NO: 16) and BAG-IB (SEQ ID NO: 18); and
  • BAG domains 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.
  • apoptosis 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.
  • tumor cell proliferation refers to the ability of tumor cells to grow and thus expand a tumor mass.
  • cell migration refers to the role cell motility plays in the invasion and potentially metastasis by tumor cells.
  • metastasis 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.
  • steroid hormone receptor function refers to physiological, cellular and molecular functioning of receptors sites that bind with steroid hormones .
  • substantially purified 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 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 refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
  • complementarity refers 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 "T-C-A” .
  • 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 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 (i.e., the hybridization) of a completely homologous sequence or probe to the target sequence under conditions of low stringency.
  • antisense refers to nucleotide sequences which are commplementary to a specific DNA or RNA sequence.
  • 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 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.
  • 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.
  • a “derivative" of a BAG protein 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, e.g., 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, e.g., 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) .
  • 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.
  • 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 L- configuration amino acid with its corresponding D- configuration counterpart.
  • mametic 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.
  • Protein nucleic acid 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)).
  • the present invention provides a family of BAG-1 related proteins from humans [BAG-IL (SEQ ID N0:2), BAG-IS beginning at residue 116 of SEQ ID NO:2, 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)], the invertebrate C. elegans [BAG-1 (SEQ ID NO:12), BAG-2 (SEQ ID NO:14)] and the fission yeast S .
  • BAG-lA (SEQ ID NO:16), BAG-IB (SEQ ID NO:18)]
  • BAG-IL SEQ ID NO:2
  • BAG-1 beginning at residue 116 of SEQ ID NO:2
  • BAG-2 SEQ ID NO : 4
  • C. elegans BAG-1 SEQ ID NO:12
  • BAG-2 SEQ ID NO:14
  • pombe BAG-lA (SEQ ID NO: 16) and BAG-IB (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.
  • 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 provides the nucleic molecule and nucleotide sequences that encode the family of BAG-1 related proteins from humans [BAG-1 (SEQ ID NO:l), 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 BAG-5 (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-lA (SEQ ID NO:15), BAG-IB (SEQ ID NO: 17) ] .
  • BAG-IL (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-IL (SEQ ID NO:2) interacts with several types of proteins, including Bcl-2, some tyrosine kinase growth factor receptors, steroid hormone receptors, and the p53- induced cell cycle regulator Siah-IA.
  • BAG-1 is a regulator of Hsc70/Hsp70 family molecular chaperones .
  • BAG-1 modulates the activity of these molecular chaperones, acting as an apparent functional antagonist of the Hsp70/Hsc70- associated protein Hip (3-5) (H ⁇ hfeld, J. and Jentsch, S., EMBO J. 16: 6209-6216, (1997); Takayama, S., Bimston, D.
  • BAG-1 appears to promote substrate release, whereas Hip stabilizes Hsp70/Hsc70 complex formation with target peptides (H ⁇ hfeld, J., Minami, Y., and Hartl, F.-U., 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.
  • Hsp70/Hsc70 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 interactions with specific protein or protein complexes.
  • BAG-1 appears to represent a novel Hsp70/Hsc70 regulator which differs functionally from all other mammalian co- chaperones 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
  • 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.
  • RARs retinoic acid receptors
  • 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 irj 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 greater inhibitor activity than BAG-1 ( Figure 4A) .
  • BAG-1, BAG-2 (SEQ ID NO:4) SEQ ID NO: 6
  • 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
  • BAG-4 SEQ ID NO: 22
  • Hsc70/ATPase Hsc70/ATPase
  • nucleotide sequence having at least about 15 nucleotides and, generally, about 25 nucleotides, preferably about 35 nucleotides, more preferably about 45 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 IB, 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,
  • R is a group of 1 to 552 independently selected amino acids;
  • R 1 is a group of 3 independently selected amino acids;
  • 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 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 s 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 1 is a group of 2 independently selected amino acids;
  • X 7 is an amino acid with a charged R group, such as arginine
  • X is an ammo 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;
  • X is an amino acid with an uncharged R group, such as glutamine;
  • R 11 is a group of 2 independently selected amino acids ;
  • X 11 is an amino acid with an apolar R group, such as leucine
  • R R c is a group of 1 to 100 independently selected amino acids
  • a nucleotide sequence of at least about 15 nucleotides and, generally, about 25 nucleotides, preferably about 35 nucleotides, more preferably about 45 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)
  • nucleotide sequence of the invention can be useful as a probe in a hybridization reaction such as
  • 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 5 ' -untranslated region or of the 5 ' -translated region of a bag-1 nucleic acid sequence in a cell.
  • 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-IL (SEQ ID NO:l), or nucleotides 79-147 of human BAG-5 (SEQ ID NO: 9) .
  • an antisense molecule directed to the 5 ' -region of a nucleic 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.
  • 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-2- related protein or Hsc70/Hsp70 protein in the cell.
  • a genetic defect in a gene encoding, for example, human BAG-1 can result in a pathology characterized by increased or decreased levels in protein folding.
  • nucleotide compound or composition as taught in the present invention can be synthesized using routine methods or can be purchased from a commercial source.
  • 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
  • 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 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.
  • 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.
  • 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.
  • 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).
  • hybridization 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.
  • 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 10 5 M "1 .
  • anti-BAG-1 antibody fragments such as Fab, F(ab') 2 and Fv fragments can retain specific binding activity for human BAG-1 (beginning at residue 116 of SEQ ID NO:2) and, thus, are included within the definition of an antibody.
  • 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.
  • 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 85 amino acids or the BAG domain of any of the human BAG 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, i.e., 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 keyhole limpet hemocyanin
  • KLH keyhole limpet hemocyanin
  • Example IV below.
  • 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.
  • This example describes methods for isolating and characterizing of BAG-family cDNA sequences from human, nematode and yeast .
  • Yeast two-hybrid library screening of a human Jurkat cell cDNA library was performed as described by Takayama et al . , EMBO J. , 16:4887-96 (1997); Matsuzawa et al., EMBO J.. 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 10 6 transformants, 112 Leu " colonies were obtained after
  • BAG-2 SEQ ID N0:4
  • BAG-3 BAG-3
  • BAG-1 SEQ ID NO:l
  • BAG-2 SEQ ID NO : 3
  • BAG-3 SEQ ID NO:5
  • cDNAs obtained by two-hybrid library screening with Hsc70/ATPase contained a conserved domain of about 40-50 amino acids which are termed the "BAG" domain and are shown in Figure 10.
  • BAG-family proteins The putative BAG-4 (SEQ ID NO: 8) and
  • BAG-5 (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
  • BAG-4 has 62% identity and ⁇ 81% similarity to BAG-3
  • BAG-5 has 51% identity and ⁇ 75% similarity to BAG-3.
  • 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
  • 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-lA all have ubiquitin-like domains near their N-termini (see Figure 10A) of unknown function.
  • the overall predicted amino acid sequences of the C. elegans BAG-1 SEQ ID NO: 12
  • S pombe BAG-lA SEQ ID NO: 12
  • proteins are ⁇ 18% identical ( ⁇ 61% similar) and ⁇ 17% identical ( ⁇ 64% similar), respectively, to human BAG-1, implying origin from a common ancestral gene.
  • the C. el egans BAG-1 protein (SEQ ID NO.-12), however, contains a
  • C. el egans 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. el egans and human BAG-2 share
  • the human BAG-2 protein (SEQ ID NO:4) , however, contains a 9 amino acid insert in its BAG-domain compared to it
  • 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 .
  • TA-BAG-2 and TA-BAG-3 demonstrated positive interactions with LexA-Hsc70/ATPase, resulting in transactivation of a lacZ reporter gene that was under the control of LexA operators ( Figure 11A) .
  • BAG-2 and BAG-3 were coexpression of BAG-2 and BAG-3 in the yeast two-hybrid assay. Coexpression of BAG-2 and BAG-3 failed to show interaction with BAG-1 or a deletion mutant of BAG-1 ( ⁇ C) which is missing part of its C-terminal domain required for Hsp70/Hsc70 binding suggest that these proteins do not form heterdimers .
  • a ⁇ -phage cDNA library was screened as follows, using hybridization probes derived from the two-hybrid screening.
  • a human jurkat T-cell ⁇ -ZapII library cDNA library (Stratagene) was screened by hybridization using 3/ P-labeled purified insert DNA from the longest of the human BAG-2 (clone #11) and human BAG-3 (clone #28) cDNA clones.
  • BAG-IL SEQ ID NO : 2
  • BAG-1 beginning at residue 116 of SEQ ID NO:2
  • BAG-2 SEQ ID NO : 4
  • BAG-3 SEQ ID NO : 6
  • search tools Prosite Search: PP search, using the Prosite pattern database, BCM Search Launcher, Baylor College of Medicine, and Blocks Search
  • the BAG-2 N- terminal region contains potential kinase phosphorylation sites but otherwise shares no apparent similarity with other proteins or known functional domains.
  • the predicted N-terminal region BAG-3 contains a WW domain as shown in Figure 10A.
  • W domains have been identified in a wide variety of signaling 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) .
  • YAP Yes kinase adaptor protein
  • Nedd4 Na * -channel regulator
  • formin-binding proteins formin-binding proteins
  • dystrophin dystrophin
  • peptidyl prolyl cis-trans-isomerase Pin-1 peptidyl prolyl cis-trans-isomerase Pin-1.
  • BAG-2 SEQ ID NO: 4
  • BAG-3 SEQ ID NO : 6
  • Hsc70/ATPase was determined by an in vi tro protein binding assay where Hsc70/ATPase or BAG-family proteins were expressed in bacteria as Glutathione S- Transferase (GST) fusion proteins.
  • GST Glutathione S- Transferase
  • a single colony was inoculated into IL of LB media containing 50 ⁇ g/ml ampicillin and grown at 37°C 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°C.
  • BAG-2 SEQ ID NO: 4
  • BAG-3 SEQ ID NO: 3
  • BAG-1 beginning at residue 116 of SEQ ID NO:2
  • BAG-2 SEQ ID NO:4
  • BAG-3 BAG-3
  • BAG-2 (SEQ 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 .
  • BAG-2 SEQ ID NO: 4
  • BAG-3 SEQ ID NO: 6
  • cDNAs encoding the ⁇ - phage cloned regions of BAG-2 and BAG-3 were subcloned in- frame 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 IOC, antiserum specific to Hsc70 detected the presence of BAG proteins associated with Hsc70, whereas control immune- complexes prepared with IgGl as well as anti-Flag immune complexes prepared from cells transfected with Flag-tagged control proteins, Daxx and Apaf-1, did not contain Hsc70 associated protein. These results further demonstrate that BAG-family proteins specifically bind to Hsc70.
  • BAG-1 (beginning at residue 116 of SEQ ID NO: 2) is known to bind tightly to the ATPase domain of Hsc70
  • 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 was produced in bacteria and purified to near homogeneity as shown in Figure 12A and described above in Example I. The purified BAG-1
  • the sensor chip was equilibrated with HK buffer (10 mM Hepes (pH 7.4), 150 mM KCL) at 5 ⁇ l/min, then activated by injecting 17 ⁇ l 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 ⁇ l 1M ethanolamine-HCL (pH8.5) . After immobilization, 5 ⁇ l of regeneration buffer (50 mM phosphate
  • Hsp70 (Sigma, H8778) was dissolved in HK buffer, and injected at 10 ⁇ l/min across the prepared surface at various concentrations. The surface was regenerated after each injection with 5 ⁇ l of regeneration buffer. The rate constants ⁇ ass and ⁇ dlss were generated with BIAevaluation softward 3.01 (Pharmacia Biosensor AB) . Addition of Hsc70 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) .
  • RU Response Units
  • Hsc70 failed to display interactions in BIAcore assays with a variety of control proteins as well as a mutant of BAG-1 lacking a C-terminal portion of the BAG domain which is required for Hsc70-binding ( Figure 3B) .
  • various control proteins such as GST, BSA and Bel -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.
  • BAG-2 SEQ ID NO:4
  • BAG-3 SEQ ID NO: 6
  • Hsp70/Hsc70-dependent refolding of denatured proteins similarly to a BAG-1 (beginning at residue 116 of SEQ ID NO: 2) protein.
  • luciferase (20 ⁇ M) was denatured in 25 mM Hepes-KOH, pH 7.2, 50 mM potassium acetate, 5 mM DTT, 6M guanidine hydrochloride at ⁇ 25°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 ⁇ M) , DnaJ (StressGen, Inc.) (0.9 ⁇ M), 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 ⁇ M. Luciferase activity was measured after 1.5 hr incubation at 35°C.
  • BAG-3 (SEQ ID NO : 6 ) to the above assays in amounts equimolar to Hsc70 (1.8 ⁇ M) resulted in striking inhibition of luciferase refolding.
  • BAG-2 (SEQ ID NO:4) and BAG-3
  • Hsc70 purified Hsc70 and human DnaJ homolog Hdj-1 (Hsp 40) were used with additional cofactors provided in reticulocyte lysates (5% v:v) to produce a system capable of refolding denatured luciferase.
  • 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.
  • BAG-1 Beginning at residue 116 of SEQ ID NO:2
  • BAG-2 SEQ ID NO:4
  • BAG-3 BAG-3
  • BAG-2 SEQ ID NO: 4
  • BAG-3 SEQ ID NO: 6
  • Hsc70/Hsp70 dependent protein refolding activity can inhibit Hsc70/Hsp70 dependent protein refolding activity to the same extent as BAG-1 (beginning at residue 116 of SEQ ID NO: 2) .
  • BAG competes with Hip for binding to Hsc70.
  • BAG-1 competes with Hip for binding to Hsc70, with these proteins exerting opposite effects on Hsc70-mediated protein refolding (Hohfeld, J. , and Jentsch, S., Embo J. , 16:6209-6216, 1997, which is incorporated herein by reference) .
  • BAG-2 SEQ ID NO: 4
  • BAG-3 SEQ ID NO: 6
  • Hip was purified as His 6 -protein.
  • the fusion protein was induced from pET28-Hip (V. Prapapanich et al . , Mol Cell Biol . , 18:944-952, 1998, which is incorporated herein by reference) with 0.1 mM IPTG at 25°C for 6h in BL21 cells.
  • Cells from IL of culture were resuspended into 50 ml of 50 mM Phosphate buffer (pH 6.8), 150 mM NaCl, and 1% (v/v) Tween-20 and then incubated with 0.5 mg/ml lysozyme at 25°C for 0.5h, followed by sonication.
  • the resulting supernatant was mixed with 15 ml nickel resin (Qiagen, Inc.) at 4°C for 3 h with 25 mM imidazol .
  • the resin was then washed with 50 mM phosphate buffer (pH 6.8), 25 mM imidazol, 150 mM NaCl and 0.1% Tween-20 until the OD280nm 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 (HRlO/10 Pharmacia) by FPLC using a linear gradient of 0.5M NaCl at pH 8.0, followed by dialysis in chaperone assay buffer.
  • 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.

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Abstract

La présente invention concerne une famille de protéines se rapportant aux BAG-1 provenant de l'homme (BAG-1L, BAG-1, BAG-2, BAG-3, BAG-4 et BAG-5), de l'invertébré C. elegans (BAG-1, BAG-2) et de la levure de fission S. pombe (BAG-1A, BAG-1B). L'invention concerne également les molécules d'acide nucléique les codant.
PCT/US1999/021053 1998-09-09 1999-09-09 Proteines bag et molecules d'acide nucleique les codant WO2000014106A1 (fr)

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AU60383/99A AU774355B2 (en) 1998-09-09 1999-09-09 Novel bag proteins and nucleic acid molecules encoding them
JP2000568863A JP2002524068A (ja) 1998-09-09 1999-09-09 新規なｂａｇタンパク質およびそれらをコードする核酸分子

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EP1323733A1 (fr) * 2001-12-28 2003-07-02 Arturo Leone Utilisation des séquences nucléotidique et protéique de BAG3 pour la recherche, le diagnostic et le traitement de maladies impliquant la mort cellulaire
US20030175958A1 (en) * 2002-03-15 2003-09-18 Reed John C. Methods for using bag expression as a cell differentiation agent and marker
WO2005054868A1 (fr) * 2003-12-05 2005-06-16 Multimmune Gmbh Compositions et procedes pour traiter et diagnostiquer des maladies neoplasiques et infectieuses
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KR102172541B1 (ko) * 2019-02-12 2020-11-02 주식회사 메드팩토 Bag2 항체를 이용한 암 진단용 조성물 및 이를 이용한 방법
KR102241558B1 (ko) * 2019-02-12 2021-04-20 주식회사 메드팩토 Bag2 폴리펩티드 또는 이의 단편에 특이적으로 결합하는 항체 또는 그의 항원 결합 단편
CN109913478A (zh) * 2019-04-09 2019-06-21 贵州大学 一种高粱E3泛素连接酶SbBAG4基因及其重组载体和表达方法

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