# Patent

## Title
BAG proteins and nucleic acid molecules encoding them

## Abstract

The present invention provides a family of BAG-1 related proteins from humans (BAG-1L, BAG-1, BAG-2, BAG-3, BAG-4 and BAG-5), the invertebrate C. elegans (BAG-1, BAG-2) and the fission yeast S. pombe (BAG-1A, BAG-1B) and the nucleic acid molecules that encode them.

## Background
This application is a continuation of U.S. Ser. No. 09/394,142, filed Sep. 9, 1999, now U.S. Pat. No. 6,696,558 which claims the benefit of U.S. Provisional Application No. 60/155,212, filed Sep. 9, 1998, which was converted from U.S. Ser. No. 09/150,489, and which is incorporated herein by reference in its entirety.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with government support under grant number CA-67329 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.


BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to the fields of molecular biology and molecular medicine and more specifically to a novel family of proteins that can regulate protein folding. The functions of these proteins are potentially diverse, including promoting tumor cell growth and metastasis.
2. Background Information
The Hsc70/Hsp70-family of molecular chaperones participate in protein folding reactions, controlling protein bioactivity; degradation, complex assembly/disassembly, and translocation across membranes. These proteins interact with hydrophobic regions within target proteins via a carboxyl (C)-terminal peptide binding domain, with substrate binding and release being controlled by the N-terminal ATP-binding domain of Hsc70/Hsp70. Hsc70/Hsp70-assisted folding reactions are accomplished by repeated cycles of peptide binding, refolding, and release, 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.
The Bcl-2 associated athanogene-1 (bag-1) is named from the Greek word athanos, which refers to anti-cell death. BAG-1 was previously referred to as Bcl-2-associated protein-1 (BAP-1) in U.S. Pat. No. 5,539,094 issued Jul. 23, 1996, which is incorporated herein by reference. In this earlier patent, BAG-1 is described as a portion of the human BAG-1 protein, absent the N-terminal amino acids 1 to 85. In addition, a human protein essentially identical to human BAG-1 was described by Zeiner and Gehring, (Proc. Natl. Acad. Sci., USA 92:11465-11469 (1995)). Subsequent to the issuance of U.S. Pat. No. 5,539,094 the N-terminal amino acid sequence from 1 to 85 of human BAG-1 was reported.
BAG-1 and its longer isoforms BAG-1M (Rap46) and BAG-1L are recently described Hsc70/Hsp70-regulating proteins. BAG-1 competes with Hip for binding to the Hsc70/Hsp70 ATPase domain and promotes substrate release. BAG-1 also reportedly stimulates Hsc70-mediated ATP 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.
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.
SUMMARY OF THE INVENTION
The present invention provides a family of BAG-1 related-proteins from humans [BAG-1L (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) and (SEQ ID NO:20), BAG-4 (SEQ ID NO:8) and (SEQ ID NO:22) and BAG-5 (SEQ ID NO:10) and (SEQ ID NO:24)], the invertebrate C. elegans [BAG-1 (SEQ-ID NO:12), BAG-2 (SEQ ID NO:14)] and the fission yeast S. pombe [BAG-1A (SEQ ID NO:16), BAG-1B (SEQ ID NO:18)] 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 NO:2), and related proteins with the Hsc70/Hsp70 Family of proteins or with other proteins that may interact with the BAG-Family proteins.
Still another aspect of the present invention is directed to methods for detecting agents that induce the dissociation of a bound complex formed by the association of BAG-Family proteins with Hsc70/Hsp70 Family molecule chaperones or other proteins.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the full length cDNA sequence for human BAG-1 (SEQ ID NO:1) protein with the corresponding amino acid sequence (SEQ ID NO:2). Within the full length sequence are included the overlapping sub-sequences of BAG-1 (beginning at nucleotide 391), BAG-1M [beginning at nucleotide 260 of (SEQ ID NO:2)], and BAG-1L [beginning at nucleotide 46 of (SEQ ID NO:2)].
FIGS. 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).
FIG. 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).
FIG. 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).
FIG. 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).
FIG. 6A shows the full length cDNA sequence for C. elegans BAG-1 protein (SEQ ID NO:11).
FIG. 6B shows the 210 amino acid sequence for C. elegans BAG-1 protein (SEQ ID NO:12).
FIG. 7A shows the full length cDNA sequence for C. elegans BAG-2 protein (SEQ ID NO:13).
FIG. 7B shows the 458 amino acid sequence for C. elegans BAG-2 protein (SEQ ID NO:14).
FIG. 8A shows the full length cDNA sequence for S. pombe BAG-1A protein (SEQ ID NO:15).
FIG. 8B shows the 195 amino acid sequence for S. pombe BAG-1A protein (SEQ ID NO:16).
FIG. 9A shows the full length cDNA sequence for S. pombe BAG-1B protein (SEQ ID NO:17).
FIG. 9B shows the 206 amino acid sequence for S. pombe BAG-1B 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 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6), BAG-4 (SEQ ID NO:8), BAG-5 (SEQ ID NO:10); S. pombe BAG-1A (SEQ ID NO:16) and BAG-1B (SEQ ID NO:18); and C. elegans BAG-1 (SEQ ID NO:12) and BAG-2 (SEQ ID NO:14). (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-1A (SEQ ID NO:16) and BAG-1B (SEQ ID NO:18), and C. elegans BAG-1 (SEQ ID NO:12) and BAG-2 (SEQ ID NO:14) are aligned demonstrating their homology. Black and gray shading represent identical and similar amino acids, respectively.
FIG. 11 shows assays demonstrating the interaction of BAG-family proteins with Hsc70/ATPase. FIG. 11A shows two-hybrid assays using yeast expressing the indicated fusion proteins. Blue color indicates a positive interaction, resulting in activation of the lacZ reporter gene. FIG11B shows in vitro protein assays using GST-fusion proteins and 35S-labeled in vitro translated proteins. FIG. 11C shows co-immunoprecipitation assays using anti-FlagTM or IgG1 control antibodies and lysates from 293T cells expressing FlagTM-tagged BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6), Daxx, or Apaf-1.
FIG. 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.
FIG. 13 shows representative SPR results for biosensor chips containing immobilized BAG-1 (beginning at residue 116 at SEQ ID NO:2), BAG-1 (ΔC), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) proteins. Hsc70/ATPase was flowed over the chips (arrow/left) until maximal binding was reached (response units), then flow was continued without Hsc70/ATPase (arrow/right). For BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6), Hsc70 was injected at 0.0175, 0.035, 0.07, 0.14, and 0.28 μM.
FIG. 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 (ΔC). (C) Hsc70/Hsp40-mediated refolding of heat-denatured luciferase was assayed in the presence of (black bars) or absence of (striped bars) of 1.8 μM Hip, with (lanes 3-10) or without (lanes 1,2) various BAG-family proteins (1.8 μM) as indicated (mean ±SE; n=3). A control (CNTL) is shown (lane 1) in which Hsc70 was replaced with an equivalent amount of BSA.
FIGS. 15A and 15B show an expanded cDNA sequence for human BAG-3 protein (SEQ ID NO:19).
FIG. 15C shows the corresponding amino acid residues for the human BAG-3 protein (SEQ ID NO:20) of FIGS. 15A and 15B.
FIGS. 15D and 15E show the expanded cDNA sequence (SEQ ID NO:19) aligned with the corresponding amino acid residues for human BAG-3 protein of FIGS. 15A and 15B (SEQ ID NO:20).
FIGS. 16A and 16B show an expanded cDNA sequence for human BAG-4 protein (SEQ ID NO:21).
FIG. 16C shows the corresponding amino acid residues for the human BAG-4 protein of FIGS. 16A and 16B (SEQ ID NO:22).
FIGS. 16D and 16E show the expanded cDNA sequence (SEQ ID NO:21) aligned with the corresponding amino acid residues for human BAG-4 protein of FIGS. 16A and 16B (SEQ ID NO:22).
FIGS. 17A, 17B, 17C, and 17D show an expanded cDNA sequence for human BAG-5 protein (SEQ ID NO:23).
FIG. 17E shows the corresponding amino acid residues for the human BAG-5 protein of FIG. 17A-17D (SEQ ID NO:24).
FIGS. 17F, 17G and 17H show the expanded cDNA sequence (SEQ ID NO:23) aligned with the corresponding amino acid residues for human BAG-5 protein of FIG. 17A-17D (SEQ ID NO:24).
FIG. 18 shows the topologies of the BAG-family proteins; human BAG proteins, BAG-1 (SEQ ID NO:2), BAG-2 (SEQ ID NO:4), expanded BAG-3 (SEQ ID NO:20), expanded BAG-4 (SEQ ID NO:22), expanded BAG-5 (SEQ ID NO:24); S. pombe BAG-1A (SEQ ID NO:16) and BAG-1B (SEQ ID NO:18); and C. elegans BAG-1 (SEQ ID NO:12) and BAG-2 (SEQ ID NO:14). The relative positions of the BAG domains are shown in black, ubiquitin-like regions are represented in gray, WW domain are represented in strips. Nucleoplasmin-like nuclear localization sequence are also shown.



DEFINITIONS
The term “apoptosis”, as used herein, refers to the process of programmed cell death, although not all programmed cell deaths occur through apoptosis, as used herein, “apoptosis” and “programmed cell death” are used interchangeably.
The term “tumor cell proliferation”, as used herein refers to the ability of tumor cells to grow and thus expand a tumor mass.
The term “cell migration”, as used herein refers to the role cell motility plays in the invasion and potentially metastasis by tumor cells.
The term “metastasis”, as used herein refers to the spread of a disease process from one part of the body to another, as in the appearance of neoplasms in parts of the body remote from the site of the primary tumor; results in dissemination of tumor cells by the lymphatics or blood vessels or by direct extension through serious cavitites or subarachnoid or other spaces.
The term “steroid hormone receptor function”, as used herein refers to physiological, cellular and molecular Functioning of receptors sites that bind with steroid hormones.
The term “substantially purified”, as used herein, refers to nucleic acid or amino acid sequence that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
“Nucleic acid molecule” as used herein refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single or double stranded, and represent the sense or antisense strand.
“Hybridization”, as used herein, refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
The terms “complementary” or “complementarity”, as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence “A-G-T binds to the complementary sequence “T-C-A”.
The term “homology”, as used herein, refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid and is referred to using the functional term “substantially homologous.” The inhibition of 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.
The term “antisense”, as used herein, refers to nucleotide sequences which are commplementary to a specific DNA or RNA sequence. The term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the “sense” strand. Antisense molecules may be produced by any method, including synthesis by ligating the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a complementary strand. Once introduced into a cell, this transcribed strand combines with natural sequences produced by the cell to form duplexes. These duplexes then block either the further transcription or translation. In this manner, mutant phenotypes may be generated. The designation “negative” is sometimes used in reference to the antisense, and “positive” is sometimes used in reference to the sense strand.
“Amino acid sequence” as used herein refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited herein this term excludes an amino acid sequence of a naturally occurring protein. “Amino acid sequence”, “polypeptide” or “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
The term “functional fragments” or “fragments”, as used herein, with regard to a protein refers to portions of that protein that are capable of exhibiting or carrying out the activity exhibited by the protein as a whole. The portions may range in size from three amino acid residues to the entire amino acid sequence minus one amino acid. For example, a protein “comprising at least a functional fragment of the amino acid sequence of SEQ ID NO:1”, encompasses the full-length of the protein of SEQ ID NO:1 and portions thereof.
A “derivative” of a BAG protein, as used herein, refers to an amino acid sequence that is alterd by one or more amino acids. The derivative may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, 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).














Abbreviations











Amino Acid
Radical
3-Letter
1-Letter










Amino Acids - Apolar R Groups










alanine
methyl
ala
A


valine
2-propyl
aal
V


leucine
2-methylpropyl
leu
L


isoleucine
2-butyl
ile
I


proline
propyl* - cyclized
pro
P


phenylalanine
benzyl
phe
F


trytophan
3-indolylmethl
tyr
W


methionine
methylthioethyl
met
M







Amino Acids - Uncharged Polar R Groups










glycine
H
gly
G


serine
hydroxymethyl
ser
S


threonine
1-hydroxyethyl
thr
T


cysteine
thiolmethyl
cys
C


tyrosine
4-hydroxyphenylmethyl
tyr
Y


asparagine
aminocarbonylmethyl
asn
N


glutamine
aminocarbonylethyl
gln
Q







Amino Acids - Charged R Groups










aspartic acid
carboxymethyl
asp
D


glutamic acid
carboxyethyl
glu
E


lysine
4-aminobutyl
lys
K


arginine
3-guanylpropyl
arg
R


histidine
4-imidazoylmethyl
his
H









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, DNASTARTM software. In addition, the derivative may also result from chemical modifications to the encoded polypeptide, including but not limited to the following, replacement of hydrogen by an alkyl, acyl, or amino group; esterification of a carboxyl group with a suitable alkyl or aryl moiety; alkylation of a hydroxyl group to form an ether derivative. Further a derivative may also result from the substitution of a L-configuration amino acid with its corresponding D-configuration counterpart.
The term “mimetic”, as used herein, refers to a molecule, the structure of which is developed from knowledge of the structure of a protein/polypeptide or portions thereof (such as BAG-1) and, as such, is able to effect some or all of the actions of BAG-1 protein.
“Peptide nucleic acid”, as used herein, refers to a molecule which comprises an oligomer to which an amino acid residue, such as lysine, and an amino group have been added. These small molecules, also designated anti-gene agents, stop transcript elongation by binding to their complementary strand of nucleic acid (Nielsen, P. E. et al., Anticancer Drug Des. 8:53-63 (1993)).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a family of BAG-1 related proteins from humans [BAG-1L (SEQ ID NO:2), BAG-1S 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. pombe [BAG-1A (SEQ ID NO:16), BAG-1B (SEQ ID NO:18)], specifically the full length amino acid sequences comprising human BAG-1L (SEQ ID NO:2), BAG-1 (beginning at residue 116 of SEQ ID NO:2), and BAG-2 (SEQ ID NO:4) C. elegans BAG-1 (SEQ ID NO:12), and BAG-2 (SEQ ID NO:14), and S. pombe BAG-1A (SEQ ID NO:16) and BAG-1B (SEQ ID NO:18); and partial sequences comprising human BAG-3 (SEQ ID NO: 6) and (SEQ ID NO:20), BAG-4 (SEQ ID NO:8) and (SEQ ID NO:22), and BAG-5 (SEQ ID NO:10) and (SEQ ID NO:24) and functional fragments thereof. In particular, the invention provides the amino acid sequences comprising human BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6) and (SEQ ID NO:20), BAG-4 (SEQ ID NO:8) and (SEQ ID NO:22), and BAG-5 (SEQ ID NO:10) and (SEQ ID NO:24) proteins.
Another aspect of the present invention provides the nucleic molecule and nucleotide sequences that encode the family of BAG-1 related proteins from humans [BAG-1 (SEQ ID NO:1), BAG-2 (SEQ ID NO:3), BAG-3 (SEQ ID NO:5) and (SEQ ID NO:19), BAG-4 (SEQ ID NO:7) and (SEQ ID NO:21) and 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-1A (SEQ ID NO:15), BAG-1B (SEQ ID NO:17)].
BAG-1L (SEQ ID NO:2) is a multifunctional protein that blocks apoptosis, promotes tumor cell metastasis, and contributes to factor-independent and p53-resistant cell growth. BAG-1L (SEQ ID NO:2) interacts with several types of proteins, including Bcl-2, some tyrosine kinase growth factor receptors, steroid hormone receptors, and the p53-induced cell cycle regulator Siah-1A.
BAG-1 is a regulator of Hsc70/Hsp70 family molecular chaperones. A carboxyl-terminal domain in this protein binds tightly to the ATPase domains of Hsc70 and Hsp70 (KD=1 nM) (Zeiner, M., Gebauer, M., and Gehring, U., EMBO J. 16: 5483-5490, (1997)). BAG-1 modulates the activity of these molecular chaperones, acting as an apparent functional antagonist of the Hsp70/Hsc70-associated protein Hip (3-5)(Höhfeld, J. and Jentsch, S., EMBO J. 16: 6209-6216, (1997); Takayama, S., Bimston, D. N., Matsuzawa, S., Freeman, B. C., Aime-Sempe, C., Xie, Z., Morimoto, R. J., and Reed, J. C., EMBO J. 16: 4887-96, (1997); Zeiner, M., Gebauer, M., and Gehring, U., EMBO J. 16: 5483-5490, (1997)). In general, protein refolding is accomplished by Hsp70/Hsc70 through repeated cycles of target peptide binding and release, coupled to ATP hydrolysis (Ellis, R., Curr Biol. 7: R531-R533, (1997)). BAG-1 appears to promote substrate release, whereas Hip stabilizes Hsp70/Hsc70 complex formation with target peptides (Höhfeld, 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.
The 70 kd 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 55 amino acids that bind the a Hsc70/Hsp70 ATPase domain. The BAG domain is situated near the C-terminus, and the ubiquitin-like domains are situated near the N-terminus.
The BAG family of proteins of the present invention contain a common conserved C-terminal domain (the “BAG” domain) that facilitates binding to the ATPase domain of Hsp70/Hsc70. The carboxyl-terminal domain of BAG-1 binds to the ATPase domain of Hsc70/Hsp70 and regulates its chaperone function by acting as a ADP-ATP exchange factor. Other domains of BAG-1 mediate interactions with proteins such as Bcl-2 and retinoic acid receptors (RARs), allowing BAG-1 to target Hsc70/Hsp70 to other proteins, presumably modulating their function by changing their conformations.
Human BAG-1 was previously shown to inhibit Hsc70/Hsp70 dependent refolding of denatured protein substrates in vitro (S. Takayama, et al., EMBO J. 16, 4887-96 (1997); M. Zeiner, M. Gebauer, U. Gehring, EMBO J. 16, 5483-5490 (1997); and J. Höhfeld, S. Jentsch, EMBO J. 16, 6209-6216 (1997)). In Example III, Part A the effects of recombinant human BAG-1, BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) were compared using in vitro protein refolding assays similar to those employed previously for assessing BAG-1. The study showed that addition of equimolar amounts of each of the recombinant proteins to Hsc70 resulted in significant inhibition of luciferase refolding, with BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) showing somewhat greater inhibitor activity than BAG-1 (FIG. 4A). In a separate luciferase folding study BAG-1, BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) once again displayed inhibition of luciferase refolding, however in this study varying amounts of BAG-1, BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) were added relative to Hsc70 which resulting in concentration-dependent inhibition of Hsc70 chaperone activity, i.e., luciferase folding (Example III Part A). Additional follow on studies using the same experimental protocols as the previous studies, as taught in Example IIA, have shown that BAG-4 (SEQ ID NO:22) also undergoes association with Hsc70/ATPase.
Yet another aspect of the present invention provides a 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 FIGS. 1-9 and FIGS. 15-17, in particular the BAG domain as shown in in FIG. 1B, e.g., nucleotides 552-593 of human BAG-3, or nucleotides 167-221 of human BAG-4.
Yet another aspect of the present invention provides a compound of the formula,

RN—R1X1R2X2R3X3R4X4R5X5R6X6R7X7X8R9X9R10X10R11X11—RC 

wherein,

RN is a group of 1 to 552 independently selected amino acids;
R1 is a group of 3 independently selected amino acids;
X1 is an amino acid with a charged or uncharged R group, such as aspartic acid, glutamic acid, asparagine, or glutamine;
R2 is a group of 7 independently selected amino acids;
X2 is an amino acid with a charged R group, such as glutamic acid;
R3 is a group of 5 independently selected amino acids;
X3 is an amino acid with an apolar R group, such as leucine, methionine, or isoleucine;
R4 is a group of 3 independently selected amino acids;
X9 is an amino acid with charged R group, such as aspartic acid or glutamine acid;
R5 is a single independently selected amino acid;
X5 is an amino acid with apolar or uncharged R group, such as leucine, valine, methionine, alanine or threonine;
R6 is a group of 15 independently selected amino acids;
X6 is an amino acid with a charged or uncharged R group, such as arginine, lysine, glutamine or aspartic acid;
R7 is a group of 2 independently selected amino acids;
X7 is an amino acid with a charged R group, such as arginine;
X8 is an amino acid with a charged R group, such as arginine or lysine;
R9 is a group of 2 independently selected amino acids;
X9 is an amino acid with an apolar R group, such as valine;
R10 is a group of 3 independently selected amino acids;
X10 is an amino acid with an uncharged R group, such as glutamine;
R11 is a group of 2 independently selected amino acids;
X11 is an amino acid with an apolar R group, such as leucine; and
RC is a group of 1 to 100 independently selected amino acids.
A nucleotide sequence of at least about 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 Protocols: A guide to methods and applications, ed. Innis et al. (Academic Press, Inc., 1990), which is incorporated herein by reference; see, for example, pages 40-41). In addition, such a nucleotide sequence of the invention can be useful as a probe in a hybridization reaction such as Southern or northern blot analysis or in a binding assay such as a gel shift assay.
A nucleotide sequence of the invention can be particularly useful as an antisense molecule, which can be DNA or RNA and can be targeted to all or a portion of the 5′-untranslated region or of the 5′-translated region of a bag-1 nucleic acid sequence in a cell. For example, an antisense molecule can be directed to at least a portion of the sequence shown as the BAG domain in FIG. 1A, e.g., nucleotides 272-319 of human BAG-1L (SEQ ID NO:11) or nucleotides 79-147 of human BAG-5 (SEQ ID NO:9). Since the 5′-region of a nucleic acid contains elements involved in the control of expression of an encoded protein, an antisense molecule directed to the 5′-region of a nucleic acid molecule can affect the levels of protein expressed in a cell.
A nucleotide sequence of the invention also can be useful as a probe to identify a genetic defect due a mutation of a gene encoding a BAG protein in a cell. Such a genetic defect can lead to aberrant expression of a BAG protein in the cell or to expression of an aberrant BAG protein, which does not properly associate with a Bcl-2-related protein or Hsc70/Hsp70 protein in the cell. As a result, 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.
Further a nucleotide compound or composition as taught in the present invention can be synthesized using routine methods or can be purchased from a commercial source. In addition, a population of such nucleotide sequences can be prepared by restriction endonuclease or mild DNAse digestion of a nucleic acid molecule that contains nucleotides as shown in the nucleotide sequences shown in FIGS. 1-9 and FIGS. 15-17 that encodes the amino acids sequences also shown in FIGS. 1-9 and FIGS. 15-17. Methods for preparing and using such nucleotide sequences, for example, as hybridization probes to screen a library for homologous nucleic acid molecules are well known in the art (see, for example, Sambrook et al., Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989); Ausubel et al., Current Protocols in Molecular Biology (Green Publ., NY 1989), each of which is incorporated herein by reference).
A particular nucleotide sequence can be designed based, for example, on a comparison of the nucleic acid molecules encoding any one of the BAG family proteins, as shown in FIGS. 1-9 and FIGS. 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. In addition, such a comparison allows the preparation of a nucleotide sequence that will hybridize to a unique region of any of the BAG family nucleotide sequences, such as those corresponding to the BAG domain, thus allowing identification of other proteins sharing this motif. In this regard, it is recognized that, while the human BAG-3 proteins shown as FIGS. 3 and 20, and human BAG-5 proteins shown as FIGS. 5 and 24, are only partial sequences, a variant human BAG-3 or BAG-5 produced, for example, by alternative splicing can exist and can be identified using an appropriately designed nucleotide sequence of the invention as a probe. Such useful probes readily can be identified by inspection of the sequences shown in the disclosed Figures by a comparison of the encoding nucleotide sequences.
If desired, a nucleotide sequence of the invention can incorporate a detectable moiety such as a radiolabel, a fluorochrome, a ferromagnetic substance, a luminescent tag or a detectable binding agent such as biotin. These and other detectable moieties and methods of incorporating such moieties into a nucleotide sequence are well known in the art and are commercially available. A population of labelled nucleotide sequences can be prepared, for example, by nick translation of a nucleic acid molecule of the invention (Sambrook et al., supra, 1989; Ausubel et al., supra, 1989).
One skilled in the art would know that a method involving hybridization of a nucleotide sequence of the invention can require that hybridization be performed under relatively stringent conditions such that nonspecific background hybridization is minimized. Such hybridization conditions can be determined empirically or can be estimated based, for example, on the relative GC content of a sequence and the number of mismatches, if known, between the probe and the target sequence (see, for example, Sambrook et al., supra, 1989).
The invention further provides antibodies specific for human BAG family protein. As used herein, the term “antibody” includes polyclonal and monoclonal antibodies, as well as polypeptide fragments of antibodies that retain a specific binding activity for human BAG-1 of at least about 1×105 M−1. One skilled in the art would know that 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. In addition, the term “antibody” as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies and fragments that retain binding activity such as chimeric antibodies or humanized antibodies. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference.
One skilled in the art would know that purified BAG family protein, which can be prepared from natural sources or synthesized chemically or produced recombinantly, or portions of a BAG family protein, including a portion of human BAG family protein such as a synthetic peptide as described above, can be used as an immunogen. Such peptides useful for raising an antibody include, for example, peptide portions of the N-terminal 85 amino acids or the BAG domain of any of the human BAG proteins (see FIG. 1B). 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) or keyhole limpet hemocyanin (KLH), as described in Example IV, below. In addition, various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art and described, for example, by Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1988), which is incorporated herein by reference.
EXAMPLES
The following examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof.
Example I
Isolation and Characterization of BAG-Family cDNA Sequences
This example describes methods for isolating and characterizing of BAG-family cDNA sequences from human, nematode and yeast.
A. Cloning of Human BAG cDNA Sequences
Yeast two-hybrid library screening of a human Jurkat cell cDNA library was performed as described by Takayama et al., EMBO 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  ̃5×106 transformants, 112 Leu+ colonies were obtained after 1 week incubation at 30° C. Assay of β-galactosidase (β-gal) activity of these colonies resulted in 96 clones. Mating tests were then performed using RFY206 yeast strain transformed with pGilda, pGilda mBAG-1 (1-219), or pGilda Hsc70/ATPase. Of these, 66 displayed specific interactions with Hsc70/ATPase. The pJG4-S cDNAs were recovered using KC8 E. coli strain which is auxotrophic for tryptophan (Trp). DNA sequencing revealed 3 partially overlapping human BAG-1, 4 identical and one overlapping cDNTAs encoding BAG-2, and 2 partially overlapping BAG-3 clones.
Using the above described yeast two-hybrid screen with the ATPase domain of Hsc70 as “bait”, several human cDNAs were cloned which encode portions of BAG-1 or of two other BAG-1-like proteins which are termed BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6). The longest of the cDNAs for BAG-2 (SEQ ID NO:3) and BAG-3 (SEQ ID NO:5) contained open reading frames (ORFs) of 207 and 162 amino acids, respectively, followed by stop codons. All BAG-1 (SEQ ID NO:1), BAG-2 (SEQ ID NO:3) and 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 FIG. 10. These results demonstrate that a family of BAG-1-related proteins all contain a conserved  ̃45 amino acid region near their C-terminus that binds Hsc70/Hsp70.
B. Identification of Additional BAG-Family Proteins
A search of the translated Genbank database using the bBLAST and FASTA search programs also identified human ESTs that provided sequences for further investigation of BAG-family proteins. The putative BAG-4 (SEQ ID NO:8) and 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 (Accession number AA693697, N74588) and BAG-5 (Accession number AA456862, N34101). BAG-4 has 62% identity and  ̃81% similarity to BAG-3, and BAG-5 has 51% identity and  ̃75% similarity to BAG-3.
Additional BAG-family orthologues or homologues were also identified using computer-based searches and resulted in BAG-family homologue in the nematode C. elegans and the fission yeast S. pombe. The C. elegans genome encodes two apparent BAG-family proteins, which are most similar in their overall sequences to the human BAG-1 (Afo39713, gi:2773211) (SEQ ID NO:12) and BAG-2 (SEQ ID NO:14) (Afo68719, gi:3168927). The S. pombe contains two BAG-family proteins that share the greatest overall sequence similarity with human BAG-1 (Alo23S54,gi/3133105 and Alo23634, gi/3150250). The human and C. elegans BAG-1 proteins as well as S. pombe BAG-1A all have ubiquitin-like domains near their N-termini (see FIG. 10A) of unknown function.
The overall predicted amino acid sequences of the C. elegans BAG-1 (SEQ ID NO:12) and S. pombe BAG-1A (SEQ ID NO:16) proteins are  ̃18% identical ( ̃61% similar) and  ̃17% identical ( ̃64% similar), respectively, to human BAG-1, implying origin from a common ancestral gene. The C. elegans BAG-1 protein (SEQ ID NO:12), however, contains a 5 to 7 amino acid insert in its BAG-domain as compared to the human, murine, and yeast BAG-1 homologues (see FIG. 10B), and is more similar to BAG-2 (SEQ ID NO:4) in regard to its BAG-domain. C. elegans and human BAG-2 also may be derived from a common ancestor as the C-terminal 225 amino acid region which encompasses both the BAG domain and upstream region of both C. elegans and human BAG-2 share 34% amino acid sequence identity and  ̃70% similarity. The human BAG-2 protein (SEQ ID NO:4), however, contains a 9 amino acid insert in its BAG-domain compared to it C. elegans counterpart (see FIG. 10B). Evolutionary-tree prediction algorithms suggest that human and C. elegans BAG-2 represent a distinct branch of the BAG-family that is more evolutionarily distant from the other BAG-family proteins. None of the predicted BAG-family proteins contain recognizable regions analogous to those found in other Hsc70 regulatory proteins, such as the J-domains and G/F-domains of DnaJ family proteins and the Tetratricopeptide Repeat (TR) domains of Hip/Hop family proteins.
C. Yeast Two-Hybrid Assay of BAG Binding to Hsc70/ATPase
The longest of the cDNAs obtained for the BAG-2 and BAG-3 proteins were expressed with N-terminal transactivation (TA) domains in yeast and tested by yeast two-hybrid assay for interactions with fusion proteins consisting of Hsp70/ATPase or a variety of unrelated proteins (Fas, Siah, Fadd) containing N-terminal LexA DNA-binding domains. 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 (FIG. 11A). No interactions with LexA-Fas (cytosolic domain), LexA-Siah, LexA-Fadd, or LexA were detected (see FIG. 1A) demonstrating that the BAG-2 and BAG-3 proteins interact specifically with Hsc70/ATPase. Specific two-hybrid interactions between Hsc70/ATPase and either BAG-2 or BAG-3 were also observed when BAG-2 and BAG-3 were expressed as LexA DNA-binding domain fusion proteins and Hsc70/ATPase was fused with a TA domain (see FIG. 11A; right panel) These results demonstrate that similarly to BAG-1, BAG-2 and BAG-3 specifically interact with Hsc70/ATPase.
In order to determine whether the BAG proteins are capable of forming heterodimers, coexpression of BAG-2 and BAG-3 in the yeast two-hybrid assay was also performed. Coexpression of BAG-2 and BAG-3 failed to show interaction with BAG-1 or a deletion mutant of BAG-1 (ΔC) which is missing part of its C-terminal domain required for Hsp70/Hsc70 binding suggest that these proteins do not form heterdimers.
D. Isolation and Characterization of the Complete Open Reading Frame Sequences of BAG-2 and BAG-3
In order to deduce the complete ORFs of BAG-2 and BAG-3, a λ-phage cDNA library was screened as follows, using hybridization probes derived from the two-hybrid screening. A human jurkat T-cell λ-ZapII library cDNA library (Stratagene) was screened by hybridization using 32P-labeled purified insert DNA from the longest of the human BAG-2 (clone #11) and human BAG-3 (clone #28) cDNA clones. From about one million clones screened, 38 BAG-2 and 23 BAG-3 clones were identified, cloned, and their cDNA inserts recovered as pSKII plasmids using a helper phage method (Stratagene). DNA sequencing of λ-phage derived human BAG-2 cDNA clones revealed an ORF encoding a predicted 211 amino acid protein, preceded by an in-frame stop codon. The longest human BAG-3 λ-phage cDNA clone contains a continuous ORF of 682 amino acids followed by a stop codon, but without an identifiable start codon (see FIG. 10A).
Although BAG-1L (SEQ ID NO:2), BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6) all contain a homologous BAG domain near their C-terminus, the N-terminal regions of these proteins are dissimilar. Using a combination of search tools (Prosite Search: PP search, using the Prosite pattern database, BCM Search Launcher, Baylor College of Medicine, and Blocks Search), it was determined that the BAG-2 N-terminal region contains potential kinase phosphorylation sites but otherwise shares no apparent similarity with other proteins or known functional domains.
In contrast, the predicted N-terminal region BAG-3 contains a WW domain as shown in FIG. 10A. WW 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).
Example II
In vitro Association of BAG Proteins and Hsc70/ATPase
This example demonstrates that BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) bind Hsc70/ATPase in various in vitro assays.
A. Solution Binding Assay of BAG-2 and BAG-3 to Hsc70/ATPase
Association of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) with Hsc70/ATPase was determine by an in vitro protein binding assay where Hsc70/ATPase or BAG-family proteins were expressed in bacteria as Glutathione S-Transferase (GST) fusion proteins. Purified cDNA sequences encoding residues 5 to 211 of human BAG-2 (clone #11) and the C-terminal 135 amino acids of human BAG-3 (clone #28) (see FIG. 10A) were subcloned into the EcoRI/Xho I sites of pGEX4T-1 prokaryotic expression plasmid (Pharmacia; Piscataway, N.J.). These plasmids as well as pGEX4T-1-BAG-1, pGEX-4T-1-BAG-1 (ΔC), and pGEX-4T-1-XL which have been described previously (Takayama et al., supra (1997); Xie et al., Biochemistry, 37:6410-6418, (1998), which are incorporated herein by reference), were expressed in XL-1 blue strain E. Coli (Stratagene, Inc., La Jolla, Calif.). Briefly, a single colony was inoculated into 1L of LB media containing 50 μg/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 0.4 mM IPTG for 6 h at 25° C.
Cells were recovered and incubated with 0.5 mg/ml lysozyme in 50 mM Tris (pH 8.0), 150 mM NaCl, 1% TweenTM-20, 0.1% 2-mercaptoethanol, 5 mM EDTA, 1 mM PMSF and a mixture of other protease inhibitors obtained from Boehringer Mannheim (1697498) at room temperature for 0.5 h, followed by sonication. Cellular debris were pelleted by centrifugation at 27,500 g for 10 min and the resulting supernatants were incubated with 30 ml of glutathionine-SepharoseTM (Pharmacia) at 4° C. overnight. The resin was then washed with 20 mM Tris (pH 8.0), 150 mM NaCl, 0.1% TweenTM-20, and 0.1% 2-mercaptoethanol until the OD 280 nm reached <0.01. For removal of GST, the resin with immobilized GST-fusion protein was incubated with 10 U of thrombin (Boehringer, Inc.) at 4° C. in 20 mM Tris (pH 8.0), 150 mM NaCl, 0.1% TweenTM-20, 0.1% 2-Mercaptoethanol, and 2.5 mM CaCl2 overnight. Released proteins were then purified on Mono QTM (HR10/10, Pharmacia) by FPLC using a linear gradient of 0.5M NaCl at pH 8.0 and dialyzed into chaperone assay buffer.
The ability of BAG-2 (SEQ ID NO:4) or BAG-3 (SEQ ID NO:6) to bind Hsc70/ATPase in solution was then examined. GST control or GST-BAG proteins were immobilized on glutathione-SepharoseTM and tested for binding to 35S-labeled in vitro translated (IVT) proteins. Immunoprecipitation and in vitro GST-protein binding assays were performed as described by Takayama et al., supra (1997), using pCI-Neo FlagTM or pcDNATM 3-HA into which human Bag-2 (clone #11) or human BAG-3 (clone #28) had been subcloned for in vitro translation of 35S-L-methionine labeled proteins or expression in 293T cells. As shown in FIG. 11B, 35S-Hsc70/ATPase bound in vitro to GST-BAG-1, GST-BAG-2, and GST-BAG-3 but not to GST-BAG-1 (ΔC) or several other control proteins. BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6) also exhibited little or no binding to themselves or to each other, demonstrating that these proteins do not strongly homo- or hetero-dimerize or oligomerize. It should be noted, however, that BAG-2 (SEQ ID NO:4) displayed weak interactions with itself in binding assays and produced a positive result in yeast two-hybrid experiments, demonstrating that it can have the ability to self-associate.
B. Binding of BAG Proteins to Hsc70 in vivo
The ability of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) proteins to interact in cells with Hsc70 was tested by expressing these proteins with N-terminal FlagTM epitope tags in 293T human epithelial cells using co-immunoprecipitation assays as described previously (Takayama et al., supra (1997)). cDNAs encoding the λ-phage cloned regions of BAG-2 and BAG-3 were subcloned in-frame into pcDNATM 3-FlagTM. Anti-FlagTM immune complexes prepared from 293T cells after transfection with plasmids encoding FlagTM-BAG-1, FlagTM-BAG-2, or FlagTM-BAG-3 were analyzed by SDS-PAGE/immunoblot assay. As shown in FIG. 10C, antiserum specific to Hsc70 detected the presence of BAG proteins associated with Hsc70, whereas control immune-complexes prepared with IgG1 as well as anti-FlagTM immune complexes prepared from cells transfected with FlagTM-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.
C. BIAcoreTM Assay of BAG Protein Binding to the ATPase Domain of Hsc70
BAG-1 (beginning at residue 116 of SEQ ID NO:2) is known to bind tightly to the ATPase domain of Hsc70 (Stuart et al., J. Biol. Chem., In Press (1998)). BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) proteins were therefore, examined for their ability to bind to Hsc70/ATPase. The affinity and binding kinetics of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) to Hsc70/ATPase was also compared to that of BAG-1 (beginning at residue 116 of SEQ ID NO:2) for Hsc70/ATPase, using a surface plasmon resonance technique (BIAcoreTM) which has been described previously (Stuart et al., supra, (1998) which is incorporated herein by reference).
BAG-family proteins were produced in bacteria and purified to near homogeneity as shown in FIG. 12A and described above in Example I. The purified BAG-1 (beginning at residue 116 of SEQ ID NO:2), -2 (SEQ ID NO:4), and -3 (SEQ ID NO:6) proteins were then immobilized on biosensor chips and tested for their interactions with Hsc7:0 in the soluble phase. Kinetic measurements were performed using a BIAcoreTM-II instrument with CM5 sensor chip and Amine Coupling Kit (Pharmacia Biosensor AB, Sweden). Briefly, for immobilization of proteins, the sensor chip was equilibrated with HK buffer (10 mM Hepes (pH 7.4), 150 mM KCL) at 5 μl/min, then activated by injecting 17 μl of 0.2M N-ethyl-N′-(3-diethylaminopropyl)-carbodiimide and 0.05M N-hydroxysuccinimide (NHS/EDC) followed by 35 μl of the protein of interest, in 10 mM acetate, pH 3.5-4.5. Excess NHS-ester on the surface was deactivated with 17 μl 1M ethanolamine-HCL (pH 8.5). After immobilization, 5 μl of regeneration buffer (50 mM phosphate (pH 6.8) and 4M GuHCl) was injected. For binding assays, Hsp70 (Sigma, H8778) was dissolved in HK buffer, and injected at 10 μl/min across the prepared surface at various concentrations. The surface was regenerated after each injection with 5 μl of regeneration buffer. The rate constants Kass and Kdiss were generated with BIAevaluation software 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 FIG. 3B). In contrast, Hsc70 failed to display interactions in BIAcoreTM 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 (FIG. 3B). Furthermore, flowing of various control proteins such as GST, BSA and Bcl-XL over the BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) chips resulted in negligible interaction. These results further demonstrate the specificity with which BAG-family proteins interact with and bind to Hsc70.
The rates of Hsc70 binding to BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6) proteins were similar, following pseudo first-order kinetics with estimated association rate constants (Ka) of 2.1, 2.1 and 2.4×105 M−1 sec−1, respectively. After allowing binding of Hsc70 to immobilized BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) to reach plateau levels, the chaperone was removed from the flow solution and the dissociation rate was monitored. BAG-1 (beginning at residue 116 at SEQ ID NO:2) and BAG-2 (SEQ ID NO:4) exhibited similar dissociation rates, with relatively slow loss of Hsc70 from the chip surface, resulting in estimated dissociation rate constants (Kd) of 3.0 and 5.0×10−4 sec−1, respectively (see FIG. 3B). In contrast, Hsc70 dissociated tore rapidly from biosensor chips containing BAG-3 (see FIG. 3B), yielding an estimated Kd of 1.7×10−3 sec−1. From the kinetic data, the apparent affinities (KD=Kd/Ka) were calculated for binding of Hsc70 to BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6) and were estimated to equal about KD=1.4 nM, KD=2.4 nM, and KD=7.4 nM, respectively. These results demonstrate that the interactions of BAG-family proteins with Hsc70 occur with apparent affinities sufficient for physiological relevance.
Example III
BAG-Family Proteins Inhibit Hsp70/Hsc70-Dependent Protein Folding
This example demonstrates that BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) proteins inhibit Hsp70/Hsc70-dependent refolding of denatured proteins similarly to a BAG-1 (beginning at residue 116 of SEQ ID NO:2) protein.
The effects of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) protein on Hsp70/Hsc70-dependent protein refolding was determined using in vitro protein refolding assays similar to those described previously by Takayama et al., supra, 1998; Terada et al., J. Cell Biol., 139:1089-1095, 1997, which are incorporated herein by reference. Briefly, luciferase (20 μM) 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 μM), DnaJ (StressGen, Inc.) (0.9 μM), and various purified recombinant proteins as indicated-were added to refolding buffer (30 mM Hepes-KOH, pH 7.6, 120 mM potassium acetate, 3 mM magnesium acetate, 2 mM DTT, 2.5 mM ATP) with 0.2 volume of diluted denatured luciferase to a final concentration of 0.1 μM. Luciferase activity was measured after 1.5 hr incubation at 35° C.
The combination of Hsc70 and DnaJ resulted in ATP-dependent refolding of chemically denatured firefly luciferase, with function of over half the denatured enzyme restored in a 90 minute reaction, as monitored by a chemiluminescence assay. In contrast, neither Hsc70 nor DnaJ alone were able to induce substantial refolding of denatured luciferase. Furthermore, little spontaneous restoration of luciferase activity was observed with control proteins, BSA, GST or Bcl-XL (see FIG. 4A).
Addition of recombinant purified BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID. NO:6) to the above assays in amounts equimolar to Hsc70 (1.8 μM) resulted in striking inhibition of luciferase refolding. BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) displayed somewhat greater inhibitory activity than BAG-1 (beginning at residue 116 of SEQ ID NO:2) as shown in FIG. 4A. In contrast, the BAG-1 (ΔC) protein, which fails to bind Hsc70 as well as several other control proteins, had no effect on luciferase refolding.
In an additional refolding assay, described previously by Minami et al., J. Biol. Chem. 271:19617-24, 1996), purified Hsc70 and human DnaJ homolog Hdj-1 (Hsp 40) were used with additional cofactors provided in reticulocyte lysates (5% v:v) to produce a system capable of refolding denatured luciferase. Briefly, additional cofactors included, recombinant Luciferase (Promega: QuantiLumTM), that had been heat denatured at 42° C. for 10 min, 1.8 μM Hsc70 (Sigma; purified from bovine brain), 0.9 μM Hsp40, and various recombinant purified proteins. Luciferase activity was measured (Promega luciferase assay kit) using a luminometer (EG&G Berthold, MicroLumat luminometer, Model #LB96P). All results were normalized relative to non-denatured luciferase that had been subjected to the same conditions. Control reactions lacking ATP, Hsc70, or Hsp40 resulted in negligible luciferase refolding.
Various amounts of purified BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6), relative to amounts of Hsc70 were used in the above-described protein refolding assay. Addition of BAG-family proteins resulted in a concentration-dependent inhibition of Hsc70 chaperone activity. Furthermore, the BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) inhibition of Hsc70 chaperone activity was demonstrated to be as potent as that observed for BAG-1 (beginning at residue 116 of SEQ ID NO:2). In contrast, the BAG-1 (ΔC) mutant as well as other control proteins did not suppress Hsc70-mediated refolding of denatured luciferase. These results indicate that BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) can inhibit Hsc70/Hsp70 dependent protein refolding activity to the same extent as BAG-1 (beginning at residue 116 of SEQ ID NO:2).
B. BAG Competes with Hip for Binding to Hsc70.
It is known that BAG-1 competes with Hip for binding to Hsc70, with these proteins exerting opposite effects on Hsc70-mediated protein refolding (Höhfeld, J., and Jentsch, S., Embo J., 16:6209-6216, 1997, which is incorporated herein by reference). In order to determine whether BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) also compete with Hip for binding to Hsc70, refolding assays were performed as described above in the presence of Hip protein.
Hip was purified as His6-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 6 h in BL21 cells. Cells from 1L of culture were resuspended into 50 ml of 50 mM Phosphate buffer (pH 6.8), 150 mM NaCl, and 1% (v/v) TweenTM-20 and then incubated with 0.5 mg/ml lysozyme at 25° C. for 0.5 h, followed by sonication. After centrifugation at 27,500 g for 10 min, 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% TweenTM-20 until the OD280 nm reached a value of <0.01. His6-Hip protein was eluted with 250 mM imidazol in washing buffer (Qiagene, Inc.) and purified on Mono QTM (HR10/10 Pharmacia) by FPLC using a linear gradient of 0.5M NaCl at pH 8.0, followed by dialysis in chaperone assay buffer.
In the refolding assay reactions, addition of purified Hip at equimolar concentrations relative to BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) (1.8 μM) completely negated the inhibitory effects of the BAG-family proteins on refolding of denatured luciferase (see FIG. 4C). These results demonstrate that the suppression of Hsc70 chaperone activity by BAG-family proteins is reversible, and that Hip antagonizes the effects of not only BAG-1 (beginning at residue 116 of SEQ ID NO:2), but also of BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6).
In summary, these results demonstrate that BAG-family proteins all contain a conserved BAG domain near their C-terminus that binds Hsc70/Hsp70, and that human BAG-family proteins can bind with high affinity to the ATPase domain of Hsc70 and inhibit its chaperone activity through a Hip-repressable mechanism.
Example IV
Expanded Nucleic Acid and Amino Acid Sequences for Human BAG-3, BAG-4 and BAG-5
Following the procedures disclosed herein, the nucleic acid and amino acids sequences to human BAG-3, BAG-4 and BAG-5 were further expanded. The expanded sequences for BAG-3, BAG-4 and BAG-5 are shown in FIGS. 15, 16 and 17, respectively, with their respective sequence identification numbers, “SEQ ID NO”s.

## Claims

1. A substantially purified nucleic acid molecule consisting of the nucleotide sequence SEQ ID NO: 1.

2. A substantially purified nucleic acid consisting of a nucleotide sequence that encodes the functionally active BAG family protein of SEQ ID NO:2.

3. The substantially purified nucleic acid of claim 2, wherein said nucleotide consists of SEQ ID NO:1.

4. A substantially purified nucleic acid consisting of nucleotide sequence that is fully complementary to a nucleotide sequence encoding the functionally active BAG family of SEQ ID NO:2.