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• W2006463550 abstract "The Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR), have been used extensively to study chaperone complex formation and maturation of a client substrate in a near native state. This chaperoning pathway can be reconstituted in vitro with the addition of five proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23. The addition of these proteins is necessary to reconstitute hormone-binding capacity to the immuno-isolated PR. It was recently shown that the first step for the recognition of PR by this system is binding by Hsp40. We compared type I and type II Hsp40 proteins and created point mutations in Hsp40 and Hsp70 to understand the requirements for this first step. The type I proteins, Ydj1 and DjA1 (HDJ2), and a type II, DjB1 (HDJ1), act similarly in promoting hormone binding and Hsp70 association to PR, while having different binding characteristics to PR. Ydj1 and DjA1 bind tightly to PR whereas the binding of DjB1 apparently has rapid on and off rates and its binding cannot be observed by antibody pull-down methods using either purified proteins or cell lysates. Mutation studies indicate that client binding, interactions between Hsp40 and Hsp70, plus ATP hydrolysis by Hsp70 are all required to promote conformational maturation of PR via the Hsp90 pathway. The Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR), have been used extensively to study chaperone complex formation and maturation of a client substrate in a near native state. This chaperoning pathway can be reconstituted in vitro with the addition of five proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23. The addition of these proteins is necessary to reconstitute hormone-binding capacity to the immuno-isolated PR. It was recently shown that the first step for the recognition of PR by this system is binding by Hsp40. We compared type I and type II Hsp40 proteins and created point mutations in Hsp40 and Hsp70 to understand the requirements for this first step. The type I proteins, Ydj1 and DjA1 (HDJ2), and a type II, DjB1 (HDJ1), act similarly in promoting hormone binding and Hsp70 association to PR, while having different binding characteristics to PR. Ydj1 and DjA1 bind tightly to PR whereas the binding of DjB1 apparently has rapid on and off rates and its binding cannot be observed by antibody pull-down methods using either purified proteins or cell lysates. Mutation studies indicate that client binding, interactions between Hsp40 and Hsp70, plus ATP hydrolysis by Hsp70 are all required to promote conformational maturation of PR via the Hsp90 pathway. Molecular chaperones are known for several vital roles in the cell. These include the folding of newly synthesized polypeptides, translocation through membranes, maturation and assembly of client proteins, prevention of aggregation, promotion of degradation, and response to cell stress (1Young J.C. Agashe V.R. Siegers K. Hartl F.U. Nat Rev Mol. Cell. Biol. 2004; 5: 781-791Crossref PubMed Scopus (941) Google Scholar). While there are several chaperone networks, we are concerned with the Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR). 2The abbreviations used are: PR, progesterone receptor; SBD, steroid binding domain; GR, glucocorticoid receptor; WT, wild type; AR, androgen receptor. More than 100 substrates or client proteins for Hsp90 have been identified (2Pratt W. Toft D. Exp. Biol. Med. (Maywood). 2003; 228: 111-133Crossref PubMed Scopus (1267) Google Scholar, 3Pratt W.B. Galigniana M.D. Morishima Y. Murphy P.J. Essays Biochem. 2004; 40: 41-58Crossref PubMed Scopus (190) Google Scholar, 4Zhao R. Davey M. Hsu Y. Kaplanek P. Tong A. Parsons A. Krogan N. Cagney G. Mai D. Greenblatt J. Boone C. Emili A. Houry W. Cell. 2005; 120: 715-727Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar). These proteins include a diverse family of kinases, transcription factors, and cell cycle regulators, many of which are involved in cancer (5Whitesell L. Lindquist S. Nat. Rev. Cancer. 2005; 5: 761-772Crossref PubMed Scopus (1975) Google Scholar, 6Workman P. Cancer Lett. 2004; 206: 149-157Crossref PubMed Scopus (231) Google Scholar, 7Chiosis G. Vilenchik M. Kim J. Solit D. Drug. Discov. Today. 2004; 9: 881-888Crossref PubMed Scopus (196) Google Scholar). Steroid receptors are a family of transcription factors chaperoned by Hsp90. Of these, PR and glucocorticoid receptor (GR) have served as models to study this pathway in detail (2Pratt W. Toft D. Exp. Biol. Med. (Maywood). 2003; 228: 111-133Crossref PubMed Scopus (1267) Google Scholar, 3Pratt W.B. Galigniana M.D. Morishima Y. Murphy P.J. Essays Biochem. 2004; 40: 41-58Crossref PubMed Scopus (190) Google Scholar). PR exists in the cell as two isoforms, PR-A and PR-B (8Conneely O. Maxwell B. Toft D. Schrader W. O'Malley B. Biochem. Biophys. Res. Commun. 1987; 149: 493-501Crossref PubMed Scopus (186) Google Scholar), which are products of a single gene and differ only in that PR-A lacks the first 164 amino acids from the N terminus. These isoforms are both ligand-activated and dimeric in their activated states. Previous studies have not shown any differences in the chaperone association of PR-A and PR-B (9Schowalter D. Sullivan W. Maihle N. Dobson A. Conneely O. O'Malley B. Toft D. J. Biol. Chem. 1991; 266: 21165-21173Abstract Full Text PDF PubMed Google Scholar, 10Smith D. Schowalter D. Kost S. Toft D. Mol. Endocrinol. 1990; 4: 1704-1711Crossref PubMed Scopus (113) Google Scholar). Chaperones appear to associate with the C-terminal domain of PR, where the steroid binding domain (SBD) is located. The primary amino acid sequence of the SBD is highly conserved throughout the steroid receptor family and a crystal structure of this domain of human PR bound to hormone is available (11Williams S. Sigler P. Nature. 1998; 393: 392-396Crossref PubMed Scopus (583) Google Scholar). Steroid receptors isolated from cell cytosol are associated with several chaperone and co-chaperone proteins, including Hsp90. When PR is stripped of its association with Hsp90, it loses its hormone binding ability in a time- and temperature-dependent manner. However, this activity can be restored or maintained in vitro through the actions of a minimum of 5 proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23 (12Kosano H. Stensgard B. Charlesworth M. McMahon N. Toft D. J. Biol. Chem. 1998; 273: 32973-32979Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 13Dittmar K.D. Banach M. Galigniana M.D. Pratt W.B. J. Biol. Chem. 1998; 273: 7358-7366Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). In vivo, other co-chaperones are involved, including HIP (14Prapapanich V. Chen S. Smith D.F. Mol. Cell. Biol. 1998; 18: 944-952Crossref PubMed Scopus (50) Google Scholar, 15Prapapanich V. Chen S. Nair S.C. Rimerman R.A. Smith D.F. Mol. Endocrinol. 1996; 10: 420-431PubMed Google Scholar) and one of several immunophilins (2Pratt W. Toft D. Exp. Biol. Med. (Maywood). 2003; 228: 111-133Crossref PubMed Scopus (1267) Google Scholar, 3Pratt W.B. Galigniana M.D. Morishima Y. Murphy P.J. Essays Biochem. 2004; 40: 41-58Crossref PubMed Scopus (190) Google Scholar, 17Smith D.F. Cell Stress Chaperones. 2004; 9: 109-121Crossref PubMed Scopus (124) Google Scholar). Incubation with the aforementioned five proteins regenerates the hormone binding ability of PR and reconstitutes a heterocomplex resembling that originally found in vivo. The hormone-binding cleft is believed to be collapsed through hydrophobic interactions in the absence of ligand, thus the SBD requires a change in conformation to bind hormone (18Gee A. Katzenellenbogen J. Mol. Endocrinol. 2001; 15: 421-428Crossref PubMed Scopus (56) Google Scholar). These five proteins work together to confer this conformational change. The Hsp90 chaperoning pathway occurs in a series of steps that include the formation of multichaperone complexes with the steroid receptor. It was recently shown that Hsp40 binding is the first step in the PR chaperoning pathway (19Hernandez M. Chadli A. Toft D. J. Biol. Chem. 2002; 277: 11873-11881Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). This binding is followed by Hsp70 association, which is ATP dependent (19Hernandez M. Chadli A. Toft D. J. Biol. Chem. 2002; 277: 11873-11881Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Hsp70 binds ATP at its N-terminal domain, and the J-domain of Hsp40 binds to Hsp70 and stimulates its ATPase activity, thus causing the tight association of Hsp70 with the substrate (20Fan C. Lee S. Cyr D. Cell Stress Chaperones. 2003; 8: 309-316Crossref PubMed Scopus (253) Google Scholar). The intermediate complex that follows is formed with the assistance of Hop, which is an adaptor protein that can simultaneously associate with Hsp70 and Hsp90 and modulate their activities (21Chen S. Smith D. J. Biol. Chem. 1998; 273: 35194-35200Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 22Lassle M. Blatch G. Kundra V. Takatori T. Zetter B. J. Biol. Chem. 1997; 272: 1876-1884Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 23Smith D. Sullivan W. Marion T. Zaitsu K. Madden B. McCormick D. Toft D. Mol. Cell. Biol. 1993; 13: 869-876Crossref PubMed Scopus (247) Google Scholar). It is capable of transporting Hsp90 into the complex. Hop senses conformational changes in Hsp70 and Hsp90, mainly brought about by ATP binding, hydrolysis, and release (24Hernandez M. Sullivan W. Toft D. J. Biol. Chem. 2002; 277: 38294-38304Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 25Johnson B. Schumacher R. Ross E. Toft D. J. Biol. Chem. 1998; 273: 3679-3686Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 26Odunuga O. Longshaw V. Blatch G. BioEssays. 2004; 26: 1058-1068Crossref PubMed Scopus (178) Google Scholar). After the intermediate complex is formed, ATP is bound to Hsp90. p23 recognizes ATP-bound Hsp90 and promotes the dissociation of the intermediate complex (27Sullivan W. Owen B. Toft D. J. Biol. Chem. 2002; 277: 45942-45948Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and a transition to a receptor complex that is able to bind hormone. The Hsp90 chaperone pathway is thus highly regulated by ATP binding and hydrolysis as well as by a series of interactions with chaperones and cofactors. We hypothesized that the initial recognition of PR by Hsp40 in one specific site is necessary for the reconstitution of hormone binding to PR. Here we characterized the assembly of the initial complex, PR-Hsp40-Hsp70, and compared type I and type II Hsp40 proteins in their association with PR. We also addressed the need for interaction between Hsp70 and Hsp40 in the context of Hsp90 chaperoning to gain insight on the requirements for client protein recognition by this chaperoning system. HeLa Cell Lysate Pull-downs—HeLa cells stably expressing PR-B (28Chadli A. Graham J. Abel M. Jackson T. Gordon D. Wood W. Felts S. Horwitz K. Toft D. Mol. Cell. Biol. 2006; 26: 1722-1730Crossref PubMed Scopus (51) Google Scholar) were grown to 70% confluency in MEM medium enriched with 5% fetal bovine serum (HyClone Laboratories, Logan, UT), 6 ng/μl insulin (Invitrogen), nonessential amino acids, penicillin, and streptomycin at 37 °C. The cells were trypsinized and washed in lysate buffer that included 20 mm Tris 7.5, 50 mm KCl, 1 mm EDTA, 2 mm dithiothreitol, 0.02% Nonidet P-40 and protease inhibitor mixture (Complete® EDTA-free from Roche). The cells were sonicated five times using the following cycle: 1 s pulse with 30 s rest on ice. The lysates were centrifuged in a microcentrifuge at maximum speed for 20 min. The lysate was added to protein A-Sepharose beads that were cross-linked using dimethylpimelimidate (DMP; Sigma-Aldrich) (29Sisson T. Castor C. J. Immunol. Methods. 1990; 127: 215-220Crossref PubMed Scopus (58) Google Scholar) with either PR-B antibody (PR6 (30Sullivan W. Beito T. Proper J. Krco C. Toft D. Endocrinology. 1986; 119: 1549-1557Crossref PubMed Scopus (151) Google Scholar)) or DjA1 antibody (Neomarkers® HDJ-2/DNAJ Ab-1). The lysate and beads were incubated on ice for 1.5 h, and washed four times with 1 ml of lysate buffer minus protease inhibitors. The beads were incubated with SDS sample buffer, boiled for 5 min at 90 °C, and proteins were resolved by 7.5% acrylamide SDS-PAGE. Construction of Mutants—Human DjA1 D36N, Hsp70 K71M and Hsp70 R171H were prepared using the QuikChange® site-directed mutagenesis kit from Stratagene. These constructs were prepared in a pET23C vector (Novagen). The Ydj1 G315D mutant was a gift from Dr. Douglas Cyr. All mutant proteins were overexpressed in the BL21 DE3 PLysS E. coli strain with the addition of 1 mm isopropyl-β-d-thiogalactopyranoside at an A600 between 0.6 and 0.8 for 3 h at room temperature. Protein Purification—Human Hsp90, human Hsp70, Ydj1, DjA1, DjB1, Hop, and p23 were all expressed and purified as described previously (12Kosano H. Stensgard B. Charlesworth M. McMahon N. Toft D. J. Biol. Chem. 1998; 273: 32973-32979Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 19Hernandez M. Chadli A. Toft D. J. Biol. Chem. 2002; 277: 11873-11881Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The mutant DjA1 D36N and the mutant Ydj1 G315D were purified as described previously (31Caplan A. Tsai J. Casey P. Douglas M. J. Biol. Chem. 1992; 267: 18890-18895Abstract Full Text PDF PubMed Google Scholar) with the following modifications. Bacterial lysates were fractionated by FPLC using first a Q-Sepharose, followed by UnoQ and Superdex 200. Both proteins were eluted early in the salt gradient of the ionic exchange columns. The Hsp70 mutations Hsp70 K71M and Hsp70 R171H were purified using the same procedure as the wild-type Hsp70 (19Hernandez M. Chadli A. Toft D. J. Biol. Chem. 2002; 277: 11873-11881Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). PR-A Expression and Cytosol Preparation—The procedure followed for the baculovirus-mediated expression of PR-A in SF9 cells included co-expression of p23, which enhances the expression of PR in a native state. This method has been described previously for GR expression (32Morishima Y. Kanelakis K.C. Murphy P.J. Lowe E.R. Jenkins G.J. Osawa Y. Sunahara R.K. Pratt W.B. J. Biol. Chem. 2003; 278: 48754-48763Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). SF9 cells were co-infected with viral vectors coding for chicken PR-A and human p23 in the Recombinant Protein Expression Proteomics Core, The Cancer Center, Baylor College of Medicine. A final concentration of 10 mm glucose was added to the cultures at 24-h postinfection. The pellets were washed in phosphate-buffered saline prior to freezing at -80 °C. The cytosol was prepared from these pellets in the following manner. A pellet containing ∼756 × 106 cells was thawed in 20 ml of lysis buffer (20 mm Tris, pH 7.5, 50 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 10% glycerol, and protease inhibitors: Complete® EDTA-free). Cells were lysed by sonication, and the lysate was then centrifuged at 40,000 rpm for 1 h and stored at -80 °C. Prior to use, the cytosol is thawed on ice and adjusted to 500 mm KCl, 5 mm MgCl2, 5 mm ATP. The lysate was salt-treated for 30 min on ice to dissociate chaperone proteins from PR. PR Immuno-isolation—We used a mouse monoclonal antibody PR22 (IgG) against chicken PR described previously (30Sullivan W. Beito T. Proper J. Krco C. Toft D. Endocrinology. 1986; 119: 1549-1557Crossref PubMed Scopus (151) Google Scholar). Antibody resin was prepared by incubating PR22 with a slurry of protein A-Sepharose CL-4B (Amersham Biosciences) in PBS for 30 min at room temperature prior to use. Proportions were 7 μl of PR22 ascites for every 20 μl of resin volume. The conjugated resin was washed three times in PBS and then resuspended as a 1:1 slurry with ice-cold stripping buffer (20 mm Tris, pH 7.5, 500 mm KCl, 5 mm MgCl2, 0.1% Nonidet P40, 1 mm dithiothreitol). When PR is isolated from SF9 cells, it is accompanied by an assortment of chaperones, mainly Hsp90, Hsp70, and p23. These chaperones are removed by treatment with high salt, ATP, and detergent (stripping buffer) while the immunoisolation is taking place. For the purification of PR, 40 μl of PR22/protein A resin slurry was added to 0.07 ml of salt-treated lysate. This mixture was incubated on ice for 1.5 h. Receptorresin complexes were washed three times with 1 ml of cold stripping buffer and once with reaction buffer, with brief centrifugation to pellet the resin. Resin pellets were used in reconstitution or binding reactions. PR Binding Assays—PR resin pellets (20 μl) were suspended with 200 μl of cold reaction buffer (20 mm Tris-HCl, 50 mm KCl, 5 mm MgCl2, 0.01% Nonidet P-40, and 2 mm dithiothreitol, pH 7.5) containing the specified amount of wild type or mutant Ydj1, DjA1, or DjB1. Reactions that assess the stimulation of Hsp70 binding to PR also include wild type or mutant Hsp70 plus 2 mm ATP. These reactions proceeded at 30 °C for 20 min; the samples were chilled on ice for 2 min, then washed four times with 1 ml of reaction buffer. The final samples were suspended in 20 μl of SDS sample buffer (2% SDS plus 5% mercaptoethanol), heated for 5 min at 95 °C, and analyzed by SDS-PAGE. The determination of association constants was performed as published previously (19Hernandez M. Chadli A. Toft D. J. Biol. Chem. 2002; 277: 11873-11881Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Progesterone Receptor Reconstitution—PR resin (20 μl) was suspended with 200 μl of cold reaction buffer containing 20 μg of Hsp70, 5 μg of Ydj1, DjA1, or DjB1, 5 μg of Hop, 20 μg of Hsp90, 5 μg of p23, and 5 mm ATP unless otherwise noted. Incubation proceeded at 30 °C for 20 min. The samples were chilled on ice for 2 min and supplemented with 100 nm [3H]progesterone (American Radiolabeled Chemicals, Inc, St. Louis, MO, 50 Ci/mmol) plus 100 nm of unlabeled progesterone. The samples were incubated for 3 h on ice with gentle resin suspension and then washed four times with 1 ml of reaction buffer. During the fourth suspension 100 μl were removed for the measurement of [3H]progesterone. The final samples were suspended in 20 μl of SDS sample buffer, heated for 5 min at 95 °C, and analyzed by SDS-PAGE. Analysis of complex formation after reconstitution assays were performed in 10% acrylamide gels, while analysis of Hsp40 and Hsp70 binding to PR used 7.5% acrylamide gels to get better resolution in the 50-40 kDa range. Type I and Type II Hsp40 Proteins Interact Differently with PR— As seen in Fig. 1A, type I and type II Hsp40 proteins contain a J-domain at the N terminus of the protein (20Fan C. Lee S. Cyr D. Cell Stress Chaperones. 2003; 8: 309-316Crossref PubMed Scopus (253) Google Scholar, 33Hennessy F. Nicoll W. Zimmermann R. Cheetham M. Blatch G. Protein Sci. 2005; 14: 1697-1709Crossref PubMed Scopus (235) Google Scholar). In some cases the J domains of type I and II Hsp40 proteins are nearly identical and interchangeable (34Fan C. Lee S. Ren H. Cyr D. Mol. Biol. Cell. 2004; 15: 761-773Crossref PubMed Scopus (91) Google Scholar, 35Yan W. Craig E. Mol. Cell. Biol. 1999; 19: 7751-7758Crossref PubMed Scopus (123) Google Scholar). This domain interacts with Hsp70 and is responsible for stimulating the ATPase activity of Hsp70. The zinc finger-like domain (ZFLD), unique to the type I Hsp40, is essential for proper recognition and delivery of substrates to Hsp70 (36Lu Z. Cyr D. J. Biol. Chem. 1998; 273: 5970-5978Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 37Fan C. Ren H. Lee P. Caplan A. Cyr D. J. Biol. Chem. 2005; 280: 695-702Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The glycine-phenylalanine (G/F)-rich region is required for the proper function of these proteins (35Yan W. Craig E. Mol. Cell. Biol. 1999; 19: 7751-7758Crossref PubMed Scopus (123) Google Scholar), while the significance of the glycine-methionine (G/M) region, unique to the type II Hsp40 proteins, is unclear (38Lee S. Fan C. Younger J. Ren H. Cyr D. J. Biol. Chem. 2002; 277: 21675-21682Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). According to a recently published tertiary structure for both type I and type II Hsp40 (39Borges J.C. Fischer H. Craievich A.F. Ramos C.H. J. Biol. Chem. 2005; 280: 13671-13681Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), the dimer of the type I forms a horseshoe with the J domains close together in space, while the dimer of the type II forms a more elongated structure where the J domains are at a distance. These peculiarities, together with the non-conserved C terminus of the Hsp40 proteins (Fig. 1A), suggest that they would behave differently and recognize different client proteins (34Fan C. Lee S. Ren H. Cyr D. Mol. Biol. Cell. 2004; 15: 761-773Crossref PubMed Scopus (91) Google Scholar). In the present study we compared the activities of two type I Hsp40 proteins, yeast Ydj1 (40Caplan A. Douglas M. J. Cell Biol. 1991; 114: 609-621Crossref PubMed Scopus (212) Google Scholar) and human DjA1 (HDJ2, HSDJ, HSJ2, HSPF4, DJ-2) (41Davis A. Alevy Y. Chellaiah A. Quinn M. Mohanakumar T. Int. J. Biochem. Cell Biol. 1998; 30: 1203-1221Crossref PubMed Scopus (32) Google Scholar), and one type II protein, human DjB1 (HDJ1, dj1, HSJ2, HSPF1) (42Raabe T. Manley J.L. Nucleic Acids Res. 1991; 19: 6645Crossref PubMed Scopus (58) Google Scholar), in the context of Hsp90 chaperoning (see Ref. 43Ohtsuka K. Hata M. Cell Stress Chaperones. 2000; 5: 98-112Crossref PubMed Scopus (126) Google Scholar for classification and nomenclature). These were compared for their interaction with PR and ability to promote hormone-binding activity. To study the cellular interaction of PR with Hsp40 proteins, we used a HeLa cell line that had been modified to express human PR-B (28Chadli A. Graham J. Abel M. Jackson T. Gordon D. Wood W. Felts S. Horwitz K. Toft D. Mol. Cell. Biol. 2006; 26: 1722-1730Crossref PubMed Scopus (51) Google Scholar). DjA1 and DjB1 are readily detected in the soluble cytosolic fraction of HeLa cells, as seen in Fig. 1B (lane 7), and both are potential PR-interacting proteins. Because the Hsp40 type that interacts with PR in the cell has not been identified, we tested for the association of DjA1 and/or DjB1 with PR in cell lysates. Using PR pull-down experiments (Fig. 1B) DjA1 showed a clear association with PR, whereas DjB1 was not detected (lane 5). When the pull-down was performed using antibody to DjA1, the co-isolation of PR was not observed (lane 6). This lack of detection may be explained by the much greater abundance of DjA1 over PR in the lysate. It is also possible that the DjA1 antibody interferes with the interaction. A pull-down of DjB1 was not able to detect PR association (data not shown). Using the PR reconstitution system, we have previously shown that the binding of Ydj1 to PR is rapid, has a high affinity, can be assembled in vitro, and is independent of nucleotide and Hsp70 (19Hernandez M. Chadli A. Toft D. J. Biol. Chem. 2002; 277: 11873-11881Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Because these conditions have been well established for the yeast Hsp40, Ydj1, we tested the human Hsp40s, DjA1, and DjB1. As seen in Fig. 1C, both type I proteins Ydj1 and DjA1 interact with PR in the absence of other proteins (lanes 1 and 2) and stimulate Hsp70 association in the presence of ATP (lanes 4 and 5). DjB1 does not visibly interact with PR (lane 3); yet, it is able to promote the association of Hsp70 with PR (lane 6). This also results in some binding of DjB1 in the complex which may represent its binding to Hsp70, PR, or both. These in vitro results agree with the in vivo analysis above in that DjA1 binds readily to PR in a pull-down experiment but DjB1 does not. However, the in vitro results suggest that DjB1 may bind transiently to PR to promote Hsp70 binding. Both Type I and II Hsp40 Proteins Are Able to Promote Hsp90 Chaperoning—In Fig. 2A we depict the hormone binding profile of PR when increasing amounts of either Ydj1 or DjA1 are used in the reconstitution reaction. In addition to Hsp40, this reaction includes purified Hsp90, Hsp70, Hop, and p23. These proteins work together to form complexes with PR to promote hormone binding through the opening of the hormone-binding site. Both Hsp40 proteins are able to promote PR chaperoning to the hormone binding state, although there are some differences in the response profiles. Ydj1 is somewhat more potent than DjA1, but it is slightly inhibitory when in excess. Fig. 2B represents the complex formation of the reaction described above. Protein association occurs in a timely and organized manner (44Smith D. Mol. Endocrinol. 1993; 7: 1418-1429Crossref PubMed Scopus (251) Google Scholar, 45Smith D. Whitesell L. Nair S. Chen S. Prapapanich V. Rimerman R. Mol. Cell. Biol. 1995; 15: 6804-6812Crossref PubMed Scopus (272) Google Scholar, 46Chen S. Prapapanich V. Rimerman R.A. Honore B. Smith D.F. Mol. Endocrinol. 1996; 10: 682-693Crossref PubMed Google Scholar). Generally, the presence of proportional quantities of Hsp90 and p23 are indicative of a mature complex that is able to bind hormone. Both Hsp40 proteins are able to promote Hsp90 binding to PR. The main difference between a reconstitution that is triggered by DjA1 or Ydj1 is the presence of Hsp70 in the isolated complexes, which is more prominent throughout when Ydj1 is used. This difference is subtle and does not appear to affect substantially the hormone binding of PR although this may account for the inhibitory effect seen when excess Ydj1 is used. The association of Ydj1 can be seen in this gel while association of DjA1 is not, because it is not resolved from the antibody heavy chain in this gel system (10% acrylamide gels). In experiments performed with beads containing cross-linked antibody or 7.5% acrylamide gels, the amounts of DjA1 and Ydj1 seen associated to the PR complexes are comparable (data not shown). Surprisingly, whereas DjB1 binding to PR is not observed using our methods, it is able to promote PR chaperoning in a similar manner to Ydj1 and DjA1. Fig. 2, C and D show that, when adding increasing amounts of DjB1 to a reconstitution reaction, we are able to achieve hormone binding by PR. As seen in Fig. 2C, the maximum potency of DjB1 is less than that of Ydj1, but very similar to that of DjA1 (Fig. 2A). In Fig. 2D, both the association of Hsp90 and Hsp70 are similar whether Ydj1 or DjB1 is used although the association of Hsp70 in the presence of Ydj1 is higher, as seen previously in Fig. 2B. Hsp40 Binding to PR Is Specific and Limited—DjA1 and Ydj1 are able to bind similarly to PR, promote Hsp70 binding, and promote hormone binding of PR when used in a reconstitution assay. The PR binding affinities for these two proteins were compared as shown in Fig. 3A. The Kd for Ydj1 is 225 nm, while the Kd for DjA1 is 278 nm. Thus, these two proteins have similar affinities for PR. In an earlier study, a higher affinity of Ydj1 for PR was reported with a Kd of 77 nm (19Hernandez M. Chadli A. Toft D. J. Biol. Chem. 2002; 277: 11873-11881Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The presence of detergent (0.01% Nonidet P-40) in the present study apparently lowers the affinity for Ydj1 binding somewhat, but enhances the efficiency of PR chaperoning overall. The Scatchard analysis in Fig. 3A depicts a linear relationship between the binding of PR and each Hsp40, Ydj1, and DjA1. Therefore, a single type of binding is expected from type I Hsp40. A binding affinity constant was not calculated for DjB1 because no measurable binding of DjB1 to PR was detected. Our laboratory has previously calculated the stoichiometry of Ydj1 binding to PR in the early complex as ∼1:1 (19Hernandez M. Chadli A. Toft D. J. Biol. Chem. 2002; 277: 11873-11881Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Since the PR exists as a monomer in chaperone complexes, Ydj1 may also bind PR in a monomeric state or the Ydj1 dimer may be able to bind two PR molecules. This stoichiometry argues for one binding site on PR for Hsp40. Thus, hypothetically, all Hsp40 proteins that are able to reconstitute PR should bind to the same site. We considered this question regarding type I Hsp40 in Fig. 3, B and C, where a competition assay between Ydj1 and DjA1 was performed. Increasing amounts of Ydj1 were added to a reaction containing a constant amount of 5 μg of DjA1. Fig. 3B shows a representative 7.5% acrylamide gel, where we can visually assess the displacement of DjA1 by Ydj1, because they have slightly different molecular weights. The graph shown in Fig. 3C, quantifies the competition experiment. Ydj1 was able to replace all DjA1 at the PR binding site. These data strengthen the hypothesis considering a single binding site for type I Hsp40. The same displacement or competition pattern occurs in the reverse experiment when DjA1 is used to occupy the site for Ydj1 binding on PR (data not shown). DjB1 does not stably bind PR when using our methods and it is not an effective competitor for Ydj1 binding. Thus, we considered a functional approach. We used a dominant negative Hsp40, DjA1 D36N, which binds to PR (see Table 1 and Fig. 5), is unable to bind Hsp70 (47Suh W. Burkholder W. Lu C. Zhao X. Gottesman M. Gross C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15223-15228Crossref PubMed Scopus (228) Google Scholar) and is also unable to reconstitute the hormone binding ability of PR (Fig. 5). Fig. 4A shows a competition experiment where Hsp90, Hsp70, Hop, DjA1, and p23 were used in a reconstitution assay. We then added increasing amounts of DjA1 D36N to this reaction. We show h" @default.
• W2006463550 created "2016-06-24" @default.
• W2006463550 creator A5037363121 @default.
• W2006463550 creator A5085587180 @default.
• W2006463550 date "2006-09-01" @default.
• W2006463550 modified "2023-09-30" @default.
• W2006463550 title "Defining the Requirements for Hsp40 and Hsp70 in the Hsp90 Chaperone Pathway" @default.
• W2006463550 cites W142850513 @default.
• W2006463550 cites W1547327928 @default.
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