Adenosine 5'-Triphosphate-Dependent Vitamin D Sterol Binding to Heat Shock Protein-70 Chaperones
http://www.100md.com
《内分泌学杂志》
Burns and Allen Research Institute and Division of Endocrinology, Diabetes ,and Metabolism (R.C., M.A.G., J.S.A.), Cedars-Sinai Medical Center, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California 90048
Division of Medical Sciences (M.H.), Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, United Kingdom
Abstract
Chaperone proteins in the heat shock protein-70 family possess endogenous ATP binding and ATPase activity and interact with intracellular protein substrates in an ATP-dependent manner; the hydrolysis of ATP to ADP results in an increase in the affinity of the chaperone for protein substrates. Heat shock protein-70s can also specifically interact with 25-hydroxylated vitamin D metabolites. Using constitutively expressed heat shock protein-70 (hsc70) as chaperone, here we demonstrate that vitamin D metabolite binding to hsc70 is also ATP dependent. Transient overexpression of an hsc70-green fluorescent protein chimeric construct in primate kidney cells resulted in a 6-fold increase in specific, extractable 25-hydroxyvitamin D3 binding. When ATPase capability of hsc70 was disabled, this increase was completely blocked. In solution, the binding of 25-hydroxylated vitamin D metabolites to hsc70 was significantly increased (P < 0.01) in the presence of ATP and a nonmetabolizable ATP analog. The ATP-directed increase in specific binding resulted from an increase in the abundance of relatively high-affinity hormone-binding sites (Kd, 0.24 nM). These results suggest that ATP hydrolysis to ADP would favor the release of vitamin D from a donor hsc70 molecule at a time when an hsc70-bound acceptor protein substrate is anchored to the chaperone with relative avidity. We theorize that the endogenous ATPase activity of hsc70 promotes the transfer of vitamin D sterols to other intracellular vitamin D binding proteins, such as the vitamin D receptor and vitamin D hydroxylases, to which hsc70 is known to bind.
Introduction
HEAT SHOCK PROTEINS have long been recognized for their ability to interact with the folding intermediates of polypeptides, disallowing proteins from forming nonspecific, nonproductive, intracellular aggregates with themselves and other proteins (1). Proteins in the heat shock protein (hsp) 70 superfamily of chaperones are also known for their ability to interact with a panoply of regulatory and/or accessory protein partners or cochaperones that govern crucial structural and functional events within the cell (2). hsp70s harbor a conserved, amino-terminal, ATP-binding, ATPase domain that supports chaperone functions of the protein (Fig. 1A). Hydrolysis of ATP by hsp70 proteins promotes more stable interaction with partner proteins in the more carboxy-terminal, protein-binding domain of the hsp70 molecule (1, 3).
In addition to their ability to interact with other proteins, members of the hsp70 family of chaperones are also known to interact with nonproteins, including metabolites of vitamin D (4, 5, 6). These chaperones provide a reliable means for the specific internalization and determination of the intracellular destination of vitamin D metabolites in target cells, thus mediating the action and metabolism of vitamin D (7). For example, augmented expression of constitutively expressed heat shock protein (hsc)70 promoted 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-vitamin D-receptor (VDR)-directed gene transactivation (8) and 1,25(OH)2D3 synthesis (9) in cultured kidney cells. Disruption or alteration of the ability of the hsp70s to bind and hydrolyze ATP interferes with their ability to bind and release substrate polypeptides from the protein-protein interaction domain of the chaperone (10). The purpose of the current set of experiments was to determine whether changes in ATP handling by hsc70 also affects its ability to interact with vitamin D sterols.
Materials and Methods
Materials and cells
Crystalline forms of vitamin D sterols, 25-hydroxyvitamin D3 (25OHD3) and 1,25(OH)2D3, were obtained from Biomol (Plymouth Meeting, PA) and cholesterol from Sigma (St. Louis, MO). ATP, ADP, and ATP analog and adenosine 5'-(, -imido) triphosphate tetralithium salt hydrate) were obtained from Sigma. Purified, recombinant bovine hsc70 was purchased from Stressgen Biotechnologies Corp. (Victoria, British Columbia, Canada). Tritiated sterols were obtained from Amersham (Piscataway, NJ); specific activities for the radiolabeled ligands were 187, 180, and 41 Ci/mmol for [3H]25(OH)2D3, [3H]1,25(OH)2D3, and [3H]cholesterol, respectively. Nonhuman and human primate kidney cell lines, COS-7 (monkey) and HKC-8 (human), were obtained from American Type Culture Collection (Manassas, VA) and the Hewison Laboratory (University of Birmingham, Birmingham, UK) (11), respectively.
Plasmids and transfection analysis
Wild-type and mutant constructs bearing a green fluorescent protein (GFP) tag at the carboxy terminus of hsc70 were developed (Fig. 1A) as part of our research program (reviewed in Refs.7 and 22) exploring the role of heat shock proteins in vitamin D transport, metabolism, and action; it is anticipated that these constructs will retain their normal functionality in the amino-terminal ATP, vitamin D interaction, and protein interaction domains (12), permitting real-time observation of the proteins. To determine whether the GFP tag impaired the functionality of hsc70, a yeast complementation assay system developed by Jones and Masison (13) was used. In this assay system, the four yeast ATPase-active, hsp70-like genes, SSA1–4, crucial for yeast survival, were inactivated by recombinant means. The resultant SSA1–4 deletion mutant yeast was viable only if one of the four functionally redundant SSA genes or a functional surrogate was reexpressed. Transfection of either the hsc70 or hsc70-GFP chimera was capable of facilitating survival and proliferation of SSA-deficient yeast, indicating that the GFP-tagged hsc70 was functional (Chun, R., and J. Adams, unpublished data).
For image analysis, COS-7 cells were transiently transfected by lipofection (Lipofectamine 2000; Invitrogen, Carlsbad, CA) overnight with 0.8 μg of the hsc70-GFP plasmid per chamber slide well. The next day, media were changed and images were taken from living cells using a Axiovert 100 fluorescent microscope (Zeiss, Gottingen, Germany) equipped with a LSM410 laser-scanning confocal unit. The 488-nm line of a krypton-argon laser with a BP 510- to 525-nm emission filter was used for GFP detection.
Ligand binding assays
Tritiated sterol binding was measured in cellular extracts of HKC-8 cells or commercially available preparations of purified hsc-70 (5). Briefly, increasing concentrations (0.05–5.0 nM) of radiolabeled sterols, in the presence or absence of 100 nM radioinert sterol, was dispensed into borosilicate glass tubes and evaporated to dryness under nitrogen. After 10 min of preexposure to 10-μl aliquots of 2.0 mM ATP, 2.0 mM ADP, or 2.0 mM ATP analog, each tube received 0.2 μg purified hsc-70 (0.28 nM), suspended in one of two buffers: high-salt buffer designed to maximize sterol binding, 0.5 M NaCl buffer [1.0 mM EDTA, 10 mM Tris-HCl (pH 7.4), 5.0 mM dithiothreitol, 1.0 mM phenylmethylsulfonyl fluoride[; or low-salt buffer [100 mM KCl, 5 mM MgCl2, 25 mM HEPES (pH 7.0)] for maximization of ATP binding. With low-salt buffer, incubation occurred at room temperature for 20 min. In high-salt buffer, tubes were incubated overnight at 4 C. After incubation, dextran-coated charcoal buffer was added to each tube, vigorously shaken, and then allowed to incubate on ice for 1 h. Tubes were centrifuged for 30 min at 3500 rpm at 4 C, and supernatant, containing protein-bound sterol, was decanted into scintillation vials and radioactivity counted.
Binding kinetics were determined by Scatchard plots (bound vs. bound/free). The slopes of these plots were used to define binding affinity and represented by the dissociation constant (Kd).
Results
Functionality of expressed hsc70-GFP chimeric proteins
When transiently transfected into cultured kidney cells, wt-hsc70-GFP overexpression resulted in the expected cytoplasmic localization (Fig. 1B). This is consistent with the results of immunohistochemical and cell fractionation studies that localize hsc70 largely to the cell cytoplasm (14) and suggests that inclusion of a carboxy-terminal GFP tail on hsc70 did not alter the intracellular localization of the chaperone. Deletion of the carboxy-terminal protein substrate and oligomerization domains (see Fig. 1A) did not significantly alter metabolite binding (8), suggesting that vitamin D sterol binding was legislated by the N-terminal portion of the hsc70 molecule. Figure 1C shows that cytoplasmic extracts of HKC-8 transfected with the wt-hsc70-GFP chimeric construct exhibited a 6-fold increase in specific 25OHD3 binding, compared with extracts of vector-alone-transfected cells. A point mutation (E175S) in the ATP-binding, ATPase domain of hsc70, known to disrupt both the ATPase and protein-protein binding activity of the chaperone (15, 16, 17), resulted in a decrease in extract-25OHD3-specific binding to levels observed in vector-alone-transfected cells. A similar significant decrease in 25OHD3 binding was measured in extracts transiently overexpressing hsc70-GFP chimeras with point mutations more proximal (K71M) and more distal (V206C) within the ATPase domain of hsc70 when compared with vector-alone-transfected cells (P < 0.005) (data not shown). Collectively, these data suggest that crippling the ATP-binding, ATPase domain of hsc70 greatly diminishes the ability of hsc70 to specifically bind physiologically relevant vitamin D metabolites; a defect in specific 1,25(OH)2D3 binding was also observed in ATPase-disabled hsc70 (data not shown).
Specificity of hsc70-vitamin D metabolite binding
Confirmation of vitamin D metabolite binding analyses with extracts of hsc70-transfected cells was sought in vitro using purified hsc70 as binding protein and [3H]1,25-(OH)2D3 and [3H]25OHD3 as displaceable ligands. In these studies, the ability of radiolabeled sterol ligands, 25-OHD3 (Fig. 2, left panel) and 1,25(OH)2D3 (Fig. 2, right panel), to compete with 100 nM radioinert sterols for binding to hsc70 was evaluated. Both 25OHD3 and 1,25(OH)2D3 competed with [3H]25OHD3 for hsc70 binding. However, cholesterol did not effectively compete with [3H]25OHD3 for hsc70 binding. When [3H]1,25(OH)2D3 was introduced as the displaceable ligand, both 25OHD3 and 1,25(OH)2D3 as well as cholesterol were effective in competing with [3H]1,25(OH)2D3 for hsc70 binding. These results confirm that hsc70 can specifically bind the two principal 25-hydroxylated metabolites, 25OHD3 and 1,25(OH)2D3 (6), and that the B-ring-closed sterol, cholesterol, is a competitive analog for 1,25(OH)2D3-hsc70 binding.
Effect of ATP on hsc70-sterol ligand binding
Next, experiments were performed to determine whether sterol-hsc70 binding was affected by the availability of ATP (Fig. 3A). hsc70-[3H]1,25(OH)2D3 binding was significantly increased above basal level in the presence of 2 mM ATP. When performed under low-salt conditions that maximize the interaction of hsc70 with ATP, hsc70-[3H]1,25(OH)2D3 binding was also significantly increased, compared with hormone binding in the absence of added ATP (P = 0.02, n = 3; data not shown). Addition of an equimolar concentration of ADP showed no increase in hormone binding, whereas the inclusion of a nonmetabolizable ATP analog was associated with a significant (compared with no nucleotides added) mean increase in hormone binding that was slightly greater than that observed in the presence of metabolizable ATP.
The [3H]1,25(OH)2D3-hsc70 binding interaction was specific as the addition of 100 nM unlabeled 1,25(OH)2D3 significantly reduced tritiated hormone binding by 70%. The nature of the [3H]1,25(OH)2D3-hsc70 binding interaction was further defined in saturation binding analysis. At any given concentration of available, free [3H]1,25(OH)2D3 there was an increase in total [3H]1,25(OH)2D3 binding to the chaperone protein (Fig. 3B, inset). Analysis of these binding data according to the method of Scatchard (Fig. 3B) suggested that the presence of ATP in solution increased the total number of available [3H]1,25(OH)2D3 binding sites on hsc70 and that this binding interaction was expressed in both elements of high affinity (Kd = 0.24 nM) and relatively low-affinity binding (Kd = 1.5 nM).
As summarized in Table 1, ATP availability increased the number of binding sites on the hsc70 molecule by 35%, with high-affinity binding sites accounting for the increase; maximal binding of [3H]1,25(OH)2D3 at the lowest concentration of free ligand (i.e. high-affinity binding) was significantly increased (P = 0.002) in the presence of ATP. By contrast, addition of ADP diminished high-affinity hsc70–1,25(OH)2D3 binding in favor of more low-affinity binding sites. In an attempt to determine whether the binding of hsc70 to 1,25(OH)2D3 was the same or similar to that for 25OHD3 and cholesterol, we compared the saturable binding of these molecules and 1,25(OH)2D3 to hsc70. As was the case for 1,25(OH)2D3, there appeared to be two elements of specific binding for 25OHD3, one with high affinity and another with relatively low affinity. Also, similar to 1,25(OH)2D3, the total number of high-affinity binding sites for 25OHD3 was increased with ATP availability. Cholesterol was also bound specifically by hsc70; incubation of hsc70 with 100 nM cholesterol and [3H]cholesterol resulted in competitive binding by 55% (P = 0.01). However, in contrast to the vitamin D metabolites, cholesterol appeared to interact solely with low-affinity binding sites on the hsc70 molecule with no alteration in binding in the presence of added ATP (data not shown).
Discussion
hsp70s were initially recognized as early responder proteins to cellular stress, recruited to provide protection against protein denaturation and promote cell survival (18). Members of the hsp70 chaperone family are known to serve a large variety of subcellular functions involved in protein folding (19). We refer to these as classical functions of hsp70 chaperones because they involve interaction of the chaperone with another protein substrate. Such classical functions include receipt of nascent polypeptides as they exit the ribosome, cytoplasmic transfer of peptide substrates among other chaperones, directed transfer of proteins to distinct intracellular compartments, and preservation of constituent members in functional oligomeric complexes (i.e. protein machines) such as those involving steroid/sterol hormone receptors (19, 20). As depicted in simplified fashion in Fig. 4, hsp70-related chaperones possess two major functional domains, a highly conserved amino-terminal ATPase domain and a carboxy-terminal protein substrate binding domain (10). The protein binding activity of the carboxy-terminal domain of hsc70 is powered by the ATPase that resides in the amino-terminal domain of hsc70 (21). In its ATP-bound form, the chaperone possesses a relatively low affinity for protein substrates resulting in rapid association and dissociation of these substrates from the carboxy terminus of the hsc70 molecule. Hydrolysis of ATP to ADP results in conversion of the protein substrate-binding domain to a state of relatively high-affinity binding with a longer dwell time of the carboxy-terminal-interacting polypeptide. It is this cycling between the ATP- and ADP-bound state of the chaperone, along with the cochaperone binding interactions (2, 27), that governs which and for how long protein substrates are bound to hsp70s.
In addition to their well-recognized classical intracellular protein-protein binding actions, members of the hsp70 family of chaperone proteins are also capable of binding sterol and steroid hormones, including 25-hydroxylated vitamin D metabolites (5, 6) and promoting vitamin D metabolite-directed signaling events (22). For example, overexpression of hsc70 in mammalian kidney cells can promote both 1,25(OH)2D3-VDR-directed transactivation events (8) and 1,25(OH)2D3 synthesis (9), presumably via increased delivery of the active vitamin D metabolite, 1,25(OH)2D3, and 1,25(OH)2D3 substrate, 25OHD3, to the VDR and vitamin D-1-hydroxylase, respectively. Knowing that the ATP-binding, ATPase domain of hsc70 encompasses the vitamin D sterol-binding domain (8) (see Fig. 1A), we sought to determine whether occupation of the ATP-binding domain also influenced the nonclassical chaperone functions of interaction with vitamin D sterols and cholesterol. Results presented in Fig. 1, B and C, confirm that when an hsc70 expression construct is transfected into cultured mammalian kidney cells, it is correctly localized to the cytoplasm and leads to an ATPase-dependent increase in specific vitamin D metabolite binding. This is the first demonstration of ATP-dependent control of vitamin D sterol binding to hsp70-related chaperone proteins.
Therefore, as in protein substrate-binding to hsc70, ATP also regulates binding of sterols. However, whereas protein substrate binding is less avid when ATP is docked within the ATPase domain of hsc70 (21), vitamin D sterols are more avidly bound when either ATP or a nonmetabolizable analog of ATP occupies the amino-terminal ATP-binding domain (Fig. 3, A and B). As such and in opposition to the protein binding events occurring at the carboxy terminus of hsc70, the presence of bound ATP appears to increase the number of relatively high-affinity binding sites for 25-hydroxylated vitamin D metabolites. Furthermore, introduction of a mutation in the ATP-binding domain, which is known to disable hsc70 ATP binding (15, 16, 17), significantly diminishes 25OHD3 binding to the molecule (Fig. 1C). By contrast, the presence of ADP appears to both decrease the number of high-affinity sterol binding sites and increase the number of relatively low-affinity hsc70 binding sites. If one merges these states of sterol and protein substrate binding to hsc70 in the presence of ADP (Fig. 4), then the high-affinity state of protein substrate binding is coincident with a time of relatively low avidity of the chaperone for substrate sterol. As previously suggested by us (22), this would permit relatively stable anchoring of a potential recipient protein for the sterol at a time when the previously avidly bound sterol substrate (i.e. in the presence of ATP) is less avidly bound (i.e. after ATP hydrolysis to ADP) and create an opportunity for sterol substrates to move from a relatively low-affinity binding site on the donor protein to a relatively high-affinity binding site on the recipient protein.
Preliminary work from our laboratories (22) (Chun, R. and J. Barsony, unpublished observations) and others (23, 24, 25) suggests that such protein-protein interaction occurs between the hsp70-related chaperones and recipient intracellular vitamin D metabolite binding proteins such as the VDR and the vitamin D-1- and 24-hydroxylases. Such an ATP-regulated intracellular relay system for protein substrates among heat shock proteins is known to exist (26). These studies of ATP-dependent hsc70-vitamin D sterol binding are not without caveat or some unexpected results. For example, in the hormone binding analyses in solution, the recombinant hsc70 used is 90% pure by SDS-PAGE (Stressgen circular for lot B111410), it is possible that an hsc70-copurifiying peptide, not hsc70 itself, is responsible for binding sterol. However, we know that the carboxy-terminal domain, the portion known to bind protein substrates, of hsc70 (see Fig. 1A) does not appear to be required for sterol binding because transfected cells overexpressing hsc70 missing this C-terminal part of hsc70 exhibit no discernible deficit in [3H]1,25(OH)2D3 binding (8). Also, using a purified hsc70 fragment (amino acids 1–386; Stressgen), we observed binding to 25OHD3, comparable with the full-length version of hsc70 (Gacad, M., unpublished observations). Because our characterization of hsc70-sterol binding was performed in solution in these studies, these findings need to be confirmed in living cells. The inclusion of cholesterol substrate in our binding analysis was a control for vitamin D metabolites of similar structure and solubility. The observation that cholesterol effectively competed with 1,25(OH)2D3 for binding to hsc70 (Fig. 2), apparently by competing for the low-affinity binding sites on hsc70 (Table 1), was unexpected. Substantial additional work will be required to determine whether hsp70s are biochemically relevant intracellular chaperones for cholesterol because they appear to be for vitamin D metabolites.
Acknowledgments
The authors thank Ms. Gloria Kiel for assistance in manuscript preparation.
Footnotes
This work was supported by National Institutes of Health Grant DK58891 (to J.S.A.).
Current address for M.H.: Cedars-Sinai Medical Center, Los Angeles, California 90048.
First Published Online September 1, 2005
Abbreviations: GFP, Green fluorescent protein; hsc, constitutively expressed heat shock protein; hsp, heat shock protein; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; 25OHD3, 25-hydroxyvitamin D3; VDR, vitamin D receptor.
Accepted for publication August 23, 2005.
References
Hartl FU, Hayer-Hartl M 2002 Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295:1852–1858
Young JC, Barral JM, Hartl F 2003 More than folding: localized functions of cytosolic chaperones. Trends Biochem Sci 28:541–547
Gassler CS, Buchberger A, Laufen T, Mayer MP, Schroder H, Valencia A, Bukau B 1998 Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone. Proc Natl Acad Sci USA 95:15229–15234
Gacad MA, Adams JS 1991 Endogenous blockade of 1,25-dihydroxyvitamin D-receptor binding in New World primate cells. J Clin Invest 87:996–1001
Gacad MA, Chen H, Arbelle JE, LeBon T, Adams JS 1997 Functional characterization and purification of an intracellular vitamin D-binding protein in vitamin D-resistant new world primate cells. Amino acid sequence homology with proteins in the hsp-70 family. J Biol Chem 272:8433–8440
Gacad MA, Adams JS 1998 Proteins in the heat shock-70 family specifically bind 25-hydroxyvitamin D3 and 17-estradiol. J Clin Endocrinol Metab 83:1264–1267
Adams JS, Chen H, Chun R, Gacad MA, Encinas C, Ren S, Nguyen L, Wu S, Hewison M, Barsony J 2004 Response element binding proteins and intracellular vitamin D binding proteins: novel regulators of vitamin D trafficking, action and metabolism. J Steroid Biochem Mol Biol 89–90:461–465
Wu S, Ren S, Chen H, Chun RF, Gacad MA, Adams JS 2000 Intracellular vitamin D binding proteins: novel facilitators of vitamin D-directed transactivation. Mol Endocrinol 14:1387–1397
Wu S, Chun R, Gacad MA, Ren S, Chen H, Adams JS 2002 Regulation of 1,25-dihydroxyvitamin D synthesis by intracellular vitamin D binding protein-1. Endocrinology 143:4135–4138
Hartl FU 1996 Molecular chaperones in cellular protein folding. Nature 381:571–579
Racusen LC, Monteil C, Sgrignoli A, Lucskay M, Marouillat S, Rhim JG, Morin JP 1997 Cell lines with extended in vitro growth potential from human renal proximal tubule: characterization, response to inducers, and comparison with established cell lines. J Lab Clin Med 129:318–329
Mayer MP, Brehmer D, Gassler CS, Bukau B 2001 hsp70 chaperone machines. Adv Protein Chem 59:1–44
Jones GW, Masison DC 2003 Saccharomyces cerevisiae hsp70 mutations affect [PSI+] prion propagation and cell growth differently and implicate hsp40 and tetratricopeptide repeat cochaperones in impairment of [PSI+]. Genetics 163:495–506
Ellis S, Killender M, Anderson RL 2000 Heat-induced alterations in the localization of hsp72 and hsp73 as measured by indirect immunohistochemistry and immunogold electron microscopy. J Histochem Cytochem 48:321–332
Flaherty KM, Wilbanks SM, DeLuca-Flaherty C, McKay DB 1994 Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. II. Structure of the active site with ADP or ATP bound to wild type and mutant ATPase fragment. J Biol Chem 269:12899–12907
Wilbanks SM, DeLuca-Flaherty C, McKay DB 1994 Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. I. Kinetic analyses of active site mutants. J Biol Chem 269:12893–12898
Johnson ER, McKay DB 1999 Mapping the role of active site residues for transducing an ATP-induced conformational change in the bovine 70-kDa heat shock cognate protein. Biochemistry 38:10823–10830
Beere HM 2004 "The stress of dying": the role of heat shock proteins in the regulation of apoptosis. J Cell Sci 117:2641–2651
Young JC, Agashe VR, Siegers K, Hartl FU 2004 Pathways of chaperone-mediated protein folding in the cytosol. Nat Rev Mol Cell Biol 5:781–791
Pratt WB, Galigniana MD, Morishima Y, Murphy PJ 2004 Role of molecular chaperones in steroid receptor action. Essays Biochem 40:41–58
Erbse A, Mayer MP, Bukau B 2004 Mechanism of substrate recognition by hsp70 chaperones. Biochem Soc Trans 32:617–621
Adams JS, Chen H, Chun RF, Nguyen L, Wu S, Ren SY, Barsony J, Gacad MA 2003 Novel regulators of vitamin D action and metabolism: lessons learned at the Los Angeles Zoo. J Cell Biochem 88:308–314
Craig TA, Lutz WH, Kumar R 1999 Association of prokaryotic and eukaryotic chaperone proteins with the human 1,25-dihydroxyvitamin D3 receptor. Biochem Biophys Res Commun 260:446–452
Swamy N, Mohr SC, Ray R 1999 Vitamin D receptor interacts with DnaK/heat shock protein 70: identification of the interaction site on the vitamin D receptor. Arch Biochem Biophys 363:219–226
Lutz W, Kohno K, Kumar R 2001 The role of heat shock protein 70 in vitamin D receptor function. Biochem Biophys Res Commun 282:1211–1219
Wegele H, Muller L, Buchner J 2004 Hsp70 and Hsp90—a relay team for protein folding. Rev Physiol Biochem Pharmacol 151:1–44
Luders J, Demand J, Hohfeld J 2000 Distinct isoforms of the cofactor BAG-1 differentially affect Hsc70 chaperone function. J Biol Chem 275:4613–4617(Rene Chun, Mercedes A. Gacad, Martin Hew)
Division of Medical Sciences (M.H.), Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, United Kingdom
Abstract
Chaperone proteins in the heat shock protein-70 family possess endogenous ATP binding and ATPase activity and interact with intracellular protein substrates in an ATP-dependent manner; the hydrolysis of ATP to ADP results in an increase in the affinity of the chaperone for protein substrates. Heat shock protein-70s can also specifically interact with 25-hydroxylated vitamin D metabolites. Using constitutively expressed heat shock protein-70 (hsc70) as chaperone, here we demonstrate that vitamin D metabolite binding to hsc70 is also ATP dependent. Transient overexpression of an hsc70-green fluorescent protein chimeric construct in primate kidney cells resulted in a 6-fold increase in specific, extractable 25-hydroxyvitamin D3 binding. When ATPase capability of hsc70 was disabled, this increase was completely blocked. In solution, the binding of 25-hydroxylated vitamin D metabolites to hsc70 was significantly increased (P < 0.01) in the presence of ATP and a nonmetabolizable ATP analog. The ATP-directed increase in specific binding resulted from an increase in the abundance of relatively high-affinity hormone-binding sites (Kd, 0.24 nM). These results suggest that ATP hydrolysis to ADP would favor the release of vitamin D from a donor hsc70 molecule at a time when an hsc70-bound acceptor protein substrate is anchored to the chaperone with relative avidity. We theorize that the endogenous ATPase activity of hsc70 promotes the transfer of vitamin D sterols to other intracellular vitamin D binding proteins, such as the vitamin D receptor and vitamin D hydroxylases, to which hsc70 is known to bind.
Introduction
HEAT SHOCK PROTEINS have long been recognized for their ability to interact with the folding intermediates of polypeptides, disallowing proteins from forming nonspecific, nonproductive, intracellular aggregates with themselves and other proteins (1). Proteins in the heat shock protein (hsp) 70 superfamily of chaperones are also known for their ability to interact with a panoply of regulatory and/or accessory protein partners or cochaperones that govern crucial structural and functional events within the cell (2). hsp70s harbor a conserved, amino-terminal, ATP-binding, ATPase domain that supports chaperone functions of the protein (Fig. 1A). Hydrolysis of ATP by hsp70 proteins promotes more stable interaction with partner proteins in the more carboxy-terminal, protein-binding domain of the hsp70 molecule (1, 3).
In addition to their ability to interact with other proteins, members of the hsp70 family of chaperones are also known to interact with nonproteins, including metabolites of vitamin D (4, 5, 6). These chaperones provide a reliable means for the specific internalization and determination of the intracellular destination of vitamin D metabolites in target cells, thus mediating the action and metabolism of vitamin D (7). For example, augmented expression of constitutively expressed heat shock protein (hsc)70 promoted 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-vitamin D-receptor (VDR)-directed gene transactivation (8) and 1,25(OH)2D3 synthesis (9) in cultured kidney cells. Disruption or alteration of the ability of the hsp70s to bind and hydrolyze ATP interferes with their ability to bind and release substrate polypeptides from the protein-protein interaction domain of the chaperone (10). The purpose of the current set of experiments was to determine whether changes in ATP handling by hsc70 also affects its ability to interact with vitamin D sterols.
Materials and Methods
Materials and cells
Crystalline forms of vitamin D sterols, 25-hydroxyvitamin D3 (25OHD3) and 1,25(OH)2D3, were obtained from Biomol (Plymouth Meeting, PA) and cholesterol from Sigma (St. Louis, MO). ATP, ADP, and ATP analog and adenosine 5'-(, -imido) triphosphate tetralithium salt hydrate) were obtained from Sigma. Purified, recombinant bovine hsc70 was purchased from Stressgen Biotechnologies Corp. (Victoria, British Columbia, Canada). Tritiated sterols were obtained from Amersham (Piscataway, NJ); specific activities for the radiolabeled ligands were 187, 180, and 41 Ci/mmol for [3H]25(OH)2D3, [3H]1,25(OH)2D3, and [3H]cholesterol, respectively. Nonhuman and human primate kidney cell lines, COS-7 (monkey) and HKC-8 (human), were obtained from American Type Culture Collection (Manassas, VA) and the Hewison Laboratory (University of Birmingham, Birmingham, UK) (11), respectively.
Plasmids and transfection analysis
Wild-type and mutant constructs bearing a green fluorescent protein (GFP) tag at the carboxy terminus of hsc70 were developed (Fig. 1A) as part of our research program (reviewed in Refs.7 and 22) exploring the role of heat shock proteins in vitamin D transport, metabolism, and action; it is anticipated that these constructs will retain their normal functionality in the amino-terminal ATP, vitamin D interaction, and protein interaction domains (12), permitting real-time observation of the proteins. To determine whether the GFP tag impaired the functionality of hsc70, a yeast complementation assay system developed by Jones and Masison (13) was used. In this assay system, the four yeast ATPase-active, hsp70-like genes, SSA1–4, crucial for yeast survival, were inactivated by recombinant means. The resultant SSA1–4 deletion mutant yeast was viable only if one of the four functionally redundant SSA genes or a functional surrogate was reexpressed. Transfection of either the hsc70 or hsc70-GFP chimera was capable of facilitating survival and proliferation of SSA-deficient yeast, indicating that the GFP-tagged hsc70 was functional (Chun, R., and J. Adams, unpublished data).
For image analysis, COS-7 cells were transiently transfected by lipofection (Lipofectamine 2000; Invitrogen, Carlsbad, CA) overnight with 0.8 μg of the hsc70-GFP plasmid per chamber slide well. The next day, media were changed and images were taken from living cells using a Axiovert 100 fluorescent microscope (Zeiss, Gottingen, Germany) equipped with a LSM410 laser-scanning confocal unit. The 488-nm line of a krypton-argon laser with a BP 510- to 525-nm emission filter was used for GFP detection.
Ligand binding assays
Tritiated sterol binding was measured in cellular extracts of HKC-8 cells or commercially available preparations of purified hsc-70 (5). Briefly, increasing concentrations (0.05–5.0 nM) of radiolabeled sterols, in the presence or absence of 100 nM radioinert sterol, was dispensed into borosilicate glass tubes and evaporated to dryness under nitrogen. After 10 min of preexposure to 10-μl aliquots of 2.0 mM ATP, 2.0 mM ADP, or 2.0 mM ATP analog, each tube received 0.2 μg purified hsc-70 (0.28 nM), suspended in one of two buffers: high-salt buffer designed to maximize sterol binding, 0.5 M NaCl buffer [1.0 mM EDTA, 10 mM Tris-HCl (pH 7.4), 5.0 mM dithiothreitol, 1.0 mM phenylmethylsulfonyl fluoride[; or low-salt buffer [100 mM KCl, 5 mM MgCl2, 25 mM HEPES (pH 7.0)] for maximization of ATP binding. With low-salt buffer, incubation occurred at room temperature for 20 min. In high-salt buffer, tubes were incubated overnight at 4 C. After incubation, dextran-coated charcoal buffer was added to each tube, vigorously shaken, and then allowed to incubate on ice for 1 h. Tubes were centrifuged for 30 min at 3500 rpm at 4 C, and supernatant, containing protein-bound sterol, was decanted into scintillation vials and radioactivity counted.
Binding kinetics were determined by Scatchard plots (bound vs. bound/free). The slopes of these plots were used to define binding affinity and represented by the dissociation constant (Kd).
Results
Functionality of expressed hsc70-GFP chimeric proteins
When transiently transfected into cultured kidney cells, wt-hsc70-GFP overexpression resulted in the expected cytoplasmic localization (Fig. 1B). This is consistent with the results of immunohistochemical and cell fractionation studies that localize hsc70 largely to the cell cytoplasm (14) and suggests that inclusion of a carboxy-terminal GFP tail on hsc70 did not alter the intracellular localization of the chaperone. Deletion of the carboxy-terminal protein substrate and oligomerization domains (see Fig. 1A) did not significantly alter metabolite binding (8), suggesting that vitamin D sterol binding was legislated by the N-terminal portion of the hsc70 molecule. Figure 1C shows that cytoplasmic extracts of HKC-8 transfected with the wt-hsc70-GFP chimeric construct exhibited a 6-fold increase in specific 25OHD3 binding, compared with extracts of vector-alone-transfected cells. A point mutation (E175S) in the ATP-binding, ATPase domain of hsc70, known to disrupt both the ATPase and protein-protein binding activity of the chaperone (15, 16, 17), resulted in a decrease in extract-25OHD3-specific binding to levels observed in vector-alone-transfected cells. A similar significant decrease in 25OHD3 binding was measured in extracts transiently overexpressing hsc70-GFP chimeras with point mutations more proximal (K71M) and more distal (V206C) within the ATPase domain of hsc70 when compared with vector-alone-transfected cells (P < 0.005) (data not shown). Collectively, these data suggest that crippling the ATP-binding, ATPase domain of hsc70 greatly diminishes the ability of hsc70 to specifically bind physiologically relevant vitamin D metabolites; a defect in specific 1,25(OH)2D3 binding was also observed in ATPase-disabled hsc70 (data not shown).
Specificity of hsc70-vitamin D metabolite binding
Confirmation of vitamin D metabolite binding analyses with extracts of hsc70-transfected cells was sought in vitro using purified hsc70 as binding protein and [3H]1,25-(OH)2D3 and [3H]25OHD3 as displaceable ligands. In these studies, the ability of radiolabeled sterol ligands, 25-OHD3 (Fig. 2, left panel) and 1,25(OH)2D3 (Fig. 2, right panel), to compete with 100 nM radioinert sterols for binding to hsc70 was evaluated. Both 25OHD3 and 1,25(OH)2D3 competed with [3H]25OHD3 for hsc70 binding. However, cholesterol did not effectively compete with [3H]25OHD3 for hsc70 binding. When [3H]1,25(OH)2D3 was introduced as the displaceable ligand, both 25OHD3 and 1,25(OH)2D3 as well as cholesterol were effective in competing with [3H]1,25(OH)2D3 for hsc70 binding. These results confirm that hsc70 can specifically bind the two principal 25-hydroxylated metabolites, 25OHD3 and 1,25(OH)2D3 (6), and that the B-ring-closed sterol, cholesterol, is a competitive analog for 1,25(OH)2D3-hsc70 binding.
Effect of ATP on hsc70-sterol ligand binding
Next, experiments were performed to determine whether sterol-hsc70 binding was affected by the availability of ATP (Fig. 3A). hsc70-[3H]1,25(OH)2D3 binding was significantly increased above basal level in the presence of 2 mM ATP. When performed under low-salt conditions that maximize the interaction of hsc70 with ATP, hsc70-[3H]1,25(OH)2D3 binding was also significantly increased, compared with hormone binding in the absence of added ATP (P = 0.02, n = 3; data not shown). Addition of an equimolar concentration of ADP showed no increase in hormone binding, whereas the inclusion of a nonmetabolizable ATP analog was associated with a significant (compared with no nucleotides added) mean increase in hormone binding that was slightly greater than that observed in the presence of metabolizable ATP.
The [3H]1,25(OH)2D3-hsc70 binding interaction was specific as the addition of 100 nM unlabeled 1,25(OH)2D3 significantly reduced tritiated hormone binding by 70%. The nature of the [3H]1,25(OH)2D3-hsc70 binding interaction was further defined in saturation binding analysis. At any given concentration of available, free [3H]1,25(OH)2D3 there was an increase in total [3H]1,25(OH)2D3 binding to the chaperone protein (Fig. 3B, inset). Analysis of these binding data according to the method of Scatchard (Fig. 3B) suggested that the presence of ATP in solution increased the total number of available [3H]1,25(OH)2D3 binding sites on hsc70 and that this binding interaction was expressed in both elements of high affinity (Kd = 0.24 nM) and relatively low-affinity binding (Kd = 1.5 nM).
As summarized in Table 1, ATP availability increased the number of binding sites on the hsc70 molecule by 35%, with high-affinity binding sites accounting for the increase; maximal binding of [3H]1,25(OH)2D3 at the lowest concentration of free ligand (i.e. high-affinity binding) was significantly increased (P = 0.002) in the presence of ATP. By contrast, addition of ADP diminished high-affinity hsc70–1,25(OH)2D3 binding in favor of more low-affinity binding sites. In an attempt to determine whether the binding of hsc70 to 1,25(OH)2D3 was the same or similar to that for 25OHD3 and cholesterol, we compared the saturable binding of these molecules and 1,25(OH)2D3 to hsc70. As was the case for 1,25(OH)2D3, there appeared to be two elements of specific binding for 25OHD3, one with high affinity and another with relatively low affinity. Also, similar to 1,25(OH)2D3, the total number of high-affinity binding sites for 25OHD3 was increased with ATP availability. Cholesterol was also bound specifically by hsc70; incubation of hsc70 with 100 nM cholesterol and [3H]cholesterol resulted in competitive binding by 55% (P = 0.01). However, in contrast to the vitamin D metabolites, cholesterol appeared to interact solely with low-affinity binding sites on the hsc70 molecule with no alteration in binding in the presence of added ATP (data not shown).
Discussion
hsp70s were initially recognized as early responder proteins to cellular stress, recruited to provide protection against protein denaturation and promote cell survival (18). Members of the hsp70 chaperone family are known to serve a large variety of subcellular functions involved in protein folding (19). We refer to these as classical functions of hsp70 chaperones because they involve interaction of the chaperone with another protein substrate. Such classical functions include receipt of nascent polypeptides as they exit the ribosome, cytoplasmic transfer of peptide substrates among other chaperones, directed transfer of proteins to distinct intracellular compartments, and preservation of constituent members in functional oligomeric complexes (i.e. protein machines) such as those involving steroid/sterol hormone receptors (19, 20). As depicted in simplified fashion in Fig. 4, hsp70-related chaperones possess two major functional domains, a highly conserved amino-terminal ATPase domain and a carboxy-terminal protein substrate binding domain (10). The protein binding activity of the carboxy-terminal domain of hsc70 is powered by the ATPase that resides in the amino-terminal domain of hsc70 (21). In its ATP-bound form, the chaperone possesses a relatively low affinity for protein substrates resulting in rapid association and dissociation of these substrates from the carboxy terminus of the hsc70 molecule. Hydrolysis of ATP to ADP results in conversion of the protein substrate-binding domain to a state of relatively high-affinity binding with a longer dwell time of the carboxy-terminal-interacting polypeptide. It is this cycling between the ATP- and ADP-bound state of the chaperone, along with the cochaperone binding interactions (2, 27), that governs which and for how long protein substrates are bound to hsp70s.
In addition to their well-recognized classical intracellular protein-protein binding actions, members of the hsp70 family of chaperone proteins are also capable of binding sterol and steroid hormones, including 25-hydroxylated vitamin D metabolites (5, 6) and promoting vitamin D metabolite-directed signaling events (22). For example, overexpression of hsc70 in mammalian kidney cells can promote both 1,25(OH)2D3-VDR-directed transactivation events (8) and 1,25(OH)2D3 synthesis (9), presumably via increased delivery of the active vitamin D metabolite, 1,25(OH)2D3, and 1,25(OH)2D3 substrate, 25OHD3, to the VDR and vitamin D-1-hydroxylase, respectively. Knowing that the ATP-binding, ATPase domain of hsc70 encompasses the vitamin D sterol-binding domain (8) (see Fig. 1A), we sought to determine whether occupation of the ATP-binding domain also influenced the nonclassical chaperone functions of interaction with vitamin D sterols and cholesterol. Results presented in Fig. 1, B and C, confirm that when an hsc70 expression construct is transfected into cultured mammalian kidney cells, it is correctly localized to the cytoplasm and leads to an ATPase-dependent increase in specific vitamin D metabolite binding. This is the first demonstration of ATP-dependent control of vitamin D sterol binding to hsp70-related chaperone proteins.
Therefore, as in protein substrate-binding to hsc70, ATP also regulates binding of sterols. However, whereas protein substrate binding is less avid when ATP is docked within the ATPase domain of hsc70 (21), vitamin D sterols are more avidly bound when either ATP or a nonmetabolizable analog of ATP occupies the amino-terminal ATP-binding domain (Fig. 3, A and B). As such and in opposition to the protein binding events occurring at the carboxy terminus of hsc70, the presence of bound ATP appears to increase the number of relatively high-affinity binding sites for 25-hydroxylated vitamin D metabolites. Furthermore, introduction of a mutation in the ATP-binding domain, which is known to disable hsc70 ATP binding (15, 16, 17), significantly diminishes 25OHD3 binding to the molecule (Fig. 1C). By contrast, the presence of ADP appears to both decrease the number of high-affinity sterol binding sites and increase the number of relatively low-affinity hsc70 binding sites. If one merges these states of sterol and protein substrate binding to hsc70 in the presence of ADP (Fig. 4), then the high-affinity state of protein substrate binding is coincident with a time of relatively low avidity of the chaperone for substrate sterol. As previously suggested by us (22), this would permit relatively stable anchoring of a potential recipient protein for the sterol at a time when the previously avidly bound sterol substrate (i.e. in the presence of ATP) is less avidly bound (i.e. after ATP hydrolysis to ADP) and create an opportunity for sterol substrates to move from a relatively low-affinity binding site on the donor protein to a relatively high-affinity binding site on the recipient protein.
Preliminary work from our laboratories (22) (Chun, R. and J. Barsony, unpublished observations) and others (23, 24, 25) suggests that such protein-protein interaction occurs between the hsp70-related chaperones and recipient intracellular vitamin D metabolite binding proteins such as the VDR and the vitamin D-1- and 24-hydroxylases. Such an ATP-regulated intracellular relay system for protein substrates among heat shock proteins is known to exist (26). These studies of ATP-dependent hsc70-vitamin D sterol binding are not without caveat or some unexpected results. For example, in the hormone binding analyses in solution, the recombinant hsc70 used is 90% pure by SDS-PAGE (Stressgen circular for lot B111410), it is possible that an hsc70-copurifiying peptide, not hsc70 itself, is responsible for binding sterol. However, we know that the carboxy-terminal domain, the portion known to bind protein substrates, of hsc70 (see Fig. 1A) does not appear to be required for sterol binding because transfected cells overexpressing hsc70 missing this C-terminal part of hsc70 exhibit no discernible deficit in [3H]1,25(OH)2D3 binding (8). Also, using a purified hsc70 fragment (amino acids 1–386; Stressgen), we observed binding to 25OHD3, comparable with the full-length version of hsc70 (Gacad, M., unpublished observations). Because our characterization of hsc70-sterol binding was performed in solution in these studies, these findings need to be confirmed in living cells. The inclusion of cholesterol substrate in our binding analysis was a control for vitamin D metabolites of similar structure and solubility. The observation that cholesterol effectively competed with 1,25(OH)2D3 for binding to hsc70 (Fig. 2), apparently by competing for the low-affinity binding sites on hsc70 (Table 1), was unexpected. Substantial additional work will be required to determine whether hsp70s are biochemically relevant intracellular chaperones for cholesterol because they appear to be for vitamin D metabolites.
Acknowledgments
The authors thank Ms. Gloria Kiel for assistance in manuscript preparation.
Footnotes
This work was supported by National Institutes of Health Grant DK58891 (to J.S.A.).
Current address for M.H.: Cedars-Sinai Medical Center, Los Angeles, California 90048.
First Published Online September 1, 2005
Abbreviations: GFP, Green fluorescent protein; hsc, constitutively expressed heat shock protein; hsp, heat shock protein; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; 25OHD3, 25-hydroxyvitamin D3; VDR, vitamin D receptor.
Accepted for publication August 23, 2005.
References
Hartl FU, Hayer-Hartl M 2002 Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295:1852–1858
Young JC, Barral JM, Hartl F 2003 More than folding: localized functions of cytosolic chaperones. Trends Biochem Sci 28:541–547
Gassler CS, Buchberger A, Laufen T, Mayer MP, Schroder H, Valencia A, Bukau B 1998 Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone. Proc Natl Acad Sci USA 95:15229–15234
Gacad MA, Adams JS 1991 Endogenous blockade of 1,25-dihydroxyvitamin D-receptor binding in New World primate cells. J Clin Invest 87:996–1001
Gacad MA, Chen H, Arbelle JE, LeBon T, Adams JS 1997 Functional characterization and purification of an intracellular vitamin D-binding protein in vitamin D-resistant new world primate cells. Amino acid sequence homology with proteins in the hsp-70 family. J Biol Chem 272:8433–8440
Gacad MA, Adams JS 1998 Proteins in the heat shock-70 family specifically bind 25-hydroxyvitamin D3 and 17-estradiol. J Clin Endocrinol Metab 83:1264–1267
Adams JS, Chen H, Chun R, Gacad MA, Encinas C, Ren S, Nguyen L, Wu S, Hewison M, Barsony J 2004 Response element binding proteins and intracellular vitamin D binding proteins: novel regulators of vitamin D trafficking, action and metabolism. J Steroid Biochem Mol Biol 89–90:461–465
Wu S, Ren S, Chen H, Chun RF, Gacad MA, Adams JS 2000 Intracellular vitamin D binding proteins: novel facilitators of vitamin D-directed transactivation. Mol Endocrinol 14:1387–1397
Wu S, Chun R, Gacad MA, Ren S, Chen H, Adams JS 2002 Regulation of 1,25-dihydroxyvitamin D synthesis by intracellular vitamin D binding protein-1. Endocrinology 143:4135–4138
Hartl FU 1996 Molecular chaperones in cellular protein folding. Nature 381:571–579
Racusen LC, Monteil C, Sgrignoli A, Lucskay M, Marouillat S, Rhim JG, Morin JP 1997 Cell lines with extended in vitro growth potential from human renal proximal tubule: characterization, response to inducers, and comparison with established cell lines. J Lab Clin Med 129:318–329
Mayer MP, Brehmer D, Gassler CS, Bukau B 2001 hsp70 chaperone machines. Adv Protein Chem 59:1–44
Jones GW, Masison DC 2003 Saccharomyces cerevisiae hsp70 mutations affect [PSI+] prion propagation and cell growth differently and implicate hsp40 and tetratricopeptide repeat cochaperones in impairment of [PSI+]. Genetics 163:495–506
Ellis S, Killender M, Anderson RL 2000 Heat-induced alterations in the localization of hsp72 and hsp73 as measured by indirect immunohistochemistry and immunogold electron microscopy. J Histochem Cytochem 48:321–332
Flaherty KM, Wilbanks SM, DeLuca-Flaherty C, McKay DB 1994 Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. II. Structure of the active site with ADP or ATP bound to wild type and mutant ATPase fragment. J Biol Chem 269:12899–12907
Wilbanks SM, DeLuca-Flaherty C, McKay DB 1994 Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. I. Kinetic analyses of active site mutants. J Biol Chem 269:12893–12898
Johnson ER, McKay DB 1999 Mapping the role of active site residues for transducing an ATP-induced conformational change in the bovine 70-kDa heat shock cognate protein. Biochemistry 38:10823–10830
Beere HM 2004 "The stress of dying": the role of heat shock proteins in the regulation of apoptosis. J Cell Sci 117:2641–2651
Young JC, Agashe VR, Siegers K, Hartl FU 2004 Pathways of chaperone-mediated protein folding in the cytosol. Nat Rev Mol Cell Biol 5:781–791
Pratt WB, Galigniana MD, Morishima Y, Murphy PJ 2004 Role of molecular chaperones in steroid receptor action. Essays Biochem 40:41–58
Erbse A, Mayer MP, Bukau B 2004 Mechanism of substrate recognition by hsp70 chaperones. Biochem Soc Trans 32:617–621
Adams JS, Chen H, Chun RF, Nguyen L, Wu S, Ren SY, Barsony J, Gacad MA 2003 Novel regulators of vitamin D action and metabolism: lessons learned at the Los Angeles Zoo. J Cell Biochem 88:308–314
Craig TA, Lutz WH, Kumar R 1999 Association of prokaryotic and eukaryotic chaperone proteins with the human 1,25-dihydroxyvitamin D3 receptor. Biochem Biophys Res Commun 260:446–452
Swamy N, Mohr SC, Ray R 1999 Vitamin D receptor interacts with DnaK/heat shock protein 70: identification of the interaction site on the vitamin D receptor. Arch Biochem Biophys 363:219–226
Lutz W, Kohno K, Kumar R 2001 The role of heat shock protein 70 in vitamin D receptor function. Biochem Biophys Res Commun 282:1211–1219
Wegele H, Muller L, Buchner J 2004 Hsp70 and Hsp90—a relay team for protein folding. Rev Physiol Biochem Pharmacol 151:1–44
Luders J, Demand J, Hohfeld J 2000 Distinct isoforms of the cofactor BAG-1 differentially affect Hsc70 chaperone function. J Biol Chem 275:4613–4617(Rene Chun, Mercedes A. Gacad, Martin Hew)