Heat-induced Oligomerization of the Molecular Chaperone Hsp90
INHIBITION BY ATP AND GELDANAMYCIN AND ACTIVATION BY TRANSITION METAL OXYANIONS*

Ahmed ChadliDagger §, Moncef M. Ladjimi, Etienne-Emile BaulieuDagger , and Maria Grazia CatelliDagger parallel **

From the Dagger  INSERM, U 488, Neurosteroïdes et Système Nerveux, 80 rue du Général Leclerc, 94276 Le Krémlin Bicêtre, the  Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, CNRS, UMR-7631, 96 Boulevard Raspail, 75006 Paris, and the parallel  Laboratoire d'Endocrinologie, Métabolisme et Développement, CNRS, UPR-1524, CHU Cochin Port Royal, 24 rue du Faubourg St. Jacques, 75014 Paris, France

    ABSTRACT
Top
Abstract
Introduction
References

It has been previously reported that heat shock protein 90 (Hsp90) oligomerizes at high temperatures and displays concomitantly a novel chaperone activity (Yonehara, M., Minami, Y., Kawata, Y., Nagai, J., and Yahara, I. (1996) J. Biol. Chem., 271, 2641-2645). In order to better define these oligomerization properties at high temperatures and to know whether they are influenced by modulators of Hsp90 function, heat-induced oligomerization of highly purified dimeric Hsp90 has been investigated over a wide range of temperature and protein concentrations by native polyacrylamide gel electrophoresis and size exclusion chromatography. Whereas below 50 °C, the dimeric form is maintained over a large range of concentrations, at the critical temperature of 50 °C, a sharp transition from dimeric to higher order oligomeric species takes place within minutes, in a highly ordered process, suggesting that a conformational change, leading to the appearance of a new oligomerization site, occurs in Hsp90 dimer. Moreover, at and above the critical temperature, the extent of oligomerization increases with Hsp90 concentration.

Formation of high order oligomers at high temperatures is sensitive to modulators of Hsp90 function. ATP and geldanamycin, both known to bind to the same pocket of Hsp90, are inhibitors of this process, whereas molybdate, vanadate, and Nonidet P-40, which are thought to increase surface hydrophobicity of the protein, are activators. Thus, oligomerization of Hsp90 at high temperatures may be mediated through hydrophobic interactions that are hindered by ligands and favored by transition metal oxyanions.

The fact that the heat-induced oligomerization of Hsp90 is affected by specific ligands that modulate its properties also suggests that this process may be involved in cell protection during heat shock.

    INTRODUCTION
Top
Abstract
Introduction
References

A wide range of adverse conditions, such as increasing temperature, trigger the synthesis of stress or heat shock proteins (Hsps),1 which participate in an essential defense mechanism, present in species from bacteria to human, that consists of a stress response and acquired stress tolerance (1-3). Even in the absence of heat induction, Hsps and their constitutively expressed cognate counterparts perform vital functions as molecular chaperones. During and after heat shock, Hsps act by preventing catastrophic protein aggregation, by assisting refolding of damaged proteins, or by targeting them toward a proteolytic pathway (4, 5).

Among Hsps, the highly conserved Hsp90, although dispensable in Escherichia coli, is essential for viability in eukaryotes (6, 7). Hsp90 constitutes 1-2% of cytosolic proteins, and this level further increases when cells are exposed to heat shock. Indeed, very high levels of Hsp90 are necessary for growth of yeast cells at extreme temperatures (6) and contribute to efficient reactivation of heat damaged proteins (8). At normal temperatures, the abundant Hsp90 seems to be only required for a specific subset of cellular proteins that need assistance to reach a native conformation (9-11). Therefore, as yet, there is little evidence indicating a general chaperone function for Hsp90 in vivo (8), and the requirement of high levels of Hsp90 in heat shock conditions is not fully understood. In vitro experiments with reticulocyte lysate and genetic experiments in vivo indicate that Hsp90 is required to assist the conformational maturation of specific targets, such as, for example, steroid hormone receptors and protein kinases, in an ATP-dependent fashion and in concert with other chaperones, such as Hsp70, Hsp40, immunophillins, and p23 (10, 12). Moreover, it has been proposed that depending on the nature of the nucleotide, Hsp90 may acquire two different functional states (13). However, ATP hydrolysis was repeatedly found to be unnecessary for Hsp90 chaperone function in vitro (14-16).

Recent crystallographic studies have shown the presence of a specific, low affinity ATP/ADP binding site in the N-terminal domain of Hsp90 (17). This ATP/ADP binding site, which has been found to be related to that of DNA gyrase B, also binds geldanamycin, a naturally occurring antitumor antibiotic (18), suggesting that geldanamycin blocks Hsp90 action through binding to the nucleotide site. Indeed, in the case of steroid receptors, geldanamycin not only accelerates receptors degradation but, most importantly, abolishes their ability to bind the specific hormone (19). The discovery of the Hsp90 ATP/geldanamycin binding site prompted further investigations of in vitro chaperone activities of Hsp90, leading to the proposal that Hsp90 bears two independent chaperone sites differing in substrate specificity and ATP dependence (20, 21). Moreover, mutations of residues implicated in ATP binding and hydrolysis abolish the ATPase activity in vitro and disrupt Hsp90 function in vivo (22).

A large body of evidence suggests that all of the members of the Hsp90 family, which in vertebrates includes Hsp90alpha , Hsp90beta , and glucose-regulated protein 94, exist as homodimeric species in crude extracts or in purified preparations (23-30). Nevertheless, some Hsp90beta has been found in monomeric form (27, 29), and a tendency of Hsp90 to self-associate has been reported when purified preparations or whole cytosols were analyzed (29, 30).

Because Hsp90 and other chaperones are heat shock proteins that have evolved to function at high temperatures, changes in structural organization of these proteins following increase in temperature are of particular interest. Such changes have been shown to occur for Hsp90 above 49 °C, the temperature at which the protein oligomerizes (31, 32) and concomitantly exhibits a newly acquired chaperone activity (33). Because this finding may have implications on the function of Hsp90 in thermoprotection, it was important to further characterize this heat-induced oligomerization process and to determine the effects of ligands known to modulate Hsp90 activity.

Results reported in this study show that oligomerization of Hsp90 occurs at a critical temperature of 50 °C regardless of the concentration and is sensitive to modulators of Hsp90 activity, such as ATP, ADP, geldanamycin, and transition metal oxyanions.

    EXPERIMENTAL PROCEDURES

Cytosol Preparation-- Decapitated 12-14-day-old chicken embryos were washed twice with PBS and once with Buffer A (20 mM triethanolamine, 50 mM potassium acetate, 2% glycerol, 1 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, pH 7.8) at 4 °C. The buffer was removed, and the embryos were homogenized in 1.5 volume (w/v) of Buffer A containing protease inhibitors (15 µg/ml leupeptin, 15 µg/ml antipain, and 15 µg/ml aprotinin). The homogenate was centrifuged for 15 min at 600 × g, and the supernatant, recovered after a 105,000 × g centrifugation (45 min at 4 °C), was aliquoted and stored at -80 °C. Cytosol contained 15-20 mg/ml protein.

Immobilization of Purified IgG, Hsp90 Immunopurification-- IgG fraction purified from BF4 ascites (34) was coupled to divinyl sulfone-activated Sepharose 4B as described (35). 2 mg of BF4 were immobilized per g of Sepharose 4B. BF4 antibody recognizes specifically Hsp90alpha .2

The cytosol was thawed, centrifuged at 4000 × g for 10 min at 4 °C, and mixed with solid KCl (to a final concentration of 400 mM). The cytosol was loaded on the immunoaffinity gel equilibrated with Buffer A containing 400 mM KCl. The column, washed with 5 volumes of the same buffer, was eluted with 43 mM diethylamine buffer, pH 10.5, as described (25). The fractions immediately brought to pH 7.5 by addition of 1 M KH2PO4, were pooled and concentrated by ultrafiltration using the AMICON system. The sedimentation coefficient, Stokes radius, and Mr of BF4-purified chicken Hsp90 were determined as previously reported (25). UV spectra of purified Hsp90 gave an A280/260 ratio of 1.8, suggesting that the protein was nucleotide free. Estrogen receptor binding activity of purified Hsp90 was tested as reported (36).

Size Exclusion Chromatography-- Size exclusion chromatography, using an FPLC system (Amersham Pharmacia Biotech), was carried out at 4 °C on Superose 6 or Superose 12 10/30 columns equilibrated with 50 mM Tris-HCl, pH 8.65. Elution was performed using the same buffer at a flow rate of 0.2 ml/min. The eluted proteins were detected by the UV absorbance at 280 nm. The columns were calibrated with high and low molecular mass calibration kit from Amersham Pharmacia Biotech. The standards used were thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa), and chymotrypsinogen A (25 kDa). Blue dextran and potassium bichromate were used to determine the void volume and the total volume, respectively.

SDS and Native PAGE-- SDS-PAGE and silver staining were performed on a Phast system (Amersham Pharmacia Biotech). The molecular mass calibration kit (Amersham Pharmacia Biotech) included phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and alpha -lactalbumin (14.4 kDa).

Native PAGE was performed at 4 °C on Multiphore II (Amersham Pharmacia Biotech) using 10% polyacrylamide Clean gel, 375 mM Tris buffer, pH 8.9. A calibration kit (Amersham Pharmacia Biotech) containing thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and bovine serum albumin (67 kDa) was used to estimate the apparent molecular mass of proteins according to the manufacturer's recommendations. Scanning of silver stained bands was performed on a Omni media Scanner apparatus using BioImage software (Millipore).

Westen Blot Analysis of the Native PAGE-- Hsp90 samples resolved on native PAGE were electrotransferred to nitrocellulose. Air-dried filters were incubated 2 h in 10% nonfat dry milk in PBS, 0.05% Tween 20 (PBST), washed with PBST, incubated for 2 h with the anti-Hsp90 antibodies Ab119 (37) (1:4000) or D7alpha (28) (1:4000), washed with PBST, incubated for 1 h with anti-rabbit or anti-mouse antibodies, washed with PBST, and revealed utilizing ECL (Amersham Pharmacia Biotech).

Heat-induced Oligomerization of Hsp90-- In standard method, 1 µg of Hsp90 in a final volume of 8 µl of 300 mM Tris, pH 8.8, was incubated at different temperatures for 20 min and then immediately cooled on ice (30 min at 4 °C) and subjected to native PAGE. Hsp90 samples (0.1 mg/ml incubated for 20 min at 50 °C and centrifuged at 105,000 × g) were devoid of precipitating material.

Effects of geldanamycine, ATP, ADP, ammonium molybdate, sodium orthovanadate, and Nonidet P-40 on Hsp90 heat-induced oligomerization were tested as follows: Hsp90 samples were mixed with the indicated concentration of each compound and kept at 4 °C for 90 min before incubation at various temperatures.

Time course experiments of Hsp90 oligomerization at a given temperature were performed using the Robocycler gradient 40 temperature cycler (Stratagene), which allows simultaneous transfer of 40 samples from the 4 °C block to the hot block. Well-to-well temperature uniformity in the block is of ±0.25 °C. At the indicated times, samples were rapidly put on ice. The same apparatus allowed the use of a precise 8-point temperature gradient in the same block.

    RESULTS

Heat-induced Oligomerization of Hsp90

It has been previously reported that purified Hsp90, when incubated above 49 °C, oligomerizes and displays a concomitant novel chaperone activity (33). In order to better define the oligomerization properties of Hsp90 at high temperatures and to know whether this process is influenced by modulators of Hsp90 function, such as ATP, geldanamycin, molybdate, vanadate, and Nonidet P-40, heat-induced oligomerization of highly purified Hsp90 has been investigated in several conditions and in the presence or the absence of these modulators.

Effect of Temperature on Hsp90 Quaternary Structure-- Hsp90 alpha  has been purified to homogeneity from chick embryos by a single immunoaffinity step. It gives a single band in SDS-PAGE, as shown in Fig. 1A, and appears to be 99.5% pure by densitometric analysis (data not shown). A sedimentation coefficient of 6.2 s, a Stokes radius of ~6.8 nm, and a Mr of 178,000 confirmed a previous report on the dimeric structure of BF4-immunopurified chicken Hsp90 (25). When the same preparation was analyzed by native PAGE, a band of apparent molecular mass of about 184 kDa was obtained (Fig. 1B and Ref. 27). Furthermore, purified chicken Hsp90 was able to bind cytosolic or purified estrogen receptor as already described for mammalian Hsp90 (36), indicating that at least the steroid receptor binding property of Hsp90 is preserved after immunoaffinity purification (data not shown).


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 1.   Electrophoretic analysis of purified Hsp90. Immunopurified Hsp90 was resolved by SDS-PAGE (A) (lane 1, 0.25 µg) or by native PAGE (B) (lane 1, 2.5 µg) and revealed by silver staining. Molecular mass markers (kDa) are indicated.

Purified Hsp90 was incubated for 20 min at different temperatures and then analyzed by native PAGE. As shown in Fig. 2A, Hsp90 remains dimeric in the temperature range of 37-45 °C. However, incubation at 50 °C leads to the appearance of well defined oligomeric forms that are shifted to even higher molecular mass oligomers at 55 and 60 °C, as indicated by the smear at the top of the gel and the progressive disappearance of the dimeric species. Experiments shown in Fig. 2B, including comparison with the migration of standard proteins indicated that in addition to the dimer, at least four different oligomeric forms, with apparent molecular masses compatible with those of the trimer, tetramer, pentamer, and hexamer, were detected. The presence of odd-numbered oligomers implies that dissociation of dimers takes place (see below).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 2.   Heat-induced oligomerization of Hsp90. A, 1-µg samples of purified Hsp90 (0.1 mg/ml) were incubated for 20 min at the indicated temperatures; equal amounts (1 µg) of protein were analyzed by native PAGE. B, determination of approximate molecular masses of oligomers resolved by native PAGE after incubation at 50 °C: black-square, relative migration of molecular mass markers ± S.D.; horizontal lines, log of molecular mass of oligomers, as determined in four independent experiments. C, elution profiles at 280 nm of Hsp90 samples (0.175 mg/ml) analyzed on Superose 6 after incubation at 37 °C (- - - - -) or 50 °C (------). DB, blue dextran.

In order to confirm that these oligomers were also present in solution before electrophoresis, Hsp90 was analyzed by size exclusion chromatography. Fig. 2C shows that in contrast with Hsp90 incubated at 37 °C, which elutes as an asymmetric dimer and shows the classical profile (23-28), Hsp90 incubated at 50 °C elutes as a mixture of different oligomeric species, as indicated by the wide peak obtained.

To determine the temperature at which the transition between the dimer and the higher order oligomers occurs, Hsp90 was incubated at different temperatures between 45 and 52 °C with a 1 °C increment and then submitted to native PAGE. As shown in Fig. 3A, whereas Hsp90 remains dimeric up to 49 °C, oligomerization occurred at 50 °C. This temperature is defined here as the critical temperature. Disappearance of the dimers into higher oligomers, as monitored by densitometry, occurred in a very narrow range of temperatures and followed a sigmoidal pattern (Fig. 3B). The critical temperature of 50 °C that defines the transition from dimers to higher order oligomers was found to be the same at low (0.1 mg/ml) and high (7.5 mg/ml) concentrations of Hsp90 (data not shown), indicating that temperature and not concentration induces oligomerization.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 3.   Hsp90 dimer disappearance during heat-induced oligomerization. A, native PAGE analysis of Hsp90 samples (1 µg) incubated for 20 min at different temperatures. Experimental points from 42 to 52 °C were obtained using a precise 8-point temperature gradient in the Robocycler apparatus (see under "Experimental Procedures"). B, the percentage of Hsp90 dimer from A was plotted as a function of the temperature after densitometric scanning.

Effect of Concentration of Hsp90 on Heat-induced Oligomerization State-- In order to know whether the heat-induced oligomerization state of Hsp90 (i.e. the abundance of high versus lower order oligomers) is dependent on its concentration, the effects of increasing concentration of Hsp90 on the oligomerization state, below and above the critical temperature of 50 °C, have been determined. As shown in Fig. 4A, increasing the concentration of Hsp90 at 55 °C affects the degree of self-association, and at low concentrations, low molecular mass oligomers are present, whereas at higher concentrations, a shift from low to higher molecular mass oligomers occurs. By contrast, below the critical temperature, only Hsp90 dimers are present for concentration up to 14 mg/ml (Fig. 4A) and even at 20 mg/ml (data not shown). This is confirmed by size exclusion chromatography, in which, after incubation at 55 °C, highly concentrated Hsp90 elutes near the void volume, whereas at low concentrations, Hsp90 elutes between 440 and 669 kDa (Fig. 4B).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of Hsp90 concentration on heat-induced oligomerization. A, purified Hsp90 at the indicated concentrations was incubated at 45 or 55 °C. Equal amounts of protein (1 µg) were analyzed by native PAGE. B, comparison of elution profiles from Superose 6 of Hsp90 heated at 37 °C (- - - - -) and 55 °C (------) at 0.175 mg/ml or at 55 °C at 7.5 mg/ml (- · - · -).

Modulation of Hsp90 Heat-induced Oligomerization by Ligands

Previous studies have shown that Hsp90 interactions with target and auxiliary proteins are affected by several low molecular weight modulators (10). Among them, the antibiotic-antitumor agent geldanamycin is a specific inhibitor of Hsp90 function that binds to the same pocket as ATP or ADP in the N-terminal region of the protein (17-19). ATP, in turn, has recently been shown to modulate the chaperone activity of the N-terminal region of HSP90 (21). On the other hand, molybdate and analogous transition metal oxyanions, such as vanadate, that bind to the Hsp90 dimer with low affinity (38), have been shown to stabilize the interaction between Hsp90 and steroid receptors (10), and non-ionic detergent Nonidet P-40 has been shown to enhance the binding of Hsp90 to p23 (13). We therefore investigated the effects of these modulators on heat-induced oligomerization of Hsp90.

Effect of Geldanamycin, ATP, and ADP-- As shown in Fig. 5, geldanamycin and ATP are powerful inhibitors of heat-induced oligomerization of Hsp90. Almost full inhibition is obtained at a concentration of 4 µM (Fig. 5, lane 3) and 8 mM (lane 4) for geldanamycin and ATP, respectively. ADP was also an inhibitor of Hsp90 heat-induced oligomerization, although with lower efficiency than ATP (data not shown). These results suggest that ATP, ADP, and geldanamycin inhibit the heat-induced oligomerization by stabilizing a dimeric structure of Hsp90 less sensitive to heat. Indeed, as shown in Fig. 5, although the dimer has almost completely disappeared in favor of higher oligomeric species at 55 °C (lane 7), it is still present with geldanamycin and ATP (lanes 8 and 9, respectively), supporting the hypothesis of dimer protection by these compounds from temperature-induced oligomerization.


View larger version (97K):
[in this window]
[in a new window]
 
Fig. 5.   Modulators of Hsp90 heat-induced oligomerization. Hsp90 samples (0.1 mg/ml) were preincubation at 4 °C for 90 min (lane 1) and then incubated for 20 min at 50 °C (lanes 2-6) or 55 °C (lanes 7-11). Preincubation was performed without (lanes 2 and 7) or with 4 µM geldanamycin (lanes 3 and 8), 8 mM ATP (lanes 4 and 9), 20 mM molybdate (lanes 5 and 10), or 0.01% Nonidet P-40 (lanes 6 and 11). Samples were analyzed by native PAGE.

Effect of Molybdate, Vanadate, and Nonidet P-40-- In contrast with ATP, ADP, and geldanamycin, molybdate and Nonidet P-40 promote the formation of high molecular mass oligomers, as shown in Fig. 5, lanes 5 and 6, respectively. Because molybdate was efficient in promoting oligomerization of Hsp90 at high temperatures, it was of interest to know whether it affected the critical temperature. As shown in Fig. 6, in the presence of molybdate, oligomerization occurred at 47 °C, 3 °C lower than in its absence (compare Fig. 6, A and B). Thus, molybdate seems to act by favoring a dimeric structure of Hsp90 more prone to oligomerization in such a way that oligomerization of the protein is induced at even lower temperatures. The same effect was observed with vanadate, another modulator of Hsp90 interactions. As shown in Fig. 7, at the nonpermissive temperature of 49 °C, where Hsp90 is stable as a dimer (lane 0), the presence of molybdate (Fig. 7A) or vanadate (Fig. 7B) induced oligomerization of the protein. Concomitantly to this oligomerization, however, Hsp90 species migrating faster than the dimer were also detected (see arrows in Fig. 7). Such faster migrating species behave in native PAGE similarly to Hsp90 mutants, with a very short deletion in the C-terminal region that cannot dimerize (28).


View larger version (72K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of molybdate on the critical temperature of Hsp90 heat-induced oligomerization. Hsp90 previously incubated for 90 min at 4 °C in the absence (A) or presence of molybdate (20 mM) (B) was induced to oligomerize by incubation for 20 min at different temperatures, and oligomers were detected by native PAGE and silver staining.


View larger version (71K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of concentrations of transition metals on Hsp90 heat-induced oligomerization. Hsp90 samples preincubated for 90 min at 4 °C with the indicated concentration of molybdate (A) or vanadate (B) were induced to oligomerize by heating at 49 °C for 20 min and then analyzed by native PAGE. The arrows indicate the appearance of monomeric species.

Two Events in Heat-induced Oligomerization of Hsp90

Formation of Hsp90 Monomers-- Because oligomers seem to be composed of even- and odd-numbered species and because, in the presence of transition metal oxyanions, Hsp90 species migrating faster than the dimer appeared, it was important to know whether these fast migrating species were the result of Hsp90 degradation at high temperatures, or of the dissociation of dimers into monomers. As shown in Fig. 8, B and C, immunoblots of Hsp90 treated with vanadate and incubated at 49 °C, using two antibodies directed against N- and C-terminal Hsp90 epitopes, reveal intact monomeric Hsp90. Furthermore, size exclusion chromatography (Fig. 8D) shows not only the presence of high molecular mass oligomers and the dimer, as expected, but also a shoulder corresponding to the monomeric form of Hsp90, confirming the tendency of the protein to dissociate into monomers under the conditions used. This is in agreement with the fact that in native PAGE, in addition to dimers, tetramers, and hexamers, odd-numbered oligomers, such as trimers and pentamers, are observed, indicating the formation of monomeric species during heat-induced oligomerization.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 8.   Hsp90 monomer formation during heat-induced oligomerization. Hsp90 samples preincubated for 90 min at 4 °C with different concentrations of vanadate (40, 30, and 14 mM, lanes 1-3, respectively) were induced to oligomerize by incubation at 50 °C and analyzed by native PAGE and Western blot. Silver staining (A) and Western blots with antibodies against the N-terminal (B) or C-terminal (C) region of Hsp90 were performed. D, elution profiles at 280 nm of Hsp90 samples (0.175 mg/ml) analyzed on Superose 12 after incubation for 90 min at 4 °C with vanadate (30 mM) followed by incubation at 37° C (- - - - -) or 50 °C (------).

Self-association of Hsp90 Dimers-- To know whether transient monomeric species may be detected in the absence of molybdate and vanadate as soon as oligomer formation occurs, a time course analysis of Hsp90 heat-induced oligomerization has been performed. As shown in Fig. 9, whether at 50 or at 55 °C, where oligomers begin to appear at 4 min at 50 °C and as early as 1 min at 55 °C, monomers could not be detected. The predominance of even-numbered oligomers at the beginning of the time course suggests that the kinetically preferred process is the self-association of dimeric species. Nevertheless, the progressive increase of odd-numbered oligomers at a later stage, so that even- and odd-numbered oligomers become almost equally represented by 20 min, still argues in favor of a slower heat-induced reaction consisting in dissociation of dimers and assembly of monomers onto larger species.


View larger version (106K):
[in this window]
[in a new window]
 
Fig. 9.   Time course of Hsp90 heat-induced oligomerization. Hsp90 samples were induced to oligomerize at 50 °C (A) or 55 °C (B) for 0-20 min. At the indicated times, the degree of oligomerization was assessed by native PAGE analysis.


    DISCUSSION

The results presented here describe the heat-induced oligomerization properties of Hsp90 and the influence of specific ligands (ATP, ADP, and geldanamycin), transition metal oxyanions (molybdate and vanadate), and the detergent Nonidet P-40 on such properties.

Properties of Heat-induced Oligomerization of Hsp90-- It appears that at the critical temperature of 50 °C, Hsp90 oligomerizes in a well ordered fashion into several species going from trimers to high molecular mass oligomers. The fact that the extent of Hsp90 oligomerization depends on protein concentration at and above the critical temperature, indicates that this oligomerization is induced by temperature and not by protein concentration. Because at low temperatures Hsp90 is stable as a dimer, its rapid disappearance above 50 °C to form oligomers may be the result of denaturation of the protein. In fact, it has already been suggested that thermal unfolding occurs around these temperatures leading to aggregation of the protein (30, 39). A disordered process of aggregation of Hsp90 under the conditions used in this work can be ruled out because formation of oligomers is an extremely ordered process, giving rise to well defined molecular mass species, as shown by the resolution of the different oligomeric species in native PAGE. Moreover, high molecular mass oligomers do not precipitate but remain in solution, as shown by gel filtration chromatography. Most importantly, formation of oligomers at high temperatures is sensitive to specific ligands modulating Hsp90 function, which either promote or inhibit this process. Thus, heat-induced oligomerization of Hsp90 may be regarded as an intrinsic and specific feature of this protein.

Because Hsp90 is known to be a stable dimer resistant to dissociation by drastic conditions, such as high salt concentration and chaotropic agents (25), the formation of trimers and pentamers during self-association of the protein was surprising and implied the formation of monomeric species. In fact, molecular forms comprising monomers, dimers, trimers, and tetramers have also been found with Hsp90 purified from Neurospora crassa cultures incubated at 48 °C (40). Although monomeric species were not detected when Hsp90 was induced to oligomerize alone at high temperatures, these forms appeared when Hsp90 was induced to oligomerize after incubation with transition metal oxyanions, suggesting that oligomer formation seems to be, at least in part, associated with a concomitant formation of Hsp90 monomer. Nevertheless, this dissociation is not the key event in Hsp90 oligomerization, because in the first minutes of the process, even-numbered oligomers are the main species observed; the odd-numbered oligomers only appear later. This is in agreement with previous data indicating that within minutes, Hsp90 oligomerizes into tetramers and hexamers at high temperatures (33). Furthermore, the even-numbered species seem to be active in binding and chaperoning unfolded proteins (33).3 Altogether, these observations suggest that heat-induced oligomerization of Hsp90 is the result of self-association of dimeric species and that formation of monomers and their subsequent incorporation into oligomers is a secondary event.

Because Hsp90 is stable as a dimer even at high concentrations, a conformational change appears to be necessary for oligomerization to occur at the critical temperature. This conformational change should lead to the unmasking, in the dimer at high temperatures, of an oligomerization site that is absent in the dimer at low temperatures, giving rise to new protein-protein interactions and new quaternary structures. The fact that the degree of oligomerization is concentration-dependent at the critical temperature suggests that at high temperatures, oligomers growing up as the Hsp90 concentration increases hold out clusters of sites that are more effective at binding and chaperoning. Whether the oligomerization site(s) is directly involved in chaperoning remains to be determined.

Because this oligomerization occurs at high temperatures, hydrophobic interactions may be involved. This is in agreement with previous observations indicating that temperature increase leads to an increase in surface hydrophobicity of Hsp90 (41) and with a recent report on glucose-regulated protein 94 suggesting a temperature-dependent conformational change accompanied by enhanced solvent accessibility to a hydrophobic binding site (42).

Effects of Specific Ligands on Hsp90 Heat-induced Oligomerization-- In this work, modulators of Hsp90 function were found to interfere with heat-induced self-association of this protein. Geldanamycin, ATP, and ADP appear to be inhibitors of the process by stabilizing a dimeric structure of Hsp90 less prone to oligomerization. This is suggested by the fact that higher temperatures are needed for oligomerization when Hsp90 has been preincubated with these ligands. This interpretation is in agreement with a recent report showing that the melting temperature of the Hsp90 is shifted to a higher temperature in the Hsp90-geldanamycin complex relative to Hsp90 alone (43).

On the contrary, molybdate and vanadate seem to lower the energy required for oligomer formation in such a way that heat-induced oligomerization of Hsp90 occurs at lower temperatures. The transition metal oxyanions may act by favoring a dimeric state of Hsp90 that is more prone to heat-induced oligomerization. Increase in surface hydrophobicity of Hsp90 is likely to be involved in this facilitated heat-induced formation of oligomeric species, because Nonidet P-40, which has similar effects on Hsp90 oligomerization, is also known to increase its surface hydrophobicity (32). This is in agreement with a recent report indicating that in the presence of ATP, Hsp90 becomes more hydrophilic, whereas in the presence of molybdate or Nonidet P-40, it is more hydrophobic (13).

In conclusion, the fact that heat-induced oligomerization of Hsp90 is sensitive to modulators, such as nucleotides and transition metal oxyanions, that are known to affect Hsp90 function suggests a biological role for oligomerization. In fact, it has been previously shown that Hsp90 oligomers display a chaperone activity at high temperatures (33), and Hsp90 oligomers seem to be formed in vivo at heat shock temperature in N. crassa (40). Whether they are formed and display a chaperone function in higher eukaryotes during heat shock remains to be determined. Because many cellular factors and other chaperones modulate Hsp90 function (10, 12), it is plausible that some of them may modulate in vivo the critical temperature of Hsp90 oligomerization to a level compatible with cell survival. Indeed, the requirement of very high concentrations of Hsp90 for yeast viability at extreme temperatures (6) may be related to the fact that such high levels favor efficient oligomers formation, which in turn may protect cellular proteins against inactivation. Similarly to Hsp70, Hsp27, and heat shock factor, for which it has been proposed that oligomerization may be involved in their function (44-46), Hsp90 may use oligomerization to trigger functions that are normally silent at physiological temperature and that counteract the deleterious effects of extreme temperatures.

    ACKNOWLEDGEMENTS

We thank M. Morange, E. Hack, K. Rajkowski, and M. Schumacher for their thoughtful comments on the manuscript and M. A. Teillet for gift of chicken embryo.

    FOOTNOTES

* This work was supported by INSERM and by grants from Association pour la Recherche sur le Cancer, Ligue contre le Cancer, and Fondation pour la Recherche Médicale (to M. G. C. and M. M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by Ligue contre le Cancer (Comité de l'Indre) and Societé de Secours des Amis des Sciences fellowships.

** To whom correspondence should be addressed. Tel.: 33-1-44-41-23-63/90; Fax: 33-1-44-41-43-92; E-mail: Catelli{at}icgm.cochin.inserm.fr.

The abbreviations used are: Hsp, heat shock protein; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

2 M. G. Catelli, unpublished results.

3 A. Chadli, unpublished results.

    REFERENCES
Top
Abstract
Introduction
References

  1. Morimoto, R. I., Tissieres, A., and Georgopoulos. (1990) in Stress Proteins in Biology and Medicine (Morimoto, R. I., Tissieres, A., and Georgopoulos, C., eds), pp. 1-36, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  2. Morimoto, R. I., Tissieres, A., and Georgopoulos, C. (1994) in The Biology of Heat Shock Proteins and Molecular Chaperones (Morimoto, R. I., Tissieres, A., and Georgopoulos, C., eds), pp. 1-30, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  3. Parsell, C. S., and Lindquist, S. (1993) Annu. Rev. Genet. 27, 437-496[CrossRef][Medline] [Order article via Infotrieve]
  4. Gething, M. J., and Sambrook, J. (1992) Nature 355, 33-45[CrossRef][Medline] [Order article via Infotrieve]
  5. Ellis, R. J. (1996) Cell Stress Chaperones 1, 155-160[Medline] [Order article via Infotrieve]
  6. Borkovich, K. A., Farrelly, F. W., Finkelstein, D. B., Taulien, J., and Lindquist, S. (1989) Mol. Cell. Biol. 9, 3919-3930[Medline] [Order article via Infotrieve]
  7. Cutforth, T., and Rubin, G. M. (1994) Cell 77, 1027-1036[Medline] [Order article via Infotrieve]
  8. Nathan, D. F., Harju Vos, M., and Lindquist, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12949-129956[Abstract/Free Full Text]
  9. Pratt, W. B. (1997) Annu. Rev. Pharmacol. Toxicol. 37, 297-326[CrossRef][Medline] [Order article via Infotrieve]
  10. Pratt, W. B., and Toft, D. (1997) Endocr. Rev. 18, 306-360[Abstract/Free Full Text]
  11. Nathan, D. F., and Lindquist, S. (1995) Mol. Cell. Biol. 15, 3917-3925[Abstract]
  12. Bohen, S. P., and Yamamoto, K. R. (1994) in The Biology of Heat Shock Proteins and Molecular Chaperones (Morimoto, R. I., Tissieres, A., and Georgopoulos, C., eds), pp. 313-334, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  13. Sullivan, W., Stensgard, B., Caucutt, G., Bartha, B., McMahon, N., Alnemri, E., Litwack, G., and Toft, D. (1997) J. Biol. Chem. 272, 8007-8012[Abstract/Free Full Text]
  14. Miyata, Y., and Yahara, I. (1992) J. Biol. Chem. 267, 7042-7047[Abstract/Free Full Text]
  15. Wiech, H., Buchner, J., Zimmermann, R., and Jakob, U. (1992) Nature 358, 169-170[CrossRef][Medline] [Order article via Infotrieve]
  16. Buchner, J. (1996) FASEB J. 10, 10-26[Abstract/Free Full Text]
  17. Prodromou, C., Roe, S. M., O'Brien, R., Ladbury, J. E., Piper, P. W., and Pearl, L. H. (1997) Cell 90, 65-75[Medline] [Order article via Infotrieve]
  18. Stebbins, C. E., Russo, A. A., Schneider, C., Rosen, N., Hartl, U. F., and Pavletich, N. (1997) Cell 89, 239-250[Medline] [Order article via Infotrieve]
  19. Segnitz, B., and Gehring, U. (1997) J. Biol. Chem. 272, 18694-18701[Abstract/Free Full Text]
  20. Young, J. C., Schneider, C., and Hartl, F. U. (1997) FEBS Lett. 418, 139-143[CrossRef][Medline] [Order article via Infotrieve]
  21. Scheibel, T., Weikl, T., and Buchner, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1495-1499[Abstract/Free Full Text]
  22. Panaretou, B., Prodomou, C., Rose, S. M., O'Brien, R., Ladbury, J. E., Piper, P. W., and Pearl, L. H. (1998) EMBO J. 17, 4829-4836[Abstract/Free Full Text]
  23. Welch, W. J., and Feramisco, J. R. (1982) J. Biol. Chem. 257, 14949-14959[Abstract/Free Full Text]
  24. Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8054-8058[Abstract]
  25. Radanyi, C., Renoir, J. M., Sabbah, M., and Baulieu, E. E. (1989) J. Biol. Chem. 264, 2568-2573[Abstract/Free Full Text]
  26. Wearsch, P. A., and Nicchitta, C. N. (1996) Biochemistry 35, 16760-16769[CrossRef][Medline] [Order article via Infotrieve]
  27. Minami, Y., Kawasaki, H., Miyata, Y., Suzuki, K., and Yahara, I. (1991) J. Biol. Chem. 266, 10099-10103[Abstract/Free Full Text]
  28. Meng, X., Devin, J., Sullivan, W. P., Toft, D., Baulieu, E. E., and Catelli, M. G. (1996) J. Cell Sci. 109, 1677-1687[Abstract/Free Full Text]
  29. Nemoto, T., and Sato, N. (1998) Biochem. J. 330, 989-995[Medline] [Order article via Infotrieve]
  30. Jakob, U., Meyer, I., Bugl, H., André, S., Bardwell, J. C. A., and Buchner, J. (1995) J. Biol. Chem. 270, 14412-14419[Abstract/Free Full Text]
  31. Lanks, K. W. (1989) J. Cell. Physiology 140, 601-607[Medline] [Order article via Infotrieve]
  32. Lanks, K. W., London, E., and Long-Yu, D. (1992) Biochem. Biophys. Res. Commun. 184, 394-399[Medline] [Order article via Infotrieve]
  33. Yonehara, M., Minami, Y., Kawata, Y., Nagai, J., and Yahara, I. (1996) J. Biol. Chem. 271, 2641-2645[Abstract/Free Full Text]
  34. Joab, I., Radanyi, C., Renoir, J. M., Buchou, T., Catelli, M. G., Binart, N., Mester, J., and Baulieu, E. E. (1984) Nature 308, 850-853[Medline] [Order article via Infotrieve]
  35. Cornillot, J. D., Caron, M., Joubert-Caron, R., and Bladier, D. (1992) Int. J. Biochem. 24, 1585-1589[Medline] [Order article via Infotrieve]
  36. Sabbah, M., Radanyi, C., Redeuilh, G., and Baulieu, E. E. (1996) Biochem. J. 314, 205-213[Medline] [Order article via Infotrieve]
  37. Lees-Miller, S. P., and Anderson, C. W. (1989) J. Biol. Chem. 264, 2431-2437[Abstract/Free Full Text]
  38. Soti, C., Radics, L., Yahara, I., and Csermely, P. (1998) Eur. J. Biochem. 255, 611-617[Abstract]
  39. Scheibel, T., Neuhofen, S., Weikl, T., Mayr, C., Reinstein, J., Vogel, P. D., and Buchner, J. (1997) J. Biol. Chem. 272, 18608-18613[Abstract/Free Full Text]
  40. Freitag, D. G., Ouimet, P. M., Girvitz, T. L., and Kapoor, M. (1997) Biochemistry 36, 10221-10229[CrossRef][Medline] [Order article via Infotrieve]
  41. Yamamoto, M., Takahashi, Y., Inano, K., Horigome, T., and Sugano, H. (1991) J. Biochem. 110, 141-145[Abstract]
  42. Wearsch, P. A., Voglino, L., and Nicchitta, C. V. (1998) Biochemistry 37, 5709-5719[CrossRef][Medline] [Order article via Infotrieve]
  43. Garnier, C., Protasevich, I., Gilli, R., Tsvetkov, P., Lobachov, V., Peyrot, V., Briand, C., and Makarov, A. (1998) Biochem. Biophys. Res. Commun. 249, 197-201[CrossRef][Medline] [Order article via Infotrieve]
  44. Benaroudj, N., Triniolles, F., and Ladjimi, M. (1996) Biochemistry 34, 15282-15290
  45. Ehrnsperger, M., Gräber, S., Gaestel, M., and Buchner, J. (1997) EMBO J. 16, 221-229[Abstract/Free Full Text]
  46. Baler, R., Welch, W. J., and Voellmy, R. (1992) J. Cell Biol. 117, 1151-1159[Abstract]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.