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J. Biol. Chem., Vol. 279, Issue 36, 37298-37303, September 3, 2004
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From the
Départment de Biologie Moléculaire Végétale, Université de Lausanne, CH-1015 Lausanne, Switzerland, Laboratoire de Biophysique Statistique, ITP-SB, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland, and ¶Laboratoire de Physique de la Matière Vivante, IPMC-SB, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
Received for publication, May 20, 2004 , and in revised form, June 14, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Hsp70 is composed of two major domains: an actin-like N-terminal ATPase domain and a C-terminal domain containing a substrate-binding cleft covered by a flexible lid. The substrate-binding domain of bacterial Hsp70 (DnaK) can typically bind short hydrophobic motives flanked by positive charges in extended peptides in partially unfolded or misfolded proteins (4–6). Hsp70 alternates between an ADP-bound substrate-bound locked state and an ATP-bound substrate-bound unlocked state (7). ATP hydrolysis, which is catalyzed by co-chaperone Hsp40 (DnaJ in bacteria), induces the tight closure (locking) of the lid and the transient increase of the chaperone affinity for the polypeptide substrate (8). ADP release from Hsp70, which may be activated by the co-chaperone GrpE in bacteria, induces the opening (unlocking) of the lid and dissociation of the Hsp70 from the substrate (7, 9).
The molecular mechanism by which a highly conserved molecule such as Hsp70 may perform a wide array of apparently dissimilar cellular functions remains unclear. Despite progress in understanding the Hsp70 cycle (7), it is not known how ATP-fueled alternate cycles of Hsp70 binding/release to and from misfolded or partially unfolded proteins can actively translocate polypeptides into organelles, drive the dissociation of native oligomers, and solubilize stable protein aggregates into native proteins (10). Here, we used sedimentation, fluorescence spectroscopy, and kinetic analysis to address the molecular mechanism by which the Escherichia coli Hsp70 (DnaK) and its co-chaperones, DnaJ and GrpE, can mediate the active solubilization and refolding of stable heat-generated protein aggregates. Our data fit a mechanism whereby local unfolding of exposed protein segments in misfolded aggregates is achieved by independent random motions of several individual DnaK molecules tightly bound to the same misfolded polypep-tide. Upon nucleotide and chaperone release, unfolded protein segments may properly refold, preventing chaperone rebinding, or improperly refold, allowing chaperone rebinding. Such iterative cycles of local unfolding by bound chaperones and spontaneous local refolding upon chaperone release may gradually solubilize and lead to the stepwise refolding of stable misfolded aggregates into native proteins.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Chaperone Reactivation Assays—Chaperone-mediated reactivation assays were carried at 25 °C in the presence of chaperones as indicated in buffer A supplemented by 4 mM ATP and an ATP regeneration system (5 mM phosphoenol pyruvate and 20 ng/ml pyruvate kinase). The enzymatic activity of G6PDH was measured at 25 °C as described previously (11). All of the protein concentrations are expressed in protomers, regardless of the oligomeric state of the chaperones or the aggregates. Chaperone/substrate ratios are protomer ratios.
Unfolding Measurements—The time-dependent unfolding of pre-aggregated G6PDH in the presence of chaperones as in Fig. 1a and of Thioflavin-T (10 µM) was monitored continuously for 1 h at 25 °C in a PerkinElmer Fluorometer (LS50B) (excitation at 400 nm and detection at 480 nm).
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Solubility Fractionation by Sedimentation—Solubility fractionation of protein aggregates was evaluated from the quantity of proteins present in the soluble fraction after a 10-min centrifugation at the indicated velocities (4,000–228,000 x g) using a Beckman ultracentrifuge with a TLA-100.2 fixed angle rotor. Soluble proteins were resolved by SDS-gel electrophoresis, and the amounts of soluble proteins were determined as above. The results in Fig. 2, a and b, are expressed as the fraction (%) of soluble protein after centrifugation compared with the total protein amount before centrifugation as described previously (11).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We next addressed the possibility that the cooperative behavior of DnaK in active aggregate refolding is carried by a discrete DnaK oligomer in which the DnaK monomers are directly associated and influence each other's activity. Hence, to preserve the stability of a presumed chaperone oligomer, we kept the chaperone concentration high and constant and the specific refolding activity was measured in the presence of decreasing concentrations of the substrate (Fig. 1b). Whereas in the absence of chaperones or ATP dilution had no effect on the stability and reactivity of the aggregates (10, 11), the presentation of diluted aggregates to a constant high concentration of DnaK (and of DnaJ, GrpE, ClpB, and ATP) resulted in a net increase of the chaperone-specific refolding activity. At equimolar (protomer to protomer) amounts of chaperone/substrate, the specific refolding activity was very low but it became optimal when the molar ratio was restored by aggregate dilution to a 5-fold chaperone excess (Fig. 1b). This behavior is contrary to a classic enzymatic reaction, in which substrate dilution may only maintain or reduce the specific activity of a fixed concentration of enzyme. The observed increase in the chaperone-specific activity could not be attributed to dilution-induced changes in the substrate, as heat-denatured G6PDH aggregates are not dynamic (10, 11). Rather, the specific refolding activity of the chaperone appeared to depend on its ratio with the substrate. Regardless of the absolute concentrations, a constant molar excess of chaperones over the substrate was essential for effective native refolding. These observations speak against an active DnaK oligomer in which DnaK subunits are in direct contact and functionally cooperate with one another as in the case of the dilution-sensitive ClpB6 and GroEL14 oligomers. At the same time, the dependence of the specific refolding activity on a molar excess of chaperone over the substrate clearly confirms that the cooperative action of several Hsp70 molecules is necessary for the active conversion of stable misfolded complexes into correctly refolded proteins. Because proteins contain hydrophobic segments on average every 30–40 residues to which DnaK can specifically bind (4), it is possible that several individual DnaK monomers can independently bind at different sites on the same misfolded polypeptide. Our results indicate that, by sharing a common polypeptide substrate, several DnaK molecules may cooperate in the active unfolding of the substrate without having necessarily to form a discrete chaperone oligomer.
Disaggregation Activity depends on the size of the aggregates—We next addressed the kinetics of two DnaK-dependent activities that precede chaperone-mediated protein refolding: active solubilization and unfolding of the stable protein aggregates. Sedimentation analysis at different velocities allowed us to follow in time the changes in the solubility profile of the aggregates at different size ranges following treatment with different DnaK concentrations. As expected for stable aggregates that are not in dynamic equilibrium, the distribution of aggregate sizes remained unchanged within 24 h following extensive dilutions or in the absence of chaperones (10, 11). Yet, a 150-min incubation with equimolar DnaK (ATP and constant co-chaperones) resulted in a significant solubilization (30–40%) of the aggregates in all of the size ranges (Fig. 2a) albeit without the recovery of active G6PDH enzymes. Gel-exclusion chromatography confirmed that partial solubilization of the aggregates took place with a significant increase of smaller inactive species, particularly species eluting at the same position as the native dimer and at a position expected for inactive monomer (double and single arrows, respectively, Fig. 2a, inset). Therefore, in the presence of equimolar amounts of DnaK (ATP and co-chaperones), large aggregates were actively fragmented and accumulated as smaller species, possibly mis-folded dimers and monomers, which were not converted further into native dimers (Fig. 1a).
In an attempt to visualize in time the DnaK-mediated dis-aggregation, we used fractionation by sedimentation at various velocities (Fig. 2b). Under treatment with a constant equimolar concentration of DnaK (and of co-chaperones and ATP), a fraction corresponding to very large aggregates (sedimenting at the lowest speed, 14,000 x g) readily became soluble. In contrast, another fraction corresponding to medium size aggregates (sedimenting at 128,000 x g) became soluble only after 40 min. The smallest aggregates, sedimenting at 228,000 x g, became more soluble only following 2 h of active chaperone treatment (Fig. 2b). This corroborates our observation that, with equimolar DnaK, small aggregates form and accumulate but do not refold (Figs. 1a and 2a) and demonstrates that the specific disaggregation activity of DnaK decreases with the size of the aggregates.
A DnaK dose-response analysis of the rates of aggregate solubilization confirmed that different degrees of cooperativity between the DnaK molecules were needed for the effective solubilization of different oligomeric states of the substrate (Fig. 2c, inset). Whereas sub-stoichiometric amounts of chaperones could optimally fragment the largest aggregates into smaller ones, the solubilization of smaller aggregates required increasing molar excess amounts of the chaperone over the substrate (Fig. 2c). This strongly indicates that the same chaperone mechanism is responsible for disaggregation and refolding, although increasingly cooperativity between Hsp70 molecules is needed to complete the reaction as the oligomeric state of the aggregates decrease. The highest molar excess of chaperones (EC50 
5) was needed to carry out the final stage of the reaction corresponding to the active conversion of stable mis-folded monomers into natively refoldable ones.
The ability of the chaperone to discriminate between small and large aggregates explains the accumulation of small inactive species observed when DnaK was at limiting concentrations. Although, during the initial phases of the reaction, the largest aggregates were first efficiently fragmented, in the later phases of the reaction, smaller aggregates were or were not further converted into smaller ones because of the size-dependent decrease in the chaperone efficiency. Increasing chaperone amounts could compensate for the size-dependent decrease in DnaK efficiency and allow the reaction to reach its final outcome of correct proper refolding.
DnaK Actively Unfolds Misfolded Structures in Aggregates— The fact that, by an ATP-dependent mechanism, DnaK can actively convert stable aggregated structures into alternatively stable native structures already suggests that the chaperone is mainly involved in unfolding misfolded proteins (1). Here, we directly demonstrated the unfolding activity for DnaK using fluorescence spectroscopy in the presence of Thioflavin-T, a dye that specifically binds 
-sheet structures, which are enriched in misfolded and aggregated proteins (12). Under the tested conditions, heat-aggregated G6PDH (1 µM) (as in Fig. 1a) bound 75% more Thioflavin-T (10 µM) than native G6PDH, confirming that the aggregated form contains a higher amount of amphiphilic intramolecular and intermolecular -sheet structures, as in the case of amyloid aggregates and prion particles (12). In the presence of a 9.4 molar excess of DnaK (with constant DnaJ and GrpE and ATP), a rapid time-dependent decrease of Thioflavin-T binding was observed in the aggregates (Fig. 3a) but not in the native controls or when the chaperone, co-chaperone, or ATP was omitted (data not shown). Moreover, indicating that DnaK can unfold a wide array of misfolded substrates, we found that an excess of DnaK (with co-chaperones and ATP) strongly decreased the signal of bound Thioflavin-T and that at the same time it increased the solubility of heat-aggregated proteins from a total E. coli extract (data not shown).
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The Role of Co-chaperones in Unfolding and Disaggregation—We next addressed the role of DnaJ, which accelerates ATP hydrolysis in DnaK and the locking of DnaK onto its substrate, and that of GrpE, which accelerates ADP release from DnaK and the release of DnaK from its substrate (7, 8), in the productive refolding of stable protein aggregates. A DnaJ dose response on the rates of DnaK-mediated refolding of stable aggregates (as in Fig. 1) was performed without and with GrpE (Fig. 4). Surprisingly, our stringent in vitro assay of DnaK-DnaJ-mediated reactivation of heat-preaggregated G6PDH was found to be active, even in the absence of GrpE albeit at slightly lower rates but not in the absence of DnaJ (Fig. 4). Therefore, ADP release from DnaK and the unlocking of DnaK from its substrate can occur spontaneously, although by accelerating ADP release, GrpE seems to optimize DnaK as an active unfolding machine. In contrast, transient DnaJ-mediated locking with very high affinity of DnaK onto its substrate is central to the unfolding, disaggregation, and refolding activities of the chaperone.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We found here that the unfolding of misfolded structures is a new ATP-dependent catalytic activity that is to be ascribed to Hsp70 chaperones. Alternating cycles of Hsp70 binding, active unfolding, and chaperone release can mediate the ATP-consuming conversion of a stable misfolded aggregated state into a transient unstable state (a partially unfolded state), which then may spontaneously acquire an alternatively stable state (the native state).
Unfolding, disaggregation, and reactivation exhibited different optima in terms of Hsp70-specific activity. This differential behavior indicated that, while only a minimal amount of optimally efficient unfolding events may readily lead to the fragmentation of large aggregates into medium-sized aggregates, the subsequent fragmentation of medium-sized aggregates into the smaller ones are gradually less efficient. Finally, we found that efficient unfolding of the smallest aggregate species into a form that can natively refold, requiring a concerted unfolding action carried by several chaperone molecules per substrate.
DnaK is known to preferentially bind unstructured peptide segments with a hydrophobic core of 4–5 residues flanked by basic residues (4), which are normally buried in native proteins. Because in aggregates some hydrophobic segments participate in oligomeric interactions, the relative amount of the exposed available DnaK-binding sites is expected to decrease proportionally to the size of the aggregate. Yet, here we found that the specific disaggregation activity of Hsp70 increased with the size of the aggregate, suggesting that the size of the aggregates plays a predominant role in the mechanism of Hsp70-mediated unfolding despite better sequestration of potential Hsp70-binding sites in larger aggregates.
Cooperative Unfolding Can Be Achieved by Individual Hsp70 Molecules—The chaperonin oligomers act as power-stroke machines in which several GroEL subunits cooperate at binding and disrupting misfolded structures within a bound substrate polypeptide, leading upon release to the proper refolding of the latter (16). We addressed the possibility that a similar mechanism is carried by a cooperative Hsp70 oligomer. However, incompatible with a mechanism involving a discrete oligomer, we found that the specific unfolding, disaggregation, and refolding activities of DnaK depended on different degrees of cooperativity among chaperone molecules, on the size of the substrate, and on the chaperone/substrate ratio. In a discrete active oligomer, the specific disaggregation activity should only depend on the affinity to the substrate and on the absolute chaperone concentration but not on the chaperone/substrate ratio nor on the size of the aggregate as in our findings.
Rather, cooperative unfolding of aggregates may be achieved by a different type of cooperative Hsp70 complex in which individually bound Hsp70 molecules indirectly cooperate with each other by a common misfolded-polypeptide substrate that they may share. Such a situation has been already suggested in the case of protein import into the mitochondria in which the cooperative ratchet action of several individual Hsp70 molecules that bind to the same imported polypeptides may produce a net unidirectional protein transport (15).
Our observation is in agreement with most in vitro studies where Hsp70 was monomeric in the presence of ATP (17, 18). Moreover, there is no structural evidence that Hsp70 molecules can form discrete functional oligomers with or without Hsp40 that would be able to bind ends of a misfolded polypep-tide and apply unfolding force upon them by a power-stroke. Although in Thermus thermophilus some DnaK is found associated in a trigonal complex with DnaJ and the assembly factor TdafA, the active form of the chaperone is the free DnaK monomer when the trigonal complexes are dissociated at physiological 55 °C (18).
The Role of Co-chaperones Supports Unfolding by Chaperone Random Motions—We found that both DnaJ and GrpE act as catalysts of the chaperone functional cycle by regulating the time DnaK spends in the tightly bound state during which productive unfolding by random motions may take place and by regulating the time DnaK is in the unbound state during which productive spontaneous local refolding may take place (Fig. 4). This is further demonstrated at higher concentrations of DnaJ (0.175:1 DnaJ:DnaK) where there is an inhibition of the chaperone activity, most likely because of a delay in DnaK release. By inducing ADP release from DnaK, GrpE was shown to mediate the dissociation of the chaperone from its substrate (9). As expected from a co-chaperone that mediates the opposite effect of DnaJ, a constant physiological ratio of GrpE inhibited both the half-activatory effect (EC50) and the half-inhibitory effect (IC50) of DnaJ (the EC50 value in DnaJ/DnaK ratio changed from 0.014 to 0.028 and the IC50 value changed from 0.175 to 0.645, Fig. 4), implying that overdue DnaK binding can be alleviated by GrpE and confirming its activity as a chaperone release factor.
The role of DnaJ and GrpE, respectively, catalyzing the tight locking of DnaK and its subsequent release to and from the misfolded substrate is fully compatible with an unfolding mechanism based on the random motions of individual DnaK molecules bound to a common misfolded substrate. By mediating the tight locking of DnaK onto the substrate, DnaJ can harness the energy from ATP hydrolysis to an efficient pulling force to be exerted by the random movements of tightly locked DnaK molecules. DnaK unlocking and dissociation, allowing the spontaneous local refolding of the substrate, are greatly optimized by GrpE, especially in the presence of high physiological concentrations of DnaJ, which would otherwise retard chaperone release (Fig. 5a).
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The Cooperative Unfolding Cycle of Hsp70 —The ability of Hsp70 to cycle between the two states by ATP hydrolysis (unlocked-to-locked) and ADP release (locked-to-unlocked) is key for its function as a cooperative protein-unfolding machine. In agreement with previous studies (7), our data suggest that the active chaperone cycle of Hsp70 involves the following steps (Fig. 5a): 1) weak binding of Hsp70-ATP to an exposed hydro-phobic motif in a misfolded polypeptide; 2) DnaJ-activated ATP hydrolysis, causing tight locking of Hsp70 onto the misfolded polypeptide; 3) unfolding of the bound segment by random movements of Hsp70; 4) GrpE-activated ADP release, causing the unlocking of Hsp70 from the unfolded segment; and 5) spontaneous refolding of the unfolded segment into a more native structure for which Hsp70 has a lower affinity. Hsp70 may then rebind to another misfolded and exposed hydrophobic motive in the same polypeptide, thus gradually unfolding and correctly refolding various misfolded regions in the aggregate. Initial unfolding can lead to polypeptide disentanglement from the aggregate. In the last step of the unfolding reaction, several Hsp70 molecules are needed to bind to the same misfolded polypeptide because only the cumulative, independent motions of several bound Hsp70 molecules randomly moving in opposite directions can sum up in the effective unfolding of single polypeptide into a form that can spontaneously refold into a native protein (Fig. 5b). Alternatively, it is possible that the last steps of the unfolding reaction may still be carried out by a minority of Hsp70 molecules when the terminal-misfolded species are maintained in a complex with other power-stroke chaperones such as ClpB (20) and GroEL (21) or holding chaperones such as small heat shock proteins (22).
Implications for Pathological Aggregates—In the cytoplasm of mammalian cells, there are no ClpB and GroEL homologues that can increase the efficiency of Hsp70 and the entire burden of aggregate detoxification must relay on Hsp70. Because Hsp70 molecules are needed to complete solubilization and refolding (Fig. 1) (1), it is essential that mammalian cells anticipate the synthesis of an excess amount of the chaperone prior to the formation of pathological aggregates. This is normally achieved by competition between heat shock factors and the Hsp70 substrates (23). However, when heat shock factor-induced chaperone synthesis is impaired as in aging or when misfolded species form too rapidly as a result of stress or mutations, pathological accumulations of misfolded species may occur.
The pathology of misfolding diseases indicates that the smallest misfolded species are more toxic (24, 25). Our results describe a situation in which insufficient amounts of Hsp70 in the cell may convert large inert aggregates into smaller, potentially more toxic and infectious particles without being able to further convert them into harmless native proteins. Therefore, the relative concentration of Hsp70 must exceed that of pathological aggregates for effective curing. Indeed, the overexpression of Hsp70 in several model systems has resulted in the clearance of pathological aggregates and the arrest of the symptoms (26). Therefore, understanding the mechanism of Hsp70 is crucial in the design of a strategy for safe and effective treatments of protein-misfolding diseases.
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FOOTNOTES |
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|| To whom correspondence should be addressed. Tel.: 41-21-692-4232; Fax: 41-21-692-4195; E-mail: Pierre.Goloubinoff{at}ie-bpv.unil.ch.
1 The abbreviation used is: G6PDH, glucose-6-phosphate dehydrogenase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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