Induction of molecular chaperones in carbon tetrachloride-treated rat liver: implications in protection against liver damage

Carbon tetrachloride (CCl4) induces liver damage, apparently through the formation of free-radical metabolites. Molecular chaperones such as heat shock protein (Hsp) of 70 kDa have been found to protect cells from various stresses. We previously found that cytosolic chaperone pairs of the Hsp70 family and their DnaJ homolog cochaperones prevent nitric oxide-mediated apoptosis and heat-induced cell death. Expression of cytosolic chaperones, including Hsp70; heat shock cognate (Hsc) 70; and DnaJ homologs dj1 (DjB1/Hsp40/hdj-1), dj2 (DjA1/HSDJ/hdj-2), dj3 (DjA2), and dj4 (DjA4), in the liver of CCl4-treated rats was analyzed. Messenger ribonucleic acids for all these chaperones were markedly induced 3-12 hours after CCl4 treatment with a maximum at 6 hours. Hsp70 and dj1 proteins were markedly induced at 6-24 hours with a maximum at 12 hours, whereas dj2 and dj4 were moderately induced at around 12 hours. Hsc70 was weakly induced after treatment, and dj3 was little induced. To better understand the significance of the induction of chaperones, the effect of preinduction of chaperones on CCl4-induced liver damage was analyzed. When chaperones were preinduced in the liver by heat treatment, increase in serum alanine aminotransferase activity after CCl4 treatment was significantly attenuated. Hsp90, another major cytosolic chaperone, also was induced by heat treatment. On the other hand, Mn- and Cu/Zn-superoxide dismutase were not induced by heat treatment or by CCl4 treatment. These results suggest that cytosolic chaperones of Hsp70 and DnaJ families or Hsp90 (or both) are induced in CCl4-treated rat liver to protect the hepatocytes from the damage being inflicted.     

Defining the requirements for Hsp40 and Hsp70 in the Hsp90 chaperone pathway

The Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR), have been used extensively to study chaperone complex formation and maturation of a client substrate in a near native state. This chaperoning pathway can be reconstituted in vitro with the addition of five proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23. The addition of these proteins is necessary to reconstitute hormone-binding capacity to the immuno-isolated PR. It was recently shown that the first step for the recognition of PR by this system is binding by Hsp40. We compared type I and type II Hsp40 proteins and created point mutations in Hsp40 and Hsp70 to understand the requirements for this first step. The type I proteins, Ydj1 and DjA1 (HDJ2), and a type II, DjB1 (HDJ1), act similarly in promoting hormone binding and Hsp70 association to PR, while having different binding characteristics to PR. Ydj1 and DjA1 bind tightly to PR whereas the binding of DjB1 apparently has rapid on and off rates and its binding cannot be observed by antibody pull-down methods using either purified proteins or cell lysates. Mutation studies indicate that client binding, interactions between Hsp40 and Hsp70, plus ATP hydrolysis by Hsp70 are all required to promote conformational maturation of PR via the Hsp90 pathway.     

Novel inhibitors of heat shock protein Hsp70-mediated luciferase refolding that bind to DnaJ

Inhibitors of both heat shock proteins Hsp90 and Hsp70 have been identified in assays measuring luciferase refolding containing rabbit reticulocyte lysate or purified chaperone components. Here, we report the discovery of a series of phenoxy-N-arylacetamides that disrupt Hsp70-mediated luciferase refolding by binding to DnaJ, the bacterial homolog of human Hsp40. Inhibitor characterization experiments demonstrated negative cooperativity with respect to DnaJ and luciferase concentration, but varying the concentration of ATP had no effect on potency. Thermal shift analysis suggested a direct interaction with DnaJ, but not with Hsp70. These compounds may be useful tools for studying DnaJ/Hsp40 in various cellular processes.     

Characterization and functional analysis of a heart-enriched DnaJ/ Hsp40 homolog dj4/DjA4

DnaJ homologs are cochaperones of the heat shock protein 70 (hsp70) family. Homologs dj1 (hsp40/hdj-1/ DjB1), dj2 (HSDJ/hdj-2/rdj-1/DjA1), and dj3 (cpr3/DNAJ3/HIRIP4/rdj2/DjA2) have been identified in the mammalian cytosol and characterized. In this paper we characterized newly found dj4 (DjA4) and compared it with other chaperones. The dj4 messenger ribonucleic acid (mRNA) and protein were expressed strongly in heart and testis, moderately in brain and ovary, and weakly in other tissues in mice. Dj4 constituted about 1% of the total protein in heart. Testis gave extraspecies of dj4 mRNA and protein in addition to those seen in other tissues. On subcellular fractionation of the mouse heart, dj4 was recovered mostly in the cytosol fraction. In immunocytochemical analysis of the H9c2 heart muscle cells, dj4 and heat shock cognate 70 (hsc70) colocalized in the cytoplasm under normal conditions, whereas they colocalized in the nucleus after heat shock. When H9c2 cells were differentiated by culturing for up to 28 days with a lowered serum concentration, dj4 was increased markedly, dj3 was increased moderately, and dj1 and dj2 were little changed. The homolog dj4 as well as hsp70, dj1, and dj2 were induced in H9c2 cells by heat treatment at 43 degrees C for 30 minutes, whereas hsc70 and dj3 were not induced. Heat pretreatment promoted survival of cells after severe heat shock at 47 degrees C for 90 minutes or 120 minutes. H9c2 cells overexpressing hsp70 were more resistant to severe heat shock, and a better survival was obtained when dj4 or dj2 was co-overexpressed with hsp70. Taking a high concentration of dj4 in heart into consideration, these results suggest that the hsc70/hsp70-dj4 chaperone pair protects the heart muscle cells from various stresses.     

D-Peptides as inhibitors of the DnaK/DnaJ/GrpE chaperone system

DnaK, a Hsp70 homolog of Escherichia coli, together with its co-chaperones DnaJ and GrpE protects denatured proteins from aggregation and promotes their refolding by an ATP-consuming mechanism. DnaJ not only stimulates the gamma-phosphate cleavage of DnaK-bound ATP but also binds polypeptide substrates on its own. Unfolded polypeptides, such as denatured luciferase, thus form ternary complexes with DnaJ and DnaK. A previous study has shown that d-peptides compete with l-peptides for the same binding site in DnaJ but do not bind to DnaK (Feifel, B., Sch?nfeld, H.-J., and Christen, P. (1998) J. Biol. Chem. 273, 11999-12002). Here we report that d-peptides efficiently inhibit the refolding of denatured luciferase by the DnaK/DnaJ/GrpE chaperone system (EC50 = 1-2 microM). The inhibition of the chaperone action is due to the binding of d-peptide to DnaJ (Kd = 1-2 microM), which seems to preclude DnaJ from forming ternary (ATP.DnaK)m.substrate.DnaJn complexes. Apparently, simultaneous binding of DnaJ and DnaK to one and the same target polypeptide is essential for effective chaperone action.     

ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation. A novel multi-chaperone system from Escherichia coli.

ClpB is a heat-shock protein from Escherichia coli with an unknown function. We studied a possible molecular chaperone activity of ClpB in vitro. Firefly luciferase was denatured in urea and then diluted into the refolding buffer (in the presence of 5 mM ATP and 0.1 mg/ml bovine serum albumin). Spontaneous reactivation of luciferase was very weak (less than 0.02% of the native activity) because of extensive aggregation. Conventional chaperone systems (GroEL/GroES and DnaK/DnaJ/GrpE) or ClpB alone did not reactivate luciferase under those conditions. However, ClpB together with DnaK/DnaJ/GrpE greatly enhanced the luciferase activity regain (up to 57% of native activity) by suppressing luciferase aggregation. This coordinated function of ClpB and DnaK/DnaJ/GrpE required ATP hydrolysis, although the ClpB ATPase was not activated by native or denatured luciferase. When the chaperones were added to the luciferase refolding solutions after 5-25 min of refolding, ClpB and DnaK/DnaJ/GrpE recovered the luciferase activity from preformed aggregates. Thus, we have identified a novel multi-chaperone system from E. coli, which is analogous to the Hsp104/Ssa1/Ydj1 system from yeast. ClpB is the only known bacterial Hsp100 protein capable of cooperating with other heat-shock proteins in suppressing and reversing protein aggregation.     

Modulation of the chaperone activities of Hsc70/Hsp40 by Hsp105alpha and Hsp105beta

Hsp105alpha and Hsp105beta are stress proteins found in various mammals including human, mouse, and rat, which belong to the Hsp105/Hsp110 protein family. To elucidate their physiological functions, we examined here the chaperone activity of these stress proteins. Hsp105alpha and Hsp105beta prevented the aggregation of firefly luciferase during thermal denaturation, whereas the thermally denatured luciferase was not reactivated by itself or by rabbit reticulocyte lysate (RRL). On the other hand, Hsp105alpha and Hsp105beta suppressed the reactivation of thermally denatured luciferase by RRL and of chemically denatured luciferase by Hsc70/Hsp40 or RRL. Furthermore, although Hsp105alpha and Hsp105beta did not show ATPase activity, the addition of Hsp105alpha or Hsp105beta to Hsc70/Hsp40 enhanced the amount of hydrolysis of ATP greater than that of the Hsp40-stimulated Hsc70 ATPase activity. These findings suggest that Hsp105alpha and Hsp105beta are not only chaperones that prevent thermal aggregation of proteins, but also regulators of the Hsc70 chaperone system in mammalian cells.     

Human DnaJ homologs dj2 and dj3, and bag-1 are positive cochaperones of hsc70

DnaJ is an essential cochaperone of mammalian heat shock cognate 70 (hsc70) protein. We previously found that dj2 (HSDJ/hdj-2/rdj1), rather than dj1 (hsp40/hdj-1), is a partner DnaJ for the hsc70-based chaperone system. Here, we compared the distribution of dj1, dj2, and the newly found dj3 (cpr3/DNJ3/HIRIP4/rdj2) in cultured cells. Both dj3 as well as dj2 were farnesylated and were ubiquitously expressed. In immunocytochemical and subfractionation studies, these two proteins colocalized with hsc70 under normal conditions. However, dj1 and hsc70 apparently colocalized in the nucleoli after heat shock. Simultaneous depletion of dj2 and dj3 from rabbit reticulocyte lysate markedly reduced mitochondrial import of pre-ornithine transcarbamylase and refolding of guanidine-denatured luciferase. Re-addition of either dj2 or dj3 led to recovery of these reactions. In a reconstituted system, both hsc70-dj2 and hsc70-dj3 were effective in protein refolding. Anti-apoptotic protein bag-1 further stimulated ATP hydrolysis and protein refolding by both pairs. Thus, dj2 and dj3 are the partner DnaJs of hsc70 within the cell, functionally similar and much more efficient than dj1, and bag-1 is a positive cochaperone of the hsc70-dj2 and hsc70-dj3 systems.     

Successive and synergistic action of the Hsp70 and Hsp100 chaperones in protein disaggregation

Proteins belonging to the B-subtype of the Hsp100/Clp chaperone family execute a crucial role in cellular thermotolerance. They cooperate with the Hsp70 chaperones in reactivation of thermally aggregated protein substrates. We investigated the initial events of the disaggregation reaction in real time using denatured, aggregated green fluorescent protein (GFP) as a substrate. Bacterial Hsp70 (DnaK), its co-chaperones (DnaJ and GrpE), and Hsp100 (ClpB) were incubated with aggregated GFP, and the increase in GFP fluorescence was monitored. Incubation of aggregated GFP with DnaK/DnaJ/GrpE but not with ClpB resulted in the rapid initiation of the disaggregation reaction. Under the same conditions a complex between DnaK, DnaJ, and GFP, but not ClpB, was formed as demonstrated by sedimentation analysis and light scattering experiments. Chaperone-dependent disaggregation of chemically denatured aggregated luciferase showed that, similar to GFP disaggregation, incubation with Hsp70 results in the rapid start of the reactivation reaction. For both aggregated GFP and luciferase, incubation with Hsp70 chaperones changes the initial rate but not the overall efficiency or rate of the refolding reaction. Our results clearly demonstrate that the interaction of DnaK and its co-chaperones with aggregated substrate is the rate-limiting reaction at the initial steps of disaggregation.     

Physical Interaction between Bacterial Heat Shock Protein (Hsp) 90 and Hsp70 Chaperones Mediates Their Cooperative Action to Refold Denatured Proteins

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.     

Chaperone network in the yeast cytosol: Hsp110 is revealed as an Hsp70 nucleotide exchange factor

The Hsp110 proteins, exclusively found in the eukaryotic cytosol, have significant sequence homology to the Hsp70 molecular chaperone superfamily. Despite this homology and the cellular abundance of these proteins, the precise functional role has remained undefined. Here, we present the intriguing finding that the yeast homologue, Sse1p, acts as an efficient nucleotide exchange factor (NEF) for both yeast cytosolic Hsp70s, Ssa1p and Ssb1p. The mechanism involves formation of a stable nucleotide-sensitive complex, but does not require ATP hydrolysis by Sse1p. The NEF activity of Sse1p stimulates in vitro Ssa1p-mediated refolding of thermally denatured luciferase, and appears to have an essential role in vivo. Overexpression of the only other described cytosolic NEF, Fes1p, can partially compensate for a lethal sse1,2Delta phenotype, however, the cells are sensitive to stress conditions. Furthermore, in the absence of Sse, the in vivo refolding of thermally denatured model proteins is affected. This is the first report of a nucleotide exchange activity for the Hsp110 class of proteins, and provides a key piece in the puzzle of the cellular chaperone network.     

Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity

Studies on the Hsp70 chaperone machine in eukaryotes have shown that Hsp70 and Hsp40/Hdj1 family proteins are sufficient to prevent protein misfolding and aggregation and to promote refolding of denatured polypeptides. Additional protein cofactors include Hip and Bag1, identified in protein interaction assays, which bind to and modulate Hsp70 chaperone activity in vitro. Bag1, originally identified as an antiapoptotic protein, forms a stoichiometric complex with Hsp70 and inhibits completely Hsp70-dependent in vitro protein refolding of an unfolded polypeptide. Given its proposed involvement in multiple cell signaling events as a regulator of Raf1, Bcl2, or androgen receptor, we wondered whether Bag1 functions in vivo as a negative regulator of Hsp70. In this study, we demonstrate that Bag1, expressed in mammalian tissue culture cells, has pronounced effects on one of the principal activities of Hsp70, as a molecular chaperone essential for stabilization and refolding of a thermally inactivated protein. The levels of Hsp70 and Bag1 were modulated either by transient transfection or conditional expression in stably transfected lines to achieve levels within the range detected in different mammalian tissue culture cell lines. For example, a twofold increase in the concentration of Bag1 reduced Hsp70-dependent refolding of denatured luciferase by a factor of 2. This effect was titratable, and higher levels of wild-type but not a mutant form of Bag1 further inhibited Hsp70 refolding by up to a factor of 5. The negative effects of Bag1 were also observed in a biochemical analysis of Bag1- or Hsp70-overexpressing cells. The ability of Hsp70 to maintain thermally denatured firefly luciferase in a soluble state was reversed by Bag1, thus providing an explanation for the in vivo chaperone-inhibitory effects of Bag1. Similar effects on Hsp70 were observed with other cytoplasmic isoforms of Bag1 which have in common the carboxyl-terminal Hsp70-binding domain and differ by variable-length amino-terminal extensions. These results provide the first formal evidence that Bag1 functions in vivo as a regulator of Hsp70 and suggest an intriguing complexity for Hsp70-regulatory events.     

Hsp105 but not Hsp70 family proteins suppress the aggregation of heat-denatured protein in the presence of ADP

Hsp105alpha and Hsp105beta are mammalian members of the Hsp105/110 family, a diverged subgroup of the Hsp70 family. Here, we show that Hsp105alpha and Hsp105beta bind non-native protein through the beta-sheet domain and suppress the aggregation of heat-denatured protein in the presence of ADP rather than ATP. In contrast, Hsc70/Hsp40 suppressed the aggregation of heat-denatured protein in the presence of ATP rather than ADP. Furthermore, the overexpression of Hsp105alpha but not Hsp70 in COS-7 cells rescued the inactivation of luciferase caused by ATP depletion. Thus, Hsp105/110 family proteins are suggested to function as a substitute for Hsp70 family proteins to suppress the aggregation of denatured proteins in cells under severe stress, in which the cellular ATP level decreases markedly.     

N-Ethylmaleimide-modified Hsp70 inhibits protein folding.

Hsp70 molecular chaperones facilitate protein folding and translocation by binding to hydrophobic regions of nascent or unfolded proteins, thereby preventing their aggregation. N-Ethylmaleimide (NEM) inhibits the ATPase and protein translocation-stimulating activities of the yeast Hsp70 Ssa1p by modifying its three cysteine residues, which are located in its ATPase domain. NEM alters the conformation of Ssa1p and disrupts the coupling between its nucleotide- and polypeptide-binding domains. Ssa1p and the yeast DnaJ homolog Ydj1p constitute a protein folding machinery of the yeast cytosol. Using firefly luciferase as a model protein to study chaperone-dependent protein refolding, we have found that NEM also inhibits the protein folding activity of Ssa1p. Interestingly, the NEM-modified protein (NEM-Ssa1p) is a potent inhibitor of protein folding. NEM-Ssa1p can prevent the aggregation of luciferase and stimulate the ATPase activity of Ssa1p suggesting that it acts as an inhibitor by binding to nonnative forms of luciferase and by competing with them for the polypeptide binding site of Ssa1p. NEM-Ssa1p inhibits Ssa1p/Ydj1p-dependent protein refolding at different stages indicating that the chaperones bind and release nonnative forms of luciferase multiple times before folding is completed.     

Effective cotranslational folding of firefly luciferase without chaperones of the Hsp70 family

Molecular chaperones of the Hsp70 family (bacterial DnaK, DnaJ, and GrpE) were shown to be strictly required for refolding of firefly luciferase from a denatured state and thus for effective restoration of its activity. At the same time the luciferase was found to be synthesized in an Escherichia coli cell-free translation system in a highly active state in the extract with no chaperone activity. The addition of the chaperones to the extract during translation did not raise the activity of the enzyme. The abrupt arrest of translation by the addition of a translational inhibitor led to immediate cessation of the enzyme activity accumulation, indicating the cotranslational character of luciferase folding. The results presented suggest that the chaperones of the Hsp70 family are not required for effective cotranslational folding of firefly luciferase.     

Structural features required for the interaction of the Hsp70 molecular chaperone DnaK with its cochaperone DnaJ.

Hsp70 family members together with their Hsp40 cochaperones function as molecular chaperones, using an ATP-controlled cycle of polypeptide binding and release to mediate protein folding. Hsp40 plays a key role in the chaperone reaction by stimulating the ATPase activity and activating the substrate binding of Hsp70. We have explored the interaction between the Escherichia coli Hsp70 family member, DnaK, and its cochaperone partner DnaJ. Our data show that the binding of ATP, subsequent conformational changes in DnaK, and DnaJ-stimulated ATP hydrolysis are all required for the formation of a DnaK-DnaJ complex as monitored by Biacore analysis. In addition, our data imply that the interaction of the J-domain with DnaK depends on the substrate binding state of DnaK.     

DnaJ dramatically stimulates ATP hydrolysis by DnaK: insight into targeting of Hsp70 proteins to polypeptide substrates

Most, if not all, of the cellular functions of Hsp70 proteins require the assistance of a DnaJ homologue, which accelerates the weak intrinsic ATPase activity of Hsp70 and serves as a specificity factor by binding and targeting specific polypeptide substrates for Hsp70 action. We have used pre-steady-state kinetics to investigate the interaction of the Escherichia coli DnaJ and DnaK proteins, and the effects of DnaJ on the ATPase reaction of DnaK. DnaJ accelerates hydrolysis of ATP by DnaK to such an extent that ATP binding by DnaK becomes rate-limiting for hydrolysis. At high concentrations of DnaK under single-turnover conditions, the rate-limiting step is a first-order process, apparently a change of DnaK conformation, that accompanies ATP binding and proceeds at 12-15 min-1 at 25 degrees C and 1-1.5 min-1 at 5 degrees C. By prebinding ATP to DnaK and subsequently adding DnaJ, the effects of this slow step may be bypassed, and the maximal rate-enhancement of DnaJ on the hydrolysis step is approximately 15 000-fold at 5 degrees C. The interaction of DnaJ with DnaK.ATP is likely a rapid equilibrium relative to ATP hydrolysis, and is relatively weak, with a KD of approximately 20 microM at 5 degrees C, and weaker still at 25 degrees C. In the presence of saturating DnaJ, the maximal rate of ATP hydrolysis by DnaK is similar to previously reported rates for peptide release from DnaK.ATP. This suggests that when DnaK encounters a DnaJ-bound polypeptide or protein complex, a significant fraction of such events result in ATP hydrolysis by DnaK and concomitant capture of the polypeptide substrate in a tight complex with DnaK.ADP. Furthermore, a broadly applicable kinetic mechanism for DnaJ-mediated specificity of Hsp70 action arises from these observations, in which the specificity arises largely from the acceleration of the hydrolysis step itself, rather than by DnaJ-dependent modulation of the affinity of Hsp70 for substrate polypeptides.     

Bag-1M accelerates nucleotide release for human Hsc70 and Hsp70 and can act concentration-dependent as positive and negative cofactor.

The cytosol of mammalian cells contains several Hsp70 chaperones and an arsenal of cochaperones, including the anti-apoptotic Bag-1M protein, which regulate the activities of Hsp70s by controlling their ATPase cycles. To elucidate the regulatory function of Bag-1M, we determined its influence on nucleotide exchange, substrate release, ATPase rate, and chaperone activity of the housekeeping Hsc70 and stress-inducible Hsp70 homologs of humans. Bag-1M and a C-terminal fragment of it are potent nucleotide exchange factors as they stimulated the ADP dissociation rate of Hsc70 and Hsp70 up to 900-fold. The N-terminal domain of Bag-1M decreased the affinity of Bag-1M for Hsc70/Hsp70 by 4-fold, indicating a modulating role of the N terminus in Bag-1M action as nucleotide exchange factor. Bag-1M inhibited Hsc70/Hsp70-dependent refolding of luciferase in the absence of P(i). Surprisingly, under physiological conditions, i.e. low Bag-1M concentrations and presence of P(i), Bag-1M activates the chaperone action of Hsc70/Hsp70 in luciferase refolding. Bag-1M accelerated ATP-triggered substrate release by Hsc70/Hsp70. We propose that Bag-1M acts as substrate discharging factor for Hsc70 and Hsp70.     

Biochemical properties of Hsp70 chaperone system from Meiothermus ruber

The guanidine-hydrochloride (Gdn-HCl) and thermally induced unfolding of Hsp70 from Meiothermus ruber (Mru.Hsp70) were analysed using tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. The ANS binding to Mru.Hsp70 showed both the increase in fluorescence intensity and a shift in emission maximum. Analysis of the unfolding profile of Mru.Hsp70 indicated that Gdn-HCl induced unfolding of Mru.Hsp70 occurred through intermediate species. The tryptophan and ANS fluorescence emission spectra revealed that ATP induced conformational change increased the thermal stability of Mru.Hsp70. The data obtained are similar to those of Escherichia coli DnaK. The ATP-ase activity of chaperones is fundamental for their biological activity. It this paper we demonstrate that, in contrast to Thermus thermophilus, both Mru.Hsp40 and Mru.Hsp22 co-chaperones affect the ATP-ase activity of Mru.Hsp70. The use of truncated Mru.Hsp40 proteins showed that full-length Mru.Hsp40 is required for stimulation of ATP-ase activity of Mru.Hsp70. E. coli GrpE could act as nucleotide exchange factor the in thermophilic Hsp70 ATP hydrolysis reaction. However, the role of E. coli DnaJ in the M. ruber ATP cycle needs further analysis. We selected the new substrate laccA suitable for determination of refolding activity of thermophilic chaperones.