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.     

Modulation of chaperone activities of Hsp70 and Hsp70-2 by a mammalian DnaJ/Hsp40 homolog, DjA4

Type I DnaJs comprise one type of Hsp70 cochaperones. Previously, we showed that two type I DnaJ cochaperones, DjA1 (HSDJ/Hdj-2/Rdj-1/dj2) and DjA2 (cpr3/DNAJ3/Rdj-2/dj3), are important for mitochondrial protein import and luciferase refolding. Another type I DnaJ homolog, DjA4 (mmDjA4/dj4), is highly expressed in heart and testis, and the coexpression of Hsp70 and DjA4 protects against heat stress-induced cell death. Here, we have studied the chaperone functions of DjA4 by assaying the refolding of chemically or thermally denatured luciferase, suppression of luciferase aggregation, and the ATPase of Hsp70s, and compared these activities with those of DjA2. DjA4 stimulates the hydrolysis of ATP by Hsp70. DjA2, but not DjA4, together with Hsp70 caused denatured luciferase to refold efficiently. Together with Hsp70, both DjA2 and DjA4 are efficient in suppressing luciferase aggregation. bag-1 further stimulates ATP hydrolysis and protein refolding by Hsp70 plus DjA2 but not by Hsp70 plus DjA4. Hsp70-2, a testis-specific Hsp70 family member, behaves very similarly to Hsp70 in all these assays. Thus, Hsp70 and Hsp70-2 have similar activities in vitro, and DjA2 and DjA4 can function as partner cochaperones of Hsp70 and Hsp70-2. However, DjA4 is not functionally equivalent in modulating Hsp70s.     

hsp70-DnaJ chaperone pairs prevent nitric oxide-mediated apoptosis in RAW 264.7 macrophages

Excess nitric oxide (NO) induces apoptosis in some cell types, including macrophages. Heat shock protein of 70 kDa (hsp70) has been reported to protect cells from various stresses, including apoptosis-inducing stimuli. Several mammalian cytosolic DnaJ homologs, partner chaperones of hsp70 family members, have been identified. We asked if a DnaJ homolog is required to prevent NO-mediated apoptosis. When mouse macrophage-like RAW 264.7 cells were treated with an NO donor, SNAP, apoptosis occurred. This apoptosis could be prevented by pretreatment of the cells with heat or a low dose of SNAP. Under these conditions, levels of hsc70 (an hsp70 member) remained unchanged, whereas hsp70 was markedly induced. Of the DnaJ homologs dj1 (hsp40/hdj-1) was strongly induced and dj2 (HSDJ/hdj-2) was moderately induced. In transfection experiments, hsp70, hsc70, dj1 or dj2 alone was ineffective in preventing NO-mediated apoptosis. In contrast, both dj1 and dj2, in combination with hsc70 or hsp70, prevented the cells from apoptosis. The hsp70-DnaJ chaperone pairs exerted their anti-apoptotic effects upstream of caspase 3 activation, and apparently upstream of cytochrome c release from mitochondria.     

The Human DnaJ Homologue dj2 Facilitates Mitochondrial Protein Import and Luciferase Refolding

DnaJ homologues function in cooperation with hsp70 family members in various cellular processes including intracellular protein trafficking and folding. Three human DnaJ homologues present in the cytosol have been identified: dj1 (hsp40/hdj-1), dj2 (HSDJ/hdj-2), and neuronal tissue-specific hsj1. dj1 is thought to be engaged in folding of nascent polypeptides, whereas functions of the other DnaJ homologues remain to be elucidated. To investigate roles of dj2 and dj1, we developed a system of chaperone depletion from and readdition to rabbit reticulocyte lysates. Using this system, we found that heat shock cognate 70 protein (hsc70) and dj2, but not dj1, are involved in mitochondrial import of preornithine transcarbamylase. Bacterial DnaJ could replace mammalian dj2 in mitochondrial protein import. We also tested the effects of these DnaJ homologues on folding of guanidine-denatured firefly luciferase. Unexpectedly, dj2, but not dj1, together with hsc70 refolded the protein efficiently. We propose that dj2 is the functional partner DnaJ homologue of hsc70 in the mammalian cytosol. Bacterial DnaJ protein could replace mammalian dj2 in the refolding of luciferase. Thus, the cytosolic chaperone system for mitochondrial protein import and for protein folding is highly conserved, involving DnaK and DnaJ in bacteria, Ssa1–4p and Ydj1p in yeast, and hsc70 and dj2 in mammals.     

Multiple Molecules of Hsc70 and a Dimer of DjA1 Independently Bind to an Unfolded Protein

Protein folding is a prominent chaperone function of the Hsp70 system. Refolding of an unfolded protein is efficiently mediated by the Hsc70 system with either type 1 DnaJ protein, DjA1 or DjA2, and a nucleotide exchange factor. A surface plasmon resonance technique was applied to investigate substrate recognition by the Hsc70 system and demonstrated that multiple Hsc70 proteins and a dimer of DjA1 initially bind independently to an unfolded protein. The association rate of the Hsc70 was faster than that of DjA1 under folding-compatible conditions. The Hsc70 binding involved a conformational change, whereas the DjA1 binding was bivalent and substoichiometric. Consistently, we found that the bound ¹⁴C-labeled Hsc70 to the unfolded protein became more resistant to tryptic digestion. The gel filtration and cross-linking experiments revealed the predominant presence of the DjA1 dimer. Furthermore, the Hsc70 and DjA1 bound to distinct sets of peptide array sequences. All of these findings argue against the generality of the widely proposed hypothesis that the DnaJ-bound substrate is targeted and transferred to Hsp70. Instead, these results suggest the importance of the bivalent binding of DjA1 dimer that limits unfavorable transitions of substrate conformations in protein folding.     

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.     

The Hsc66-Hsc20 Chaperone System in Escherichia coli: Chaperone Activity and Interactions with the DnaK-DnaJ-GrpE System

Hsc66, a stress-70 protein, and Hsc20, a J-type accessory protein, comprise a newly described Hsp70-type chaperone system in addition to DnaK-DnaJ-GrpE in Escherichia coli. Because endogenous substrates for the Hsc66-Hsc20 system have not yet been identified, we investigated chaperone-like activities of Hsc66 and Hsc20 by their ability to suppress aggregation of denatured model substrate proteins, such as rhodanese, citrate synthase, and luciferase. Hsc66 suppressed aggregation of rhodanese and citrate synthase, and ATP caused effects consistent with complex destabilization typical of other Hsp70-type chaperones. Differences in the activities of Hsc66 and DnaK, however, suggest that these chaperones have dissimilar substrate specificity profiles. Hsc20, unlike DnaJ, did not exhibit intrinsic chaperone activity and appears to function solely as a regulatory cochaperone protein for Hsc66. Possible interactions between the Hsc66-Hsc20 and DnaK-DnaJ-GrpE chaperone systems were also investigated by measuring the effects of cochaperone proteins on Hsp70 ATPase activities. The nucleotide exchange factor GrpE did not stimulate the ATPase activity of Hsc66 and thus appears to function specifically with DnaK. Cross-stimulation by the cochaperones Hsc20 and DnaJ was observed, but the requirement for supraphysiological concentrations makes it unlikely that these interactions occur significantly in vivo. Together these results suggest that Hsc66-Hsc20 and DnaK-DnaJ-GrpE comprise separate molecular chaperone systems with distinct, nonoverlapping cellular functions.     

Low resolution structural study of two human HSP40 chaperones in solution. DJA1 from subfamily A and DJB4 from subfamily B have different quaternary structures

Proteins that belong to the heat shock protein (Hsp) 40 family assist Hsp70 in many cellular functions and are important for maintaining cell viability. A knowledge of the structural and functional characteristics of the Hsp40 family is therefore essential for understanding the role of the Hsp70 chaperone system in cells. In this work, we used small angle x-ray scattering and analytical ultracentrifugation to study two representatives of human Hsp40, namely, DjA1 (Hdj2/dj2/HSDJ/Rdj1) from subfamily A and DjB4 (Hlj1/DnaJW) from subfamily B, and to determine their quaternary structure. We also constructed low resolution models for the structure of DjA1-(1-332), a C-terminal-deleted mutant of DjA1 in which dimer formation is prevented. Our results, together with the current structural information of the Hsp40 C-terminal and J-domains, were used to generate models of the internal structural organization of DjA1 and DjB4. The characteristics of these models indicated that DjA1 and DjB4 were both dimers, but with substantial differences in their quaternary structures: whereas DjA1 consisted of a compact dimer in which the N and C termini of the two monomers faced each other, DjB4 formed a dimer in which only the C termini of the two monomers were in contact. The two proteins also differed in their ability to bind unfolded luciferase. Overall, our results indicate that these representatives of subfamilies A and B of human Hsp40 have different quaternary structures and chaperone functions.     

BAG-1 modulates the chaperone activity of Hsp70/Hsc70.

The 70 kDa heat shock family of molecular chaperones is essential to a variety of cellular processes, yet it is unclear how these proteins are regulated in vivo. We present evidence that the protein BAG-1 is a potential modulator of the molecular chaperones, Hsp70 and Hsc70. BAG-1 binds to the ATPase domain of Hsp70 and Hsc70, without requirement for their carboxy-terminal peptide-binding domain, and can be co-immunoprecipitated with Hsp/Hsc70 from cell lysates. Purified BAG-1 and Hsp/Hsc70 efficiently form heteromeric complexes in vitro. BAG-1 inhibits Hsp/Hsc70-mediated in vitro refolding of an unfolded protein substrate, whereas BAG-1 mutants that fail to bind Hsp/Hsc70 do not affect chaperone activity. The binding of BAG-1 to one of its known cellular targets, Bcl-2, in cell lysates was found to be dependent on ATP, consistent with the possible involvement of Hsp/Hsc70 in complex formation. Overexpression of BAG-1 also protected certain cell lines from heat shock-induced cell death. The identification of Hsp/Hsc70 as a partner protein for BAG-1 may explain the diverse interactions observed between BAG-1 and several other proteins, including Raf-1, steroid hormone receptors and certain tyrosine kinase growth factor receptors. The inhibitory effects of BAG-1 on Hsp/Hsc70 chaperone activity suggest that BAG-1 represents a novel type of chaperone regulatory proteins and thus suggest a link between cell signaling, cell death and the stress response.     

In vitro evidence of Hsc70 functioning as a molecular chaperone during cold stress

Hsp70 molecular chaperones have been shown to play an important role in helping cells to cope with adverse environments, especially in response to high temperatures. The molecular chaperone function of Hsc70 at low temperature was investigated. A cold-inducible spinach cytosolic Hsc70 was subcloned into a protein expression vector and the recombinant protein was expressed in bacterial cells. Recombinant Hsc70 bound a permanently unfolded substrate: alpha-carboxymethylated lactalbumin (CMLA) in the presence of 3 mM ATP and MgCl(2) at low temperature (4 and -4 degrees C). Radiolabeling with (35)S-Met and (35)S-Cys and immunoprecipitation with cytosolic Hsc70 monoclonal antibodies showed that there were several proteins co-immunoprecipitated at low temperature (4 and -4 degrees C) but not at room temperature. Enhanced co-purification of sHsp17.7 with Hsc70 at low temperature was observed and suggests that co-chaperone interactions can contribute to molecular chaperone function during cold stress. These results suggest that the molecular chaperone Hsc70 may have a functional role in plants during low temperature stress.     

Function of cytosolic chaperones in Tom70-mediated mitochondrial import

The great majority of mitochondrial proteins are synthesized by cytosolic ribosomes and then imported into the organelle post-translationally. The translocase of the outer membrane (TOM) is a proteinaceous machinery that contains surface receptors for preprotein recognition and also serves as the main entry gateway into mitochondria. Mitochondrial targeting requires various cytosolic factors, in particular the molecular chaperones Hsc70/Hsp70 and Hsp90. The chaperone activity of Hsc70/Hsp70 and Hsp90 occurs in coordinated cycles of ATP hydrolysis and substrate binding, and is regulated by a number of co-chaperone proteins. The import receptor Tom70 is a member of the tetratricopeptide repeat (TPR) co-chaperone family and contains a conserved TPR clamp domain for interaction with Hsc70 and Hsp90. Such interaction is essential for the initiation of the import process. This review will discuss the roles of Hsc70 and Hsp90 in mitochondrial import and summarize recent progress in understanding these pathways.     

Co-immunoprecipitation of Hsp101 with cytosolic Hsc70.

In animals and yeast, cytosolic Hsp70s function in concert with other molecular chaperones. Hsp70 is a major chaperone in the Hsp90 multi-chaperone complexes that participate in maturation of steroid receptors and several other proteins. Hsp70s also appear to form a complex with Hsp90 and Hsp110/sHsp. A 100 kDa protein was co-immunoprecipitated with cytosolic Hsc70 from maize seedlings (Zea mays). The presence of this complex was further confirmed using gel-filtration chromatography. Mass spectrometric analysis showed that the 100 kDa protein is homologous with Arabidopsis Hsp101. Treatment with apyrase enhanced the co-immunoprecipitation of Hsp101 with Hsc70, while ATP had the opposite effect. In the presence of carboxymethylated alpha-lactalbumin (CMLA), which is permanently unfolded, the complex dissociated. Based on these observations, it is concluded that Hsc70 and Hsp101 are present in a complex in the plant cytosol.     

A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein

Small heat shock proteins (sHsps) are a diverse group of heat-induced proteins that are conserved in prokaryotes and eukaryotes and are especially abundant in plants. Recent in vitro data indicate that sHsps act as molecular chaperones to prevent thermal aggregation of proteins by binding non-native intermediates, which can then be refolded in an ATP-dependent fashion by other chaperones. We used heat-denatured firefly luciferase (Luc) bound to pea (Pisum sativum) Hsp18.1 as a model to define the minimum chaperone system required for refolding of a sHsp-bound substrate. Heat-denatured Luc bound to Hsp18.1 was effectively refolded either with Hsc/Hsp70 from diverse eukaryotes plus the DnaJ homologs Hdj1 and Ydj1 (maximum = 97% Luc reactivation with k(ob) = 1.0 x 10(-2)/min), or with prokaryotic Escherichia coli DnaK plus DnaJ and GrpE (100% Luc reactivation, k(ob) = 11.3 x 10(-2)/min). Furthermore, we show that Hsp18.1 is more effective in preventing Luc thermal aggregation than the Hsc70 or DnaK systems, and that Hsp18.1 enhances the yields of refolded Luc even when other chaperones are present during heat inactivation. These findings integrate the aggregation-preventive activity of sHsps with the protein-folding activity of the Hsp70 system and define an in vitro system for further investigation of the mechanism of sHsp action.     

Binding to chaperones allows import of a purified mitochondrial precursor into mitochondria

Refolding of the acid-unfolded precursor to mitochondrial aspartate aminotransferase (pmAAT) is inhibited when cytosolic Hsc70 is included in the refolding reaction (Artigues, A., Iriarte, A., and Martinez-Carrion, M. (1997) J. Biol. Chem. 272, 16852-16861). At low molar excess of Hsc70 pmAAT is recovered in insoluble aggregates containing equal amounts of Hsc70. However, in the presence of a large excess of Hsc70, refolding of pmAAT is still arrested, but the enzyme remains in solution. Similar behavior was observed with two other cytosolic chaperones, bovine Hsp90 and yeast Ydj1. Coimmunoprecipitation of pmAAT using Hsc70 antibodies confirmed the formation of soluble Hsc70-pmAAT complexes at high concentrations of the chaperone. Data from analytical centrifugation, sedimentation in glycerol gradients, and partial purification of the soluble complexes indicate that multiple Hsc70 molecules bind per pmAAT polypeptide chain. The absence of catalytic activity together with the protease susceptibility of pmAAT bound to Hsc70, Hsp90, or Ydj1 suggest that these chaperones bind and maintain pmAAT in a partially unfolded state, analogous to the import-competent conformation of the protein synthesized in cell-free extracts. Remarkably, the purified pmAAT bound to Hsc70 or Ydj1, but not to Hsp90, is imported by isolated mitochondria in a reticulocyte lysate-dependent manner. Thus, both Hsc70 and Ydj1 can trap an import-competent folding intermediate of pmAAT, but productive binding and import into mitochondria require the collaboration of additional cytosolic factors from the lysate.     

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.     

The membrane-bound DnaJ protein located at the cytosolic site of glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species.

DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase-Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.     

DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.

Members of the conserved Hsp70 chaperone family are assumed to constitute a main cellular system for the prevention and the amelioration of stress-induced protein damage, though little direct evidence exists for this function. We investigated the roles of the DnaK (Hsp70), DnaJ and GrpE chaperones of Escherichia coli in prevention and repair of thermally induced protein damage using firefly luciferase as a test substrate. In vivo, luciferase was rapidly inactivated at 42 degrees C, but was efficiently reactivated to 50% of its initial activity during subsequent incubation at 30 degrees C. DnaK, DnaJ and GrpE did not prevent luciferase inactivation, but were essential for its reactivation. In vitro, reactivation of heat-inactivated luciferase to 80% of its initial activity required the combined activity of DnaK, DnaJ and GrpE as well as ATP, but not GroEL and GroES. DnaJ associated with denatured luciferase, targeted DnaK to the substrate and co-operated with DnaK to prevent luciferase aggregation at 42 degrees C, an activity that was required for subsequent reactivation. The protein repair function of DnaK, GrpE and, in particular, DnaJ is likely to be part of the role of these proteins in regulation of the heat shock response.     

Heat shock proteins, substrate specificity and modulation of function

Hsp70 is a universally conserved essential protein chaperone. In addition to its roles in many cellular process, Hsp70 protects cells from stress by binding partially unfolded proteins. Therefore, Hsp70 prevents protein aggregation and prion formation. Prions are infectious agents and are responsible for several fatal neurodegenerative diseases. Eukaryotic cells have several cytosolic Hsp70 isoforms, some constitutively expressed (Hsc70s), and others expressed only when cells are exposed to stress (Hsp70s). To determine which factors conferred functional specificity, we constructed hybrid Hsc/Hsp chaperones. All hybrids supported growth except those that contained the ATPase domain derived from inducible Hsp70. Thus, regulation of peptide binding by ATP hydrolysis must differ significantly between Hsc- and Hsp70 isoforms. In this work, nucleotide and peptide binding domain communication of Hsp70 proteins during their interaction with nucleotides and peptide substrates were investigated in vitro by using hybrid constructs.