Structural and functional analysis of the Hsp70/Hsp40 chaperone system

As one of the most abundant and highly conserved molecular chaperones, the 70‐kDa heat shock proteins (Hsp70s) play a key role in maintaining cellular protein homeostasis (proteostasis), one of the most fundamental tasks for every living organism. In this role, Hsp70s are inextricably linked to many human diseases, most notably cancers and neurodegenerative diseases, and are increasingly recognized as important drug targets for developing novel therapeutics for these diseases. Hsp40s are a class of essential and universal partners for Hsp70s in almost all aspects of proteostasis. Thus, Hsp70s and Hsp40s together constitute one of the most important chaperone systems across all kingdoms of life. In recent years, we have witnessed significant progress in understanding the molecular mechanism of this chaperone system through structural and functional analysis. This review will focus on this recent progress, mainly from a structural perspective.     

Maintenance of structure and function of mitochondrial Hsp70 chaperones requires the chaperone Hep1

Hsp70 chaperones mediate folding of proteins and prevent their misfolding and aggregation. We report here on a new kind of Hsp70 interacting protein in mitochondria, Hep1. Hep1 is a highly conserved protein present in virtually all eukaryotes. Deletion of HEP1 results in a severe growth defect. Cells lacking Hep1 are deficient in processes that need the function of mitochondrial Hsp70s, such as preprotein import and biogenesis of proteins containing FeS clusters. In the mitochondria of these cells, Hsp70s, Ssc1 and Ssq1 accumulate as insoluble aggregates. We show that it is the nucleotide-free form of mtHsp70 that has a high tendency to self-aggregate. This process is efficiently counteracted by Hep1. We conclude that Hep1 acts as a chaperone that is necessary and sufficient to prevent self-aggregation and to thereby maintain the function of the mitochondrial Hsp70 chaperones.     

Proteotoxicity is not the reason for the dependence of cancer cells on the major chaperone Hsp70

Several years ago a hypothesis was proposed that the survival of cancer cells depend on elevated expression of molecular chaperones because these cells are prone to proteotoxic stress. A critical prediction of this hypothesis is that depletion of chaperones in cancer cells should lead to proteotoxicity. Here, using the major chaperone Hsp70 as example, we demonstrate that its depletion does not trigger proteotoxic stress, thus refuting the model. Accordingly, other functions of chaperones, e.g., their role in cell signaling, might define the requirements for chaperones in cancer cells, which is critical for rational targeting Hsp70 in cancer treatment.     

Effect of evolution of the C-terminal region on chaperone activity of Hsp70

Dynamic interdomain interactions within the Hsp70 protein is the basis for the allosteric and functional properties of Hsp70s. While Hsp70s are generally conserved in terms of structure, allosteric behavior, and some overlapping functions, Hsp70s also contain variable sequence regions which are related to distinct functions. In the Hsp70 sequence, the part with the greatest sequence variation is the C-terminal α-helical lid subdomain of substrate-binding domain (SBDα) together with the intrinsically disordered region. Dynamic interactions between the SBDα and β-sandwich substrate-binding subdomain (SBDβ) contribute to the chaperone functions of Hsp70s by tuning kinetics of substrate binding. To investigate how the C-terminal region of Hsp70 has evolved from prokaryotic to eukaryotic organisms, we tested whether this region can be exchanged among different Hsp70 members to support basic chaperone functions. We found that this region from eukaryotic Hsp70 members cannot substitute for the same region in Escherichia coli DnaK to facilitate normal chaperone activity of DnaK. In contrast, this region from E. coli DnaK and Saccharomyces cerevisiae Hsp70 (Ssa1 and Ssa4) can partially support some roles of human stress inducible Hsp70 (hHsp70) and human cognate Hsp70 (hHsc70). Our results indicate that the C-terminal region from eukaryotic Hsp70 members cannot properly support SBDα–SBDβ interactions in DnaK, but this region from DnaK/Ssa1/Ssa4 can still form some SBDα–SBDβ interactions in hHsp70 or hHsc70, which suggests that the mode for SBDα–SBDβ interactions is different in prokaryotic and eukaryotic Hsp70 members. This study provides new insight in the divergency among different Hsp70 homologs and the evolution of Hsp70s.     

Hsp70 localizes differently from chaperone Hsc70 in mouse mesoangioblasts under physiological growth conditions

Mouse A6 mesoangioblasts express Hsp70 even in the absence of cellular stress. Its expression and its intracellular localization were investigated under normal growth conditions and under hyperthermic stress. Immunofluorescence assays indicated that without any stress a fraction of Hsp70 co-localized with actin microfilaments, in the cell cortex and in the contractile ring of dividing cells, while the Hsc70 chaperone did not. Hsp70 immunoprecipitation assays confirmed that a portion of Hsp70 binds actin. Immunoblot assays showed that both proteins were present in the nucleus. After heat treatment Hsp70 and actin continued to co-localize in the leading edge of A6 cells but not on microfilaments. Although Hsp70 and Hsc70 are both basally synthesized they showed different cellular distribution, suggesting an Hsp70 different activity respect to the Hsc70 chaperone. Moreover, we found Hsp70 in the culture medium as it has been described in other cell types.     

Hsp104, Hsp70 and Hsp40 interplay regulates formation, growth and elimination of Sup35 prions

Self-templating amyloid forms of Sup35 constitute the yeast prion [PSI(+)]. How the protein-remodelling factor, Hsp104, collaborates with other chaperones to regulate [PSI(+)] inheritance remains poorly delineated. Here, we report how the Ssa and Ssb components of the Hsp70 chaperone system directly affect Sup35 prionogenesis and cooperate with Hsp104. We identify the ribosome-associated Ssb1:Zuo1:Ssz1 complex as a potent antagonist of Sup35 prionogenesis. The Hsp40 chaperones, Sis1 and Ydj1, preferentially interact with Sup35 oligomers and fibres compared with monomers, and facilitate Ssa1 and Ssb1 binding. Various Hsp70:Hsp40 pairs block prion nucleation by disassembling molten oligomers and binding mature oligomers. By binding fibres, Hsp70:Hsp40 pairs occlude prion recognition elements and inhibit seeded assembly. These inhibitory activities are partially relieved by the nucleotide exchange factor, Fes1. Low levels of Hsp104 stimulate prionogenesis and alleviate inhibition by some Hsp70:Hsp40 pairs. At high concentrations, Hsp104 eliminates Sup35 prions. This activity is reduced when Ssa1, or enhanced when Ssb1, is incorporated into nascent prions. These findings illuminate several facets of the chaperone interplay that underpins [PSI(+)] inheritance.     

FK228 inhibits Hsp90 chaperone function in K562 cells via hyperacetylation of Hsp70

Some pan-histone-deacetylase (HDAC) inhibitors have recently been reported to exert their anti-leukemia effect by inhibiting the activity of class IIB HDAC6, which is the deacetylase of Hsp90 and α-tubulin, thereby leading to hyperacetylation of Hsp90, disruption of its chaperone function and apoptosis. In this study, we compared the effect of a class I HDAC inhibitor FK228 with the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on the Hsp90 chaperone function of K562 cells. We demonstrated that, although having a weaker inhibitory effect on HDAC6, FK228 mediated a similar disruption of Hsp90 chaperone function compared to SAHA. Unlike SAHA, FK228 did not mediate hyperacetylation of Hsp90, instead the acetylation of Hsp70 was increased and Bcr-Abl was increasingly associated with Hsp70 rather than Hsp90, forming an unstable complex that promotes Bcr-Abl degradation. These results indicated that FK228 may disrupt the function of Hsp90 indirectly through acetylation of Hsp70 and inhibition of its function.     

A novel method for removing contaminant Hsp70 molecular chaperones from recombinant proteins

The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affects the quality of the obtained proteins. The ability of chaperones to bind to unfolded proteins or to regions where the polypeptide chain is exposed make the removal of the contamination during purification challenging to achieve. This work aimed to develop a strategy to remove contaminating DnaK, one of the homologous Hsp70 molecular chaperones found in Escherichia coli, from purified recombinant proteins. For this purpose, we developed a methodology that captures the DnaK from the contaminating proteins by co‐incubation with a GST‐cleanser protein that has free functional binding sites for the chaperone. The cleanser protein can then be easily removed together with the captured DnaK. Here, we demonstrated the utility of our system by decontaminating a Histidine‐tagged recombinant protein in a batch process. The addition of the GST‐cleanser protein in the presence of ATP‐Mg eliminates the DnaK contamination substantially. Thus, our decontaminant strategy results versatile and straightforward and can be applied to proteins obtained with different expression and purifications systems as well as to small samples or large volume preparations.     

A hypoxia-induced decrease of either MICA/B or Hsp70 on the membrane of tumor cells mediates immune escape from NK cells

Recent findings suggest that hypoxia of the tumor microenvironment contributes to immune escape from natural killer (NK) cell-mediated cytotoxicity. Heat shock protein 70 (Hsp70) and the stress-regulated major histocompatibility class I chain-related protein A and B (MICA/B) both serve as ligands for activated NK cells when expressed on the cell surface of tumor cells. Herein, we studied the effects of hypoxia and hypoxia-inducible factor-1α (HIF-1α) on the membrane expression of these NK cell ligands in H1339 with high and MDA-MB-231 tumor cells with low basal HIF-1α levels and its consequences on NK cell-mediated cytotoxicity. We could show that a hypoxia-induced decrease in the membrane expression of MICA/B and Hsp70 on H1339 and MDA-MB-231 cells, respectively, is associated with a reduced sensitivity to NK cell-mediated lysis. A knockdown of HIF-1α revealed that the decreased surface expression of MICA/B under hypoxia is dependent on HIF-1α in H1339 cells with high basal HIF-1α levels. Hypoxia and HIF-1α did not affect the MICA/B expression in MDA-MB-231 cells but reduced the Hsp70 membrane expression which in turn also impaired NK cell recognition. Furthermore, we could show that the hypoxia-induced decrease in membrane Hsp70 is independent of HIF-1α in MDA-MB-231. Our data indicate that hypoxia-induced downregulation of both NK cell ligands MICA/B and Hsp70 impairs NK cell-mediated cytotoxicity, whereby only MICA/B appears to be regulated by HIF-1α.     

The switch from client holding to folding in the Hsp70/Hsp90 chaperone machineries is regulated by a direct interplay between co-chaperones

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The structural and functional diversity of Hsp70 proteins from Plasmodium falciparum

It is becoming increasingly apparent that heat shock proteins play an important role in the survival of Plasmodium falciparum against temperature changes associated with its passage from the cold-blooded mosquito vector to the warm-blooded human host. Interest in understanding the possible role of P. falciparum Hsp70s in the life cycle of the parasite has led to the identification of six HSP70 genes. Although most research attention has focused primarily on one of the cytosolic Hsp70s (PfHsp70-1) and its endoplasmic reticulum homolog (PfHsp70-2), further functional insights could be inferred from the structural motifs exhibited by the rest of the Hsp70 family members of P. falciparum. There is increasing evidence that suggests that PfHsp70-1 could play an important role in the life cycle of P. falciparum both as a chaperone and immunogen. In addition, P. falciparum Hsp70s and Hsp40 partners are implicated in the intracellular and extracellular trafficking of proteins. This review summarizes data emerging from studies on the chaperone role of P. falciparum Hsp70s, taking advantage of inferences gleaned from their structures and information on their cellular localization. The possible associations between P. falciparum Hsp70s with their cochaperone partners as well as other chaperones and proteins are discussed.     

NudC guides client transfer between the Hsp40/70 and Hsp90 chaperone systems

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Protein folding rates and thermodynamic stability are key determinants for interaction with the Hsp70 chaperone system

The Hsp70 family of molecular chaperones participates in vital cellular processes including the heat shock response and protein homeostasis. E. coli''s Hsp70, known as DnaK, works in concert with the DnaJ and GrpE co-chaperones (K/J/E chaperone system), and mediates cotranslational and post-translational protein folding in the cytoplasm. While the role of the K/J/E chaperones is well understood in the presence of large substrates unable to fold independently, it is not known if and how K/J/E modulates the folding of smaller proteins able to fold even in the absence of chaperones. Here, we combine experiments and computation to evaluate the significance of kinetic partitioning as a model to describe the interplay between protein folding and binding to the K/J/E chaperone system. First, we target three nonobligatory substrates, that is, proteins that do not require chaperones to fold. The experimentally observed chaperone association of these client proteins during folding is entirely consistent with predictions from kinetic partitioning. Next, we develop and validate a computational model (CHAMP70) that assumes kinetic partitioning of substrates between folding and interaction with K/J/E. CHAMP70 quantitatively predicts the experimentally measured interaction of RNase H^D as it refolds in the presence of various chaperones. CHAMP70 shows that substrates are posed to interact with K/J/E only if they are slow-folding proteins with a folding rate constant k_f <50 s⁻¹, and/or thermodynamically unstable proteins with a folding free energy ΔG⁰_UN ≥−2 kcal mol⁻¹. Hence, the K/J/E system is tuned to use specific protein folding rates and thermodynamic stabilities as substrate selection criteria.     

The Four Hydrophobic Residues on the Hsp70 Inter-Domain Linker Have Two Distinct Roles

The ubiquitous molecular chaperone 70-kDa heat shock proteins (Hsp70) play key roles in maintaining protein homeostasis. Hsp70s contain two functional domains: a nucleotide binding domain and a substrate binding domain. The two domains are connected by a highly conserved inter-domain linker, and allosteric coupling between the two domains is critical for chaperone function. The auxiliary chaperone 40-kDa heat shock proteins (Hsp40) facilitate all the biological processes associated with Hsp70s by stimulating the ATPase activity of Hsp70s. Although an overall essential role of the inter-domain linker in both allosteric coupling and Hsp40 interaction has been suggested, the molecular mechanisms remain largely unknown. Previously, we reported a crystal structure of a full-length Hsp70 homolog, in which the inter-domain linker forms a well-ordered β strand. Four highly conserved hydrophobic residues reside on the inter-domain linker. In DnaK, a well-studied Hsp70, these residues are V389, L390, L391, and L392. In this study, we biochemically dissected their roles. The inward-facing side chains of V389 and L391 form extensive hydrophobic contacts with the nucleotide binding domain, suggesting their essential roles in coupling the two functional domains, a hypothesis confirmed by mutational analysis. On the other hand, L390 and L392 face outward on the surface. Mutation of either abolishes DnaK's in vivo function, yet intrinsic biochemical properties remain largely intact. In contrast, Hsp40 interaction is severely compromised. Thus, for the first time, we separated the two essential roles of the highly conserved Hsp70 inter-domain linker: coupling the two functional domains through V389 and L391 and mediating the interaction with Hsp40 through L390 and L392.     

Ydj1 but not Sis1 stabilizes Hsp70 protein under prolonged stress in vitro

Yeast cytosol has two important co-chaperons; Ydj1 and Sis1. Genetic experiments showed that Ydj1 is not essential for viability; however, cells lacking it grow very poorly at 30 degrees C or unable to grow at extreme temperatures. On the other hand, Sis1 is an essential protein and apparently plays a functional role at assembly or disassembly of protein complexes. Stability experiments revealed that only Ydj1-protected Hsp70 proteins can hydrolyze ATP under prolonged stress.     

Keep the traffic moving: mechanism of the Hsp70 motor

Hsp70s are ubiquitous chaperones that use ATP hydrolysis to drive a variety of protein processing reactions, including a number of steps in protein trafficking. Recent studies have shed light on how ATP might generate conformational changes in an Hsp70 molecule and how such changes might be harnessed to drive processes as diverse as protein import into subcellular organelles and uncoating of clathrin-coated vesicles.     

Molecular Mechanism of J-Domain-Triggered ATP Hydrolysis by Hsp70 Chaperones

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