Partner proteins determine multiple functions of Hsp70

The 70 kDa heat shock proteins (Hsp70s) are ubiquitous molecular chaperones that are best known for their participation in protein folding. However, evidence is accumulating that Hsp70s perform several other cellular functions in cooperation with specific soluble or membrane-bound partner proteins. While the basic function of Hsp70s is explained by their ability to bind unfolded polypeptide segments, the partner proteins appear to customize them for specific roles such as involvement in protein traffic and folding, translocation of preproteins across membranes, and gene regulation.     

Arabidopsis thaliana J-class heat shock proteins: cellular stress sensors

Plants are sessile organisms that have evolved a variety of mechanisms to maintain their cellular homeostasis under stressful environmental conditions. Survival of plants under abiotic stress conditions requires specialized group of heat shock protein machinery, belonging to Hsp70:J-protein family. These heat shock proteins are most ubiquitous types of chaperone machineries involved in diverse cellular processes including protein folding, translocation across cell membranes, and protein degradation. They play a crucial role in maintaining the protein homeostasis by reestablishing functional native conformations under environmental stress conditions, thus providing protection to the cell. J-proteins are co-chaperones of Hsp70 machine, which play a critical role by stimulating Hsp70s ATPase activity, thereby stabilizing its interaction with client proteins. Using genome-wide analysis of Arabidopsis thaliana, here we have outlined identification and systematic classification of J-protein co-chaperones which are key regulators of Hsp70s function. In comparison with Saccharomyces cerevisiae model system, a comprehensive domain structural organization, cellular localization, and functional diversity of A. thaliana J-proteins have also been summarized.     

Hsp70 in oncology

Hsp70 classes of molecular chaperones are highly conserved in all organisms and play an essential role in the maintenance of cellular homeostasis. Hsp70s assist nascent chain protein folding and denatured proteins, as well as the import of proteins to the organelles, and solubilization of aggregated proteins. ATPase function is required for Hsp70 function. Hsp70s use ATP hydrolysis driven mechanism for substrate protein binding and release. Various Hsps are unregulated in cancers but their significance for tumor growth is poorly understood. Studies have linked Hsp70 to several types of carcinoma. Human Hsp70s allow proliferation of cancer cells and suppress apoptotic and senescence pathways. This review presents Hsp70s role for growth of transformed cells and the current state of Hsp70 as a drug target along with recent patents in humans in this particular area.     

Conserved,Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

The 70-kDa heat shock proteins (Hsp70s) function as molecular chaperones through the allosteric coupling of their nucleotide- and substrate-binding domains, the structures of which are highly conserved. In contrast, the roles of the poorly structured, variable length C-terminal regions present on Hsp70s remain unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD tetrapeptide sequence associates with co-chaperones via binding to tetratricopeptide repeat domains. It is not known whether this is the only function for this region in eukaryotic Hsp70s and what roles this region performs in Hsp70s that do not form complexes with tetratricopeptide repeat domains. We compared C-terminal sequences of 730 Hsp70 family members and identified a novel conservation pattern in a diverse subset of 165 bacterial and organellar Hsp70s. Mutation of conserved C-terminal sequence in DnaK, the predominant Hsp70 in Escherichia coli, results in significant impairment of its protein refolding activity in vitro without affecting interdomain allostery, interaction with co-chaperones DnaJ and GrpE, or the binding of a peptide substrate, defying classical explanations for the chaperoning mechanism of Hsp70. Moreover, mutation of specific conserved sites within the DnaK C terminus reduces the capacity of the cell to withstand stresses on protein folding caused by elevated temperature or the absence of other chaperones. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.     

Not all J domains are created equal: implications for the specificity of Hsp40-Hsp70 interactions

Heat shock protein 40s (Hsp40s) and heat shock protein 70s (Hsp70s) form chaperone partnerships that are key components of cellular chaperone networks involved in facilitating the correct folding of a broad range of client proteins. While the Hsp40 family of proteins is highly diverse with multiple forms occurring in any particular cell or compartment, all its members are characterized by a J domain that directs their interaction with a partner Hsp70. Specific Hsp40-Hsp70 chaperone partnerships have been identified that are dedicated to the correct folding of distinct subsets of client proteins. The elucidation of the mechanism by which these specific Hsp40-Hsp70 partnerships are formed will greatly enhance our understanding of the way in which chaperone pathways are integrated into finely regulated protein folding networks. From in silico analyses, domain swapping and rational protein engineering experiments, evidence has accumulated that indicates that J domains contain key specificity determinants. This review will critically discuss the current understanding of the structural features of J domains that determine the specificity of interaction between Hsp40 proteins and their partner Hsp70s. We also propose a model in which the J domain is able to integrate specificity and chaperone activity.     

The molecular chaperone Hsp70 family members function by a bidirectional heterotrophic allosteric mechanism

The Hsp70 family is one of the most important and conserved molecular chaperone families. It is well documented that Hsp70 family members assist many cellular processes involving protein quality control, as follows: protein folding, transport through membranes, protein degradation, escape from aggregation, intracellular signaling, among several others. The Hsp70 proteins act as a cellular pivot capable of receiving and distributing substrates among the other molecular chaperone families. Despite the high identity of the Hsp70 proteins, there are several homologue Hsp70 members that do not have the same role in the cell, which allow them to develop and participate in such large number of activities. The Hsp70 proteins are composed of two main domains: one that binds ATP and hydrolyses it to ADP and another which directly interacts with substrates. These domains present bidirectional heterotrophic allosteric regulation allowing a fine regulated cycle of substrate binding and release. The general mechanism of the Hsp70s cycle is under the control of ATP hydrolysis that modulates the low (ATP-bound state) and high (ADP-bound state) affinity states of Hsp70 for substrates. An important feature of the Hsp70s cycle is that they have several co-chaperones that modulate their cycle and that can also interact and select substrates. Here, we review some known details of the bidirectional heterotrophic allosteric mechanism and other important features for Hsp70s regulating cycle and function.     

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.     

Energy landscape remodeling mechanism of Hsp70-chaperone-accelerated protein folding

Hsp70 chaperone is one of the key protein machines responsible for the quality control of protein production in cells. Facilitating in vivo protein folding by counteracting misfolding and aggregation is the essence of its biological function. Although the allosteric cycle during its functional actions has been well characterized both experimentally and computationally, the mechanism by which Hsp70 assists protein folding is still not fully understood. In this work, we studied the Hsp70-mediated folding of model proteins with rugged energy landscape by using molecular simulations. Different from the canonical scenario of Hsp70 functioning, which assumes that folding of substrate proteins occurs spontaneously after releasing from chaperones, our results showed that the substrate protein remains in contacts with the chaperone during its folding process. The direct chaperone-substrate interactions in the open conformation of Hsp70 tend to shield the substrate sites prone to form non-native contacts, which therefore avoids the frustrated folding pathway, leading to a higher folding rate and less probability of misfolding. Our results suggest that in addition to the unfoldase and holdase functions widely addressed in previous studies, Hsp70 can facilitate the folding of its substrate proteins by remodeling the folding energy landscape and directing the folding processes, demonstrating the foldase scenario. These findings add new, to our knowledge, insights into the general molecular mechanisms of chaperone-mediated protein folding.     

Structural basis for the cooperation of Hsp70 and Hsp110 chaperones in protein folding

Protein folding by Hsp70 is tightly controlled by cochaperones, including J-domain proteins that trigger ATP hydrolysis and nucleotide exchange factors (NEFs) that remove ADP from Hsp70. Here we present the crystal structure of the yeast NEF Sse1p (Hsp110) in complex with the nucleotide-binding domain (NBD) of Hsp70. Hsp110 proteins are homologous to Hsp70s and consist of an NBD, a beta sandwich domain, and a three helix bundle domain (3HBD). In the complex, the NBD of Sse1p is ATP bound, and together with the 3HBD it embraces the NBD of Hsp70, inducing opening and the release of bound ADP from Hsp70. Mutations that abolish NEF activity are lethal, thus defining nucleotide exchange on Hsp70 as an essential function of Sse1p. Our data suggest that Sse1p does not employ the nucleotide-dependent allostery and peptide-binding mode of canonical Hsp70s, and that direct interactions of substrate with Sse1p may support Hsp70-assisted protein folding in a cooperative process.     

Molecular chaperones of the Hsp110 family act as nucleotide exchange factors of Hsp70s

Hsp70 molecular chaperones function in protein folding in a manner dependent on regulation by co-chaperones. Hsp40s increase the low intrinsic ATPase activity of Hsp70, and nucleotide exchange factors (NEFs) remove ADP after ATP hydrolysis, enabling a new Hsp70 interaction cycle with non-native protein substrate. Here, we show that members of the Hsp70-related Hsp110 family cooperate with Hsp70 in protein folding in the eukaryotic cytosol. Mammalian Hsp110 and the yeast homologues Sse1p/2p catalyze efficient nucleotide exchange on Hsp70 and its orthologue in Saccharomyces cerevisiae, Ssa1p, respectively. Moreover, Sse1p has the same effect on Ssb1p, a ribosome-associated isoform of Hsp70 in yeast. Mutational analysis revealed that the N-terminal ATPase domain and the ultimate C-terminus of Sse1p are required for nucleotide exchange activity. The Hsp110 homologues significantly increase the rate and yield of Hsp70-mediated re-folding of thermally denatured firefly luciferase in vitro. Similarly, deletion of SSE1 causes a firefly luciferase folding defect in yeast cells under heat stress in vivo. Our data indicate that Hsp110 proteins are important components of the eukaryotic Hsp70 machinery of protein folding.     

Positive natural selection has driven the evolution of the Hsp70s in Diguetia spiders

Hsp70s are a ubiquitous family of highly conserved proteins. Hsp70s are chaperones and have important roles in both protein folding and thermotolerance. It has been widely assumed that Hsp70 sequence evolution is governed by the strong functional constraints imposed by its crucial cellular functions. In this study of cytosolic heat-inducible Hsp70s from three spider families, we have found clear evidence of positive natural selection altering Hsp70s in desert-dwelling and heat-loving Diguetidae spiders. These spiders are a small family restricted to deserts. They display heat-tolerant behaviours not seen in their closest relatives, the Pholcidae and Plectreuridae.     

Distinct but overlapping functions of Hsp70, Hsp90, and an Hsp70 nucleotide exchange factor during protein biogenesis in yeast

Hsp70 and Hsp90 molecular chaperones play essential roles in protein expression and maturation, and while catalyzing protein folding they can "decide" to target mis-folded substrates for degradation. In this report, we show for the first time distinct but partially overlapping requirements for Hsp90, Hsp70, and an Hsp70 nucleotide exchange factor (NEF) at different steps during the biogenesis of a model substrate, firefly luciferase (FFLux), in yeast. By examining the inducible expression of FFLux in wild type cells and in specific yeast mutants, we find that the Fes1p NEF is required for efficient FFLux folding, whereas the Hsp70, Ssa1p, is required for both protein folding and stability, and to maintain maximal FFLux mRNA levels. In contrast, Hsp90 function was primarily necessary to express the FFLux-encoding gene from an inducible promoter. Together, these data indicate previously unknown roles for these proteins and point to the complexity with which chaperones and cochaperones function in the cell.     

Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1.

The Hsp70 family of molecular chaperones has an essential role in the synthesis, folding and translocation of the nascent peptide chain. While the general features of these activities are well documented, less is understood about the regulation of these activities. The ATPase rate is stimulated by non-native proteins, furthermore, interaction with ATP leads to the release of protein substrate concurrent with a conformational change in Hsp70. One interpretation of these data is that the two domains of Hsp70 interact. In the process of mapping the carboxyl-terminal boundary of the substrate binding domain for human Hsp70, we identified a regulatory motif, EEVD, which is conserved at the extreme carboxyl terminus among nearly all cloned cytosolic eukaryotic Hsp70s. Deletion or mutation of EEVD affects the ATPase activity, the ability to interact with substrates, and interferes with the ability of the mutant Hsp70 to interact with HDJ-1 in the refolding of denatured firefly luciferase. Examination of the biophysical properties of the mutant Hsp70s reveals a change in the overall shape and conformation of the protein consistent with reduced interactions between the two domains. These data suggest that the EEVD motif is involved in the intramolecular regulation of Hsp70 function and intermolecular interactions with HDJ-1.     

Control of cell fate by Hsp70: more than an evanescent meeting

During their lifetime, proteins inevitably expose hydrophobic segments within the polypeptide chains on a molecule's surface, which may be otherwise buried inside the molecules in the proper conformation. This potentially dangerous situation is managed with the aid of the 70-kDa heat shock proteins (Hsp70s) and other molecular chaperones. Although a major function of Hsp70 is assisting in efficient folding of anonymous proteins in unfolded states, recent studies have revealed that Hsp70 plays a variety of specific roles, sometimes deciding the cell fate. These multiple activities are based on the specific binding of Hsp70 to proteins in native states, which regulate cell growth and/or death. It is now well recognized that unfolding of some proteins may cause serious diseases, especially those associated with neurodegeneration, such as Alzheimer's disease. It is suggested that Hsp70 might be a potential drug against these diseases, but caution should be taken because Hsp70 exerts multiple effects by binding to specific proteins.     

Substrate transfer from the chaperone Hsp70 to Hsp90

Hsp90 is an essential chaperone protein in the cytosol of eukaryotic cells. It cooperates with the chaperone Hsp70 in defined complexes mediated by the adaptor protein Hop (Sti1 in yeast). These Hsp70/Hsp90 chaperone complexes play a major role in the folding and maturation of key regulatory proteins in eukaryotes. Understanding how non-native client proteins are transferred from one chaperone to the other in these complexes is of central importance. Here, we analyzed the molecular mechanism of this reaction using luciferase as a substrate protein. Our experiments define a pathway for luciferase folding in the Hsp70/Hsp90 chaperone system. They demonstrate that Hsp70 is a potent capture device for unfolded protein while Hsp90 is not very efficient in this reaction. When Hsp90 is absent, in contrast to the in vivo situation, Hsp70 together with the two effector proteins Ydj1 and Sti1 exhibits chaperone activity towards luciferase. In the presence of the complete chaperone system, Hsp90 exhibits a specific positive effect only in the presence of Ydj1. If this factor is absent, the transferred luciferase is trapped on Hsp90 in an inactive conformation. Interestingly, identical results were observed for the yeast and the human chaperone systems although the regulatory function of human Hop is completely different from that of yeast Sti1.     

Lipid interaction differentiates the constitutive and stress-induced heat shock proteins Hsc70 and Hsp70

Heat shock proteins play a major role in the process of protein folding, and they have been termed molecular chaperones. Two members of the Hsp70 family, Hsc70 and Hsp70, have a high degree of sequence homology. But they differ in their expression pattern. Hsc70 is constitutively expressed, whereas Hsp70 is stress inducible. These 2 proteins are localized in the cytosol and the nucleus. In addition, they have also been observed in close proximity to cellular membranes. We have recently reported that Hsc70 is capable of interacting with a lipid bilayer forming ion-conductance channels. In the present study, we found that both Hsc70 and Hsp70 interact with lipids and can be differentiated by their characteristic induction of liposome aggregation. These proteins promote the aggregation of phosphatidylserine liposomes in a time- and protein concentration-dependent manner. Although both proteins are active in this process, the level and kinetics of aggregation are different between them. Calcium ions enhance Hsc70 and Hsp70 liposome aggregation, but the effect is more dramatic for Hsc70 than for Hsp70. Addition of adenosine triphosphate blocks liposome aggregation induced by both proteins. Adenosine diphosphate (ADP) also blocks Hsp70-mediated liposome aggregation. Micromolar concentrations of ADP enhance Hsc70-induced liposome aggregation, whereas at millimolar concentrations the nucleotide has an inhibitory effect. These results confirm those of previous studies indicating that the Hsp70 family can interact with lipids directly. It is possible that the interaction of Hsp70s with lipids may play a role in the folding of membrane proteins and the translocation of polypeptides across membranes.     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> Hsp70-z,an Hsp110 homologue,exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion

The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival.