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.     

Plant Hsp70 molecular chaperones: Protein structure, gene family, expression and function

The Hsp70 molecular chaperones of plants are encoded by a multi-gene family whose members are developmentally regulated and differentially expressed in response to temperature stress and other conditions that interrupt normal protein folding or favor protein denaturation. Under non-stressful conditions, Hsp70 cognates function in concert with a variety of co-chaperones to facilitate folding of de novo synthesized proteins, assist in transport of precursor proteins into organelles and to help target damaged proteins for degradation. Stress-induced Hsp70s function to mitigate aggregation of stress-denatured proteins and to refold non-native proteins restoring their biological function through iterative cycles of adenine nucleotide hydrolysis-dependent peptide binding and release. Much of what is known about how plant Hsp70s function comes from the study of Hsp70s from other types of organisms. Owing to their unique biology, much remains to be learned about the many functions Hsp70s play in plants.     

Structure of the Hsp110:Hsc70 nucleotide exchange machine

Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family. We report the structure of an Hsp110:Hsc70 nucleotide exchange complex. The complex is characterized by extensive protein:protein interactions and symmetric bridging interactions between the nucleotides bound in each partner protein's NBD. An electropositive pore allows nucleotides to enter and exit the complex. The role of nucleotides in complex formation and dissociation, and the effects of the protein:protein interactions on nucleotide exchange, can be understood in terms of the coupled effects of the nucleotides and protein:protein interactions on the open-closed isomerization of the NBDs. The symmetrical interactions in the complex may model other Hsp70 family heterodimers in which two Hsp70s reciprocally act as NEFs.     

A Trypanosoma cruzi heat shock protein 40 is able to stimulate the adenosine triphosphate hydrolysis activity of heat shock protein 70 and can substitute for a yeast heat shock protein 40

The process of assisted protein folding, characteristic of members of the heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) molecular chaperone families, is important for maintaining the structural integrity of cellular protein machinery under normal and stressful conditions. Hsp70 and Hsp40 cooperate to bind non-native protein conformations in a process of adenosine triphosphate (ATP)-regulated assisted protein folding. We have analysed the molecular chaperone activity of the cytoplasmic inducible Hsp70 from Trypanosoma cruzi (TcHsp70) and its interactions with its potential partner Hsp40s (T. cruzi DnaJ protein 1 [Tcj1] and T. cruzi DnaJ protein 2 [Tcj2]). Histidine-tagged TcHsp70 (His-TcHsp70), Tcj1 (Tcj1-His) and Tcj2 (His-Tcj2) were over-produced in Escherichia coli and purified by nickel affinity chromatography. The in vitro basal specific ATP hydrolysis activity (ATPase activity) of His-TcHsp70 was determined as 40 nmol phosphate/min/mg protein, significantly higher than that reported for other Hsp70s. The basal specific ATPase activity was stimulated to a maximal level of 60 nmol phosphate/min/mg protein in the presence of His-Tcj2 and a model substrate, reduced carboxymethylated alpha-lactalbumin. In vivo complementation assays showed that Tcj2 was able to overcome the temperature sensitivity of the ydj1 mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 homologue (yeast DnaJ protein 1, Ydj1). These data suggest that Tcj2 is involved in cytoprotection in a similar fashion to Ydj1, and that TcHsp70 and Tcj2 may interact in a nucleotide-regulated process of chaperone-assisted protein folding.     

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.     

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.     

More than folding: localized functions of cytosolic chaperones

Compared with other chaperone systems, heat shock proteins Hsp70 and Hsp90 interact with a larger variety of co-chaperone proteins that regulate their activity or aid in the folding of specific substrate proteins. Although many co-chaperones are soluble cytosolic proteins, co-chaperone domains are also found in modular adaptor proteins, which are often localized to intracellular membranes or elements of the cytoskeleton. These specialized co-chaperones include auxilin, cysteine string protein, Tom70, UNC-45 and homologs of Bag-1. The localized co-chaperones can harness the ATP-dependent mechanisms of Hsp70 and Hsp90 to do conformational work in diverse functional contexts, including vesicle secretion and recycling, protein transport and the regulated assembly and/or disassembly of protein complexes. Such flexibility is unique to the cytosolic Hsp70 and Hsp90 chaperone system.     

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.     

Causal links between protein folding in the ER and events along the secretory pathway

The 70-kDa heat shock protein (Hsp70) family comprises the most abundant and important group of molecular chaperones. Hsp70s cooperate with a number of cofactors, which define their functions. We recently reported that a yeast protein, Rot1, is a putative cofactor of BiP, an endoplasmic reticulum (ER)-localized Hsp70. Rot1 is an essential ER membrane protein and may be involved in protein folding. Mutation of the ROT1 gene caused defects in cell wall synthesis and lysis of autophagic bodies. We suggest that Rot1 is required for folding of proteins engaged in these cellular processes.     

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.     

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.     

The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins

To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.     

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.     

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.     

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.     

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.     

Protein quality control: U-box-containing E3 ubiquitin ligases join the fold

Molecular chaperones act with folding co-chaperones to suppress protein aggregation and refold stress damaged proteins. However, it is not clear how slowly folding or misfolded polypeptides are targeted for proteasomal degradation. Generally, selection of proteins for degradation is mediated by E3 ubiquitin ligases of the mechanistically distinct HECT and RING domain sub-types. Recent studies suggest that the U-box protein family represents a third class of E3 enzymes. CHIP, a U-box-containing protein, is a degradatory co-chaperone of heat-shock protein 70 (Hsp70) and Hsp90 that facilitates the polyubiquitination of chaperone substrates. These data indicate a model for protein quality control in which the interaction of Hsp70 and Hsp90 with co-chaperones that have either folding or degradatory activity helps to determine the fate of non-native cellular proteins.