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.     

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.     

Chaperone discovery

Molecular chaperones assist de novo protein folding and facilitate the refolding of stress‐denatured proteins. The molecular chaperone concept was coined nearly 35 years ago, and since then, tremendous strides have been made in understanding how these factors support protein folding. Here, we focus on how various chaperone proteins were first identified to play roles in protein folding. Examples are used to illustrate traditional routes of chaperone discovery and point out their advantages and limitations. Recent advances, including the development of folding biosensors and promising methods for the stabilization of proteins in vivo, provide new routes for chaperone discovery.     

Regulation of molecular chaperones through post-translational modifications: Decrypting the chaperone code

Molecular chaperones and their associated cofactors form a group of highly specialized proteins that orchestrate the folding and unfolding of other proteins and the assembly and disassembly of protein complexes. Chaperones are found in all cell types and organisms, and their activity must be tightly regulated to maintain normal cell function. Indeed, deregulation of protein folding and protein complex assembly is the cause of various human diseases. Here, we present the results of an extensive review of the literature revealing that the post-translational modification (PTM) of chaperones has been selected during evolution as an efficient mean to regulate the activity and specificity of these key proteins. Because the addition and reciprocal removal of chemical groups can be triggered very rapidly, this mechanism provides an efficient switch to precisely regulate the activity of chaperones on specific substrates. The large number of PTMs detected in chaperones suggests that a combinatory code is at play to regulate function, activity, localization, and substrate specificity for this group of biologically important proteins. This review surveys the core information currently available as a starting point toward the more ambitious endeavor of deciphering the “chaperone code”.     

Detection of a very rapid first phase in complex formation of DnaK and peptide substrate

Complex formation of the Hsp70 chaperone DnaK with the fluorescence-labeled peptide ALLLSAPRR shows a very rapid first phase that has as yet not been observed with other peptides. This first phase is completed within the dead time (1–2 ms) of the stopped-flow instrument and corresponds to two thirds of the total increase in fluorescence. It occurs both in the presence and in the absence of ATP and is followed by a fast, a slow and a very slow step. These binding kinetics that are vastly different from those observed with other peptides might indicate the existence of a second substrate-binding site of DnaK.     

叶绿体J蛋白研究进展

J蛋白是广泛存在于细胞内的一种分子伴侣。它作为Hsp70的辅伴侣分子有着广泛而复杂的生物学功能。本文概述了J蛋白的相关概念、结构、种类、分布及其作用机制,并重点讨论了其在叶绿体内的功能。最后对有关J蛋白研究中需要解决的问题做了展望。     

Single-point mutation in a conserved TPR domain of Hip disrupts enhancement of glucocorticoid receptor signaling

The Hsp70-interacting protein Hip has been identified as a transient participant in the assembly of both glucocorticoid (GR) and progesterone receptor complexes. Although it has been difficult to identify a physiological role for Hip, it is believed to have intrinsic chaperoning properties and has been identified as a potential anti-apoptotic target of Granzyme B. In vitro assays have provided evidence that Hip may interact with GR complexes in an Hsp70 independent manner and can enhance the function of GR in hormone based reporter assays. In this study, a cDNA for human Hip was used in mutational analysis to map Hip function to critical structural elements. A single amino acid substitution (L211S) resulted in a loss of Hip function. This mutation also appears to disrupt the interaction of Hip with Hsp70 in vitro. Failure to recover Hip-L211S constructs in co-immunoprecipitation assays with an Hsp70 monoclonal antibody suggests that the mutation is unlikely to result in a misfolded substrate.     

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.     

分子伴侣介导的细胞自噬作用机制及其功能

分子伴侣介导的细胞自噬(chaperone-mediated autophagy,CMA)是通过溶酶体途径选择性降解胞质中带KFERQ-序列的蛋白质。CMA不仅为细胞在持久饥饿状态下提供能量,还在氧化性损伤保护、维持细胞内环境稳态等方面发挥作用。此外,CMA功能障碍还与某些疾病的发生有关。该文简要综述了这方面的研究进展。     

Effect of hsp70 chaperone on the folding and misfolding of polypeptides modeling an elongating protein chain

Virtually nothing is known about the interaction of co-translationally active chaperones with nascent polypeptides and the resulting effects on peptide conformation and folding. We have explored this issue by NMR analysis of apomyoglobin N-terminal fragments of increasing length, taken as models for different stages of protein biosynthesis, in the absence and presence of the substrate binding domain of Escherichia coli Hsp70, DnaK-beta. The incomplete polypeptides misfold and self-associate under refolding conditions. In the presence of DnaK-beta, however, formation of the original self-associated species is completely or partially prevented. Chaperone interaction with incomplete protein chains promotes a globally unfolded dynamic DnaK-beta-bound state, which becomes folding-competent only upon incorporation of the residues corresponding to the C-terminal H helix. The chaperone does not bind the full-length protein at equilibrium. However, its presence strongly disfavors the kinetic accessibility of misfolding side-routes available to the full-length chain. This work supports the role of DnaK as a "holder" for incomplete N-terminal polypeptides. However, as the chain approaches its full-length status, the tendency to intramolecularly bury non-polar surface efficiently outcompetes chaperone binding. Under these conditions, DnaK serves as a "folding enhancer" by supporting folding of a population of otherwise folding-incompetent full-length protein chains.     

Characterisation of the <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> Hsp70–Hsp90 organising protein (PfHop)

Malaria is caused by Plasmodium species, whose transmission to vertebrate hosts is facilitated by mosquito vectors. The transition from the cold blooded mosquito vector to the host represents physiological stress to the parasite, and additionally malaria blood stage infection is characterised by intense fever periods. In recent years, it has become clear that heat shock proteins play an essential role during the parasite's life cycle. Plasmodium falciparum expresses two prominent heat shock proteins: heat shock protein 70 (PfHsp70) and heat shock protein 90 (PfHsp90). Both of these proteins have been implicated in the development and pathogenesis of malaria. In eukaryotes, Hsp70 and Hsp90 proteins are functionally linked by an essential adaptor protein known as the Hsp70–Hsp90 organising protein (Hop). In this study, recombinant P. falciparum Hop (PfHop) was heterologously produced in E. coli and purified by nickel affinity chromatography. Using specific anti-PfHop antisera, the expression and localisation of PfHop in P. falciparum was investigated. PfHop was shown to co-localise with PfHsp70 and PfHsp90 in parasites at the trophozoite stage. Gel filtration and co-immunoprecipitation experiments suggested that PfHop was present in a complex together with PfHsp70 and PfHsp90. The association of PfHop with both PfHsp70 and PfHsp90 suggests that this protein may mediate the functional interaction between the two chaperones.     

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 cell-based screen for inhibitors of protein folding and degradation

Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.     

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.     

The Hsp70 chaperones of the Tritryps are characterized by unusual features and novel members

Proteins belonging to the Hsp70 class of molecular chaperones are highly conserved and ubiquitous, performing an essential role in the maintenance of cellular homeostasis in almost all known organisms. Trypanosoma brucei, Trypanosoma cruzi and Leishmania major are human parasites collectively known as the Tritryps. The Tritryps undergo extensive morphological changes during their life cycles, largely triggered by the marked differences between conditions in their insect vector and human host. Hsp70s are synthesised in response to these marked changes in environment and are proposed to be required for these parasites to successfully transition between differentiation stages while remaining viable and infective. While the Tritryps Hsp70 complement consists of homologues of all the major eukaryotic Hsp70s, there are a number of novel members, and some unique structural features. This review critically evaluates the current knowledge on the Tritryps Hsp70 proteins with an emphasis on T. brucei, and highlights some novel and previously unstudied aspects of these multifaceted molecular chaperones.     

Distinguishing integral and receptor-bound heat shock protein 70 (Hsp70) on the cell surface by Hsp70-specific antibodies

Cell Stress & Chaperones journal has become a major outlet for papers and review articles about anti-heat shock protein (HSP) antibodies. In the last decade, it became evident that apart from their intracellular localization, members of the heat shock protein 90 (Hsp90; HSPC) and Hsp70 (HSPA) family are also found on the cell surface. In this review, we will focus on Hsp70 (HSPA1A), the major stress-inducible member of the human Hsp70 family. Depending on the cell type, the membrane association of Hsp70 comes in two forms. In tumor cells, Hsp70 appears to be integrated within the plasma membrane, whereas in non-malignantly transformed (herein termed normal) cells, Hsp70 is associated with cell surface receptors. This observation raises the question whether or not these two surface forms of Hsp70 in tumor and normal cells can be distinguished using Hsp70 specific antibodies. Presently a number of Hsp70 specific antibodies are commercially available. These antibodies were generated by immunizing mice either with recombinant or HeLa-derived human Hsp70 protein, parts of the Hsp70 protein, or with synthetic peptides. This review aims to characterize the binding of different anti-human Hsp70 antibodies and their capacity to distinguish between integrated and receptor-bound Hsp70 in tumor and normal cells.