Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding

Small heat shock proteins (sHsps) are an evolutionary conserved class of ATP-independent chaperones that protect cells against proteotoxic stress. sHsps form assemblies with aggregation-prone misfolded proteins, which facilitates subsequent substrate solubilization and refolding by ATP-dependent Hsp70 and Hsp100 chaperones. Substrate solubilization requires disruption of sHsp association with trapped misfolded proteins. Here, we unravel a specific interplay between Hsp70 and sHsps at the initial step of the solubilization process. We show that Hsp70 displaces surface-bound sHsps from sHsp–substrate assemblies. This Hsp70 activity is unique among chaperones and highly sensitive to alterations in Hsp70 concentrations. The Hsp70 activity is reflected in the organization of sHsp–substrate assemblies, including an outer dynamic sHsp shell that is removed by Hsp70 and a stable core comprised mainly of aggregated substrates. Binding of Hsp70 to the sHsp/substrate core protects the core from aggregation and directs sequestered substrates towards refolding pathway. The sHsp/Hsp70 interplay has major impact on protein homeostasis as it sensitizes substrate release towards cellular Hsp70 availability ensuring efficient refolding of damaged proteins under favourable folding conditions.     

Role of sHsps in organizing cytosolic protein aggregation and disaggregation

Small heat shock proteins (sHsps) exhibit an ATP-independent chaperone activity to prevent the aggregation of misfolded proteins in vitro. The seemingly conflicting presence of sHsps in insoluble protein aggregates in cells obstructs a precise definition of sHsp function in proteostasis networks. Recent findings specify sHsp activities in protein quality control systems. The sHsps of yeast, Hsp42 and Hsp26, interact with early unfolding intermediates of substrates, keeping them in a ready-to-refold conformation close to the native state. This activity facilitates substrate refolding by ATP-dependent Hsp70-Hsp100 disaggregating chaperones. Hsp42 can actively sequester misfolded proteins and promote their deposition at specific cellular sites. This aggregase activity represents a cytoprotective protein quality control strategy. The aggregase function of Hsp42 controls the formation of cytosolic aggregates (CytoQs) under diverse stress regimes and can be reconstituted in vitro, demonstrating that Hsp42 is necessary and sufficient to promote protein aggregation. Substrates sequestered at CytoQs can be dissociated by Hsp70-Hsp100 disaggregases for subsequent triage between refolding and degradation pathways or are targeted for destruction by selective autophagy termed proteophagy.     

Refolding of substrates bound to small Hsps relies on a disaggregation reaction mediated most efficiently by ClpB/DnaK

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that bind denatured proteins in vitro, thereby facilitating their subsequent refolding by ATP-dependent chaperones. The mechanistic basis of this refolding process is poorly defined. We demonstrate that substrates complexed to sHsps from various sources are not released spontaneously. Dissociation and refolding of sHsp bound substrates relies on a disaggregation reaction mediated by the DnaK system, or, more efficiently, by ClpB/DnaK. While the DnaK system alone works for small, soluble sHsp/substrate complexes, ClpB/DnaK-mediated protein refolding is fastest for large, insoluble protein aggregates with incorporated sHsps. Such conditions reflect the situation in vivo, where sHsps are usually associated with insoluble proteins during heat stress. We therefore propose that sHsp function in cellular protein quality control is to promote rapid resolubilization of aggregated proteins, formed upon severe heat stress, by DnaK or ClpB/DnaK.     

Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation.

The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers.     

Disassembling protein aggregates in the yeast cytosol. The cooperation of Hsp26 with Ssa1 and Hsp104

In all organisms studied, elevated temperatures induce the expression of a variety of stress proteins, among them small Hsps (sHsp). sHsps are chaperones that prevent the unspecific aggregation of proteins by forming stable complexes with unfolded polypeptides. Reactivation of captured proteins requires the assistance of other ATP-dependent chaperones. How sHsps and ATP-dependent chaperones work together is poorly understood. Here, we analyzed the interplay of chaperones present in the cytosol of Saccharomyces cerevisiae. Specifically, we characterized the influence of Hsp104 and Ssa1 on the disassembly of Hsp26 x substrate complexes in vitro and in vivo. We show that recovery of proteins from aggregates in the cell requires the chaperones to work together with defined but overlapping functions. During reactivation, proteins are transferred from a stable complex with Hsp26 to Hsp104 and Hsp70. The need for ATP-dependent chaperones depends on the type of sHsp x substrate complex. Although Ssa1 is able to release substrate proteins from soluble Hsp26 x substrate complexes, Hsp104 is essential to dissociate substrate proteins from aggregates with incorporated sHsps. Our results are consistent with a model of several interrelated defense lines against protein aggregation.     

Two Bacterial Small Heat Shock Proteins,IbpA and IbpB,Form a Functional Heterodimer

Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones which in stress conditions bind to unfolded protein substrates and prevent their irreversible aggregation. Substrates trapped in sHsps-containing aggregates are efficiently refolded into native structures by ATP-dependent Hsp70 and Hsp100 chaperones. Most γ-proteobacteria possess a single sHsp (IbpA), while in a subset of Enterobacterales, as a consequence of ibpA gene duplication event, a two-protein sHsp (IbpA and IbpB) system has evolved. IbpA and IbpB are functionally divergent. Purified IbpA, but not IbpB, stably interacts with aggregated substrates, yet both sHsps are required to be present at the substrate denaturation step for subsequent efficient Hsp70-Hsp100-dependent substrate refolding. IbpA and IbpB interact with each other, influence each other’s expression levels and degradation rates. However, the crucial information on how these two sHsps interact and what is the basic building block required for proper sHsps functioning was missing. Here, based on NMR, mass spectrometry and crosslinking studies, we show that IbpA-IbpB heterodimer is a dominating functional unit of the two sHsp system in Enterobacterales. The principle of heterodimer formation is similar to one described for homodimers of single bacterial sHsps. β-hairpins formed by strands β5 and β7 of IbpA or IbpB crystallin domains associate with the other one's β-sandwich in the heterodimer structure. Relying on crosslinking and molecular dynamics studies, we also propose the orientation of two IbpA-IbpB heterodimers in a higher order tetrameric structure.     

Some like it hot: the structure and function of small heat-shock proteins

Small heat-shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. Recent evidence suggests that they maintain protein homeostasis by binding proteins in non-native conformations, thereby preventing substrate aggregation. Some members of the sHsp family are inactive or only partially active under physiological conditions, and transition toward the active state is induced by specific triggers, such as elevated temperature. Release of substrate proteins bound to sHsps requires cooperation with ATP-dependent chaperones, suggesting that sHsps create a reservoir of non-native proteins for subsequent refolding.     

Hsp42 is the general small heat shock protein in the cytosol of Saccharomyces cerevisiae

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the unspecific aggregation of proteins. So far, Hsp26 was the only unambiguously identified member of the sHsp family in Saccharomyces cerevisiae. We show here that the sHsp system in the cytosol of S. cerevisiae consists of two proteins, Hsp26 and Hsp42. Hsp42 forms large dynamic oligomers with a barrel-like structure. In contrast to Hsp26, which functions predominantly at heat shock temperatures, Hsp42 is active as a chaperone under all conditions tested in vivo and in vitro. Under heat shock conditions, both Hsp42 and Hsp26 suppress the aggregation of one-third of the cytosolic proteins. This subset is about 90% overlapping for Hsp42 and Hsp26. The sHsp substrates belong to different biochemical pathways. This indicates a general protective function of sHsps for proteome stability in S. cerevisiae. Consistent with this observation, sHsp knockout strains show phenotypical defects. Taken together, our results define Hsp42 as an important player for protein homeostasis at physiological and under stress conditions.     

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.     

Structural dynamics of archaeal small heat shock proteins

Small heat shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. In vivo, sHsps contribute to thermotolerance. Recent evidence suggests that their function in the cellular chaperone network is to maintain protein homeostasis by complexing a variety of non-native proteins. One of the most characteristic features of sHsps is their organization into large, sphere-like structures commonly consisting of 12 or 24 subunits. Here, we investigated the functional and structural properties of Hsp20.2, an sHsp from Archaeoglobus fulgidus, in comparison to its relative, Hsp16.5 from Methanocaldococcus jannaschii. Hsp20.2 is active in suppressing the aggregation of different model substrates at physiological and heat-stress temperatures. Electron microscopy showed that Hsp20.2 forms two distinct types of octahedral oligomers of slightly different sizes, indicating certain structural flexibility of the oligomeric assembly. By three-dimensional analysis of electron microscopic images of negatively stained specimens, we were able to reconstitute 3D models of the assemblies at a resolution of 19 Å. Under conditions of heat stress, the distribution of the structurally different Hsp20.2 assemblies changed, and this change was correlated with an increased chaperone activity. In analogy to Hsp20.2, Hsp16.5 oligomers displayed structural dynamics and exhibited increased chaperone activity under conditions of heat stress. Thus, temperature-induced conformational regulation of the activity of sHsps may be a general phenomenon in thermophilic archaea.     

Sequestration of toxic oligomers by HspB1 as a cytoprotective mechanism

Small heat shock proteins (sHsps) are molecular chaperones that protect cells from cytotoxic effects of protein misfolding and aggregation. HspB1, an sHsp commonly associated with senile plaques in Alzheimer's disease (AD), prevents the toxic effects of Aβ aggregates in vitro. However, the mechanism of this chaperone activity is poorly understood. Here, we observed that in two distinct transgenic mouse models of AD, mouse HspB1 (Hsp25) localized to the penumbral areas of plaques. We have demonstrated that substoichiometric amounts of human HspB1 (Hsp27) abolish the toxicity of Aβ oligomers on N2a (mouse neuroblastoma) cells. Using biochemical methods, spectroscopy, light scattering, and microscopy methods, we found that HspB1 sequesters toxic Aβ oligomers and converts them into large nontoxic aggregates. HspB1 was overexpressed in N2a cells in response to treatment with Aβ oligomers. Cultured neurons from HspB1-deficient mice were more sensitive to oligomer-mediated toxicity than were those from wild-type mice. Our results suggest that sequestration of oligomers by HspB1 constitutes a novel cytoprotective mechanism of proteostasis. Whether chaperone-mediated cytoprotective sequestration of toxic aggregates may bear clues to plaque deposition and may have potential therapeutic implications must be investigated in the future.     

A model for heterooligomer formation in the heat shock response of Escherichia coli

Small heat shock proteins (sHsp) are widely distributed molecular chaperones that bind to misfolded proteins to prevent irreversible aggregation and aid in refolding to a competent state. The sHsps characterized thus far all contain a conserved α-crystallin, and variable N- and C-termini critical for chaperone activity and oligomerization. The Escherichia coli sHsps IbpA and IbpB share 48% sequence homology, are induced by heat shock and oxidative stress, and each requires the presence of the other to effect protein protection. Molecular Dynamics (MD) simulations of homology-modeled monomers and heterooligomers of these sHsps identify a possible mechanism for cooperation between IbpA and IbpB.     

Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21

Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25° in relation to each other, suggesting a role for global dynamics in dodecamer function.     

Ubiquitin conjugation triggers misfolded protein sequestration into quality control foci when Hsp70 chaperone levels are limiting

Ubiquitin accumulation in amyloid plaques is a pathological marker observed in the vast majority of neurodegenerative diseases, yet ubiquitin function in these inclusions is controversial. It has been suggested that ubiquitylated proteins are directed to inclusion bodies under stress conditions, when both chaperone-mediated refolding and proteasomal degradation are compromised or overwhelmed. Alternatively, ubiquitin and chaperones may be recruited to preformed inclusions to promote their elimination. We address this issue using a yeast model system, based on expression of several mildly misfolded degradation substrates in cells with altered chaperone content. We find that the heat shock protein 70 (Hsp70) chaperone pair Ssa1/Ssa2 and the Hsp40 cochaperone Sis1 are essential for degradation. Substrate ubiquitylation is strictly dependent on Sis1, whereas Ssa1 and Ssa2 are dispensable. Remarkably, in Ssa1/Ssa2-depleted cells, ubiquitylated substrates are sequestered into detergent-insoluble, Hsp42-positive inclusion bodies. Unexpectedly, sequestration is abolished by preventing substrate ubiquitylation. We conclude that Hsp40 is required for the targeting of misfolded proteins to the ubiquitylation machinery, whereas the decision to degrade or sequester ubiquitylated proteins is mediated by the Hsp70s. Accordingly, diminished Hsp70 levels, as observed in aging or certain pathological conditions, might be sufficient to trigger ubiquitin-dependent sequestration of partially misfolded proteins into inclusion bodies.     

The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. Proteostasis is preserved in the face of stress by a complex network of cellular machinery, including the small heat shock molecular chaperone proteins (sHsps), which act to inhibit the aggregation and deposition of misfolded protein intermediates. Despite this, the pathogenesis of several neurodegenerative diseases has been inextricably linked with the amyloid fibrillar aggregation and deposition of α-synuclein (α-syn). The sHsps are potent inhibitors of α-syn aggregation in vitro. However, the limited availability of a robust, cell-based model of α-syn aggregation has, thus far, restricted evaluation of sHsp efficacy in the cellular context. 相似文献   

Small heat shock proteins potentiate amyloid dissolution by protein disaggregases from yeast and humans

How small heat shock proteins (sHsps) might empower proteostasis networks to control beneficial prions or disassemble pathological amyloid is unknown. Here, we establish that yeast sHsps, Hsp26 and Hsp42, inhibit prionogenesis by the [PSI+] prion protein, Sup35, via distinct and synergistic mechanisms. Hsp42 prevents conformational rearrangements within molten oligomers that enable de novo prionogenesis and collaborates with Hsp70 to attenuate self-templating. By contrast, Hsp26 inhibits self-templating upon binding assembled prions. sHsp binding destabilizes Sup35 prions and promotes their disaggregation by Hsp104, Hsp70, and Hsp40. In yeast, Hsp26 or Hsp42 overexpression prevents [PSI+] induction, cures [PSI+], and potentiates [PSI+]-curing by Hsp104 overexpression. In vitro, sHsps enhance Hsp104-catalyzed disaggregation of pathological amyloid forms of α-synuclein and polyglutamine. Unexpectedly, in the absence of Hsp104, sHsps promote an unprecedented, gradual depolymerization of Sup35 prions by Hsp110, Hsp70, and Hsp40. This unanticipated amyloid-depolymerase activity is conserved from yeast to humans, which lack Hsp104 orthologues. A human sHsp, HspB5, stimulates depolymerization of α-synuclein amyloid by human Hsp110, Hsp70, and Hsp40. Thus, we elucidate a heretofore-unrecognized human amyloid-depolymerase system that could have applications in various neurodegenerative disorders.     

A novel small heat shock protein of <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>: characterization,structure modeling,and expression profiles under environmental stresses

Small heat shock proteins (sHsps) are a class of chaperones with low molecular weight, feathered by a C-terminal α-crystallin domain (ACD). They participate in reestablishing the stability of partially denatured proteins and therefore contribute to cellular homeostasis. In this work, we identified a sHsp homolog (designated as sHsp19) from Haliotis discus hannai, an economically important farmed mollusk in East Asia. sHsp19 possesses a sHsp hallmark domain, which exhibits the typical fold of ACD as revealed by a three-dimensional model constructed through an iterative threading assembly refinement method. The amino acid sequence sHsp19 shares low identities with any other known sHsps, with percentages below 35 %. Besides, sHsp19 shows relatively distant phylogenetic relationships with sHsps of various mollusks, including two other identified sHsps of abalone subspecies. qRT-PCR analysis indicated that the expression of sHsp19 occurred in multiple tissues. Upon exposure to thermal, oxidative, and multiple toxic metal stresses, the level of sHsp19 mRNA was rapidly elevated in a persistent fashion, with the maximum increase up to 170.58-, 405.84-, and 361.96-fold, respectively. These results indicate sHsp is a novel sHsp that possesses the distinguishing structural feature of sHsps but has remote homologies with known sHsps. It is likely to be important in stress adaptation of abalone and may be applied as a bioindicator for monitoring pollution or detrimental changes of environment in abalone culture.     

Chaperone activity of human small heat shock protein-GST fusion proteins

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.     

Preventing α-synuclein aggregation: The role of the small heat-shock molecular chaperone proteins

Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies.     

The chloroplast‐localized small heat shock protein Hsp21 associates with the thylakoid membranes in heat‐stressed plants

The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity of sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps with a single conserved β‐sandwich fold domain defining the family, the α‐crystallin domain, whereas the N‐terminal and C‐terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non‐metazoan sHsp expressed in the chloroplasts in green plants which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat‐stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic ¹⁵N‐isotope labeling of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat‐stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered.