The functional roles of the unstructured N- and C-terminal regions in αB-crystallin and other mammalian small heat-shock proteins

Small heat-shock proteins (sHsps), such as αB-crystallin, are one of the major classes of molecular chaperone proteins. In vivo, under conditions of cellular stress, sHsps are the principal defence proteins that prevent large-scale protein aggregation. Progress in determining the structure of sHsps has been significant recently, particularly in relation to the conserved, central and β-sheet structured α-crystallin domain (ACD). However, an understanding of the structure and functional roles of the N- and C-terminal flanking regions has proved elusive mainly because of their unstructured and dynamic nature. In this paper, we propose functional roles for both flanking regions, based around three properties: (i) they act in a localised crowding manner to regulate interactions with target proteins during chaperone action, (ii) they protect the ACD from deleterious amyloid fibril formation and (iii) the flexibility of these regions, particularly at the extreme C-terminus in mammalian sHsps, provides solubility for sHsps under chaperone and non-chaperone conditions. In the eye lens, these properties are highly relevant as the crystallin proteins, in particular the two sHsps αA- and αB-crystallin, are present at very high concentrations.     

Microbial small heat shock proteins and their use in biotechnology

Small heat shock proteins (sHsps) exist in almost all organisms. Most organisms have more than one sHsp, but their number can be as high as 65, as found in the eukaryote, Vitis vinifera. The function of sHsps is well-known; they confer thermotolerance to cellular cultures and proteins in cellular extracts during prolonged incubations at elevated temperatures. This demonstrates the ability of sHsps to protect cellular proteins, and to maintain cellular viability under conditions of intensive stress, such as heat shock or chemical treatments. sHsps have several properties that distinguish them from heat shock proteins (Hsps): they function as ATP-independent chaperones, require the flexible assembly and reassembly of oligomeric complex structures for their activation, and exhibit a wide range of substrate-binding capacities. Recent studies indicate that sHsps have important biological functions in thermostability, disaggregation, and proteolysis inhibition. These functions can be harnessed for various applications, including nanobiotechnology, proteomics, bioproduction, and bioseparation. In this review, we discuss the properties and diversity of microbial sHsps, as well as their potential uses in the biotechnology industry.     

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.     

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.     

Expression of low molecular weight heat-shock proteins and total antioxidant activity in the Mediterranean tree Quercus ilex L. in relation to seasonal and diurnal changes in physiological parameters

The total antioxidant activity (TAA) and the accumulation of small heat shock proteins (sHsps) were analysed under field conditions in Quercus ilex with regard to organ ontogeny and the physiological state of the plant. The results point toward the participation of sHsps and an increase of TAA in the general adaptation syndrome (GAS) of woody Mediterranean evergreens. In leaves and stems, there was a definite TAA seasonal pattern but no effect from diurnal variations or from the current stage of organ ontogeny. TAA was about twice as large in summer as in spring and winter and the hydrophilic antioxidant content was about 16 times greater than that of lipophilic antioxidants. The accumulation of sHsps in leaves also showed a seasonal pattern, but no effect from diurnal variations or from leaf ontogeny. In summer days, the sHsps content remained invariable even during the daylight hours in which the leaves were physiologically recovered. However, the accumulation of sHsps in stems did vary in relation to organ ontogeny. Old stems had a high accumulation of sHsps throughout the year, whereas in young stems, accumulation of sHsps was detected only in summer. This is probably due to a higher quantity of lignified and suberized tissues in the older stems. Using two‐dimensional immunodetection for leaves and stems, two sets of Hs protein species (17 and 10 kDa regions) were observed. In stems, there was an increase in 10 kDa proteins from winter to summer. These results are discussed and the possible role of the two types of polypeptides in the face of environmental and endogenous oxidative stress are considered.     

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.     

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.     

Reaction of small heat-shock proteins to different kinds of cellular stress in cultured rat hippocampal neurons

Upregulation of small heat-shock proteins (sHsps) in response to cellular stress is one mechanism to increase cell viability. We previously described that cultured rat hippocampal neurons express five of the 11 family members but only upregulate two of them (HspB1 and HspB5) at the protein level after heat stress. Since neurons have to cope with many other pathological conditions, we investigated in this study the expression of all five expressed sHsps on mRNA and protein level after sublethal sodium arsenite and oxidative and hyperosmotic stress. Under all three conditions, HspB1, HspB5, HspB6, and HspB8 but not HspB11 were consistently upregulated but showed differences in the time course of upregulation. The increase of sHsps always occurred earlier on mRNA level compared with protein levels. We conclude from our data that these four upregulated sHsps (HspB1, HspB5, HspB6, HspB8) act together in different proportions in the protection of neurons from various stress conditions.     

The Diverse Functions of Small Heat Shock Proteins in the Proteostasis Network

The protein quality control (PQC) system maintains protein homeostasis by counteracting the accumulation of misfolded protein conformers. Substrate degradation and refolding activities executed by ATP-dependent proteases and chaperones constitute major strategies of the proteostasis network. Small heat shock proteins represent ATP-independent chaperones that bind to misfolded proteins, preventing their uncontrolled aggregation. sHsps share the conserved α-crystallin domain (ACD) and gain functional specificity through variable and largely disordered N- and C-terminal extensions (NTE, CTE). They form large, polydisperse oligomers through multiple, weak interactions between NTE/CTEs and ACD dimers. Sequence variations of sHsps and the large variability of sHsp oligomers enable sHsps to fulfill diverse tasks in the PQC network. sHsp oligomers represent inactive yet dynamic resting states that are rapidly deoligomerized and activated upon stress conditions, releasing substrate binding sites in NTEs and ACDs Bound substrates are usually isolated in large sHsp/substrate complexes. This sequestration activity of sHsps represents a third strategy of the proteostasis network. Substrate sequestration reduces the burden for other PQC components during immediate and persistent stress conditions. Sequestered substrates can be released and directed towards refolding pathways by ATP-dependent Hsp70/Hsp100 chaperones or sorted for degradation by autophagic pathways. sHsps can also maintain the dynamic state of phase-separated stress granules (SGs), which store mRNA and translation factors, by reducing the accumulation of misfolded proteins inside SGs and preventing unfolding of SG components. This ensures SG disassembly and regain of translational capacity during recovery periods.     

Involvement of small heat shock proteins,trehalose, and lipids in the thermal stress management in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Changes in the levels of three structurally and functionally different important thermoprotectant molecules, namely small heat shock proteins (sHsps), trehalose, and lipids, have been investigated upon heat shock in Schizosaccharomyces pombe. Both α-crystallin-type sHsps (Hsp15.8 and Hsp16) were induced after prolonged high-temperature treatment but with different kinetic profiles. The shsp null mutants display a weak, but significant, heat sensitivity indicating their importance in the thermal stress management. The heat induction of sHsps is different in wild type and in highly heat-sensitive trehalose-deficient (tps1Δ) cells; however, trehalose level did not show significant alteration in shsp mutants. The altered timing of trehalose accumulation and induction of sHsps suggest that the disaccharide might provide protection at the early stage of the heat stress while elevated amount of sHsps are required at the later phase. The cellular lipid compositions of two different temperature-adapted wild-type S. pombe cells are also altered according to the rule of homeoviscous adaptation, indicating their crucial role in adapting to the environmental temperature changes. Both Hsp15.8 and Hsp16 are able to bind to different lipids isolated from S. pombe, whose interaction might provide a powerful protection against heat-induced damages of the membranes. Our data suggest that all the three investigated thermoprotectant macromolecules play a pivotal role during the thermal stress management in the fission yeast.     

Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation.

Small heat shock proteins (sHsps) are a conserved and ubiquitous protein family. Their ability to convey thermoresistance suggests their participation in protecting the native conformation of proteins. However, the underlying functional principles of their protective properties and their role in concert with other chaperone families remain enigmatic. Here, we analysed the influence of Hsp25 on the inactivation and subsequent aggregation of a model protein, citrate synthase (CS), under heat shock conditions in vitro. We show that stable binding of several non-native CS molecules to one Hsp25 oligomer leads to an accumulation of CS unfolding intermediates, which are protected from irreversible aggregation. Furthermore, a number of different proteins which bind to Hsp25 can be isolated from heat-shocked extracts of cells. Under permissive folding conditions, CS can be released from Hsp25 and, in cooperation with Hsp70, an ATP-dependent chaperone, the native state can be restored. Taken together, our findings allow us to integrate sHsps functionally in the cellular chaperone system operating under heat shock conditions. The task of sHsps in this context is to efficiently trap a large number of unfolding proteins in a folding-competent state and thus create a reservoir of non-native proteins for an extended period of time, allowing refolding after restoration of physiological conditions in cooperation with other chaperones.     

Evidence for a novel set of small heat-shock proteins that associates with the mitochondria of murine PC12 cells and protects NADH:ubiquinone oxidoreductase from heat and oxidative stress.

Several previously unreported small heat-shock proteins (sHsps) were detected in mitochondria from heat-stressed rat PC12 cells, but not in unstressed controls. Functional inactivation of the mitochondrial sHsps with murine Hsp25 antibody indicated that these sHsps protect NADH:ubiquinone oxidoreductase and NADH dehydrogenase activity (i.e., complex I) in submitochondrial vesicles during heat and oxidative stress. These results (i) confirm the existence of multiple sHsps in mammals and indicate that several of these sHsps associate with the mitochondria, (ii) indicate a conserved function between plant and mammalian mitochondrial sHsps in protecting electron transport during stress, and (iii) suggest that these sHsps may play an important role in diseases whose etiology is based upon oxidative damage of complex I.     

Stress- and mitogen-induced phosphorylation of the small heat shock protein Hsp25 by MAPKAP kinase 2 is not essential for chaperone properties and cellular thermoresistance.

Small heat shock proteins (sHsps) show a very rapid stress- and mitogen-dependent phosphorylation by MAPKAP kinase 2. Based on this observation, phosphorylation of sHsps was thought to play a key role in mediating thermoresistance immediately after heat shock, before the increased synthesis of heat shock proteins becomes relevant. We have analysed the phosphorylation dependence of the chaperone and thermoresistance-mediating properties of the small heat shock protein Hsp25. Surprisingly, overexpression of Hsp25 mutants, which are not phosphorylated in the transfected cells, confers the same thermoresistant phenotype as overexpression of wild type Hsp25, which is either mono- or bis-phosphorylated at serine residues 15 and 86 within the cells. Furthermore, in vitro phosphorylated Hsp25 shows the same oligomerization properties and the same chaperone activity as the nonphosphorylated protein. No differences between phosphorylated and nonphosphorylated Hsp25 are detected in preventing thermal aggregation of unfolding proteins and assisting refolding of denatured proteins. The results suggest that chaperone properties of the small heat shock proteins contribute to the increased cellular thermoresistance in a phosphorylation-independent manner.     

Mouse Hsp25, a small shock protein. The role of its C-terminal extension in oligomerization and chaperone action.

Under conditions of cellular stress, small heat shock proteins (sHsps), e.g. Hsp25, stabilize unfolding proteins and prevent their precipitation from solution. 1H NMR spectroscopy has shown that mammalian sHsps possess short, polar and highly flexible C-terminal extensions. A mutant of mouse Hsp25 without this extension has been constructed. CD spectroscopy reveals some differences in secondary and tertiary structure between this mutant and the wild-type protein but analytical ultracentrifugation and electron microscopy show that the proteins have very similar oligomeric masses and quaternary structures. The mutant shows chaperone ability comparable to that of wild-type Hsp25 in a thermal aggregation assay using citrate synthase, but does not stabilize alpha-lactalbumin against precipitation following reduction with dithiothreitol. The accessible hydrophobic surface of the mutant protein is less than that of the wild-type protein and the mutant is also less stable at elevated temperature. 1H NMR spectroscopy reveals that deletion of the C-terminal extension of Hsp25 leads to induction of extra C-terminal flexibility in the molecule. Monitoring complex formation between Hsp25 and dithiothreitol-reduced alpha-lactalbumin by 1H NMR spectroscopy indicates that the C-terminal extension of Hsp25 retains its flexibility during this interaction. Overall, these data suggest that a highly flexible C-terminal extension in mammalian sHsps is required for full chaperone activity.     

Small heat shock proteins--role in apoptosis, cancerogenesis and diseases associated with protein aggregation

Small heat shock proteins (sHsps) belong to molecular chaperones, which protect prokaryotic and eukaryotic cells against deleterious effects, of stress. sHsps prevent stress induced, irreversible aggregation of damaged proteins and facilitate renaturation of bound substrates cooperating with other molecular chaperones. This review summarizes recent studies focused mainly on the involvement of sHsps in diseases related to protein aggregation. sHsps are often a component of protein aggregates forming during progress of neurodegenerative disorders. Mutation in sHsps genes have been identified, which are responsible for development of cataract, desmin related myopathy and neuropathies. sHsps protect cells against oxidative stress resulting from ischemia/reperfusion during heart or brain stroke. Several studies indicate that sHsp participate in regulation of apoptosis and are involved in cancerogenesis. Uncovering the sHsps role in diseases enable to develop new therapeutic strategies.     

The sperm outer dense fiber protein is the 10th member of the superfamily of mammalian small stress proteins

Nine proteins have been assigned to date to the superfamily of mammalian small heat shock proteins (sHsps): Hsp27 (HspB1, Hsp25), myotonic dystrophy protein kinase-binding protein (MKBP) (HspB2), HspB3, alphaA-crystallin (HspB4), alphaB-crystallin (HspB5), Hsp20 (p20, HspB6), cardiovascular heat shock protein (cvHsp [HspB7]), Hsp22 (HspB8), and HspB9. The most pronounced structural feature of sHsps is the alpha-crystallin domain, a conserved stretch of approximately 80 amino acid residues in the C-terminal half of the molecule. Using the alpha-crystallin domain of human Hsp27 as query in a BLAST search, we found sequence similarity with another mammalian protein, the sperm outer dense fiber protein (ODFP). ODFP occurs exclusively in the axoneme of sperm cells. Multiple alignment of human ODFP with the other human sHsps reveals that the primary structure of ODFP fits into the sequence pattern that is typical for this protein superfamily: alpha-crystallin domain (conserved), N-terminal domain (less conserved), central region (variable), and C-terminal tails (variable). In a phylogenetic analysis of 167 proteins of the sHsp superfamily, using Bayesian inference, mammalian ODFPs form a clade and are nested within previously identified sHsps, some of which have been implicated in cytoskeletal functions. Both the multiple alignment and the phylogeny suggest that ODFP is the 10th member of the superfamily of mammalian sHsps, and we propose to name it HspB10 in analogy with the other sHsps. The C-terminal tail of HspB10 has a remarkable low-complexity structure consisting of 10 repeats of the motif C-X-P. A BLAST search using the C-terminal tail as query revealed similarity with sequence elements in a number of Drosophila male sperm proteins, and mammalian type I keratins and cornifin-alpha. Taken together, the following findings suggest a specialized role of HspB10 in cytoskeleton: (1) the exclusive location in sperm cell tails, (2) the phylogenetic relationship with sHsps implicated in cytoskeletal functions, and (3) the partial similarity with cytoskeletal proteins.     

The plant sHSP superfamily: five new members in Arabidopsis thaliana with unexpected properties

The small heat shock proteins (sHsps), which are ubiquitous stress proteins proposed to act as chaperones, are encoded by an unusually complex gene family in plants. Plant sHsps are classified into different subfamilies according to amino acid sequence similarity and localization to distinct subcellular compartments. In the whole Arabidopsis thaliana genome, 19 genes were annotated to encode sHsps, of which 14 belong to previously defined plant sHsp families. In this paper, we report studies of the five additional sHsp genes in A. thaliana, which can now be shown to represent evolutionarily distinct sHsp subfamilies also found in other plant species. While two of these five sHsps show expression patterns typical of the other 14 genes, three have unusual tissue specific and developmental profiles and do not respond to heat induction. Analysis of intracellular targeting indicates that one sHsp represents a new class of mitochondrion-targeted sHsps, while the others are cytosolic/nuclear, some of which may cooperate with other sHsps in formation of heat stress granules. Three of the five new proteins were purified and tested for chaperone activity in vitro. Altogether, these studies complete our basic understanding of the sHsp chaperone family in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.