Analysis of Chaperone Function and Formation of Hetero-oligomeric Complexes of Hsp18.1 and Hsp17.7, Representing Two Different Cytoplasmic sHSP Classes in Pisum sativum

Small heat shock proteins (sHSPs) are the most abundant stress proteins in plants. Usually not expressed under permissive conditions, they can accumulate to more than 2% of the total cellular protein content during heat stress. At present several points of evidence indicate that these proteins act as molecular chaperones by keeping partially denatured proteins in a folding-competent state. In plants sHSPs are encoded by a multigene family, which can be segregated into several classes according to their subcellular position and/or sequence homology. Curiously, two different classes appear in the cytoplasm. Their specific role during heat shock remains elusive. Here we present some evidence that both classes of sHSPs enhance recovery of reporter protein activity in the presence of HSP70. Applying peptide arrays prepared by SPOT synthesis and in situ analysis by confocal laser scanning microscopy, we could further show that the two classes of sHSP are attached to each other and are able to interact with non-native proteins both in vivo and in vitro. Although both of the sHSPs act similarly as molecular chaperones, immunohistochemistry experiments support the hypothesis that the two have different cellular functions in the development of heat-induced cytoplasmic heat shock granules under elevated temperatures. Daniela Wagner Deceased 24 Feburary 2004.     

The role of small heat shock proteins in parasites

The natural life cycle of many protozoan and helminth parasites involves exposure to several hostile environmental conditions. Under these circumstances, the parasites arouse a cellular stress response that involves the expression of heat shock proteins (HSPs). Small HSPs (sHSPs) constitute one of the main families of HSPs. The sHSPs are very divergent at the sequence level, but their secondary and tertiary structures are conserved and some of its members are related to α-crystallin from vertebrates. They are involved in a variety of cellular processes. As other HSPs, the sHSPs act as molecular chaperones; however, they have shown other activities apparently not related to chaperone action. In this review, the diverse activities of sHSPs in the major genera of protozoan and helminth parasites are described. These include stress response, development, and immune response, among others. In addition, an analysis comparing the sequences of sHSPs from some parasites using a distance analysis is presented. Because many parasites face hostile conditions through its life cycles the study of HSPs, including sHSPs, is fundamental.     

ATP causes small heat shock proteins to release denatured protein.

Small heat-shock proteins (sHSPs) are a ubiquitous family of low molecular mass (15-30 kDa) stress proteins that have been found in all organisms. Under stress, sHSPs such as alpha-crystallin can act as chaperones binding partially denatured proteins and preventing further denaturation and aggregation. Recently, it has been proposed that the function of sHSPs is to stabilize stress-denatured protein and then act cooperatively with other HSPs to renature the partially denatured protein in an ATP-dependent manner. However, the process by which this occurs is obscure. As no significant phosphorylation of alpha-crystallin was observed during the renaturation, the role of ATP is not clear. It is now shown that ATP at normal physiological concentrations causes sHSPs to change their confirmation and release denatured protein, allowing other molecular chaperones such as HSP70 to renature the protein and renew its biological activity. In the absence of ATP, sHSPs such as alpha-crystallin are more efficient than HSP70 in preventing stress-induced protein aggregation. This work also indicates that in mammalian systems at normal cellular ATP concentrations, sHSPs are not effective chaperones.     

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.     

Developmental control of small heat shock protein expression during pea seed maturation

Plants synthesize four classes of small heat shock proteins (sHSPs); two classes are targeted to the plastid and endoplasmic reticulum, respectively, and two are found in the cytoplasm. In this paper, we describe a new role for the two classes of cytoplasmic HSPs in maturing embryos of developing seeds. The expression of each class of sHSPs was examined in pea seeds grown under non-stress conditions using Western and Northern analysis. Class I and class II cytoplasmic sHSPs are coordinately expressed in the embryo and accumulate to levels seen in moderately heat-stressed leaves. Their induction in cotyledons coincides with the mid-maturation phase of seed development, and induction in axes roughly coincides with abscission of the seed from the ovary wall. Both classes of sHSPs persisted in cotyledons for 4 days after the onset of imbibition, but disappeared from axes shortly after germination. Neither class of cytoplasmic sHSP is expressed in non-embryonic organs associated with the seed. The timing and organ specificity of sHSP expression is paralleled by the expression of the corresponding mRNAs. Neither the plastid nor the endoplasmic reticulum sHSPs were consistently expressed during seed development, but both could be induced by heat-stressing the developing seed. Developmental regulation of the cytoplasmic sHSPs is evidence that these proteins function not only in responding to heat-stress but also during seed development and/or germination.     

Developmentally and stress-induced small heat shock proteins in cork oak somatic embryos

The timing and tissue localization of small heat shock proteins (sHSPs) during cork oak somatic embryo development was investigated under normal growing culture conditions and in response to stress. Western blot analyses using polyclonal antibodies raised against cork oak recombinant HSP17 showed a transient accumulation of class I sHSPs during somatic embryo maturation and germination. Moreover, the amount of protein increased at all stages of embryo development in response to exogenous stress. The developmentally accumulated proteins localized to early differentiating, but not the highly dividing, regions of the root and shoot apical meristems. By contrast, these highly dividing regions were strongly immunostained after heat stress. Findings support the hypothesis of a distinct control for developmentally and stress-induced accumulation of class I sHSPs. The possible role of sHSPs is discussed in relation to their tissue specific localization.     

The small heat shock proteins in plants are members of an ancient family of heat induced proteins

In response to high temperature stress, plants express numerous small heat shock proteins (sHSPs) belonging to at least five related gene families. in vitro studies suggest sHSPs act as molecular chaperones to prevent irreversible heat denaturation of other proteins. The diversity of sHSPs in plants is unique among eukaryotes and makes it of interest to understand the origins of these proteins. sHSP-related proteins have now been identified in 13 prokaryotes, and in many of these prokaryotes the sHSPs are heat-regulated as seen higher plants. The prokaryotic sHSPs were analyzed by pairwise and mutliple sequence alignments with each other and with plant sHSPs. The higher plant class I cytosolic sHSPs are shown to be most similar to a subset of the prokaryotic sHSPs, including HSP 16.6 from the cyanobacterium Synechocystis. Genetic studies in this model cyanobacterium may provide insight into sHSP function in vivo, and into potential roles of sHSPs in higher plant cells.     

The small heat shock protein (sHSP) genes in the silkworm, Bombyx mori, and comparative analysis with other insect sHSP genes

Background  

Small heat shock proteins (sHSPs) are products of heat shock response and of other stress responses, and ubiquitous in all three domains of life, archaea, bacteria, and eukarya. They mainly function as molecular chaperones to protect proteins from being denatured in extreme conditions. Study on insect sHSPs could provide some insights into evolution of insects that have adapted to diverse niches in the world.     

The emerging role of HSP20 as a multifunctional protective agent

The small heat shock proteins (sHSPs) are a highly conserved family of molecular chaperones that are ubiquitously expressed throughout nature. They are transiently upregulated in many tissue types following stressful stimuli. Recently, one member of the sHSP family, HSP20 (HspB6), has been shown to be highly effective as a protective mediator against a number of debilitating pathological conditions, including cardiac hypertrophy and Alzheimer's disease. Hsp20 is also an important modulator of vital physiological processes, such as smooth muscle relaxation and cardiac contractility. This review focuses on the molecular mechanisms employed by HSP20 that allow it to act as an innate protector in the context of cardiovascular and neurological diseases. Emerging evidence for a possible role as an anti-cancer agent is also presented.     

The growing world of small heat shock proteins: from structure to functions

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world’s experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12–15, 2016).     

Comparison of the small heat shock proteins alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP in heart and skeletal muscle

Seven members of the small heat shock protein (sHSP) family are exceptional with respect to their constitutive high abundance in muscle tissue. It has been suggested that sHSPs displaying chaperone-like properties may stabilize myofibrillar proteins during stress conditions and prevent them from loss of function. In the present study five sHSPs (alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP) were investigated with respect to similarities and differences of their expression in heart and skeletal muscle under normal and ischemic conditions. In ischemic heart and skeletal muscle these five sHSPs translocated from cytosol to the Z-/I-area of myofibrils. Myofibrillar binding of all sHSPs was very tight and resisted for the most part extraction with 1 M NaSCN or 1 M urea. MKBP and HSP20 became extracted by 1 M NaSCN to a significant extent indicating that these two sHSPs may bind partially to actin-associated proteins which were completely extracted by this treatment. Ultrastructural localization of alphaB-crystallin showed diffuse distribution of immunogold label throughout the entire I-band in skeletal muscle fibers whereas in cardiomyocytes alphaB-crystallin was preferentially located at the N-line position of the I-band. These observations indicate different myofibrillar binding sites of alphaB-crystallin in cardiomyocytes versus skeletal muscle fibers. Further differences of the properties of sHSPs could be observed regarding fiber type distribution of sHSPs. Thus sHSPs form a complex stress-response system in striated muscle tissue with some common as well as some distinct functions in different muscle types.     

Small heat shock proteins and α-crystallins: dynamic proteins with flexible functions

The small heat shock proteins (sHSPs) and the related α-crystallins (αCs) are virtually ubiquitous proteins that are strongly induced by a variety of stresses, but that also function constitutively in multiple cell types in many organisms. Extensive research has demonstrated that a majority of sHSPs and αCs can act as ATP-independent molecular chaperones by binding denaturing proteins and thereby protecting cells from damage due to irreversible protein aggregation. As a result of their diverse evolutionary history, their connection to inherited human diseases, and their novel protein dynamics, sHSPs and αCs are of significant interest to many areas of biology and biochemistry. However, it is increasingly clear that no single model is sufficient to describe the structure, function or mechanism of action of sHSPs and αCs. In this review, we discuss recent data that provide insight into the variety of structures of these proteins, their dynamic behavior, how they recognize substrates, and their many possible cellular roles.     

Identification of mammalian protein quality control factors by high-throughput cellular imaging

Protein Quality Control (PQC) pathways are essential to maintain the equilibrium between protein folding and the clearance of misfolded proteins. In order to discover novel human PQC factors, we developed a high-content, high-throughput cell-based assay to assess PQC activity. The assay is based on a fluorescently tagged, temperature sensitive PQC substrate and measures its degradation relative to a temperature insensitive internal control. In a targeted screen of 1591 siRNA genes involved in the Ubiquitin-Proteasome System (UPS) we identified 25 of the 33 genes encoding for 26S proteasome subunits and discovered several novel PQC factors. An unbiased genome-wide siRNA screen revealed the protein translation machinery, and in particular the EIF3 translation initiation complex, as a novel key modulator of misfolded protein stability. These results represent a comprehensive unbiased survey of human PQC components and establish an experimental tool for the discovery of genes that are required for the degradation of misfolded proteins under conditions of proteotoxic stress.     

Phylogenetic and Biochemical Studies Reveal a Potential Evolutionary Origin of Small Heat Shock Proteins of Animals from Bacterial Class A

Small heat shock proteins (sHSPs), as one subclass of molecular chaperones, are important for cells to protect proteins under stress conditions. Unlike the large HSPs (represented by Hsp60 and Hsp70), sHSPs are highly divergent in both primary sequences and oligomeric status, with their evolutionary relationships being unresolved. Here the phylogenetic analysis of a representative 51 sHSPs (covering the six subfamilies: bacterial class A, bacterial class B, archae, fungi, plant, and animal) reveals a close relationship between bacterial class A and animal sHSPs which form an outgroup. Accumulating data indicate that the oligomers from bacterial class A and animal sHSPs appear to exhibit polydispersity, while those from the rest exhibit monodispersity. Together, the close evolutionary relationship and the similarity in oligomeric polydispersity between bacterial class A and animal sHSPs not only suggest a potential evolutionary origin of the latter from the former, but also imply that their oligomeric polydispersity is somehow a property determined by their primary sequences. [Reviewing Editor: Dr. Martin Kreitman]     

TRIM11 cooperates with HSF1 to suppress the anti-tumor effect of proteotoxic stress drugs

Cells mainly rely on stress proteins, such as heat-shock proteins (HSPs), to respond to various proteotoxic conditions. These proteins protect tumor cells and enhance their survive. However, the regulation of stress proteins involved in protein quality control (PQC) is still poorly understood. Here, we report that the expression of TRIM11, an important regulator of PQC, is positively correlated with tumor cell surviaval during the proteotoxic conditions induced by anti-tumor drugs. In addition, HSF1 is required for TRIM11-mediated removal of protein aggregates and resistance of proteotoxic stress. During these processes, TRIM11 interacts with and stabilizes HSF1, increaseing HSF1 levels in the nucleus. These findings identify that TRIM11, through cooperation with HSF1, protects cells against the proteotoxic stress and promotes tumor cell survival.     

Dodecameric structure of a small heat shock protein from Mycobacterium marinum M

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones present ubiquitously in all kingdoms of life. Their low molecular weight subunits associate to form higher order structures. Under conditions of stress, sHSPs prevent aggregation of substrate proteins by undergoing rapid changes in their conformation or stoichiometry. Polydispersity and dynamic nature of these proteins have made structural investigations through crystallography a daunting task. In pathogens like Mycobacteria, sHSPs are immuno-dominant antigens, enabling survival of the pathogen within the host and contributing to disease persistence. We characterized sHSPs from Mycobacterium marinum M and determined the crystal structure of one of these. The protein crystallized in three different conditions as dodecamers, with dimers arranged in a tetrahedral fashion to form a closed cage-like architecture. Interestingly, we found a pentapeptide bound to the dodecamers revealing one of the modes of sHSP-substrate interaction. Further, we have observed that ATP inhibits the chaperoning activity of the protein.     

Evolution and functional diversification of the small heat shock protein/α-crystallin family in higher plants

Small heat shock proteins (sHSPs) are chaperones that play an important role in stress tolerance. They consist of an alpha-crystallin domain (ACD) flanked by N- and C-terminal regions. However, not all proteins that contain an ACD, hereafter referred to as ACD proteins, are sHSPs because certain ACD proteins are known to have different functions. Furthermore, since not all ACD proteins have been identified yet, current classifications are incomplete. A total of 17 complete plant proteomes were screened for the presence of ACD proteins by HMMER profiling and the identified ACD protein sequences were classified by maximum likelihood phylogeny. Differences among and within groups were analysed, and levels of functional constraint were determined. There are 29 different classes of ACD proteins, eight of which contain classical sHSPs and five likely chaperones. The other classes contain proteins with uncharacterised or poorly characterised functions. N- and C-terminal sequences are conserved within the phylogenetic classes. Phylogenetics suggests a single duplication of the CI sHSP ancestor that occurred prior to the speciation of mono- and dicotyledons. This was followed by a number of more recent duplications that resulted in the presence of many paralogues. The results suggest that N- and C-terminal sequences of sHSPs play a role in class-specific functionality and that non-sHSP ACD proteins have conserved but unexplored functions, which are mainly determined by subsequences other than that of the ACD.     

植物小分子热休克蛋白

在热刺激下,真核和原核细胞都能产生一类小分子热休克蛋白(smallheatshockproteins,sHSPs),植物sHSPs数量、种类众多而且十分重要。目前已经发现植物中至少存在6种细胞内sHSPs。植物在特定的发育阶段或受到逆境条件的刺激产生sHSPs。大量研究表明sHSPs通过分子伴侣机制保护逆境中的细胞,在植物逆境忍耐中起着重要作用。     

Small heat shock proteins and the cytoskeleton: An essential interplay for cell integrity?

The cytoskeleton is a highly complex network of three major intracellular filaments, microfilaments (MFs), microtubules (MTs) and intermediate filaments (IFs). This network plays a key role in the control of cell shape, division, functions and interactions in animal organs and tissues. Dysregulation of the network can contribute to numerous human diseases. Although small HSPs (sHSPs) and in particular HSP27 (HSPB1) or αB-crystallin (HSPB5) display a wide range of cellular properties, they are mostly known for their ability to protect cells under stress conditions. Mutations in some sHSPs have been found to affect their ability to interact with cytoskeleton proteins, leading to IF aggregation phenotypes that mimick diseases related to disorders in IF proteins (i.e. desmin, vimentin and neuro-filaments). The aim of this review is to discuss new findings that point towards the possible involvement of IFs in the cytoprotective functions of sHSPs, both in physiological and pathological settings, including the likelihood that sHSPs such as HSPB1 may play a role during epithelial-to-mesenchymal transition (EMT) during fibrosis or cancer progression. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.