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.     

Methylglyoxal and small heat shock proteins

Methylglyoxal is a highly reactive dicarbonyl compound formed during glucose metabolism and able to modify phospholipids, nucleic acids, and proteins belonging to the so-called dicarbonyl proteome. Small heat shock proteins participating in protection of the cell against different unfavorable conditions can be modified by methylglyoxal. The probability of methylglyoxal modification is increased in the case of distortion of glucose metabolism (diabetes), in the case of utilization of glycolysis as the main source of energy (malignancy), and/or at low rate of modified protein turnover. We have analyzed data on modification of small heat shock protein HspB1 in different tumors and under distortion of carbohydrate metabolism. Data on the effect of methylglyoxal modification on stability, chaperone-like activity, and antiapoptotic activity of HspB1 were analyzed. We discuss data on methylglyoxal modifications of lens α-crystallins. The mutual dependence and mutual effects of methylglyoxal modification and other posttranslational modifications of lens crystallins are analyzed. We conclude that although there is no doubt that the small heat shock proteins undergo methylglyoxal modification, the physiological significance of this process remains enigmatic, and new experimental approaches should be developed for understanding how this type of modification affects functioning of small heat shock proteins in the cell.     

Chaperone activity of cytosolic small heat shock proteins from wheat.

Small Hsps (sHsps) and the structurally related eye lens alpha-crystallins are ubiquitous stress proteins that exhibit ATP-independent molecular chaperone activity. We studied the chaperone activity of dodecameric wheat TaHsp16.9C-I, a class I cytosolic sHsp from plants and the only eukaryotic sHsp for which a high resolution structure is available, along with the related wheat protein TaHsp17.8C-II, which represents the evolutionarily distinct class II plant cytosolic sHsps. Despite the available structural information on TaHsp16.9C-I, there is minimal data on its chaperone activity, and likewise, data on activity of the class II proteins is very limited. We prepared purified, recombinant TaHsp16.9C-I and TaHsp17.8C-II and find that the class II protein comprises a smaller oligomer than the dodecameric TaHsp16.9C-I, suggesting class II proteins have a distinct mode of oligomer assembly as compared to the class I proteins. Using malate dehydrogenase as a substrate, TaHsp16.9C-I was shown to be a more effective chaperone than TaHsp17.8C-II in preventing heat-induced malate dehydrogenase aggregation. As observed by EM, morphology of sHsp/substrate complexes depended on the sHsp used and on the ratio of sHsp to substrate. Surprisingly, heat-denaturing firefly luciferase did not interact significantly with TaHsp16.9C-I, although it was fully protected by TaHsp17.8C-II. In total the data indicate sHsps show substrate specificity and suggest that N-terminal residues contribute to substrate interactions.     

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.     

Mutations of small heat shock proteins and human congenital diseases

The structure and properties of different members of a large family of small heat shock proteins (sHsp) playing an important role in cell homeostasis are described. Participation of the N-terminal domain in formation of large oligomers and chaperone activity of sHsp is analyzed. The structure of the α-crystallin domain of sHsp is characterized and the role of this domain in sHsp dimerization and chaperone activity is discussed. The properties of the C-terminal region of sHsp are described, and its participation in formation of large oligomers and chaperone activity are analyzed. The data from the literature on HspB1 and HspB3 mutations are presented, and involvement of these mutations in development of certain neurodegenerative diseases is discussed. Mutations of HspB4 are described and data on involvement of these mutations in development of cataract are presented. Multiple effects of HspB5 mutations are analyzed, and data are presented indicating that mutations of this protein are accompanied by development of different congenital diseases, such as cataract and different types of myopathies. The data on HspB6 and HspB8 mutations are presented, and feasible effects of these mutations on proteins structure are analyzed. Probable mechanisms underlying sHsp mutation-induced development of different congenital diseases are discussed.     

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.     

A mechanism of action for small heat shock proteins

Molecular dynamics simulations of a fitted multimeric structure of Mycobacterium tuberculosis α-crystallin (Mtb Acr) identify solvent exclusion from the β(4)-β(8) hydrophobic groove as a critical factor driving subunit assembly. Dehydration is also implicated as a determinant factor governing the chaperone activity of the dimer upon its dissociation from the oligomer. Two exposed hydrogen bonds, responsible for stabilizing the β(8)-β(9) fold are identified as key mechanistic elements in this process. Based on the overproduction of the chemokine CXCL16, observed after macrophage exposure to Mtb Acr, the proteases ADAM10 and ADAM17 are mooted as possible targets of this chaperone activity.     

Biotechnical applications of small heat shock proteins from bacteria

The stress responses of most bacteria are thought to involve the upregulation of small heat shock proteins. We describe here some of the most pertinent aspects of small heat shock proteins, to highlight their potential for use in various applications. Bacterial species have between one and 13 genes encoding small heat shock proteins, the precise number depending on the species considered. Major efforts have recently been made to characterize the protein protection and membrane stabilization mechanisms involving small heat shock proteins in bacteria. These proteins seem to be involved in the acquisition of cellular heat tolerance. They could therefore potentially be used to maintain cell viability under unfavorable conditions, such as heat shock or chemical treatments. This review highlights the potential roles of applications of small heat shock proteins in stabilizing overproduced heterologous proteins in Escherichia coli, purified bacterial small heat shock proteins in protein biochip technology, proteomic analysis and food technology and the potential impact of these proteins on some diseases. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.     

Pressure activation of the chaperone function of small heat shock proteins.

Small heat shock proteins play an important role in the stress response of cells and in several other cellular functions. They possess chaperone-like activity; i.e. they can bind and protect damaged proteins from aggregation and maintain them in a folding-competent state. Two members of this family were investigated in this work: bovine alpha-crystallin and heat shock protein (HSP)16.5 from the thermophilic archaebacteria Methanococcus jannaschii. We reported earlier the enhancement of chaperone potency of alpha-crystallin by high pressure. We now report the completion of the work with results on HSP16.5. The chaperone potency of both proteins can be enhanced significantly by applying high pressure. Evidence by light scattering, Fourier transform infrared (FT-IR) and tryptophan fluorescence experiments show that while the secondary and tertiary structure of these proteins are not influenced by high pressure, their quatemary structure becomes affected: H bonds between subunits are weakened or broken, tryptophan environments become more polar, oligomers dissociate to some extent. We conclude that the oligomeric structure of both proteins is loosened, resulting in stronger dynamics and in more accessible hydrophobic surfaces. These properties lead to increased chaperone potency.     

Extracellular small heat shock proteins: exosomal biogenesis and function

Small heat shock proteins (sHsps) belong to the family of heat shock proteins (Hsps): some are induced in response to multiple stressful events to protect the cells while others are constitutively expressed. Until now, it was believed that Hsps, including sHsps, are present inside the cells and perform intracellular functions. Interestingly, several groups recently reported the extracellular presence of Hsps, and sHsps have also been detected in sera/cerebrospinal fluids in various pathological conditions. Secretion into the extracellular milieu during many pathological conditions suggests additional or novel functions of sHsps in addition to their intracellular properties. Extracellular sHsps are implicated in cell-cell communication, activation of immune cells, and promoting anti-inflammatory and anti-platelet responses. Interestingly, exogenous administration of sHsps showed therapeutic effects in multiple disease models implying that extracellular sHsps are beneficial in pathological conditions. sHsps do not possess signal sequence and, hence, are not exported through the classical Endoplasmic reticulum-Golgi complex (ER-Golgi) secretory pathway. Further, export of sHsps is not inhibited by ER-Golgi secretory pathway inhibitors implying the involvement of a nonclassical secretory pathway in sHsp export. In lieu, lysoendosomal and exosomal pathways have been proposed for the export of sHsps. Heat shock protein 27 (Hsp27), αB-crystallin (αBC), and Hsp20 are shown to be exported by exosomes. Exosomes packaged with sHsps have beneficial effects in in vivo disease models. However, secretion mechanisms and therapeutic use of sHsps have not been elucidated in detail. Therefore, this review aimed at highlighting the current understanding of sHsps (Hsp27, αBC, and Hsp20) in the extracellular medium.     

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.     

昆虫小分子量热激蛋白的研究进展

昆虫小分子量热激蛋白(Small heat shock proteins,s HSPs)是最早被发现的热激蛋白,但是有关它们的研究相对较少。本文对昆虫小分子量热激蛋白的最新研究成果进行了总结,旨在引起人们对该类蛋白的关注,以便进一步研究其功能,探讨其可能的应用前景。目前研究表明:昆虫小分子量热激蛋白是其所有热激蛋白中最不保守的家族。同时,它们通常拥有一个α-晶状体结构域;分子量范围一般在12~43 ku;具有分子伴侣的活性。每种昆虫体内拥有多种s HSPs,而且其功能也各不相同。这些热激蛋白在昆虫的生长发育、生殖以及滞育等重要生命活动中起着重要的作用;同时在抵御不良环境以及适应性进化中也具有重要意义。随着研究的深入,还将会有更多的昆虫s HSPs被鉴定,它们更多的功能也将被逐渐发掘。     

Analysis of the interaction of small heat shock proteins with unfolding proteins

The ubiquitous small heat shock proteins (sHsps) are efficient molecular chaperones that interact with nonnative proteins, prevent their aggregation, and support subsequent refolding. No obvious substrate specificity has been detected so far. A striking feature of sHsps is that they form large complexes with nonnative proteins. Here, we used several well established model chaperone substrates, including citrate synthase, alpha-glucosidase, rhodanese, and insulin, and analyzed their interaction with murine Hsp25 and yeast Hsp26 upon thermal unfolding. The two sHsps differ in their modes of activation. In contrast to Hsp25, Hsp26 undergoes a temperature-dependent dissociation that is required for efficient substrate binding. Our analysis shows that Hsp25 and Hsp26 reacted in a similar manner with the nonnative proteins. For all substrates investigated, complexes of defined size and shape were formed. Interestingly, several different nonnative proteins could be incorporated into defined sHsp-substrate complexes. The first substrate protein bound seems to determine the complex morphology. Thus, despite the differences in quaternary structure and mode of activation, the formation of large uniform sHsp-substrate complexes seems to be a general feature of sHsps, and this unique chaperone mechanism is conserved from yeast to mammals.     

Contribution of small heat shock proteins to muscle development and function

Investigations undertaken over the past years have led scientists to introduce the concept of protein quality control (PQC) systems, which are responsible for polypeptide processing. The PQC system monitors proteostasis and involves activity of different chaperones such as small heat shock proteins (sHSPs). These proteins act during normal conditions as housekeeping proteins regulating cellular processes, and during stress conditions. They also mediate the removal of toxic misfolded polypeptides and thereby prevent development of pathogenic states. It is postulated that sHSPs are involved in muscle development. They could act via modulation of myogenesis or by maintenance of the structural integrity of signaling complexes. Moreover, mutations in genes coding for sHSPs lead to pathological states affecting muscular tissue functioning.     

Transglutaminase catalyzes differential crosslinking of small heat shock proteins and amyloid-beta

Crosslinking of proteins by tissue transglutaminase (tTG) is enhanced in amyloid (Abeta) deposits characteristic of Alzheimer's disease and sporadic inclusion body myositis. Small heat shock proteins (sHsps) also occur in amyloid deposits. We here report the substrate characteristics for tTG of six sHsps. Hsp27, Hsp20 and HspB8 are both lysine- and glutamine-donors, alphaB-crystallin only is a lysine-donor, HspB2 a glutamine-donor, and HspB3 no substrate at all. Close interaction of proteins stimulates crosslinking efficiency as crosslinking between different sHsps only takes place within the same heteromeric complex. We also observed that alphaB-crystallin, Hsp27 and Hsp20 associate with Abeta in vitro, and can be readily crosslinked by tTG.     

The potential role of small heat shock proteins in mitochondria

Mitochondria play a central role in cellular metabolism, calcium homeostasis, redox signaling and cell fates. Mitochondrial homeostasis is tightly regulated, and mitochondrial dysfunction is frequently associated with severe human pathologies. Small heat shock proteins are molecular chaperones that play major roles in development, stress responses, and diseases, and have been envisioned as targets for therapy. The mechanisms that lie behind the cytoprotection of small heat shock proteins are related to the regulation of mitochondrial functions. This review recapitulates the current knowledge of the expression of various small heat shock proteins in mitochondria and discusses their implication in the role of mitochondria and their regulation. Based on their involvement in mitochondrial normal physiology and pathology, a better understanding of their roles and regulation will pave the way for innovative approaches for the successful treatment of a range of stress-related syndromes whose etiology is based upon dysfunction of mitochondria.     

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.