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.     

Chaperone activity and homo- and hetero-oligomer formation of bacterial small heat shock proteins

Rhizobia are the only bacteria known to induce a multitude of small heat shock proteins (sHsps) upon temperature upshift. The sHsps of Bradyrhizobium japonicum fall into two different classes, class A and class B. Here, we studied the chaperone activity and oligomeric features of two representative members of each class. The purified sHsps were efficient chaperones, as demonstrated by their ability to prevent thermally induced aggregation of citrate synthase in vitro. Homo-oligomer formation of all four sHsps was demonstrated by gel filtration and by two independent co-purification approaches. Mixed oligomers were readily observed between members of the same class, even when these proteins originated from different species such as Escherichia coli and B. japonicum. The chaperone activity of purified hetero-oligomers was indistinguishable from the activity of homo-oligomers. Heteromeric complexes were never obtained between class A and class B sHsps, indicating that hetero-oligomer formation is restricted to sHsps of the same class.     

The diversification of plant cytosolic small heat shock proteins preceded the divergence of mosses.

A cDNA library was constructed with mRNA isolated from heat-stressed cell cultures of Funaria hygrometrica (Bryophyta, Musci, Funariaceae). cDNA clones encoding six cytosolic small heat shock proteins (sHSPs) were identified using differential screening. Phylogenetic analysis of these sHSP sequences with other known sHSPs identified them as members of the previously described higher plant cytosolic class I and II families. Four of the F. hygrometrica sHSPs are members of the cytosolic class I family, and the other two are members of the cytosolic class II family. The presence of members of the cytosolic I and II sHSP families in a bryophyte indicates that these gene families are ancient, and evolved at least 450 MYA. This result also indicates that the plant sHSP gene families duplicated much earlier than did the well-studied phytochrome gene family. Members of the cytosolic I and II sHSP families are developmentally regulated in seeds and flowers in higher plants. Our findings show that the two cytosolic sHSP families evolved before the appearance of these specialized structures. Previous analysis of angiosperm sHSPs had identified class- or family-specific amino acid consensus regions and determined that rate heterogeneity exists among the different sHSP families. The analysis of the F. hygrometrica sHSP sequences reveals patterns and rates of evolution distinct from those seen among angiosperm sHSPs. Some, but not all, of the amino acid consensus regions identified in seed plants are conserved in the F. hygrometrica sHSPs. Taken together, the results of this study illuminate the evolution of the sHSP gene families and illustrate the importance of including representatives of basal land plant lineages in plant molecular evolutionary studies.     

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.     

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.     

Clusterin has chaperone-like activity similar to that of small heat shock proteins

Clusterin is a highly conserved protein which is expressed at increased levels by many cell types in response to a broad variety of stress conditions. A genuine physiological function for clusterin has not yet been established. The results presented here demonstrate for the first time that clusterin has chaperone-like activity. At physiological concentrations, clusterin potently protected glutathione S-transferase and catalase from heat-induced precipitation and alpha-lactalbumin and bovine serum albumin from precipitation induced by reduction with dithiothreitol. Enzyme-linked immunosorbent assay data showed that clusterin bound preferentially to heat-stressed glutathione S-transferase and to dithiothreitol-treated bovine serum albumin and alpha-lactalbumin. Size exclusion chromatography and SDS-polyacrylamide gel electrophoresis analyses showed that clusterin formed high molecular weight complexes (HMW) with all four proteins tested. Small heat shock proteins (sHSP) also act in this way to prevent protein precipitation and protect cells from heat and other stresses. The stoichiometric subunit molar ratios of clusterin:stressed protein during formation of HMW complexes (which for the four proteins tested ranged from 1.0:1.3 to 1.0:11) is less than the reported ratios for sHSP-mediated formation of HMW complexes (1.0:1.0 or greater), indicating that clusterin is a very efficient chaperone. Our results suggest that clusterin may play a sHSP-like role in cytoprotection.     

Heat causes oligomeric disassembly and increases the chaperone activity of small heat shock proteins from sugarcane

Small heat shock proteins (sHsp) constitute an important chaperone family linked to conformational diseases. In plants, sHsps prevent protein aggregation by acting as thermosensors and to enhance cell stress tolerance. SsHsp17.2 and SsHsp17.9 are the most highly expressed class I sHsps in sugarcane. They exist as dodecamers at 20 °C and have distinct substrate specificities. Therefore, they are useful models to study how class I SHsps work. Here we present data on the effects of heat on the oligomerization and chaperone activity of SsHsp17.2 and SsHsp17.9. Using several biophysical and biochemical probes, we show that the effects of heat are completely reversible, an important property for proteins that act at heat shock temperatures. SsHsp17.2 and SsHsp17.9 dodecamers dissociated to dimers at temperatures ranging from 40 to 45 °C and this dissociation was followed by enhanced chaperone activity. We conclude that high temperature affects the oligomeric state of these chaperones, resulting in enhanced chaperone activity.     

Heterooligomeric complexes of human small heat shock proteins

Oligomeric association of human small heat shock proteins HspB1, HspB5, HspB6 and HspB8 was analyzed by means of size-exclusion chromatography, analytical ultracentrifugation and chemical cross-linking. Wild-type HspB1 and Cys mutants of HspB5, HspB6 and HspB8 containing a single Cys residue in position homologous to that of Cys137 of human HspB1 were able to generate heterodimers cross-linked by disulfide bond. Cross-linked heterodimers between HspB1/HspB5, HspB1/HspB6 and HspB5/HspB6 were easily produced upon mixing, whereas formation of any heterodimers with participation of HspB8 was significantly less efficient. The size of heterooligomers formed by HspB1/HspB6 and HspB5/HspB6 was different from the size of the corresponding homooligomers. Disulfide cross-linked homodimers of small heat shock proteins were unable to participate in heterooligomer formation. Thus, monomers can be involved in subunit exchange leading to heterooligomer formation and restriction of flexibility induced by disulfide cross-linking prevents subunit exchange.     

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.     

Temperature and concentration-controlled dynamics of rhizobial small heat shock proteins.

A hallmark of alpha-crystallin-type small heat shock proteins (sHsps) is their highly dynamic oligomeric structure which promotes intermolecular interactions involved in subunit exchange and substrate binding (chaperone-like activity). We studied the oligomeric features of two classes of bacterial sHsps by size exclusion chromatography and nanoelectrospray mass spectrometry. Proteins of both classes formed large complexes that rapidly dissociated upon dilution and at physiologically relevant heat shock temperatures. As the secondary structure was not perturbed, temperature- and concentration-dependent dissociations were fully reversible. Complexes formed between sHsps and the model substrate citrate synthase were stable and exceeded the size of sHsp oligomers. Small Hsps, mutated in a highly conserved glycine residue at the C-terminal end of the alpha-crystallin domain, formed labile complexes that disassembled more readily than the corresponding wild-type proteins. Reduced complex stability coincided with reduced chaperone activity.     

Chaperone action of a versatile small heat shock protein from Methanococcoides burtonii, a cold adapted archaeon

The Methanococcoides burtonii small heat shock protein (Mb-sHsp) is an alphaB-crystallin homolog that delivers protein stabilizing and protective functions to model enzymes, presumably reflecting its role as a molecular chaperone in vivo. Although the gene encoding Mb-shsp was cloned from a cold-adapted microorganism, the Mb-sHsp is an efficient protein chaperone at temperatures far above the optimum growth temperature of M. burtonii. We show that Mb-sHsp can prevent aggregation in E. coli cell free extracts at 60 degrees C for 4 h and can stabilize bovine liver glutamate dehydrogenase for 3 h at 50 degrees C. Surface plasmon resonance was used to determine the binding affinity of Mb-sHsp for denatured proteins. Mb-sHsp bound tightly to denatured lysozyme but not to the native form. When Mb-Cpn and Mg(2+)-ATP were added to the reaction, bound lysozyme was released from Mb-sHsp establishing that Mb-Cpn is able to off-load folding intermediates from Mb-sHsp. In addition, Mb-sHsp and Mb-Cpn also function cooperatively to protect an enzyme substrate. Through characterization of these M. burtonii chaperones, we were able to reconstitute a key heat shock regulated protein folding function of this cold adapted organism in vitro.     

Genetic variability for heat shock proteins in common wheat

Summary The response of the common wheat line Chinese Spring to heat shocks of different time lengths was studied by the two-dimensional (2D) electrophoresis of denatured proteins. After a heat shock of 5 h, 33 heat shock proteins (HSPs) accumulated in an amount sufficient to be revealed by silver stain. Two other wheat lines (Moisson and Selkirk) were then submitted to a heat shock of 5 h, and the responses of the 3 lines were compared: of a total of 35 HSPs, 13 (37.1%) were quantitatively or qualitatively variable. This variability concerns low-molecular-weight and high-molecular-weight HSPs. The three genotypes showed thermal tolerance but Chinese Spring's response to heat treatments was slightly different from those of the other two lines The possibility of a relationship between HSP patterns and thermal sensitivity is discussed.     

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.     

Purification and characterization of two small heat shock proteins from Anabaena sp. PCC 7120

Two small heat shock proteins (sHsps), Hsp17.8 and Hsp17.1, were identified in the cyanobacterium Anabaena sp. PCC 7120. Recombinant Hsp17.8 and Hsp17.1 were overexpressed in Escherichia coli and characterized here. Hsp17.8 was purified by sequential chromatography on DEAE-Sepharose and Superose 6 10/300 column, and Hsp17.1 was purified by Superose 6 10/300 column in 4M urea. Size exclusion chromatography demonstrated that both purified proteins form large oligomers approximately 420kDa and 410kDa, respectively. Both Hsp17.8 and Hsp17.1 showed chaperone-like activity to protect citrate synthase (CS) from thermal aggregation at 43 degrees C. Furthermore, both proteins were found to form complexes with denatured CS at 45 degrees C. Our study also demonstrated that despite a high degree of sequence homology and similar subunit size, Hsp17.1 showed higher hydrophobicity indicated by 8-anilino-1-naphthalene sulfonate fluorescence and thus greater chaperone-like activity. This is the first report of characterization and comparison of an sHsp system containing two chaperones in cyanobacteria.     

Translation of some maize small heat shock proteins is initiated from internal in-frame AUGs.

Etiolated maize radicles (inbred Oh43) subjected to a brief heat shock synthesize a family of small heat shock proteins (approximately 18 kD) that is composed of at least 12 members. We previously described the cDNA-derived sequence of three maize shsp mRNAs (cMHSP18-1, cMHSP18-3, and cMHSP18-9). In this report, we demonstrate that the mRNA transcribed in vitro from one of these cDNAs (cMHSP18-9) is responsible for the synthesis of three members of the shsp family, and we suggest that cMHSP18-3 may be responsible for the synthesis of three additional members and cMHSP18-1 for the synthesis of two other members of this family. The fact that these genes do not contain introns, coupled with the observations reported herein, suggest that maize may have established another method of using a single gene to produce a number of different proteins.     

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.     

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.