A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state.

The small heat shock proteins (sHSPs) recently have been reported to have molecular chaperone activity in vitro; however, the mechanism of this activity is poorly defined. We found that HSP18.1, a dodecameric sHSP from pea, prevented the aggregation of malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase heated to 45 degrees C. Under conditions in which HSP18.1 prevented aggregation of substrates, size-exclusion chromatography and electron microscopy revealed that denatured substrates coated the HSP18.1 dodecamers to form expanded complexes. SDS-PAGE of isolated complexes demonstrated that each HSP18.1 dodecamer can bind the equivalent of 12 MDH monomers, indicating that HSP18.1 has a large capacity for non-native substrates compared with other known molecular chaperones. Photoincorporation of the hydrophobic probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) into a conserved C-terminal region of HSP18.1 increased reversibly with increasing temperature, but was blocked by prior binding of MDH, suggesting that bis-ANS incorporates proximal to substrate binding regions and that substrate-HSP18.1 interactions are hydrophobic. We also show that heat-denatured firefly luciferase bound to HSP18.1, in contrast to heat-aggregated luciferase, can be reactivated in the presence of rabbit reticulocyte or wheat germ extracts in an ATP-dependent process. These data support a model in which sHSPs prevent protein aggregation and facilitate substrate refolding in conjunction with other molecular chaperones.     

Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity

Small heat shock proteins protect cells from stress presumably by acting as molecular chaperones. Here we report on the functional characterization of a developmentally regulated, heat-inducible member of the Xenopus small heat shock protein family, Hsp30C. An expression vector containing the open reading frame of the Hsp30C gene was expressed in Escherichia coli. These bacterial cells displayed greater thermoresistance than wild type or plasmid-containing cells. Purified recombinant protein, 30C, was recovered as multimeric complexes which inhibited heat-induced aggregation of either citrate synthase or luciferase as determined by light scattering assays. Additionally, 30C attenuated but did not reverse heat-induced inactivation of enzyme activity. In contrast to an N-terminal deletion mutant, removal of the last 25 amino acids from the C-terminal end of 30C severely impaired its chaperone activity. Furthermore, heat-treated concentrated solutions of the C-terminal mutant formed nonfunctional complexes and precipitated from solution. Immunoblot and gel filtration analysis indicated that 30C binds with and maintains the solubility of luciferase preventing it from forming heat-induced aggregates. Coimmunoprecipitation experiments suggested that the carboxyl region is necessary for 30C to interact with target proteins. These results clearly indicate a molecular chaperone role for Xenopus Hsp30C and provide evidence that its activity requires the carboxyl terminal region.     

Spatial pattern of constitutive and heat shock-induced expression of the small heat shock protein gene family, Hsp30, in Xenopus laevis tailbud embryos

We employed whole-mount in situ hybridization and immunohistochemistry to study the spatial pattern of hsp30 gene expression in normal and heatshocked embryos during Xenopus laevis development. Our findings revealed that hsp30 mRNA accumulation was present constitutively only in the cement gland of early and midtailbud embryos, while hsp30 protein was detected until at least the early tadpole stage. Heat shock-induced accumulation of hsp30 mRNA and protein was first observed in early and midtailbud embryos with preferential enrichment in the cement gland, somitic region, lens placode, and proctodeum. In contrast, cytoskeletal actin mRNA displayed a more generalized pattern of accumulation which did not change following heat shock. In heat shocked midtailbud embryos the enrichment of hsp30 mRNA in lens placode and somitic region was first detectable after 15 min of a 33 degrees C heatshock. The lowest temperature capable of inducing this pattern was 30 degrees C. Placement of embryos at 22 degrees C following a 1-h 33 degrees C heat shock resulted in decreased hsp30 mRNA in all regions with time, although enhanced hsp30 mRNA accumulation still persisted in the cement gland after 11 h compared to control. In late tailbud embryos the basic midtailbud pattern of hsp30 mRNA accumulation was enhanced with additional localization to the spinal cord as well as enrichment across the embryo surface. These studies demonstrate that hsp30 gene expression can be detected constitutively in the cement gland of tailbud embryos and that heat shock results in a preferential accumulation of hsp30 mRNA and protein in certain tissues.     

Some properties of complexes formed by small heat shock proteins with denatured actin

We applied different methods to analyze the effects of the recombinant wild-type small heat shock protein with an apparent molecular mass of 27 kD (Hsp27-wt) and its S15,78,82D mutant (Hsp27-3D), which mimics the naturally occurring phosphorylation of this protein, on the thermal denaturation and aggregation of F-actin. It has been shown that, at the weight ratio of Hsp27/actin equal to 1/4, both Hsp27-wt and Hsp27-3D do not affect the thermal unfolding of F-actin but effectively prevent the aggregation of F-actin by forming soluble complexes with denatured actin. The formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. It is known that Hsp27-wt forms high-molecular-mass oligomers, whereas Hsp27-3D forms small dimers or tetramers. However, the complexes formed by Hsp27-wt and Hsp27-3D with denatured actin did not differ in their size, as measured by dynamic light scattering, and demonstrated the same hydrodynamic radius of 17-18 nm. On the other hand, the sedimentation coefficients of these complexes were distributed within the range 10-45 S in the case of Hsp27-3D and 18-60 S in the case of Hsp27-wt. Thus, the ability of Hsp27 to form soluble complexes with denatured actin does not significantly depend on the initial oligomeric state of Hsp27.     

Analysis of interactions between domains of a small heat shock protein,Hsp30 of Neurospora crassa

The alpha-crystallin-related, small heat shock proteins (sHsps), despite their overall variability in sequence, have discrete regions of conserved sequence that are involved in structural organization, as well as nonconserved regions that may perform similar roles in each protein. Recent X-ray diffraction analyses of an archeal and a plant sHsp have revealed both similarities and differences in how they are organized, suggesting that there is variability, particularly in the oligomeric organization of sHsps. As an adjunct to crystallographic analysis of sHsp structure, we employed the yeast 2-hybrid system to detect interactions between peptide regions of the sHsp of Neurospora crassa, Hsp30. We found that the conserved alpha-crystallin domain can be divided into N-terminal and C-terminal subdomains that interact strongly with one another. This interaction likely represents the tertiary contacts of the monomer that were visualized in the crystallographic structures of MjHsp16.5 and wheat Hsp16.9. The conserved sHsp monomeric fold is apparently determined by these regions of conserved sequence. We found that the C-terminal portion of the alpha-crystallin domain also interacts with itself in 2-hybrid assays; however, this interaction requires peptide extension into the semiconserved carboxyl tail. This C-terminal association may represent a principal contact site between dimers that contributes to higher-order assembly, as seen for the crystallized sHsps.     

Intracellular localization of Xenopus small heat shock protein, hsp30, in A6 kidney epithelial cells

Small heat shock proteins (shsps) are molecular chaperones that are inducible by environmental stress. In this study, immunocytochemical analysis and laser scanning confocal microscopy revealed that the shsp family, hsp30, was localized primarily in the cytoplasm of Xenopus A6 kidney epithelial cells after heat shock or sodium arsenite treatment. Heat shock-induced hsp30 was enriched in the perinuclear region with some immunostaining in the nucleus but not in the nucleolus. In sodium arsenite-treated cells hsp30 was enriched towards the cytoplasmic periphery as well as showing some immunostaining in the nucleus. At higher heat shock temperatures (35 degrees C) or after 10 microM sodium arsenite treatment, the actin cytoskeleton displayed some disorganization that co-localized with areas of hsp30 enrichment. Treatment of A6 cells with 50 microM sodium arsenite induced a collapse of the cytoskeleton around the nucleus. These results coupled with previous studies suggest that stress-inducible hsp30 acts as a molecular chaperone primarily in the cytoplasm and may interact with cytoskeletal proteins.     

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.     

Characterization of a small heat shock protein, Mx Hsp16.6, of Myxococcus xanthus

A number of heat shock proteins in Myxococcus xanthus were previously identified by two-dimensional (2D) gel electrophoresis. One of these protein was termed Mx Hsp16.6, and the gene encoding Mx Hsp16.6 was isolated. Mx Hsp16.6 consists of 147 amino acid residues and has an estimated molecular weight of 16,642, in accordance with the apparent molecular mass in the 2D gel. An alpha-crystallin domain, typically conserved in small heat shock proteins, was found in Mx Hsp16.6. Mx Hsp16.6 was not detected during normal vegetative growth but was immediately induced after heat shock. Expression of the hsp16.6 gene was not induced by other stresses, such as starvation, oxidation, and high osmolarity. Mx Hsp16.6 was mostly localized in particles formed after heat shock and precipitated by low-speed centrifugation. Furthermore, Mx Hsp16.6 was detected in highly electron-dense particles in heat-shocked cells by immunoelectron microscopy, suggesting that it forms large complexes with heat-denatured proteins. An insertion mutation in the hsp16.6 gene resulted in lower viability during heat shock and lower acquired thermotolerance. Therefore, it is likely that Mx Hsp16.6 plays critical roles in the heat shock response in M. xanthus.     

StHsp14.0, a small heat shock protein of Sulfolobus tokodaii strain 7, protects denatured proteins from aggregation in the partially dissociated conformation

The small heat shock protein (sHsp), categorized into a class of molecular chaperones, binds and stabilizes denatured proteins for the purpose of preventing aggregation. The sHsps undergo transition between different oligomeric states to control their nature. We have been studying the function of sHsp of Sulfolobus tokodaii, StHsp14.0. StHsp14.0 exists as 24meric oligomer, and exhibits oligomer dissociation and molecular chaperone activity over 80°C. We constructed and characterized StHsp14.0 mutants with replacement of the C-terminal IKI to WKW, IKF, FKI and FKF. All mutant complexes dissociated into dimers at 50°C. Among them, StHsp14.0FKF is almost completely dissociated, probably to dimers. All mutants protected citrate synthase (CS) from thermal aggregation at 50°C. But, the activity of StHsp14.0FKF was the lowest. Then, we examined the complexes of StHsp14.0 mutants with denatured CS by SAXS. StHsp14.0WKW protects denatured CS by forming the globular complexes of 24 subunits and a substrate. StHsp14.0FKF also formed similar complex but the number of subunits in the complex is a little smaller. These results suggest that the dimer itself exhibits low chaperone activity, and a partially dissociated oligomer of StHsp14.0 protects a denatured protein from interacting with other molecules by surrounding it.     

Differential expression of small heat shock protein genes Hsp23 and Hsp40, and heat shock gene Hsr-omega in fruit flies (Drosophila melanogaster) along a microclimatic gradient

We examined the role of small Hsp genes (Hsp23 and Hsp40) and heat shock gene Hsr-omega in the thermoadaptation of Drosophila melanogaster inhabiting a highly heterogeneous microsite (Nahal Oren canyon, Carmel massif, Israel). We tested whether interslope differences in Drosophila thermoadaptation, revealed in our previous studies, are associated with the differential expression of these genes. Our results demonstrate an increased expression of the Hsp40 gene in thermotolerant lines subjected to mild heat shock treatment (P < 10(-6), analysis of variance test). A high positive correlation was found between the levels of Hsp40 expression and scores of basal (R = 0.74; P < 0.001, based on the Spearman rank correlation test) and induced thermotolerance (R = 0.78; P < 0.0001), implying a significant contribution of Hsp40 gene in thermoadaptation.     

Heterooligomeric complexes formed by human small heat shock proteins HspB1 (Hsp27) and HspB6 (Hsp20)

Formation of heterooligomeric complexes of human small heat shock proteins (sHsp) HspB6 (Hsp20) and HspB1 (Hsp27) was analyzed by means of native gel electrophoresis, analytical ultracentrifugation, chemical cross-linking and size-exclusion chromatography. HspB6 and HspB1 form at least two different complexes with apparent molecular masses 100–150 and 250–300 kDa, and formation of heterooligomeric complexes is temperature dependent. These complexes are highly mobile, easily exchange their subunits and are interconvertible. The stoichiometry of HspB1 and HspB6 in both complexes is close to 1/1 and smaller complexes are predominantly formed at low, whereas larger complexes are predominantly formed at high protein concentration. Formation of heterooligomeric complexes does not affect the chaperone-like activity of HspB1 and HspB6 if insulin or skeletal muscle F-actin was used as model protein substrates. After formation of heterooligomeric complexes the wild type HspB1 inhibits the rate of phosphorylation of HspB6 by cAMP-dependent protein kinase. The 3D mutant mimicking phosphorylation of HspB1 also forms heterooligomeric complexes with HspB6, but is ineffective in inhibition of HspB6 phosphorylation. Inside of heterooligomeric complexes HspB6 inhibits phosphorylation of HspB1 by MAPKAP2 kinase. Thus, in heterooligomeric complexes HspB6 and HspB1 mutually affect the structure of each other and formation of heterooligomeric complexes might influence diverse processes depending on small heat shock proteins.     

The heat shock proteins,Hsp70 and Hsp83, of Leishmania infantum are mitogens for mouse B cells

Extending earlier studies, this report demonstrates that Leishmania infantum heat shock proteins (Hsps), Hsp70 and Hsp83, expressed as recombinant proteins fused to the Escherichia coil maltose-binding protein (MBP), are potent mitogens for murine splenocytes. The response was not due to lipopolysaccharide (LPS) because the stimulatory activity of Hsp preparations was sensitive to boiling and trypsin treatments, whereas the corresponding activity of LPS was resistant to both treatments. It was found that in vitro incubation of spleen cells with the Leishmania Hsps leads to the expansion of CD220-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. In fact, splenocytes from B cell-deficient mice did not proliferate in response to the Leishmania Hsps. In contrast, spleen cells from athymic nude mice were significantly stimulated by these recombinant proteins as an indication that the MBP-Hsp70 and MBP-Hsp83 recombinant proteins behave as T cell-independent mitogens of B cells. Furthermore, both proteins were able to induce proliferation on B cell populations purified from BALB/c spleen.     

Ordered assembly of heat shock proteins, Hsp26, Hsp70, Hsp90, and Hsp104, on expanded polyglutamine fragments revealed by chemical probes

In Saccharomyces cerevisae, expanded polyglutamine (polyQ) fragments are assembled into discrete cytosolic aggregates in a process regulated by the molecular chaperones Hsp26, Hsp70, Hsp90, and Hsp104. To better understand how the different chaperones might cooperate during polyQ aggregation, we used sequential immunoprecipitations and mass spectrometry to identify proteins associated with either soluble (Q25) or aggregation-prone (Q103) fragments at both early and later times after induction of their expression. We found that Hsp26, Hsp70, Hsp90, and other chaperones interact with Q103, but not Q25, within the first 2 h. Further, Hsp70 and Hsp90 appear to be partially released from Q103 prior to the maturation of the aggregates and before the recruitment of Hsp104. To test the importance of this seemingly ordered process, we used a chemical probe to artificially enhance Hsp70 binding to Q103. This treatment retained both Hsp70 and Hsp90 on the polyQ fragment and, interestingly, limited subsequent exchange for Hsp26 and Hsp104, resulting in incomplete aggregation. Together, these results suggest that partial release of Hsp70 may be an essential step in the continued processing of expanded polyQ fragments in yeast.     

Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21

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

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like G?6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.     

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.     

The small heat shock proteins Hsp20 and alphaB-crystallin in cultured cardiac myocytes: differences in cellular localization and solubilization after heat stress.

Hsp20, a recently described new member of the small heat shock protein superfamily, is abundant in heart, skeletal muscle types and smooth muscle. We investigated the intracellular localization of Hsp20 in cultured rat neonatal cardiac myocytes, under normal conditions and after stress. These cellular characteristics of Hsp20 were compared with those of its closest relative, alphaB-crystallin, which is also highly expressed in heart. Like alphaB-crystallin, Hsp20 is normally located in the cytoplasm of the cardiac myocytes. After a heat stress, a subpopulation of Hsp20 migrates into the nucleus, while another part remains in the cytoplasm. In very few cells a faint sarcomeric association of Hsp20 is observed. In contrast, as previously reported, alphaB-crystallin displays a very distinct cross-striated sarcomeric staining after the heat shock, but no nuclear migration. Also at the level of Triton solubility, differences exist between the two related proteins; while alphaB-crystallin, like other small heat shock proteins, becomes insoluble upon heat stress, Hsp20 remains largely soluble. Our results indicate that Hsp20 and alphaB-crystallin, despite their structural similarities, display conspicuous functional differences.