The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers. Small heat shock proteins (sHsps)1 are constitutively expressed in virtually all organisms and exhibit a monomeric molecular mass of 15-42 kDa (for a recent review see Ref. 1). Within the cell they can form oligomeric complexes of up to 1 MDa (2). Overexpression of different mammalian sHsps increases cellular thermoresistance to a significant degree (3, 4). sHsps can, furthermore, function in different, seemingly unrelated processes like RNA stabilization (5), interaction with the cytoskeleton (6, 7), or apoptosis (8, 9). In vitro sHsps act as molecular chaperones preventing unfolded proteins from irreversible aggregation (10 -12) and, in cooperation with other factors, e.g. Hsp70 and ATP, facilitating productive refolding of unfolded proteins (13,14).In mammalian cells certain sHsps, e.g. mouse Hsp25 or human Hsp27, form a converging element of the cellular stress response since they show both a stress-induced increase in expression and phosphorylation. Under heat shock conditions increased phosphorylation can be detected after several minutes while changes in expression are detected after several hours (15). The rapid stress-induced phosphorylation is the result of stimulation of the p38 MAP kinase cascade and subsequent activation of MAPKAP kinases 2 and 3 which directly phosphorylate mammalian sHsps (16, 17) at several distinct sites (18,19). Since sHsp phosphorylation and stress-induced expression show different kinetics, it is assumed that phosphorylation of the pre-existing constitutively expressed sHsps is a first phase of the stress response while the elevated expression at a time w...

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

Small heat shock proteins (sHsps) are a conserved protein family, with members found in all organisms analysed so far. Several sHsps have been shown to exhibit chaperone activity and protect proteins from irreversible aggregation in vitro. Here we show that Hsp26, an sHsp from Saccharomyces cerevisiae, is a temperature-regulated molecular chaperone. Like other sHsps, Hsp26 forms large oligomeric complexes. At heat shock temperatures, however, the 24mer chaperone complex dissociates. Interestingly, chaperone assays performed at different temperatures show that the dissociation of the Hsp26 complex at heat shock temperatures is a prerequisite for efficient chaperone activity. Binding of non-native proteins to dissociated Hsp26 produces large globular assemblies with a structure that appears to be completely reorganized relative to the original Hsp26 oligomers. In this complex one monomer of substrate is bound per Hsp26 dimer. The temperature-dependent dissociation of the large storage form of Hsp26 into a smaller, active species and the subsequent re-association to a defined large chaperone-substrate complex represents a novel mechanism for the functional activation of a molecular chaperone.

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, ␣-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.In response to environmental stress such as heat shock, which leads to the accumulation of nonnative proteins, cells increase the expression of several classes of proteins (1). The major conserved families of these heat shock proteins (Hsps) 1 have been shown to be involved in protein folding as molecular chaperones (2).The most divergent of these chaperone classes are the small heat shock proteins (sHsps). sHsps have been found in almost all organisms investigated so far, with the number of members varying from species to species. They share conserved regions mostly in the C-terminal part of the protein, whereas the Nterminal part differs in sequence and length, leading to molecular masses of 16 -42 kDa for sHsps in different organisms (3). The conserved C-terminal domain of ϳ100 amino acids shares sequence homology with the major eye lens protein ␣A-crystallin (4). Almost all sHsps assemble into large oligomeric complexes of 9 to Ͼ30 subunits, and complexes in the range of 125 kDa to 2 MDa have been found (5-11). Some sHsps such as those from plants form assemblies with well defined stoichiometries, whereas other sHsps, including the mammalian proteins, form a range of oligomeric sizes (12, 13). This polydispersity has limited the amount of structural information available. The crystal structure of an archaeal sHsp (14) and the cryo-electron microscopy reconstruction of ␣B-crystallin (15) revealed that the overall organization is that of a hollow globular sphere. A variation of this scheme is the three-dimensional structure of wheat Hsp16.9, which assembles into a dodecameric double disk, where each disk is organized as a trimer of dimers (16). Many sHsps have dynamic and variable quaternary s...

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Small heat shock proteins (sHsps), including ␣-crystallin, represent a conserved and ubiquitous family of proteins. They form large oligomers, ranging in size from 140 to more than 800 kDa, which seem to be important for the interaction with non-native proteins as molecular chaperones. Here we analyzed the stability and oligomeric structure of murine Hsp25 and its correlation with function. Upon unfolding, the tertiary and quaternary structure of Hsp25 is rapidly lost, whereas the secondary structure remains remarkably stable. Unfolding is completely reversible, leading to native hexadecameric structures. These oligomers are in a concentration-dependent equilibrium with tetramers and dimers, indicating that tetramers assembled from dimers represent the basic building blocks of Hsp25 oligomers. At high temperatures, the Hsp25 complexes increase in molecular mass, consistent with the appearance of "heat shock granules" in vivo after heat treatment. This high molecular mass "heat shock form" of Hsp25 is in a slow equilibrium with hexadecameric Hsp25. Thus, it does not represent an off-pathway reaction. Interestingly, the heat shock form exhibits unchanged chaperone activity even after incubation at 80°C. We conclude that Hsp25 is a dynamic tetramer of tetramers with a unique ability to refold and reassemble into its active quaternary structure after denaturation. So-called heat shock granules, which have been reported to appear in response to stress, seem to represent a novel functional species of Hsp25.Small heat shock proteins (sHsps), 1 exhibiting a monomeric molecular mass of 9 -42 kDa, are expressed in all organisms investigated so far. Because of functional as well as structural homologies, ␣-crystallin, a major mammalian eye lens protein, which is also expressed in non-lenticular tissue, is a member of this protein family (1-4). Although the overall homology between different sHsps is rather low, they are grouped together based on conserved sequences in the C-terminal half of the protein and short, conserved, phenylalanine-rich stretches near the N terminus of the protein (5, 6). Mammalian sHsps are expressed constitutively even under physiological conditions. However, stress factors such as heat shock induce a strong up-regulation of protein levels by 10 -20-fold to maximum concentrations of 0.1% of the cellular protein (7, 8). Overexpression of different mammalian sHsps increases cellular thermoresistance significantly (9, 10). Furthermore, sHsps have been suggested to function in different, seemingly unrelated processes like RNA stabilization (11), interaction with the cytoskeleton (12, 13), or apoptosis (14). Interestingly, sHsps are also overexpressed in several cancers and neurodegenerative diseases like Alzheimer's disease or multiple sclerosis (15-17). In plants, five different classes of sHsps have been identified, which are partly localized in organella (8,18).In vitro sHsps act as molecular chaperones in preventing unfolded proteins from irreversible aggregation (3,4,19) and, in cooperation with o...

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