Analysis of the Interaction of Small Heat Shock Proteins with Unfolding Proteins

Abstract

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...

The Selective Inhibition of Serpin Aggregation by the Molecular Chaperone, α-Crystallin, Indicates a Nucleation-dependent Specificity

“…␣-lactalbumin (36). In this case, and as with all of the other substrate proteins shown previously to interact with ␣-crystallin (37)(38)(39)(40)(41)(42)(43)(44), the aggregation profile is sigmoidal (Figs. 1a and 2a), indicative of a nucleation-dependent mechanism.…”

Analysis of the Regulation of the Molecular Chaperone Hsp26 by Temperature-induced Dissociation

“…In the absence of urea, the Hsp26⅐substrate complexes eluted at the position expected (21). When the samples were incubated in the presence of urea, a decrease in the fluorescence intensity of this peak was observed; concomitantly, peaks at the positions of the components of the substrate complex appeared.…”

“…Complexes-A striking feature of the chaperone mechanism of Hsp26 is its ability to form large, well-defined complexes with substrates (13,21). Analysis of electron micrographs suggested that the structural organization of the substrate complexes is different from that of the Hsp26 24-mers.…”

“…Analysis of electron micrographs suggested that the structural organization of the substrate complexes is different from that of the Hsp26 24-mers. The finding that substrate complexes can be isolated by SEC already suggested a stable interaction (13,21). In the context of this study, we were interested to compare the stability of the substrate complexes with that of the native Hsp26 complex.…”

“…A comparison of Hsp26 with Hsp25, a cytosolic sHsps from mouse, concerning the interaction with non-native proteins showed that the general chaperone properties are conserved although the mechanisms of activation are completely different (21). As mentioned above, Hsp26 requires dissociation for activation, whereas Hsp25 did not show any detectable dissociation.…”