Small heat-shock proteins (sHSPs) are one of the major families of heat-shock proteins and are present in all forms of life. sHSPs have a low monomeric molecular mass, ranging from 15 to 40 kDa and are characterized by the presence of an ~90 residue central domain termed the alpha-crystallin domain (ACD). The N- and C- terminal regions that flank the ACD are poorly conserved and vary in length across the sHSP family. Metazoan sHSPs are structurally diverse, existing as polydisperse oligomers that can range from monomers to multimers containing as many as 40 subunits. As a result of this polydispersity, relatively few high-resolution structures of eukaryotic sHSPs have been solved and the mechanisms by which sHSPs inhibit amyloid fibril formation remain elusive.
In order to better understand how and why sHSPs interact with proteins prone to amyloid fibril formation, we assayed the relative chaperone activities of full-length, truncated, and mutated sHSP constructs from two ubiquitously expressed human sHSPs. Full-length but not ACD-only constructs inhibited the early stages amyloid formation and disaggregated pre-formed amyloid fibrils, revealing the importance of the N- and/or C- terminal regions for these chaperone functions. However, the ACD-only constructs were highly effective inhibitors of amyloid fibril elongation and naturally occurring fibril end-to-end joining. These data indicate that the ACD may interact specifically with the ends of elongating amyloid fibrils to prevent further growth. Disruption of an inter-chain disulfide bridge in the ACD of Hsp27 using reducing agents or via mutagenesis significantly enhanced its chaperone activity. Furthermore, an engineered disulfide at the same location in the αB-C ACD impaired its chaperone activity. Therefore, we identified both a potential mechanism for the redox-regulation of Hsp27 and key determinants of the chaperone activity of the ACD. Our results provide novel insights into the mechanism of action of human sHSPs.