Amyloid fibrils and their misfolded precursors are defining features of numerous age-related diseases including Alzheimer’s disease, Parkinson’s disease, type II diabetes and atherosclerosis. Small heat-shock proteins (sHsps) are stress-inducible molecular chaperones that not only prevent amyloid formation but are also found associated with amyloid deposits isolated post mortem. Despite their importance for maintaining proteostasis, the structure-function relationship of sHsps remains poorly understood. Wild-type (WT) sHsps populate a polydisperse ensemble of oligomers that exchange dimers at equilibrium. Heat-shock protein 27 (Hsp27) and αB-crystallin (αB-C) are ubiquitously expressed human sHsps that share structural features and substrates. However, subunits within Hsp27 dimers are linked via a disulfide bridge that is absent in other sHsps. In this study, we investigate the importance of this disulfide bridge for the structure and chaperone activity of Hsp27.
Three mutations mimicking phosphorylation of serine residues (Hsp27-3D) cause Hsp27 to form dimers, rather than higher order oligomers (1). Additionally, a Hsp27 construct truncated of its N- and C-terminal domains (Hsp27CD) forms only dimers. Mass-spectrometry and gel electrophoresis indicate that the disulfide bridge in WT Hsp27, Hsp27-3D and Hsp27CD is stable under denaturing conditions, but can be easily reduced. Analytical ultracentrifugation and small angle X-ray scattering indicate that reducing agents destabilise the Hsp27-3D dimer and promote the formation of small oligomers. Furthermore, WT Hsp27, Hsp27-3D and Hsp27CD all exhibit increased chaperone activity in the presence of reducing agents. Reducing agents did not affect the structure or chaperone activity of αB-C, providing evidence that the change in structure and chaperone activity of Hsp27 is due to reduction of the intra-dimer disulfide. Interestingly, several disease-relevant mutations in Hsp27 are located immediately adjacent to the disulfide bridge, providing evidence for the importance of the redox state of Hsp27 in human diseases.