Small Heat Shock Proteins (sHSPs) form an integral part of the cellular chaperone network whereby their levels of expression increase under conditions of stress such as heat, ischemia, and pH. sHSPs bind nascent or stress-induced unfolded proteins and maintain them in a soluble state until rescued by ATP-dependent chaperones. aB-Crystallin (aB) and HSP27 are two of the ten sHSPs found in humans and they are implicated in a number of diseases. Both aB and HSP27 are involved in the apoptotic pathway and they have been shown to protect against cardiac ischemia and reperfusion. Inherited mutations in aB and HSP27 are associated with muscular diseases such as desmin-related myopathy and distal hereditary motor neuropathy (Type 2 Charcot-Marie-Tooth disease). Despite growing information on their biological roles, structural and functional aspects of aB and HSP27 remain poorly understood. aB and HSP27 form polydisperse macromolecular assemblies and are refractory targets for structure determination by X-ray crystallography, the technique most suited for molecules of this size. We propose a hierarchical approach to obtain structural information. The a-crystallin domain, which is shared by all sHSPs, forms a homodimer, believed to be the building block of sHSP oligomers. We propose to study the dimeric a-crystallin domains from aB and HSP27 by solution state NMR (Aims 1A and 3A). Structural information on higher order multimers in aB will be obtained using (i) solid state NMR (Aim 1B) and (ii) protein chimeras that may be amenable to crystallography (Aim 1C). Proposed models for sHSP action include disassembly of sHSP oligomers into dimeric subunits before binding denatured protein substrates.
In Aim 2, we will investigate the mechanism of aB chaperone activity by protein aggregation assays, FRET measurements of subunit exchange, and solution-state NMR to map the binding site of denatured protein on aB. The role of HSP27 in apoptosis (Aim 3) will be explored by investigating its binding to cellular targets, ubiquitin and cytochrome c. Finally, insights into the consequences of inherited mutations in aB and HSP27 associated with disease will be sought by comparison of the structures and functional properties of mutant proteins with their wild-type counterparts. ? ? ?
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