Structural characterization of the heat shock protein HSPB8 by nuclear magnetic resonance and its interaction with amyloidogenic alpha-synuclein

Abstract

Neurodegeneration in Parkinson's disease is correlated with the occurrence of Lewy bodies, intracellular inclusions containing aggregates of the intrinsically disordered protein (IDP) a-synuclein (a-syn). In the cell, however, a-syn aggregation can be prevented by a class of proteins known as molecular chaperones, also called heat shock proteins. While numerous chaperones interact with a-syn, little is known regarding the mechanisms by which these prevent a-syn aggregation. Small heat shock proteins (sHSP) from the HSPB8 family have been demonstrated to be the most potent in inhibiting mature fibril formation of both wild-type and mutant a-syn. However, the recombinant human protein is not easy to study because it can form large molecular-weight oligomers at high concentrations. To overcome these disadvantages, we propose to use the HSPB8 homolog from Aedes aegypti (AaHSPB8), which remains monomeric at high concentration, in structural and a-syn interaction studies. Using solution Nuclear Magnetic Resonance (NMR), we aim to solve the 3D structure of AaHSPB8 in solution and study the interaction of AaHSPB8 with both monomeric and fibrillar a-syn. Combining NMR and hydrostatic pressure, we will monitor the structural transitions occurring upon unfolding and determine the kinetic properties of these processes. From these studies, we aim to understand the mechanism of HSPB8s to counteract the formation and propagation of toxic a-syn aggregates. As such, our project will let us learn how sHSPs regulate a-syn aggregation and, therefore, how to optimize its interaction with a-syn to develop therapeutic approaches to battle the pathogenesis of a-synucleinopathies. (AU)