Characterizing the Structural and Functional Impacts and Regulation of HSPB4 T148 Phosphorylation

Abstract

Small heat shock proteins (sHSPs) are chaperones which generally exhibit large, heterogenous, and polydisperse oligomeric assemblies. In part through their chaperone function, sHSPs exert protective effects in several contexts. Post-translational modifications (PTMs) of sHSPs, and in particular phosphorylation, are tightly linked to alterations in their structure and function. Specifically, we have previously shown that HSPB4 T148 phosphorylation, which is highly diminished in diabetic retinopathy, controls its protective capabilities in retinal neurons. Thus, we sought to characterize the structural and functional impacts of T148 phosphorylation on HSPB4, as well as the regulatory mechanisms underpinning this modification. In the introductory chapter of this dissertation, a review of sHSPs is presented. The roles of each protein domain and specific motifs, as well as of phosphorylation and other PTMs in regulating sHSP structure and function are first discussed, followed by an overview of disease associations, involvement in retinal neuroprotection, and regulation of sHSP phosphorylation. In chapter two, we have shown that T148 phosphorylation is associated with reduced stress-induced HSPB4 insolubility, altered oligomeric profile, improved chaperone function, and neuroprotective capacity in metabolic stress conditions. We have also identified paracrine protective roles of MGC-secreted HSPB4, and particularly of the functionally enhanced T148D HSPB4. In chapter three, we have identified several kinases, including mTORC2, which phosphorylate T148 in vitro. In addition to identifying mTORC2 as a bona fide T148-phosphorylating kinase, we have shown that its interaction with HSPB4 is strengthened by HSPB4 chaperone function. The findings presented in this dissertation further characterize T148 phosphorylation-mediated neuroprotection and the mechanistic underpinnings of this PTM. This fills several critical gaps in our knowledge of HSPB4 and lays the groundwork for further interrogation of its capabilities as a therapeutic agent.