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Title: Regulating proteostasis : a study of interactions between HSP70 proteins, HSP40 proteins, and the sigma 1 receptor
Author: Ossa, Felipe
ISNI:       0000 0004 7653 6557
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Proteostasis encompasses a number of cellular processes that are essential for maintaining cellular protein machinery, ranging from synthesis and folding through to degradation, prevention of aggregation and even the mediation of constitutive function. This fundamental cellular process is linked to several clinically important phenomena, in particular neurodegeneration, cancer and ageing. Understanding the proteostatic machinery requires not just an individual examination of the key players in the system, namely the protein chaperones, but also an examination of the vast network of regulatory machinery that underpins their function. The work presented here examines two types of regulators: the HSP40 proteins and the Sigma 1 Receptor, in the context of their impact on the activity of HSP70 proteins, one of the key hubs of the proteostatic network. The Sigma 1 Receptor is a small (25 kDa) protein of significant clinical importance, and recent work has highlighted its links to the proteostatic machinery and its interaction with HSP70 proteins. With its unique pharmacology, Sigma 1 is now emerging as a novel ligand-operated regulator of proteostasis. Our work on the Sigma 1 Receptor was hindered by the difficulties of expressing and isolating a functional integral human membrane protein; despite extensive optimisation of the construct and procedures for expression in E. coli and reconstitution into detergent we remained unable to demonstrate functionality. The data presented here on balance argues that even under our optimised conditions the receptor structure is significantly disrupted, and that activity is largely absent. More successful studies in cellulo show the response of the oligomeric state of Sigma 1 to its ligands using a novel number and brightness-based methodology. We next turned to HSP40 regulation of HSP70s. HSP40s are canonical regulators of HSP70s and act by stimulating ATPase activity using J-domains. We hypothesised that because of their different roles in the cell, the human cytosolic HSP70 and HSC70 may preferentially interact with or be stimulated by J-domains from different HSP40s. Using the J-domains of DNAJB2, DNAJB6, and DNAJC5, we show that the rank order of ATPase stimulation is preserved for HSP70 and HSC70, but that the stimulated activity of HSP70 is less than that of HSC70. Furthermore, we present data indicating that the HSP70 C-terminal domain plays a role in determining the response of HSC70 and HSP70 to regulation by HSP40 co-chaperone J-domains. Finally, we confirm the importance of the G/F rich region for the structure and function of a B class J-domain from humans, DNAJB6.
Supervisor: Schnell, Jason ; Redfield, Christina ; Padilla-Parra, Sergi Sponsor: Wellcome Trust ; Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Protein Expression and Purification ; NMR ; Protein-Protein Interactions ; Biophysics ; Protein Chaperones ; Fluorescence Microscopy