• Login
• Admin

Scott, Daniel (2016) Recognition of unanchored polyubiquitin by natural and engineered ubiquitin-binding proteins. PhD thesis, University of Nottingham.

PDF (Thesis - as examined) - Repository staff only - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader Download (5MB) Request Thesis

Abstract

The covalent post-translational modification of selected substrates with the ubiquitin protein has emerged as a central regulatory mechanism, governing protein stability, activity and localisation, and accordingly an array of cellular processes. Ubiquitin signalling versatility arises owing to the diverse nature of (poly)ubiquitin modification, with distinct modifications subsequently transduced in a specific manner by ubiquitin-binding domains found in ubiquitin-binding proteins. In recent years the notion that ubiquitin exerts influence solely via the covalent modification of substrates has been challenged, with unanchored, or substrate-free polyubiquitin chains emerging as key regulators of cellular physiology.

The investigations described in this thesis seek to exploit the inherent selectivity of natural and engineered ubiquitin-binding proteins, to afford the purification of endogenous unanchored polyubiquitin, probing the molecular composition and interactions of this biologically significant ubiquitin pool. In chapter 3 by utilising the deubiquitinating enzyme USP5, which contains multiple ubiquitin-binding domains, and is normally responsible for the selective disassembly of unanchored ubiquitin, we purify unanchored polyubiquitin from mammalian cell extracts. Subsequently, we apply both ubiquitin-selective antibodies and mass spectrometry-based analyses to examine the polyubiquitin profile of the mammalian unanchored ubiquitin pool. In chapter 4 we then assess the mechanisms of ubiquitin recognition by USP5, presenting a structural mass spectrometry-based framework to probe and quantify ubiquitin: ubiquitin-binding domain interactions. Finally in chapter 5, based on the conclusions we draw from USP5-ubiquitin recognition in chapter 4, that multiple domains in suitable arrangement yield specificity for polyubiquitin chains, we design and synthesize a synthetic protein to favour the capture of unanchored polyubiquitin chains of defined topology, namely Lys-48 linked diubiquitin (and longer polyubiquitin chains), from mammalian cell extracts.

We conclude that strategies for the rational design and engineering of polyubiquitin chain-selective binding in non-biological polymers are possible, paving the way for the generation of reagents to probe the unanchored polyubiquitin chains of defined topology, and more widely the ‘ubiquitome’.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Layfield, R. Oldham, N.
Keywords:	Ubiquitin signaling, Polyubiquitin modification, Ubiquitin binding domains, Ubiquitin binding proteins
Subjects:	Q Science > QP Physiology QS-QZ Preclinical sciences (NLM Classification) > QU Biochemistry
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	37671
Depositing User:	Scott, Daniel
Date Deposited:	15 Dec 2016 06:40
Last Modified:	27 Mar 2018 14:59
URI:	https://eprints.nottingham.ac.uk/id/eprint/37671

Actions (Archive Staff Only)

Edit View