Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.685918
Title: The role of membrane-associated UCH-L1 and a screen for Cullin-RING ligase substrates in neurons
Author: Bishop, Paul Nicholas
ISNI:       0000 0004 5917 155X
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2015
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Abstract:
Ubiquitination is a dynamic and reversible post-translational modification that is fundamentally important in controlling multiple aspects of neuronal function, including turnover, trafficking and synaptic plasticity. Dysfunction of ubiquitin metabolism is a hallmark of many neurodegenerative diseases, and abnormal accumulation of ubiquitinated proteins is associated with Parkinson's disease, and Alzheimer's disease. Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) is a deubiquitinating enzyme (DUB) that is highly expressed in neurons. A possible role for UCH-LI in neurodegeneration has been highlighted because of its presence in Lewy Bodies associated with Parkinson's disease and neurofibrillary tangles observed in Alzheimer's disease. UCHL1 exists in two forms in neurons, a soluble cytoplasmic form (UCH-LI c) and a membrane-associated form (UCH-L1M). Alzheimer's brains show reduced levels of soluble UCH-L1 C correlating with the formation of UCH-L1-immunoreactive tau tangles whereas UCH-L1 M has been implicated in α-synuclein dysfunction in Parkinson's disease. Given these reports of divergent roles, this thesis investigates the properties of UCHL1 membrane-association. Surprisingly, the results indicate that UCH-L1 does not partition to the membrane in the clonal cell lines tested. Fmihermore, in primary cultured neurons, a proportion of UCH-L1M does partition to the membrane, but, contrary to a previous report, this does not require farnesylation. Deletion of the four C-terminal residues caused the loss of protein solubility, abrogation of substrate binding, increased cell death and an abnormal intracellular distribution, consistent with protein misfolding and aggregation. These data indicate that UCH-L1 is differently processed in neurons compared to clonal cell lines and that farnesylation does not account for the membrane association in neurons. The cullin family of proteins act as molecular structures that scaffold the components of the Cullin-RING ligase (CRL) class of E3 ubiquitin ligases. Cullins undergo a conformational change upon neddylation which activates the ligase activity of the CRL complex, leading to mono- and poly-ubiquitination of specific sets of substrate proteins. The neddylation E1 enzyme inhibitor MLN4924 blocks activation of the cullin family of proteins and leads to loss of substrate ubiquitination, including polyubiquitinated substrates destined for degradation by the 26S proteasome. The last few years have seen a rise in the use of high-throughput proteomics screens to quantify global protein changes in response to certain manipulations. Proteomics screens have already been conducted using MLN4924 to identify potential CRL substrates in clonal cell lines, but this thesis presents the first time a screen has been conducted to identify neuronal substrates. The work in this thesis has identified a number of candidate neuronal proteins and represents a first step toward identifying a CRL-mediated neuronal ubiquitome and determining how CRL-mediated degradation impacts synaptic structure, transmission and plasticity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.685918  DOI: Not available
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