Identification of deubiquitylases involved in the regulation of adherens junction components

Wong, Jia Identification of deubiquitylases involved in the regulation of adherens junction components. Doctor of Philosophy thesis, University of Liverpool.

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Abstract

Protein ubiquitylation represents a versatile mechanism to regulate multiple cellular processes, including protein turnover, transcription, cell signaling, membrane trafficking and DNA damage repair. The attachment of ubiquitin moieties to a target substrate is sequentially catalysed by three enzymes, namely E1, E2 and E3, and this process can be reversed by the deubiquitylases (DUBs). This research project seeks to decipher the involvement of DUBs in regulation of the components of the adherens junction (AJ), E-cadherin and β-catenin, in particular, which are the core components of the AJ. It is found in MCF7 cells, a breast cancer cell line, that E-cadherin undergoes constitutive turnover via the lysosomal pathway, as indicated by the accumulation of an 80kDa fragment of E-cadherin degradative fragment following treatment of MCF7 cells with Folimycin, a lysosomal v-ATPase inhibitor. Using the ratio of the full length and 80kDa degradative fragment as a biochemical readout for the trafficking status of E-cadherin, a siRNA human DUB library screen was performed and USP38 was identified as a leading candidate. siRNA depletion of USP38 led to increase in the full length to 80kDa E-cadherin ratio. Notably, the loss of USP38 led to significant loss of total E-cadherin level (sum of full length E-cadherin and 80kDa E-cadherin fragment). The loss of E-cadherin at the protein level following USP38 knockdown was not accompanied by a decrease in its mRNA level or increase in degradation rate, suggesting that translational effects could operate. Preliminary characterisation of this enzyme showed that it has both nuclear and cytosolic localisation. Mapping of nuclear localisation determinants revealed a role for a 46 amino acid insertion within the core catalytic domain, and the last 100 amino acid on C-terminus of USP38. In a separate siRNA DUB library screen performed on A549 cells, the established tumour suppressor BAP1 was identified as a DUB regulating β-catenin. siRNA depletion of BAP1 resulted in a loss of β-catenin and loss of cell-cell contacts, whereas its overexpression led to an increase in β-catenin level, indicating a positive regulatory role of BAP1 on β-catenin level. To facilitate further screening efforts and to provide an extra layer of validation to my own screens, a human DUB endonuclease-prepared small interfering RNA (esiRNA) library was developed. EsiRNA is a complex mixture of siRNAs generated by random cleavage of long double-stranded RNA and is argued to be able to circumvent the problem of siRNA-induced off-target effects. EsiRNAs were made for 91 out of the currently predicted 93 human DUBs. Quality control experiments were carried out upon a sample of selected DUBs and optimisation of experimental conditions for efficient knockdown in various cell lines was explored.

Item Type:	Thesis (Doctor of Philosophy)
Additional Information:	Date: 2012-10 (completed)
Subjects:	?? QH301 ??
Divisions:	Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User:	Symplectic Admin
Date Deposited:	10 Feb 2014 11:27
Last Modified:	16 Dec 2022 04:38
DOI:	10.17638/00010733
Supervisors:	Clague, Michael Urbe, Sylvie
URI:	https://livrepository.liverpool.ac.uk/id/eprint/10733