Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.706555
Title: Identification of deubiquitylases involved in parkin-mediated mitophagy
Author: Liang, Jin Rui
ISNI:       0000 0004 6057 7688
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2014
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Abstract:
Ubiquitylation is a post-translational modification of proteins with a broad range of downstream effects, ranging from protein turnover to transcriptional regulation, membrane trafficking and DNA damage repair. The conjugation of ubiquitin to its substrate is a sequential process mediated by three enzymes, termed the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme and the E3 ubiquitin ligase. Conversely, the removal of ubiquitin is mediated by deubiquitylases (DUBs). My project aims to identify DUBs that are involved in the selective removal of mitochondria via autophagy (mitophagy), a process that is regulated by a serine/threonine kinase, PINK1, and an E3 ligase, Parkin. Loss-of-function mutations in both PINK1 and Parkin have been reported to cause autosomal recessive-juvenile Parkinsonism (AR-JP), a form of early onset Parkinson’s Disease (PD). During mitochondrial depolarisation, PINK1 is stabilized at the outer mitochondrial membrane (OMM) where it recruits and activates Parkin. Subsequently, Parkin ubiquitylates a range of OMM proteins either for their proteasomal degradation or for the recruitment of the autophagic machinery. I first characterised this process in two different cell lines expressing either low endogenous levels of Parkin (SH-SY5Y neuroblastoma cells) or high exogenous levels of YFP-Parkin (hTERT-RPE1-YFP-Parkin, retinal pigment epithelial cells). I developed two different assays to assess Parkin-mediated mitophagy: (1) real time monitoring of YFP-Parkin by live-cell imaging (only in hTERT-RPE1-YFP-Parkin cells), and (2) western blot analysis of the cleavage, ubiquitylation and loss of proteins during mitophagy (in both hTERT-RPE1-YFP-Parkin and SH-SY5Y cells). I used these two assays to perform siRNA screens to identify DUBs, for which depletion resulted in altered Parkin recruitment and/or ubiquitylation and degradation of Parkin substrates. I identified several DUBs, including USP10, USP30, USP38, USP42, USP43, USP49, BAP1, OTUD4 and TRABID, for which siRNA depletion resulted in delayed Parkin recruitment to the mitochondria. Further characterisation of the effects of USP42 and USP43 siRNA depletions suggests that the loss of these DUBs could either desensitize the cells or affect the cellular response towards mitochondrial depolarisation. Depletion of another DUB, USP31, partially inhibited the later stages of mitophagy, i.e. the degradation of mitochondria via the lysosomal pathway. I discovered by chance that high expression levels of Parkin resulted in a small percentage of mitophagic cell death that was dependent on both PINK1 and Parkin. This cell death was prevented or delayed by inhibiting the proteasome. Interestingly, depletion of USP30 exacerbated this mitophagic cell death. This effect of USP30 depletion is of particular interest as USP30 is the only mitochondria-localised DUB. Further characterisation revealed that USP30 opposes TOM20 ubiquitylation and degradation, a previously described Parkin substrate. I speculate that USP30 counteracts disproportionate Parkin-dependent ubiquitylation and proteasomal degradation of outer mitochondrial membrane proteins, and its depletion enhances apoptosis by promoting Cytochome-C release from damaged mitochondria. Importantly, USP30 depletion also enhances apoptosis induced by BH3-mimetics ABT-737 and ABT-263, suggesting a more general role for USP30 in determining the threshold for mitochondrial cell death. As USP30 depletion enhances the degradation of Parkin substrate and sensitize cells to apoptosis, this data suggest that USP30 is a potential drug target not only in Parkinson’s Disease but also in cancer.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.706555  DOI: Not available
Keywords: Q Science (General) ; QP Physiology
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