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Title: Characterising the function of the mitochondrial deubiquitylase USP30 in mitophagy
Author: Jardine, Jane
ISNI:       0000 0005 0288 1662
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2021
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Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder after Alzheimer's disease. The causes of this disorder are not fully understood, as 95% of cases are sporadic forms of PD. However, the genes involved in familial forms of the disease have been intensely studied and have highlighted the importance of mitochondrial quality control in PD. Indeed, PARK2/PRKN and PARK6/PINK1 are core regulators of mitophagy. Mitophagy is triggered by mitochondrial impairment through the engagement of the mitochondrial kinase PINK1. Upon mitochondrial membrane depolarisation, PINK1 promotes both the activation of the ubiquitin E3 ligase PRKN and its recruitment to mitochondria. In turn, PRKN tags mitochondrial proteins with ubiquitin. The ubiquitincoated mitochondrion is then recognised by autophagy receptors that bind to the autophagosomal membrane for safe disposal of the defective organelle. My project aims at gaining a deeper understanding of the PINK1-PRKN pathway of mitophagy and finding regulators of this pathway. As described above, mitophagy is regulated through ubiquitylation, which itself is controlled by opposing E3 ligases and deubiquitylating enzymes (DUBs). Those enzymes are of particular interest as they could be chemically targeted to prevent mitochondrial failure. In this thesis I have focussed on USP30, a mitochondrial DUB, that is thought to remove ubiquitin from PRKN substrates. Mitophagy reporters enabled me to measure spontaneously occurring, "basal" or constitutive, mitophagy events in live cell imaging experiments. I found that USP30 regulates both basal and depolarisation-induced mitophagy in cells expressing endogenous PRKN. Serendipitously, I discovered that a pool of USP30 was localised at peroxisomes where it regulates pexophagy. In parallel, I made use of those same reporters to characterise alternative means to induce mitophagy. I also contributed to the generation and characterisation of a new in vivo mitophagy reporter model, the mt-Keima fly. This tool enabled me to study mitophagy events in a genetically tractable organism. Preliminary results show that USP30 knockout induces an increase in mitophagy events occurring in the fly brain. I further engineered USP30 knockout neuroblastoma cells using the CRISPR-Cas9 technology and found, through western blot analysis that TOMM20 was one of the main substrates of USP30. It thus appeared that USP30 removes ubiquitin from TOMM20 to prevent the Parkin-feedforward loop. Accordingly, I found that USP30 deletion and inhibition results in an increase of pS65-Ub generation by PINK1. I performed a series of proteomic and ubiquitylomic experiments using SILAC based mass spectrometry to further search for substrates of USP30. I found that many outer mitochondrial membrane (OMM) proteins are more ubiquitylated in the absence of USP30, including members of the translocase outer membrane (TOM) complex, the very abundant VDAC proteins, and other less well characterised OMM proteins, many of which are known Parkin substrates. Although many OMM protein were ubiquitylated, I did not measure a global loss of mitochondrial or peroxisomal proteins. Intriguingly, the ubiquitylome datasets also suggested that USP30 loss or inhibition may impact on ribosomal quality control. Overall, this thesis discloses new exciting roles of USP30 in the context of pexophagy, basal and induced-mitophagy, and further highlights the relevance of USP30 as a therapeutic target for PD.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral