Kallinos, Andreas 
ORCID: 0000-0001-8771-963X
(2022)
The role of the mitochondrial deubiquitylase USP30 in
cellular fitness and death.
PhD thesis, University of Liverpool.
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Abstract
Ubiquitylation is a protein post-translational modification that involves the covalent conjugation of ubiquitin onto protein substrates through the collective activities of E1, E2 and E3 ubiquitin ligases and is reversed by deubiquitylating enzymes (DUBs). Ubiquitylation serves a plethora of cellular functions, including modes of selective autophagy such as mitophagy as well as protein homeostasis by the proteasome. Defects in the different pathways that ubiquitylation is involved in are associated with a number of pathologies including neurodegeneration. Parkinson’s disease (PD) is the second most common neurodegenerative disease, with approximately 10% of all cases having a genetic component to their aetiology. Mutations in PINK1 (PARK6) and Parkin (PARK2) are known causes of early onset juvenile parkinsonism. PINK1 and Parkin operate in unison in the mitochondrial quality control pathway of mitophagy, preventing accumulation of dysfunctional and potentially harmful mitochondria. PINK1 responds to instances of mitochondrial damage and phosphorylates ubiquitin (S65), which recruits and activates Parkin. Activated Parkin decorates outer mitochondrial membrane (OMM) proteins with more ubiquitin, which results in the recruitment of the autophagophore membrane through autophagy receptors. The mitochondrial DUB USP30 opposes the PINK1/Parkin pathway and USP30 depletion rescues PD-associated phenotypes caused by loss of PINK1 and Parkin in the fly. The above presented the very first evidence that USP30 silencing may be a valid therapeutic strategy in the treatment of PD. I contributed to a body of work from my host lab that showed that USP30 suppresses the PINK1-dependent component of basal mitophagy. This suggests USP30 inhibition may indeed have protective effects in the long-term by enhancing the basal rate of mitophagy. I have also shown that a pool of USP30 localises to peroxisomes independently of mitochondria. Previous work from our lab has shown USP30 depletion enhanced the effect of BH3 mimetics in cells. I followed up on with this work and introduced other BH3 mimetic compounds, which are more selective in terms of which anti-apoptotic proteins they inhibit. I utilised CRISPR/Cas9 technology to generate USP30KO in the HCT116 FlpIn TRex cell line. I characterised the metabolic parameters of these cells using Seahorse Technology, measured intracellular levels and sources of ATP, and assessed their proliferation and colony forming potential in different substrates. I also performed a transcriptome analysis and a small-scale proteome in the same cells to get an understanding of USP30 function in cells. I investigated the generation of pS65-Ub in cells that lacked detectable Parkin utilising a global ubiquitylation inhibitor and in parallel investigated the role of USP30 in this process. Lastly, I assisted in the characterisation of a USP30 inhibitor in Parkin over-expressing cells as well as cells expressing endogenous Parkin. I have shown that USP30 inhibition phenocopied USP30KO in terms of enhanced TOMM20 ubiquitylation and enhanced pS65-Ub generation. My work has led to the generation and characterisation of new tools that allow us to gain a deeper understanding of USP30 biology in PD and other pathologies.
| Item Type: | Thesis (PhD) |
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| Divisions: | Faculty of Health and Life Sciences |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 11 Mar 2022 16:05 |
| Last Modified: | 18 Jan 2023 21:13 |
| DOI: | 10.17638/03148453 |
| Supervisors: |
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| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3148453 |
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