AbstractParkinson’s disease (PD) is the second most common neurodegenerative disorder. Discoveries of heritable, disease associated mutations in multiple genes has linked mitochondrial dysfunction with PD. Two of these gene products, PINK1 and Parkin, function together in a common pathway. The PINK1 kinase acts as a sensor for mitochondrial damage, catalysing downstream phosphorylation of both Parkin and ubiquitin (Ub) at Ser65, thereby activating Parkin E3 Ub ligase activity. Upon activation, Parkin ubiquitylates multiple proteins at the mitochondrial surface, triggering degradation of mitochondria or mitochondrial membrane proteins.
Our understanding of the PINK1-Parkin pathway is mainly derived from artefactprone overexpression studies. A key aim of my thesis was to therefore address both regulation and downstream function of PINK1 and Parkin under endogenous conditions. Towards this goal, I characterised multiple antibodies to enable the robust assessment of endogenous signalling of key components of the PINK1-Parkin pathway. I then identified and characterised the ovarian cancer cell lines SK-OV-3 as a cell system enabling endogenous analysis of PINK1-Parkin signalling. Utilising CRISPR/Cas9 technology, I generated and characterised PINK1 and Parkin knock-out SK-OV-3 cells and employed both quantitative mass spectrometry and biochemical assays to assess endogenous signalling downstream of PINK1 and Parkin. I demonstrate that, upon mitochondrial depolarisation and PINK1-Parkin-dependent activation, K63-linked Ub is the major Ub-linkage chain type that becomes upregulated. Furthermore, I describe that these Ub chain types contain Ser65-phosphorylated Ub (p(Ser65)-Ub), and that PINK1 is essential for Parkin activation and generation of p(Ser65)-Ub. In contrast, I observe that Parkin is dispensable for p(Ser65)-Ub accumulation, indicating redundant regulation of p(Ser65)-Ub by additional E3 ligases in SK-OV-3 cells. Using siRNA and CRISPR/Cas9, I provide genetic evidence that knock-down of the mitochondrial E3 Ub protein ligase 1 (MUL1), causes further reduction in p(Ser65)-Ub in Parkin knock-out SK-OV-3 cells. This suggests MUL1 is required for optimal generation of p(Ser65)-Ub upon mitochondrial damage and PINK1 activation. However, p(Ser65)-Ub production is not fully abolished, suggesting the contribution of other as yet to be determined E3 ligases.
Overall these studies have elaborated a cellular system to robustly assess endogenous PINK1-Parkin signalling and revealed new insights into p(Ser65)-Ub architecture and regulation.
|Date of Award||2019|
|Sponsors||J. Macdonald Menzies Charitable Trust|
|Supervisor||Miratul Muqit (Supervisor) & Dario Alessi (Supervisor)|