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Determination of the Composition and Architecture of the Mitochondrial Ubiquitome in PINK1/Parkin Mitophagy


Type

Thesis

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Authors

Abstract

Parkinson’s Disease is the second most common age-related neurodegenerative disorder, with multiple lines of evidence suggesting that mitochondrial dysfunction may underlie the etiology of the disease. The kinase PINK1, and the ubiquitin (Ub) E3 ligase Parkin, function in a pathway to maintain mitochondrial quality control through the engulfment of mitochondria by the autophagy system, in a process termed mitophagy. Through a positive feedback loop centred on the production of S65-phosphorylated Ub, PINK1 and Parkin work in concert to ubiquitinate proteins on the outer mitochondrial membrane, leading to the recognition, engulfment, and eventual destruction of the damaged mitochondria by the autophagy machinery. While significant work has been done to establish the substrates and chain type composition of the Ub placed on mitochondrial substrates by Parkin, little work has been done to establish the broader Ub chain architecture: the degree of chain branching, and the relationship between phosphorylation and chain formation in the context of a single ubiquitin moiety. Further, while Drosophila melanogaster has been successfully used as a model organism to understand that PINK1 and Parkin function in a common pathway, the chain composition of mitochondrial Ub has not been explored in this model. Using a novel mass spectrometry-based technique, Ub-clipping, combined with biochemical methods for understanding Ub biology, this thesis explores the composition and architecture of the mitochondrial ubiquitome. Using a depolarisation-induced HeLa cell model, it was found that Parkin predominantly produces mono- and short chain-ubiquitination of integral mitochondrial membrane proteins, and that mono- and distal Ub moieties are preferentially phosphorylated. Further, significant method development was performed to enable detection of the lowly abundant mitochondrial Ub in Drosophila, and it was found that treatment of flies with the oxidant paraquat robustly induces formation of pS65-Ub in the presence of endogenous levels of PINK1 and Parkin. These results further our understanding of the precise nature of Parkin-mediated ubiquitination events, and provide the first evidence that the feedforward production of pS65-Ub occurs in response to the induction of mitochondrial damage in Drosophila. The paraquat model for inducing PINK1 and Parkin activation will form a basis for future studies understanding the role of PINK1 and Parkin, in enabling the study of other players in the PINK1/Parkin pathway, and in determining the downstream fate of pS65-ubiquitinated mitochondria, in the context of an organism.

Description

Date

2020-07-29

Advisors

Whitworth, Alexander
Komander, David

Keywords

Ubiquitin, Mitochondria, Mitophagy

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Sponsorship
Gates Cambridge, MRC Strauss