Discovery and characterisation of novel mitochondrial damage-induced E3 ligases that regulate p-Ub

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PINK1 and Parkin act in the same pathway to orchestrate mitophagy upon mitochondrial damage. Parkinson's disease patients with mutations in PINK1 and Parkin display a large range of symptoms with varying age of onset, indicating that additional proteins are involved in the mitochondrial damage pathway. The pathway operates a feedforward mechanism whereby PINK1 phosphorylates ubiquitin which is required to activate Parkin which provides further ubiquitin for phosphorylation by PINK1. Previous work has demonstrated that ubiquitin is still conjugated and phosphorylated to mitochondrialproteins in absence of Parkin, signifying that additional E3 ligases are involved in this pathway. Previous screens have identified 3 potential E3 ligases that need to be validated.Additionally, a negative CRISPRi screen of E3 ligases will be employed to identify novel candidates in the mitochondrial damage response pathway. The identified E3 ligases will be biochemically validated by defining their activation, localisation, and regulation of phospho-ubiquitin upon mitochondrial damage.