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

PINK1和Parkin在线粒体损伤时通过相同的途径协调线粒体自噬。PINK1和Parkin基因突变的帕金森病患者表现出不同年龄的大范围症状，表明有额外的蛋白质参与线粒体损伤途径。该途径通过一种前馈机制运作，即PINK1磷酸化激活Parkin所需的泛素，而Parkin则为PINK1磷酸化提供进一步的泛素。先前的研究表明，在没有Parkin的情况下，泛素仍然被偶联并磷酸化为线粒体蛋白，这表明该途径涉及额外的E3连接酶。先前的筛选已经确定了3个需要验证的潜在E3连接酶。此外，E3连接酶的阴性CRISPRi筛选将用于鉴定线粒体损伤反应途径中的新候选基因。确定的E3连接酶将通过定义其激活，定位和磷酸化泛素对线粒体损伤的调节进行生化验证。

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.