Substitution of PINK1 Gly411 modulates substrate receptivity and turnover

The ubiquitin (Ub) kinase-ligase pair PINK1-PRKN mediates the degradation of damaged mitochondria by macroautophagy/autophagy (mitophagy). PINK1 surveils mitochondria and upon stress accumulates on the mitochondrial surface where it phosphorylates serine 65 of Ub to activate PRKN and to drive mitochondrial turnover. While loss of either PINK1 or PRKN is genetically linked to Parkinson disease (PD) and activating the pathway seems to have great therapeutic potential, there is no formal proof that stimulation of mitophagy is always beneficial. Here we used biochemical and cell biological methods to study single nucleotide variants in the activation loop of PINK1 to modulate the enzymatic function of this kinase. Structural modeling and in vitro kinase assays were used to investigate the molecular mechanism of the PINK1 variants. In contrast to the PD-linked PINK1G411S mutation that diminishes Ub kinase activity, we found that the PINK1G411A variant significantly boosted Ub phosphorylation beyond levels of PINK1 wild type. This resulted in augmented PRKN activation, mitophagy rates and increased viability after mitochondrial stress in midbrain-derived, gene-edited neurons. Mechanistically, the G411A variant stabilizes the kinase fold of PINK1 and transforms Ub to adopt the preferred, C-terminally retracted conformation for improved substrate turnover. In summary, we identify a critical role of residue 411 for substrate receptivity that may now be exploited for drug discovery to increase the enzymatic function of PINK1. The genetic substitution of Gly411 to Ala increases mitophagy and may be useful to confirm neuroprotection in vivo and might serve as a critical positive control during therapeutic development. Abbreviations: ATP: adenosine triphosphate; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Ub-CR: ubiquitin with C-terminally retracted tail; CTD: C-terminal domain (of PINK1); ELISA: enzyme-linked immunosorbent assay; HCI: high-content imaging; IB: immunoblot; IF: immunofluorescence; NPC: neuronal precursor cells; MDS: molecular dynamics simulation; PD: Parkinson disease; p-S65-Ub: ubiquitin phosphorylated at Ser65; RMSF: root mean scare fluctuation; TOMM: translocase of outer mitochondrial membrane; TVLN: ubiquitin with T66V and L67N mutation, mimics Ub-CR; Ub: ubiquitin; WT: wild-type.泛素（ubiquitin, Ub）激酶-连接酶对PINK1-PRKN通过巨自噬/自噬（mitophagy，线粒体自噬）介导受损线粒体的降解。PINK1可监视线粒体状态，在遭遇应激时于线粒体外膜积累，通过磷酸化泛素的丝氨酸65位以激活PRKN，进而推动线粒体周转。 尽管PINK1或PRKN的缺失在遗传学上与帕金森病（Parkinson disease, PD）相关，且激活该通路似乎具备巨大的治疗潜力，但目前尚无正式证据表明刺激线粒体自噬总是有益的。 本研究采用生化与细胞生物学方法，探究PINK1激活环中的单核苷酸变异以调控该激酶的酶学功能；通过结构建模与体外激酶实验，分析PINK1变异体的分子机制。 与会削弱泛素激酶活性的PD相关突变PINK1G411S不同，我们发现PINK1G411A变体的泛素磷酸化水平显著高于野生型PINK1。该变体可增强中脑来源的基因编辑神经元中的PRKN激活程度与线粒体自噬速率，并提升线粒体应激后的细胞存活率。 机制层面，G411A变体可稳定PINK1的激酶折叠构象，并使泛素采取更优的C端回缩构象，从而提升底物周转效率。 综上，我们鉴定出411位残基对底物接纳具有关键作用，这一发现可用于开发增强PINK1酶学功能的药物。将Gly411替换为Ala的遗传学变异可提升线粒体自噬水平，或可用于在体内验证神经保护作用，同时也可作为治疗开发过程中的关键阳性对照。 缩写说明： ATP：三磷酸腺苷（adenosine triphosphate）； CCCP：羰基氰化物间氯苯腙（carbonyl cyanide m-chlorophenyl hydrazone）； Ub-CR：C端尾回缩的泛素（ubiquitin with C-terminally retracted tail）； CTD：PINK1的C端结构域（C-terminal domain）； ELISA：酶联免疫吸附实验（enzyme-linked immunosorbent assay）； HCI：高内涵成像（high-content imaging）； IB：免疫印迹（immunoblot）； IF：免疫荧光（immunofluorescence）； NPC：神经前体细胞（neuronal precursor cells）； MDS：分子动力学模拟（molecular dynamics simulation）； PD：帕金森病（Parkinson disease）； p-S65-Ub：丝氨酸65位磷酸化泛素（ubiquitin phosphorylated at Ser65）； RMSF：均方根波动（root mean square fluctuation，原文疑似笔误，应为root mean square fluctuation）； TOMM：线粒体外膜转位酶（translocase of outer mitochondrial membrane）； TVLN：携带T66V与L67N突变的泛素，可模拟Ub-CR； Ub：泛素（ubiquitin）； WT：野生型（wild-type）。