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 <i>in vitro</i> kinase assays were used to investigate the molecular mechanism of the PINK1 variants. In contrast to the PD-linked PINK1^G411S mutation that diminishes Ub kinase activity, we found that the PINK1^G411A 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 <i>in vivo</i> and might serve as a critical positive control during therapeutic development. <b>Abbreviations</b>: 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(Ub)激酶-连接酶配对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可监测线粒体状态，当线粒体遭遇应激时，PINK1会积累在线粒体外膜表面，在此处磷酸化泛素的丝氨酸65位点以激活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(PD，Parkinson disease)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（in vitro）激酶实验，我们分析了PINK1变异体的分子机制。与帕金森病相关的PINK1^G411S突变会削弱泛素激酶活性，与之相反，我们发现PINK1^G411A变异体可显著提升泛素磷酸化水平，远超野生型PINK1的活性。这一效应使得中脑来源的基因编辑神经元中PRKN激活水平升高、线粒体自噬速率加快，且在线粒体应激后细胞存活率得到提升。

从机制层面来看，G411A变异体可稳定PINK1的激酶折叠构象，并促使泛素采取更优的C端回缩构象，以提升底物周转效率。综上，我们鉴定出411位残基对底物结合亲和力具有关键调控作用，这一发现可被用于药物开发以增强PINK1的酶学功能。将甘氨酸411替换为丙氨酸的遗传变异可提升线粒体自噬水平，或可用于在体内（in vivo）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端回缩尾泛素(ubiquitin with C-terminally retracted tail)；CTD：PINK1的C端结构域(C-terminal domain (of PINK1))；ELISA：酶联免疫吸附试验(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：均方根涨落(root mean square fluctuation，原文疑似笔误scare应为square)；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(ubiquitin with T66V and L67N mutation, mimics Ub-CR)；Ub：泛素(ubiquitin)；WT：野生型(wild-type)