Regulation of PINK1 and PARKIN-dependent mitophagy

摘要大量的研究，包括我们实验室的关键工作，揭示了 由两个帕金森病（PD）基因编码的蛋白质-PINK 1蛋白激酶和PARKIN 泛素（Ub）连接酶-促进受损线粒体的泛素化和自噬捕获， 通过线粒体自噬清除最近，我们将定量蛋白质组学平台与干细胞- 衍生的诱导神经元（iNeurons）携带通路突变，以阐明PARKIN和PINK 1泛素化 在内源性条件下的目标，并确定了线粒体去泛素化酶USP 30的作用 以及PARKIN和线粒体吞噬流量调节中的p97分离酶。然而，我们对这种程度的理解 在PD中突变的其他蛋白质与PARKIN-PINK 1系统协作以促成疾病病因 仍然是有限的，因为我们的理解是如何PINK 1激活阈值对线粒体易位 是机械控制的。在这里，我们提出了一系列的实验，解决这两个知识 差距。首先，在我们最近的线粒体吞噬通量CRISPR筛选中出现的最引人注目的基因之一是 FBXO 7，PD（PARK 15）中突变的基因，形成SCF Ub的F-Box蛋白家族成员 连接酶FBXO 7的关键功能和靶点，以及其突变如何易患PD，目前尚不清楚。 通过相互作用蛋白质组学，我们发现FBXO 7与多个调控组件的相关性。 蛋白酶体，并提出FBXO 7可能发挥核心作用，通过整合线粒体自噬和蛋白酶体的控制 支持细胞器稳态的机制。在目标1中，我们将使用我们的iNeuron系统来检查FBXO 7的 使用一系列定量测定法检查途径中的顺序步骤， 我们将从基因和功能上剖析泛素化的目标和调控机制作为第一步 了解FBXO 7中的患者突变如何导致PD。第二，我们的初步数据， 在该领域的工作，表明PINK 1和USP 30都与线粒体易位子物理相关， 将易位子置于PARKIN调节的中心。我们的数据显示USP 30在控制 通过从易位子中去除Ub激活PARKIN的阈值， 在translocon本身的进口质量控制中发挥了不受重视的作用。在目标2中，我们将系统地研究 转位子成分和泛素化在通过Ub设置PARKIN激活阈值中的作用 磷酸化同时，我们将阐明USP 30在这一新认可的进口质量中的作用 用于从易位子输入底物中去除Ub链的对照（IQC）途径。最后，我们的工作导致了 使用单粒子电子显微镜首次观察到PINK 1与易位子的关联，以及 我们寻求进一步发展对这种复合物如何组装的生物化学和结构的理解， 监管.总之，这些集中在这些关键分子如何与PARKIN交叉的机制研究 系统将提供线粒体质量控制的更深层次的理解。

SUMMARY. Numerous studies, including critical work from our lab, has revealed the fundamental mechanisms by which proteins encoded by two Parkinson’s Disease (PD) genes – the PINK1 protein kinase and PARKIN ubiquitin (Ub) ligase – promote the ubiquitylation and autophagic capture of damaged mitochondria to promote their clearance by mitophagy. Recently, we have merged a quantitative proteomics platform with stem cell- derived, induced neurons (iNeurons) harboring pathway mutations to elucidate PARKIN and PINK1 ubiquitylation targets under endogenous conditions, and have determined the role of the mitochondrial deubiquitylase USP30 and the p97 segregase in PARKIN and mitophagic flux regulation. Yet, our understanding of the extent to which other proteins mutated in PD collaborate with the PARKIN-PINK1 system to contribute to disease etiology remains limited, as is our understanding of how the PINK1 activation threshold on the mitochondrial translocon is mechanistically controlled. Here, we propose a series of experiments that address both of these knowledge gaps. First, among the most compelling genes to emerge from our recent mitophagic flux CRISPR screen is FBXO7, a gene mutated in PD (PARK15) and a member of the F-Box family of proteins that forms an SCF Ub ligase. FBXO7’s critical functions and targets, as well as how its mutation predisposes to PD, are unknown. Through interaction proteomics, we find that FBXO7 associates with multiple regulatory components of the proteasome, and propose that FBXO7 may play a central role by integrating mitophagy and proteasomal control mechanisms to support organelle homeostasis. In Aim 1, we will use our iNeuron system to examine FBXO7’s role in mitophagic flux using an array of quantitative assays that examine sequential steps in the pathway, and we will genetically and functionally dissect ubiquitylation targets and regulatory mechanisms as an initial step toward understanding how patient mutations in FBXO7 may contribute to PD. Second, our preliminary data, and work in the field, indicate that both PINK1 and USP30 are physically associated with the mitochondrial translocon, placing the translocon at the nexus of PARKIN regulation. Our data show that USP30 has a role in controlling both the threshold for PARKIN activation by removing Ub from the translocon and also may have a previously unappreciated role in import quality control at the translocon itself. In Aim 2, we will systematically examine translocon components and ubiquitylation for their roles in setting the threshold for PARKIN activation via Ub phosphorylation. In parallel, we will elucidate how USP30 functions in this newly recognized Import Quality Control (IQC) pathway for removal of Ub chains from translocon import substrates. Finally, our work has led to the first visualization of PINK1 in association with the translocon using single-particle electron microscopy, and we seek to further develop a biochemical and structural understanding of how this complex is assembled and regulated. Together, these focused mechanistic studies on how these key molecules intersect with the PARKIN system will provide a deeper understanding of mitochondrial quality control.