Regulation of PINK1 and PARKIN-dependent mitophagy

总结。许多研究，包括我们实验室的关键工作，已经揭示了基本的机制 帕金森病(PD)两个基因--PINK1蛋白激酶和Parkin通过哪些蛋白编码 泛素(Ub)连接酶促进受损线粒体的泛素化和自噬捕获 他们通过有丝分裂获得的清除权。最近，我们将一个定量蛋白质组学平台与干细胞合并- 诱导派生的神经元(INeurons)具有阐明Parkin和PINK1泛素化的途径突变 内源条件下的靶标，并确定了线粒体去泛素化酶USP30的作用 以及p97分离酶在Parkin和丝裂原体通量调节中的作用。然而，我们对这一问题的理解 帕金森病患者中突变的其他蛋白质与Parkin-PINK1系统合作对疾病病因学做出贡献 仍然是有限的，我们对线粒体易位子上的PINK1激活阈值的理解也是有限的 是机械控制的。在这里，我们提出了一系列实验，以解决这两个知识 差距。首先，在我们最近的有丝分裂通量CRISPR筛查中出现的最引人注目的基因之一是 Fbxo7，PD突变基因(PARK15)，F-Box家族成员，形成SCF Ub 连接酶。Fbxo7的S关键功能和靶点，以及它的突变如何易患帕金森病，目前尚不清楚。 通过相互作用蛋白质组学，我们发现Fbxo7与多个调控成分有关 并提出Fbxo7可能通过整合有丝分裂和蛋白酶体调控而发挥核心作用。 维持细胞器动态平衡的机制。在目标1中，我们将使用我们的iNeuron系统来检查Fbxo7的S 在有丝分裂通量中的作用，使用一系列定量分析来检查途径中的连续步骤，以及 作为第一步，我们将从基因和功能上剖析泛素化靶点和调控机制。 了解患者Fbxo7基因突变如何导致帕金森病。第二，我们的初步数据，以及 在现场的研究表明，PINK1和USP30在物理上都与线粒体易位子有关， 将易位子置于帕金规则的结合点。我们的数据表明，USP30在控制 通过从易位子中移除Ub来激活Parkin的阈值以及也可能具有先前的 在运输环节本身的进口质量控制方面发挥了未被重视的作用。在目标2中，我们将系统地检查 转位基因及其泛素化在设定Ub激活Parkin阈值中的作用 磷酸化。同时，我们将阐明USP30如何在这一新认可的进口质量中发挥作用 从转运子输入底物上移除Ub链的控制(IQC)途径。最后，我们的工作导致了 首次使用单粒子电子显微镜显示与易位子相关的PINK1，以及 我们寻求进一步发展对这个复合体如何组装和结构的生化和结构的理解 受监管的。总而言之，这些关于这些关键分子如何与公园相交的机制研究 该系统将提供对线粒体质量控制的更深层次的了解。

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.