Regulation of PINK1 and PARKIN-dependent mitophagy

PINK1 和 PARKIN 依赖性线粒体自噬的调节

• 批准号: 10401903
• 负责人: JEFFREY W HARPER
• 金额: $ 44.81万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-25 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10401903
• 关键词: Address; Alleles; Autophagocytosis; Autophagosome; Biochemical; Biochemical Genetics; Biological; Biological Assay; Biotinylation; CRISPR screen; Cells; Complex; Coupled; Cytoplasm; Data; Disease; Electron Microscopy; Engineering; Etiology; Excision; F-Box Proteins; FBXO7 gene; Genes; Homeostasis; Individual; Knowledge; Link; Maps; Membrane; Membrane Proteins; Methods; Mitochondria; Mitochondrial DNA; Molecular; Mutate; Mutation; Neurons; Organelles; Outer Mitochondrial Membrane; PINK1 gene; Parkinson Disease; Pathway interactions; Patients; Phosphorylation; Physiological; Play; Protein Kinase; Proteins; Proteomics; Quality Control; Regulation; Reporter; Role; SKP Cullin F-Box Protein Ligases; Series; Signal Transduction; Site; Synapses; System; Ubiquitin; Visualization; Work; base; cohort; dopaminergic neuron; embryonic stem cell; experimental study; human embryonic stem cell; member; multicatalytic endopeptidase complex; mutant; novel; particle; receptor; recruit; stem cells; ubiquitin ligase; ubiquitin-protein ligase

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.

概括。大量研究，包括我们实验室的关键工作，揭示了基本机制 由两个帕金森病 (PD) 基因（PINK1 蛋白激酶和 PARKIN）编码的蛋白质 泛素（Ub）连接酶——促进受损线粒体的泛素化和自噬捕获，以促进 它们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合并- 衍生的诱导神经元 (iNeurons) 含有通路突变，可阐明 PARKIN 和 PINK1 泛素化 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 PARKIN 和线粒体自噬通量调节中的 p97 分离酶。然而，我们对这种程度的理解 PD 中突变的其他蛋白质与 PARKIN-PINK1 系统协同作用，促成疾病病因学 仍然有限，我们对线粒体易位子上 PINK1 激活阈值的理解也是有限的 是机械控制的。在这里，我们提出了一系列解决这些知识的实验 差距。首先，我们最近的线粒体自噬流 CRISPR 筛选中出现的最引人注目的基因是 FBXO7，PD 突变基因 (PARK15)，是形成 SCF Ub 的 F-Box 蛋白家族的成员 连接酶。 FBXO7 的关键功能和靶点，以及其突变如何导致帕金森病，目前尚不清楚。 通过相互作用蛋白质组学，我们发现 FBXO7 与多种调节成分相关联。 蛋白酶体，并提出 FBXO7 可能通过整合线粒体自噬和蛋白酶体控制发挥核心作用 支持细胞器稳态的机制。在目标 1 中，我们将使用 iNeuron 系统来检查 FBXO7 使用一系列定量分析来检查途径中的连续步骤，以了解线粒体自噬流中的作用，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 旨在了解患者 FBXO7 突变如何导致 PD。其次，我们的初步数据，以及 该领域的工作表明 PINK1 和 USP30 都与线粒体易位子物理相关， 将易位子置于 PARKIN 调节的枢纽。我们的数据表明 USP30 具有控制作用 通过从易位子中去除 Ub 来激活 PARKIN 的阈值，也可能具有先前的 易位子本身在进口质量控制中的作用未被重视。在目标 2 中，我们将系统地考察 易位子成分及其泛素化在通过 Ub 设置 PARKIN 激活阈值中的作用 磷酸化。与此同时，我们将阐明 USP30 如何在这一新认可的进口质量中发挥作用 用于从易位子输入底物中去除 Ub 链的控制 (IQC) 途径。最后，我们的工作导致 使用单粒子电子显微镜首次可视化 PINK1 与易位子的关系，以及 我们寻求进一步发展对这种复合物如何组装和形成的生化和结构理解 受监管。总之，这些重点机制研究集中在这些关键分子如何与 PARKIN 相交 系统将提供对线粒体质量控制的更深入的了解。