Regulation of PINK1 and PARKIN-dependent mitophagy

PINK1 和 PARKIN 依赖性线粒体自噬的调节

• 批准号: 10212467
• 负责人: JEFFREY W HARPER
• 金额: $ 43.94万
• 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/10212467
• 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; Gap Junctions; 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; Structure; 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，包括我们实验室的重要工作，揭示了基本机制 蛋白质由两个帕金森氏病（PD）基因编码 - Pink1蛋白激酶和帕克蛋白 泛素（UB）连接酶 - 促进对线粒体受损的泛素化和自噬捕获以促进 它们通过线粒体清除。最近，我们将一个定量蛋白质组学平台与干细胞合并 衍生的，诱导的神经元（无神经元）具有途径突变以阐明帕金和粉红色的泛素化 靶标在内源性条件下，并确定了线粒体去偶联性地板USP30的作用 以及Parkin和线粒体通量调节中的P97分离酶。然而，我们对在多大程度上的理解 PD中突变的其他蛋白质与Parkin-Pink1系统合作，为疾病病因做出贡献 仍然有限 是机械控制的。在这里，我们提出了一系列实验，以解决这两种知识 空白。首先，我们最近从我们最近的线粒体通量CRISPR屏幕中出现的最引人注目的基因之一是 FBXO7，一种在PD中突变（PARK15）突变的基因和形成SCF UB的F-box家族的成员 连接酶。 FBXO7的关键功能和目标以及其突变如何易于PD。 通过相互作用蛋白质组学，我们发现FBXO7与多个调节组件相关联 蛋白酶体和提议FBXO7可能通过整合线粒体和蛋白酶体控制起着核心作用 支持细胞器稳态的机制。在AIM 1中，我们将使用我们的Ineuron系统检查FBXO7 使用一系列定量测定法检查途径中的顺序步骤，在线粒体通量中的作用， 我们将普遍且功能地剖析泛素化目标和调节机制作为初始步骤 旨在了解FBXO7中的患者突变如何有助于PD。其次，我们的初步数据和 该领域的工作表明PINK1和USP30都与线粒体易位相关， 将易位放置在帕金法规的联系中。我们的数据表明，USP30在控制中起作用 帕金激活的阈值是通过从转运量中删除UB的阈值，并且可能具有以前的 在转运本身的进口质量控制中未欣赏的作用。在AIM 2中，我们将系统地检查 易位组件和泛素化在设定parkin通过UB的阈值中的作用 磷酸化。同时，我们将阐明USP30在这种新认识的进口质量中的功能 控制（IQC）途径，用于从转运导入底物中删除UB链的途径。最后，我们的工作导致了 使用单粒子电子显微镜与易位的pink1的第一个可视化 我们试图进一步对这种复合物的组装方式发展生化和结构性理解和 受监管。这些关键分子如何与Parkin相交的这些重点机械研究在一起 系统将对线粒体质量控制有更深入的了解。