Systematic discovery and functional analysis of the PARKIN modified proteome

PARKIN修饰蛋白质组的系统发现和功能分析

• 批准号: 8629086
• 负责人: JEFFREY W HARPER
• 金额: $ 42.38万
• 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 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8629086
• 关键词: Active Sites; Address; Affect; Architecture; Autophagocytosis; Autophagosome; Beryllium; Biochemical; Biological Assay; CUL3 gene; Cells; Complex; Cytoplasm; Cytoplasmic Protein; DNA Damage; Data; Defect; Disease; Elements; Engineering; Event; Face; Funding; Genes; Genetic; Goals; Grant; Guanosine Triphosphate Phosphohydrolases; Health; Homeostasis; Image; In Vitro; Inherited; Measures; Membrane; Membrane Proteins; Metabolic; Methods; Microtubules; Mitochondria; Modeling; Molecular; Monitor; Mutate; Mutation; Neurons; Outer Mitochondrial Membrane; PARK2 protein; PARK6 gene; PINK1 gene; Parkinson Disease; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Play; Polyubiquitin; Population; Process; Production; Protein Kinase; Proteins; Proteome; Proteomics; Quality Control; RNA Interference; Reaction; Resolution; Role; Series; Side; Signal Transduction; Signal Transduction Pathway; Site; Specificity; Substrate Specificity; System; Technology; Testing; Transducers; Ubiquitin; Ubiquitin Ligase Gene; Work; base; cohort; density; early onset; in vivo; multicatalytic endopeptidase complex; mutant; novel; parkin gene/protein; protein degradation; public health relevance; receptor; research study; response; sensor; tool; trafficking; ubiquitin ligase

Project Abstract Ubiquitin (Ub) ligases are components of dynamic signaling systems whose activation leads to re-sculpting of the proteome through degradative and non-degradative mechanisms. The Ub ligase PARKIN and its upstream regulatory kinase PINK1 are key components of a signal transduction pathway that controls mitochondrial homeostasis in response to mitochondrial damage via, for example, depolarization. Both of these genes are mutated in early onset Parkinson's Disease (PD). Mitochondrial quality control via this pathway occurs, in part, by altering mitochondrial dynamics and by promoting the degradation of damaged mitochondria by mitophagy. PINK1, a mitochondrially localized kinase, is required for recruitment of PARKIN to the mitochondrial outer membrane (MOM) through a phosphorylation dependent mechanism that is poorly understood at the molecular level. Once associated with the MOM, PARKIN is known to ubiquitylate several MOM proteins including mitofusin and Miro GTPases to alter mitochondrial fission-fusion cycles and trafficking on microtubules, respectively. In the previous funding cycle, we have developed quantitative diGLY capture proteomics as a means by which to identify targets of the Ub system and precisely elucidate the sites of ubiquitylation. Using this method, we have performed a series of studies that have revealed the PARKIN-modified proteome, including hundreds of ubiquitylation sites on dozens of proteins, including known and novel targets. The many candidate PARKIN targets located on the MOM are ubiquitinated on their cytoplasmic face, while other PARKIN targets appear to be primarily cytoplasmic. Parallel interaction proteomic and in vivo functional studies revealed signal dependent association of PARKIN with a cohort of MOM proteins in a manner that depends upon the integrity of the active site of PARKIN. Thus, this work provides the first topological and molecular framework for understanding the mechanisms by which PARKIN controls mitochondrial fate and by which damage activates PARKIN activity. In this renewal, we propose two thematic, yet integrated, aims that exploit both the PARKIN target landscape we have elucidated and several proteomics tools that allow quantitative decoding of signaling mechanisms. AIM 1 seeks to understand how site-specific ubiquitylation of proteins on the MOM control mitochondrial clustering and recruitment to autophagosomes. AIM 2 seeks to employ in vivo and in vitro systems to elucidate the mechanistic basis for PARKIN activation through what appears to be a multi-step mechanism, using engineered and patient-derived mutations, and to discover the functional basis for chain-linkage specific poly-Ub synthesis by PARKIN using proteomic and genetic approaches. Together, these studies will provide a much deeper understanding of the molecular mechanisms underlying PARKIN function and how disease mutants affect mitochondrial homeostasis.

项目摘要 泛素 (Ub) 连接酶是动态信号系统的组成部分，其激活会导致信号重塑 通过降解和非降解机制的蛋白质组。 Ub连接酶PARKIN及其上游 调节激酶 PINK1 是控制线粒体的信号转导途径的关键组成部分 通过例如去极化来应对线粒体损伤的稳态。这两个基因都是 早发型帕金森病 (PD) 中发生突变。通过该途径进行的线粒体质量控制部分地发生， 通过改变线粒体动力学并通过线粒体自噬促进受损线粒体的降解。 PINK1 是一种线粒体定位激酶，是招募 PARKIN 至线粒体外层所必需的 膜（MOM）通过磷酸化依赖性机制，但在分子水平上人们对此知之甚少。 等级。一旦与 MOM 结合，PARKIN 就会泛素化多种 MOM 蛋白，包括 mitofusin 和 Miro GTPases 改变线粒体裂变融合周期和微管运输， 分别。在上一个融资周期中，我们开发了定量二甘氨酸捕获蛋白质组学作为 识别 Ub 系统靶标并精确阐明泛素化位点的方法。使用 通过这种方法，我们进行了一系列研究，揭示了 PARKIN 修饰的蛋白质组， 包括数十种蛋白质上的数百个泛素化位点，包括已知和新的靶标。许多 位于 MOM 上的候选 PARKIN 靶标在其细胞质表面被泛素化，而其他 PARKIN 靶标似乎主要是细胞质的。并行相互作用蛋白质组学和体内功能研究 揭示了 PARKIN 与一组 MOM 蛋白的信号依赖性关联，其方式取决于 取决于 PARKIN 活性位点的完整性。因此，这项工作提供了第一个拓扑和分子 了解 PARKIN 控制线粒体命运的机制的框架 损伤会激活 PARKIN 活性。在这次更新中，我们提出了两个综合性的主题目标： 我们已经阐明的 PARKIN 目标景观和一些允许定量分析的蛋白质组学工具 信号机制的解码。 AIM 1 旨在了解蛋白质的位点特异性泛素化如何 MOM 控制线粒体聚集和招募至自噬体。 AIM 2 寻求在体内应用 和体外系统来阐明 PARKIN 激活的机制基础 多步骤机制，使用工程突变和患者衍生的突变，并发现功能基础 PARKIN 使用蛋白质组学和遗传学方法进行链连接特异性多聚泛素合成。在一起，这些 研究将更深入地了解 PARKIN 功能的分子机制 以及疾病突变体如何影响线粒体稳态。