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: 8742018
• 负责人: JEFFREY W HARPER
• 金额: $ 41.95万
• 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 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8742018
• 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

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：泛素（Ub）连接酶是动态信号传导系统的组分，其激活通过降解和非降解机制导致蛋白质组的重新塑造。Ub连接酶PARKIN及其上游调节激酶PINK 1是信号转导途径的关键组分，该信号转导途径通过例如去极化响应线粒体损伤来控制线粒体稳态。这两种基因在早发性帕金森病（PD）中都发生了突变。通过这一途径的线粒体质量控制部分地通过改变线粒体动力学和通过促进线粒体自噬降解受损的线粒体而发生。PINK 1是一种位于脑内的激酶，通过磷酸化依赖性机制将PARKIN募集到线粒体外膜（线粒体）是必需的，但在分子水平上了解甚少。PARKIN一旦与线粒体结合，已知可使几种线粒体蛋白泛素化，包括线粒体融合蛋白和Miro GTP酶，分别改变线粒体分裂-融合周期和微管运输。在上一个资助周期中，我们已经开发了定量的diGLY捕获蛋白质组学，作为识别Ub系统靶点并精确阐明泛素化位点的一种手段。使用这种方法，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遍在化位点，其中包括已知和新型靶点。位于胞浆的许多候选PARKIN靶点在其胞质表面被泛素化，而其他PARKIN靶点似乎主要在胞质。平行相互作用蛋白质组学和体内功能研究揭示了PARKIN与一组P2P蛋白的信号依赖性缔合，其方式取决于PARKIN活性位点的完整性。因此，这项工作为理解PARKIN控制线粒体命运和损伤激活PARKIN活性的机制提供了第一个拓扑和分子框架。在这次更新中，我们提出了两个主题，但综合的目标，利用我们已经阐明的PARKIN目标景观和几个蛋白质组学工具，允许定量解码的信号传导机制。目的1试图了解如何在特定位点的泛素化的蛋白质的控制线粒体集群和招聘的自噬体。目的2旨在采用体内和体外系统，通过似乎是一个多步骤的机制，使用工程和患者衍生的突变，阐明PARKIN激活的机制基础，并发现链连接特异性聚Ub合成PARKIN使用蛋白质组学和遗传学方法的功能基础。总之，这些研究将为PARKIN功能的分子机制以及疾病突变体如何影响线粒体稳态提供更深入的了解。