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

基本信息

批准号：
9104225
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
项目类别：
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/9104225
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 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 Work base cohort density early onset genetic approach imaging platform in vivo multicatalytic endopeptidase complex mutant novel parkin gene/protein protein degradation receptor research study response sensor tool trafficking ubiquitin ligase

项目摘要

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）连接酶是动态信号系统的组成部分，其激活导致通过降解和非降解机制对蛋白质组进行重新雕刻。 UB连接酶Parkin及其上游调节激酶PINK1是信号转导途径的关键组成部分，该途径可控制线粒体稳态以响应线粒体损伤，例如通过去极化。这两个基因在早期发作帕金森氏病（PD）中都被突变。线粒体质量控制通过该途径部分通过改变线粒体动力学并通过线粒体促进损坏的线粒体降解而发生。 PINK1是一种线粒体局部局部的激酶，是通过在分子水平上熟悉的磷酸化依赖机制募集Parkin到线粒体外膜（MOM）所必需的。一旦与妈妈相关，Parkin就会被泛素盐水泛滥，包括丝线脂蛋白和miro gtpases，分别改变了线粒体裂变融合周期和对微管上的运输。在上一个融资周期中，我们开发了定量挖掘蛋白质组学，作为识别UB系统目标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，这些研究揭示了Parkin修饰的蛋白质组，包括数百个蛋白质上的泛素化位点，包括已知和新靶标。位于妈妈身上的许多候选帕克蛋白靶标在其细胞质面上泛素化，而其他帕金靶标似乎主要是细胞质。平行相互作用的蛋白质组学和体内功能研究表明，帕金蛋白与同类MOM蛋白的信号依赖性相关性，其方式取决于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框架，用于理解帕金控制线粒体命运的机制以及损害激活parkin活性的机制。在此续约中，我们提出了两个主题但综合的目标，以利用我们已经阐明的Parkin目标景观和几种允许对信号机制进行定量解码的蛋白质组学工具。 AIM 1试图了解蛋白质在MOM控制线粒体聚类和募集自噬体上的特定位点特异性泛素化。 AIM 2试图利用体内和体外系统，通过使用蛋白质组学和遗传学方法，使用工程和患者衍生的突变，使用工程和患者衍生的突变来阐明帕克蛋白激活的机理基础，并使用工程和患者衍生的突变，并发现链球蛋白通过parkin通过parkin进行链条链接特定的多重AB合成的功能基础。总之，这些研究将对帕金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。