Systematic discovery and functional analysis of the PARKIN modified proteome

项目摘要 泛素（Ub）连接酶是动态信号传导系统的组分，其激活导致细胞内蛋白质的重新塑造。 蛋白质组通过降解和非降解机制。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成部分， 通过例如去极化来响应线粒体损伤的稳态。这两种基因都是 帕金森氏病（PD）的早期症状。线粒体质量控制通过这一途径发生，部分， 通过改变线粒体动力学和促进线粒体自噬引起的受损线粒体的降解。 PINK 1是一种脑内定位的激酶，是PARKIN募集到线粒体外膜所必需的。 膜（cytoplasmic membrane，cytoplasmic membrane）是通过磷酸化依赖性的机制，在分子水平上知之甚少。 水平PARKIN一旦与β-淀粉样蛋白结合，已知可使几种β-淀粉样蛋白泛素化，包括 线粒体融合素和Miro GTP酶改变线粒体分裂-融合周期和微管上的运输， 分别在上一个资助周期中，我们开发了定量的diGLY捕获蛋白质组学， 通过这种方法来识别Ub系统的靶点并精确阐明泛素化的位点。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，包括已知的和新的靶点。的许多 定位在细胞质表面的候选PARKIN靶点在其细胞质表面被泛素化，而其他靶点在其细胞质表面被泛素化。 PARKIN靶点似乎主要是细胞质。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cardiac蛋白的信号依赖性关联，其方式依赖于 对PARKIN活性位点的完整性的影响。因此，这项工作提供了第一个拓扑和分子 理解PARKIN控制线粒体命运的机制以及 损伤激活PARKIN活动。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目标景观和几种蛋白质组学工具， 信号机制的解码。AIM 1试图了解蛋白质的位点特异性泛素化是如何在细胞表面发生的。 MOM控制线粒体聚集和自噬体的招募。AIM 2寻求在体内使用 和体外系统，以阐明PARKIN激活的机制基础， 多步骤机制，使用工程和患者衍生的突变，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poly-Ub合成。所有这些 这些研究将为PARKIN功能的分子机制提供更深入的理解 以及疾病突变体如何影响线粒体内稳态。

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.