Systematic discovery and functional analysis of the PARKIN modified proteome

项目摘要 泛素(Ub)连接酶是动态信号系统的组成部分，它的激活会导致 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PINK1是控制线粒体的信号转导途径的关键组成部分 例如，通过去极化反应线粒体损伤的动态平衡。这两个基因都 在早发性帕金森病(PD)中发生突变。通过这种途径进行线粒体质量控制，在一定程度上， 通过改变线粒体动力学和通过吞噬线粒体来促进受损线粒体的降解。 PINK1是一种线粒体定位的激酶，是Parkin募集到线粒体外部所必需的 膜(MOM)通过磷酸化依赖的机制，在分子上了解得很少 水平。一旦与MOM联系在一起，Parkin就被认为泛素化了几种MoM蛋白，包括 Mitofusin和Miro GTP酶改变线粒体分裂-融合周期和微管运输， 分别进行了分析。在之前的资金周期中，我们开发了定量的diGLY捕获蛋白质组学作为一种 识别Ub系统的靶标和精确阐明泛素化位点的手段。vbl.使用 通过这种方法，我们进行了一系列研究，揭示了帕金修饰的蛋白质组， 包括数十种蛋白质上的数百个泛素化位点，包括已知和新的靶点。众多人 位于MOM上的候选Parkin靶点在其细胞质表面泛素化，而其他 帕金靶标似乎主要是细胞质的。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MoM蛋白之间的信号依赖关联，这种关联依赖于 帕金活跃地点的完整性。因此，这项工作提供了第一个拓扑和分子 理解帕金控制线粒体命运的机制及其机制的框架 伤害会激活帕金的活动。在这次更新中，我们提出了两个主题但综合的目标，这两个目标利用 我们已经阐明的Parkin靶向和几种蛋白质组学工具都允许 信令机制的解码。目标1试图了解蛋白质的位点特异性泛素化是如何在 MOM控制线粒体的聚集和自噬小体的募集。AIM 2寻求在体内使用 以及体外系统，以阐明Parkin激活的机制基础，似乎是通过一种 多步骤机制，使用工程和患者衍生的突变，并发现 用蛋白质组学和遗传学方法由Parkin合成链状特异的多聚Ub。加在一起，这些 研究将对帕金森氏症的分子机制有更深入的了解 以及疾病突变如何影响线粒体的动态平衡。

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.