Systematic discovery and functional analysis of the PARKIN modified proteome

描述(申请人提供)：泛素(Ub)连接酶是动态信号系统的组成部分，它的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PINK1是信号转导通路的关键组成部分，该信号转导通路通过去极化等途径控制线粒体内稳态，以应对线粒体损伤。在早发性帕金森病(PD)中，这两个基因都发生了突变。通过这一途径进行的线粒体质量控制，部分是通过改变线粒体动力学和通过有丝分裂促进受损线粒体的降解来实现的。PINK1是一种线粒体定位的激酶，它通过一种在分子水平上鲜为人知的磷酸化依赖机制将Parkin重新募集到线粒体外膜(MOM)。一旦与MOM相关，已知Parkin泛素化几种MOM蛋白，包括丝裂霉素和Miro GTP酶，分别改变线粒体的分裂-融合周期和微管上的运输。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系统的靶点和精确阐明泛素化位点的手段。使用这种方法，我们已经进行了一系列的研究，揭示了Parkin修饰的蛋白质组，包括数十个蛋白质上的数百个泛素化位点，包括已知和新的靶点。位于MOM上的许多候选Parkin靶点在它们的细胞质表面泛素化，而其他Parkin靶点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表明，Parkin与一组MoM蛋白之间的信号依赖关系取决于Parkin活性部位的完整性。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靶点图景和几个蛋白质组学工具，允许定量解码信号机制。目的1试图了解MOM上蛋白质的位置特异性泛素化如何控制线粒体聚集和向自噬小体募集。目的2试图利用体内和体外系统，利用工程和患者来源的突变，通过似乎是多步骤的机制来阐明Parkin激活的机制基础，并使用蛋白质组学和遗传学方法发现Parkin合成链状特异性PolyUb的功能基础。总之，这些研究将提供对Parkin功能潜在的分子机制以及疾病突变如何影响线粒体动态平衡的更深入的理解。

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.