Systematic discovery and functional analysis of the PARKIN modified proteome

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，其激活通过降解和非降解机制导致蛋白质组的重新雕刻。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，该信号转导途径控制线粒体稳态，以应对线粒体损伤，例如去极化。这两种基因在早发性帕金森病（PD）中都会发生突变。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线粒体自噬促进受损线粒体的降解来实现的。PINK1是一种线粒体定位激酶，通过磷酸化依赖机制将PARKIN募集到线粒体外膜（MOM），这一机制在分子水平上知之甚少。一旦与MOM相关，PARKIN就会使几种MOM蛋白泛素化，包括有丝分裂蛋白和Miro gtpase，分别改变线粒体分裂融合周期和微管运输。在之前的融资周期中，我们已经开发了定量的diGLY捕获蛋白质组学，作为识别Ub系统靶点和精确阐明泛素化位点的一种手段。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，包括几十种蛋白质上的数百个泛素化位点，包括已知的和新的靶标。位于MOM上的许多候选PARKIN靶点在其细胞质表面泛素化，而其他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与一系列MOM蛋白的信号依赖性关联，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，但整合的目标，利用我们已经阐明的PARKIN靶点景观和几个蛋白质组学工具，允许定量解码信号机制。AIM 1旨在了解MOM上蛋白的位点特异性泛素化如何控制线粒体聚集和自噬体的招募。AIM 2旨在利用体内和体外系统阐明PARKIN激活的机制基础，这似乎是一个多步骤的机制，利用工程和患者衍生的突变，并利用蛋白质组学和遗传学方法发现PARKIN链连锁特异性多ub合成的功能基础。总之，这些研究将提供对PARKIN功能的分子机制和疾病突变如何影响线粒体稳态的更深入的理解。

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.