Genetic Analysis of the PINK1-Parkin Pathway

描述（由申请人提供）：在过去的几年中，帕金森病相关因子PINK1和Parkin的遗传和细胞生物学研究已经开始描述选择性检测和降解受损线粒体的机制。这项工作已经建立了一个模型，即PINK1，一种线粒体靶向丝氨酸/苏氨酸激酶，选择性地稳定在受损线粒体表面，在那里它招募Parkin，一种细胞质E3泛素连接酶。然后，Parkin使特定的线粒体蛋白泛素化，以分离受损的线粒体，并通过自噬促进其最终降解。虽然这项工作极大地促进了我们对受损线粒体检测和降解机制的理解，但许多关键问题仍未得到解答。特别是，调节PINK1和Parkin的稳定性、定位和活性的因素尚不清楚。此外，虽然已知分离受损线粒体所需的一些Parkin底物，但这些线粒体随后自噬降解所需的Parkin底物尚不清楚。最后，PINK1-Parkin通路是否也以不依赖自噬的方式影响线粒体完整性尚不清楚。为了解决这些问题和其他问题，我们正在果蝇中进行全面的遗传筛选，以确定PINK1-Parkin通路的新成分。我们建议的目标是确定我们屏幕上的一些因素是如何影响这一途径的。具体来说，我们将努力实现四个目标。首先，我们将验证在我们的筛选中发现的三个关键的线粒体生物发生促进因子是PINK1和Parkin调节的重要下游靶点的假设。其次，我们将验证我们筛选中发现的两种去泛素化酶通过直接作用于PINK1、Parkin或Parkin底物mitofusin、miro或PARIS来影响PINK1-Parkin途径的假设。第三，我们将测试在我们筛选中发现的两种线粒体蛋白酶通过促进其递送到基质降解来影响PINK1活性的模型。第四，我们将使用一种新的蛋白质组学方法来检测蛋白质周转，以及简单的细胞生物学方法来对我们收集的剩余修饰剂进行分类。从我们的研究中获得的见解应该与帕金森病的病因和治疗直接相关，以及与线粒体功能障碍有关的许多其他疾病。

DESCRIPTION (provided by applicant): Over the past several years, genetic and cell biological studies of the Parkinson's disease- related factors PINK1 and Parkin have begun to delineate the mechanisms by which damaged mitochondria are selectively detected and degraded. This work has led to the model that PINK1, a mitochondrially targeted serine/threonine kinase, is selectively stabilized on the surface of damaged mitochondria where it recruits Parkin, a cytosolic E3 ubiquitin ligase. Parkin then ubiquitinates particular mitochondrial proteins to isolate the damaged mitochondria, and to promote their eventual degradation through autophagy. While this work has tremendously advanced our understanding of the mechanisms by which damaged mitochondria are detected and degraded, many critical questions remain unanswered. In particular, the factors that regulate the stability, localization and activity of PINK1 and Parkin are poorly understood. Additionally, while some of the Parkin substrates required to isolate damaged mitochondria are known, the Parkin substrates required for the subsequent autophagic degradation of these mitochondria are unknown. Finally, whether the PINK1-Parkin pathway also acts in an autophagy-independent manner to influence mitochondrial integrity is unclear. To address these and other matters, we are performing a comprehensive genetic screen in Drosophila to identify novel components of the PINK1-Parkin pathway. The goal of our proposal is to determine how some of the factors from our screen influence this pathway. Specifically, we will pursue four Aims. First, we will test the hypothesis that three key mitochondrial biogenesis-promoting factors identified in our screen are important downstream targets of regulation by PINK1 and Parkin. Second, we will test the hypothesis that two deubiquitinating enzymes identified in our screen influence the PINK1-Parkin pathway by acting directly on PINK1, Parkin, or the Parkin substrates mitofusin, miro, or PARIS. Third, we will test the model that two mitochondrial proteases identified in our screen influence the activit of PINK1 by promoting its delivery to the matrix for degradation. Fourth, we will use a novel proteomic assay of protein turnover, and simple cell biological assays to categorize the remaining modifiers in our collection. Insight from our studies should be directly relevant to the etiology and treatment of Parkinson's disease, as well as the many other diseases in which mitochondrial dysfunction is implicated.