Proteomic analyses of Drosophila Parkinson's disease model

描述（由申请人提供）：编码泛素蛋白连接酶的parkin基因的功能丧失突变是早发性帕金森病的主要原因，越来越多的证据表明，parkin功能障碍也可能在晚发性典型帕金森病中起作用。为了探索帕金森氏病的生物学作用，以及帕金森氏病功能丧失导致神经退行性变的机制，我们最近通过对高度保守的果蝇帕金森氏病同源基因的突变失活，建立了一个帕金森病的果蝇模型。果蝇帕金突变体是半活的，表现出精子形成的晚期发育缺陷，飞行肌的广泛凋亡变性和中枢神经系统中多巴胺神经元亚群的变性。线粒体病理是帕金突变体组织变性的一个突出的早期特征，帕金的很大一部分定位于线粒体。根据这些发现，我们假设Parkin通过直接泛素化特定的线粒体目标来促进线粒体完整性。为了鉴定Parkin介导线粒体和组织完整性的底物，我们建议使用体内蛋白质组学方法鉴定Parkin结合成分和在Parkin突变体中显示泛素化降低的蛋白质。在这些研究中使用果蝇的一个优势是，我们的蛋白质组学研究结果可以在需要Parkin才能存活的组织中快速验证，包括多巴胺神经元。这项工作将阐明帕金森氏病的生物学作用，并将为未来帕金森氏病发病机制的假设驱动研究奠定基础。

DESCRIPTION (provided by applicant): Loss-of-function mutations of the parkin gene, which encodes a ubiquitin-protein ligase, are a major cause of early-onset Parkinson's disease, and increasing evidence suggests that parkin dysfunction may also play a role in late-onset typical Parkinson's disease. To explore the biological role of parkin, and the mechanism by which loss of parkin function results in neurodegeneration, we recently created a Drosophila model of Parkinson's disease through mutational inactivation of a highly-conserved Drosophila parkin ortholog. Drosophila parkin mutants are semi-viable and display a late developmental defect in spermatid formation, widespread apoptotic degeneration of flight muscle, and degeneration of a subset of dopamine neurons in the central nervous system. Mitochondrial pathology is a prominent and early characteristic of tissue degeneration in parkin mutants and a significant fraction of Parkin localizes to mitochondria. From these findings we hypothesize that Parkin promotes mitochondrial integrity by directly ubiquitinating particular mitochondrial targets. To identify the substrates of Parkin that mediate mitochondrial and tissue integrity we propose to use in vivo proteomic approaches to identify Parkin-binding components and proteins displaying reduced ubiquitination in parkin mutants. An advantage of using Drosophila for these studies is that findings from our proteomic studies can be rapidly validated in tissues that require Parkin for viability, including dopamine neurons. This work should clarify the biological role of Parkin and will serve as a foundation for future hypothesis-driven investigation of parkin pathogenesis.