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发挥作用。为了探索parkin的生物学作用，以及parkin功能丧失导致神经退行性变的机制，我们最近通过高度保守的果蝇parkin直系同源物的突变失活创建了帕金森病的果蝇模型。果蝇帕金突变体是半可行的，并显示在精子细胞形成，广泛的凋亡变性的飞行肌肉，在中枢神经系统中的多巴胺神经元的一个子集的退化的晚期发育缺陷。线粒体病理学是帕金突变体中组织变性的一个突出和早期特征，并且帕金的显著部分定位于线粒体。根据这些发现，我们推测帕金通过直接泛素化特定的线粒体靶点来促进线粒体完整性。为了确定介导线粒体和组织完整性的帕金底物，我们建议使用体内蛋白质组学方法来鉴定帕金结合组分和帕金突变体中显示泛素化减少的蛋白质。使用果蝇进行这些研究的一个优点是，我们的蛋白质组学研究结果可以在需要Parkin生存能力的组织中快速验证，包括多巴胺神经元。这项工作应该澄清帕金的生物学作用，并将作为未来假设驱动的帕金发病机制的调查的基础。

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.