A proteomic approach to identify substrates of the AAA+ mitochondrial proteases

 描述(由申请人提供)：最近对帕金森氏症因子PINK1和Parkin的研究表明，它们通过线粒体选择性的自噬形式(称为有丝分裂吞噬)在受损线粒体的降解过程中发挥关键作用。这一进展促进了人们对线粒体质量控制对人类健康重要性的更大认识，并导致了对有丝分裂吞噬的研究大幅增加。然而，有丝分裂只是线粒体质量控制的几种机制之一，我们最近的工作表明，在模式生物果蝇中，有丝分裂只占线粒体蛋白质降解的不到一半。这一意想不到的发现表明，非有丝分裂降解过程是线粒体蛋白质质量控制的主要原因。我们从这一发现和其他发现中推测，线粒体中驻留的蛋白水解酶是线粒体中蛋白质降解质量控制的主要原因。尽管对酵母中线粒体不同细胞活性相关蛋白(AAA+)家族的遗传研究表明，它们在线粒体蛋白质质量控制中起着重要作用，但对它们的底物知之甚少。此外，尽管编码其中几种酶的基因突变会导致人类疾病，但对这些酶的后生动物对应的研究很少。我们建议使用稳定的同位素蛋白质组学方法在果蝇体内鉴定四种后生动物AAA+线粒体蛋白的底物，以比较WT果蝇和那些承受针对这些酶的遗传扰动的果蝇线粒体蛋白的半衰期。此外，我们将测试这些蛋白酶是否会降解线粒体蛋白质，这些蛋白质是由两种经常被引用的线粒体压力破坏的：氧化和蛋白质错误折叠。我们的工作将通过确定这些酶的生物学作用来促进对线粒体质量控制的基本理解，并将为研究编码这些酶的基因突变导致疾病的机制提供基础。

 DESCRIPTION (provided by applicant): Recent studies of the Parkinson's disease factors PINK1 and Parkin indicate that they play a critical role in the degradation of damaged mitochondria through a mitochondrial selective form of autophagy, termed mitophagy. This advance has fostered a greater appreciation of the importance of mitochondrial quality control to human health, and has led to a dramatic increase in research on mitophagy. However, mitophagy is only one of several mechanisms of mitochondrial quality control, and our recent work indicates that mitophagy accounts for less than half of all mitochondrial protein degradation in the model organism Drosophila. This unexpected finding suggests that non- mitophagic degradative processes are primarily responsible for mitochondrial protein quality control. We hypothesize from this and other findings that mitochondrial-resident proteases account for the majority of the protein degradative quality control that occurs in mitochondria. While genetic studies of the mitochondrial ATPase Associated with diverse cellular Activities (AAA+) family of proteases in yeast suggest that they play an important role in mitochondrial protein quality control, few of their substrates are known. Furthermore, the metazoan counterparts of these proteases have been little studied, despite the fact that mutations in the genes encoding several of them cause human disease. We propose to identify in vivo substrates of the four metazoan AAA+ mitochondrial proteases in Drosophila by using a stable isotope proteomic method to compare the half-lives of mitochondrial proteins in WT flies with those in flies that bear genetic perturbations targeting these proteases. Moreover, we will test whether these proteases degrade mitochondrial proteins that are damaged by two oft-cited mitochondrial stresses: oxidation and protein misfolding. Our work will advance the basic understanding of mitochondrial quality control by defining the biological roles of these proteases, and will provide a foundation to study the mechanisms by which mutations in the genes encoding these proteases cause disease.