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

 描述（由申请人提供）：最近对帕金森病因子PINK1和Parkin的研究表明，它们通过线粒体选择性自噬形式（称为线粒体自噬）在受损线粒体的降解中发挥关键作用。这一进展促进了对线粒体质量控制对人类健康重要性的更高认识，并导致对线粒体自噬的研究急剧增加。然而，线粒体自噬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，线粒体自噬占所有线粒体蛋白质降解的不到一半。这一出乎意料的发现表明，非线粒体吞噬降解过程是线粒体蛋白质质量控制的主要原因。我们假设从这个和其他的发现，线粒体驻留蛋白酶占大多数的蛋白质降解的质量控制，发生在线粒体。虽然对酵母中与多种细胞活性相关的线粒体ATP酶（AAA+）蛋白酶家族的遗传研究表明，它们在线粒体蛋白质质量控制中起重要作用，但很少有人知道它们的底物。此外，这些蛋白酶的后生动物对应物很少被研究，尽管事实上编码其中几种蛋白酶的基因突变会导致人类疾病。我们建议确定在体内基板的四个后生动物AAA+线粒体蛋白酶在果蝇中，通过使用稳定的同位素蛋白质组学方法比较的半衰期的线粒体蛋白质在野生型苍蝇与那些在苍蝇承担遗传干扰，针对这些蛋白酶。此外，我们将测试这些蛋白酶是否降解被两种经常引用的线粒体应激损伤的线粒体蛋白：氧化和蛋白质错误折叠。我们的工作将通过定义这些蛋白酶的生物学作用来推进对线粒体质量控制的基本理解，并将为研究编码这些蛋白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.