Control of mitochondrial proteostasis by AAA-ATPase p97

描述(由申请人提供)：线粒体功能障碍是与大量人类衰老相关疾病相关的基本问题。针对线粒体功能障碍的各种保护策略大多旨在清除或抑制有毒活性氧物种(ROS)的产生。然而，这些方法虽然部分成功，但将受益于对导致与衰老相关的功能障碍线粒体积累的其他机制的理解。一个关键的问题是线粒体质量是如何维持/调节的。我们(和其他人)最近表明，泛素/蛋白酶体系统控制着线粒体的功能完整性。与此一致，我们的研究结果表明，线粒体膜外膜的蛋白质被泛素偶联修饰，其降解是由蛋白酶体介导的。此外，我们还发现，AAA-ATPase p97是内质网相关降解(ERAD)途径的关键蛋白，对于调节依赖泛素化的外膜蛋白的周转和自噬介导的功能受损线粒体的降解都是必不可少的。这些数据将p97置于两条基本分解代谢途径的中心，i)泛素依赖的蛋白质降解和ii)线粒体特异性自噬(有丝分裂)。这里提出的研究将进一步仔细审查泛素/蛋白酶体系统和p97的机制和范围，特别是在调节线粒体内稳态方面。将解决三个问题：1)p97在泛素依赖的线粒体蛋白降解中的机制和范围是什么？这些研究将包括与线粒体上的p97一起作用的辅因子的特征。我们还将询问非OMM线粒体蛋白是否以p97依赖的方式从线粒体移位。2)泛素/蛋白酶体系统和p97是否作为线粒体的质量控制机制？将仔细研究p97在线粒体相关突变蛋白降解中的作用。3)p97是如何激活线粒体特异性自噬的？这些实验将包括对p97如何调控有丝分裂的不同模型的分析。将进行使用培养细胞和体外分析的研究，以及使用时间推移方法和新的荧光工具的成像研究，包括光激活荧光蛋白和最近由美国荧光泛素化链传感器开发的。还将利用诱变和RNAi方法。

DESCRIPTION (provided by applicant): Mitochondrial dysfunction is a fundamental problem associated with a significant number of human aging-associated diseases. Various protective strategies targeting mitochondrial dysfunctions are aimed mostly at scavenging or inhibiting generation of toxic reactive oxygen species (ROS). However, these approaches, while partly successful, would benefit from an understanding of other mechanisms leading to aging-related accumulation of dysfunctional mitochondria. A critical question is how mitochondrial quality is maintained/ regulated. We (and others) have recently shown that the ubiquitin/proteasome system controls the functional integrity of the mitochondria. Consistent with this, our data demonstrate that proteins of the outer mitochondrial membrane are modified by ubiquitin conjugation, and their degradation is mediated by the proteasome. Furthermore, we also found that AAA- ATPase p97, a critical protein for the endoplasmic reticulum associated degradation (ERAD) pathway, is essential for both regulation of ubiquitination-dependent turnover of the outer mitochondrial membrane proteins and for autophagy-mediated degradation of functionally compromised mitochondria. These data place p97 in the center of two fundamental catabolic pathways, i) ubiquitin-dependent protein degradation and ii) mitochondria-specific autophagy (mitophagy). Studies proposed here will further scrutinize the mechanisms and scope of the ubiquitin/proteasome system and p97, in particular, in the regulation of mitochondrial homeostasis. Three questions will be addressed: 1) what is the mechanism and scope of p97 in ubiquitin-dependent mitochondrial protein degradation? These studies will include characterization of cofactors acting together with p97 on the mitochondria. We will also ask whether non-OMM mitochondrial proteins are dislocated from the mitochondria in p97-dependent manner. 2) Do ubiquitin/proteasome system and p97 serve as a mitochondrial quality control mechanism? The role for p97 in degradation of mitochondria-associated mutant proteins will be scrutinized. 3) How does p97 activate mitochondrial specific autophagy? The experiments will include analyses of distinct models of how p97 regulates mitophagy. Studies using cultured cells and in vitro assays will be carried out, along with imaging investigations using time- lapse methods and new fluorescent tools, including photoactivated fluorescent proteins and recently developed by us fluorescent ubiquitination chain sensors. Mutagenesis and RNAi methods will also be exploited.