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主要针对清除或抑制有毒活性氧（ROS）的产生。然而，这些方法虽然部分成功，但将受益于对导致衰老相关的功能失调线粒体积累的其他机制的理解。一个关键的问题是如何维持/调节线粒体质量。我们（和其他人）最近表明，泛素/蛋白酶体系统控制线粒体的功能完整性。与此一致，我们的数据表明，线粒体外膜的蛋白质被泛素偶联修饰，它们的降解是由蛋白酶体介导的。此外，我们还发现AAA- atp酶p97是内质网相关降解（ERAD）途径的关键蛋白，对于调节线粒体外膜蛋白泛素化依赖性的转换和自噬介导的功能受损线粒体的降解都是必不可少的。这些数据表明p97处于两个基本分解代谢途径的中心，1)泛素依赖性蛋白质降解和2)线粒体特异性自噬（mitophagy）。本文提出的研究将进一步仔细研究泛素/蛋白酶体系统和p97的机制和范围，特别是在线粒体稳态的调节中。将解决三个问题：1)p97在泛素依赖性线粒体蛋白降解中的机制和范围是什么？这些研究将包括表征与p97一起作用于线粒体的辅因子。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脱位。2)泛素/蛋白酶体系统和p97是否作为线粒体质量控制机制？p97在线粒体相关突变蛋白降解中的作用将被仔细研究。3) p97如何激活线粒体特异性自噬？实验将包括分析p97如何调节有丝分裂的不同模型。使用培养细胞和体外实验进行研究，同时使用延时方法和新的荧光工具进行成像研究，包括光激活荧光蛋白和美国最近开发的荧光泛素化链传感器。诱变和RNAi方法也将被开发。

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. PUBLIC HEALTH RELEVANCE: Dysfunctions of mitochondria are linked to numerous debilitating pathologies including Parkinson's disease (PD), Huntington's disease (HD), Alzheimer's disease (AD), amyotrophic lateral sclerosis, and diabetic complications. The goal is to define the role and mechanism of AAA-ATPase p97 with mitochondrial protein ubiquitination in the regulation of mitochondrial function and integration of mitochondrial quality control pathways. We believe that characterizing this process in its molecular detail will lead us to important insights into new aspects of mitochondrial biology, and ultimately to novel therapeutic targets for mitochondria dysfunction-linked diseases.