Control of mitochondrial proteostasis by AAA-ATPase p97

AAA-ATPase p97 对线粒体蛋白质稳态的控制

• 批准号: 8342402
• 负责人: MARIUSZ KARBOWSKI
• 金额: $ 29.17万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8342402
• 关键词: Address; Aging; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Area; Autophagocytosis; Biochemical; Biochemical Genetics; Biological Assay; Biology; Cellular biology; Complications of Diabetes Mellitus; Cultured Cells; Data; Degradation Pathway; Disease; Endoplasmic Reticulum; Environment; Excision; Functional disorder; Generations; Goals; Homeostasis; Housekeeping; Human; Huntington Disease; Image; Investigation; Lead; Life; Link; Mammalian Cell; Mediating; Membrane Proteins; Methods; Mitochondria; Mitochondrial Membrane Protein; Mitochondrial Proteins; Modeling; Molecular; Mutagenesis; Outer Mitochondrial Membrane; Parkinson Disease; Pathology; Pathway interactions; Peripheral Nervous System Diseases; Process; Proteins; Quality Control; RNA Interference; Reactive Oxygen Species; Regulation; Research; Role; Scientist; Specificity; System; Testing; Ubiquitin; Ubiquitination; age related; cellular imaging; cofactor; design; gain of function; human disease; imaging modality; in vitro Assay; insight; instrumentation; mitochondrial dysfunction; multicatalytic endopeptidase complex; mutant; new therapeutic target; novel; p97 ATPase; protein degradation; protein misfolding; research study; sensor; time use; tool

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.

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旨在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，但将受益于对导致与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/蛋白酶体系统控制着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通过泛素缀合进行修饰，并且它们的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，对于调节泛素化依赖性线粒体外膜蛋白周转以及自噬介导的功能受损的降解至关重要。线粒体。这些数据将 p97 置于两个基本分解代谢途径的中心：i) 泛素依赖性蛋白质降解和 ii) 线粒体特异性自噬（线粒体自噬）。这里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？这些研究将包括与 p97 一起作用于线粒体的辅因子的表征。我们还将询问非 OMM 线粒体蛋白是否以 p97 依赖性方式从线粒体中脱位。 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受到仔细研究。 3）p97如何激活线粒体特异性自噬？实验将包括对 p97 如何调节线粒体自噬的不同模型的分析。将使用培养细胞和体外测定进行研究，同时使用延时方法和新荧光工具（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）进行成像研究。诱变和 RNAi 方法也将被利用。 公共健康相关性：线粒体功能障碍与许多使人衰弱的疾病有关，包括帕金森病 (PD)、亨廷顿病 (HD)、阿尔茨海默病 (AD)、肌萎缩侧索硬化症和糖尿病并发症。目标是明确 AAA-ATPase p97 与线粒体蛋白泛素化在线粒体功能调节和线粒体质量控制途径整合中的作用和机制。我们相信，从分子细节上表征这一过程将使我们对线粒体生物学的新方面产生重要见解，并最终找到线粒体功能障碍相关疾病的新治疗靶点。