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: 8710273
• 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/8710273
• 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.

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是针对清除或抑制有毒活性氧（ROS）的产生。但是，这些方法虽然部分成功，但将从对导致与衰老相关的线粒体衰老积累的其他机制的理解中受益。 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。与此相一致，我们的数据表明，线粒体外膜的蛋白质通过泛素结合而修饰，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是内质网的关键蛋白相关的降解（ERAD）途径，对于调节泛素化依赖性依赖性转换的线粒体膜膜蛋白的泛素化周转至关重要。这些数据将p97置于两个基本分解代谢途径的中心，即ubiquitin依赖性蛋白质降解和ii）线粒体特异性自噬（Mitophagy）。这里提出的研究将进一步审查泛素/蛋白酶体系统和p97的机制和范围，尤其是在调节线粒体稳态时。将解决三个问题：1）在泛素依赖性线粒体蛋白降解中，p97的机理和范围是什么？这些研究将包括在线粒体上与P97一起作用的辅因子的表征。我们还将询问是否以p97依赖性方式将非点线粒体蛋白与线粒体脱位。 2）泛素/蛋白酶体系统和p97是否充当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检查。 3）P97如何激活线粒体特异性自噬？这些实验将包括对P97如何调节线粒体的不同模型的分析。使用培养细胞和体外测定的研究将进行，并使用延时方法和新的荧光工具（包括光活化的荧光蛋白）进行成像研究，并最近由美国荧光泛素化链传感器开发。诱变和RNAi方法也将被利用。