Characterization of effectors and ER in Drp1-mediated mitochondrial division

Drp1 介导的线粒体分裂中效应器和 ER 的表征

• 批准号: 8525242
• 负责人: Megan Wemmer
• 金额: $ 2.27万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2013-09-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8525242
• 关键词: Action Potentials; Address; Alzheimer' s Disease; Apoptosis; Apoptotic; Behavior; Binding; Biochemical; Biological Assay; Biology; Cell Culture Techniques; Cells; Complex; Coupled; Cultured Cells; Data; Diabetes Mellitus; Disease; Dynamin; Dynamin I; Electron Microscopy; Endoplasmic Reticulum; Event; Frequencies; Functional disorder; Genetic; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Health; Heart Diseases; Homeostasis; Hot Spot; Huntington Disease; Hydrolysis; Induction of Apoptosis; Learning; Link; Lipids; Mammalian Cell; Mammals; Maps; Mass Spectrum Analysis; Measures; Mediating; Membrane; Membrane Proteins; Methods; Microscopic; Mitochondria; Modeling; Monitor; Nature; Negative Staining; Nerve Degeneration; Orthologous Gene; Parkinson Disease; Pathogenesis; Pathologic; Pathway interactions; Phospholipids; Play; Post-Translational Protein Processing; Process; Production; Proteins; Proteomics; Recruitment Activity; Regulation; Research; Role; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Stress; Stroke; Structure; Techniques; Testing; Tissues; Training; Work; Yeasts; base; biological adaptation to stress; cell type; constriction; endoplasmic reticulum stress; human disease; in vitro Assay; insight; lipid transport; meetings; membrane assembly; mitochondrial membrane; prevent; public health relevance; response; therapy development; yeast two hybrid system

DESCRIPTION (provided by applicant): We propose a detailed analysis of the mechanisms regulating mitochondrial division dynamics. The core mitochondrial fission regulator, Drp1 is a dynamin-related GTPase that also plays an important regulatory role in intrinsic apoptosis. Drp1 assembles on the mitochondrial membrane to execute division; however the mechanisms regulating these events remain largely unknown. We are working to identify and characterize Drp1 effectors to determine how these proteins mediate Drp1 recruitment and assembly for execution of mitochondrial division (Aim 1). The recent observation that the endoplasmic reticulum (ER) contacts mitochondria, and these contacts are sites of mitochondrial constriction and division, suggests that the ER strongly influences mitochondrial dynamics. However the mechanisms that coordinate this process are uncharacterized. We will identify components of the ER-mitochondrial tethering complexes using mass spectrometry and proteomics approaches, and identify how these sites influence mitochondrial division dynamics (Aim 2). We will use established microscopic and cell-based approaches, as well as siRNA methods to study how effectors function in a pathway of mitochondrial division in cells, and in vitro assays to specifically address the mechanism by which effectors influence Drp1 assembly and GTP hydrolysis, and determine the structure of assembled Drp1-effector complexes using negative-stain electron microscopy. We will use candidate proteins that localize to ER-mitochondrial contacts to identify new tethering components, and characterize their influence on mitochondrial division dynamics. Given the fact that the ER influences mitochondrial division, we will study the relationship between the Drp1 division effectors and ER-mitochondrial tethering complexes in order to understand the mechanisms that regulate the site and execution of mitochondrial division. Both ER and mitochondria undergo dynamic remodeling under stressed conditions; we will apply what we learn regarding the regulation of mitochondrial division under healthy conditions to learn about the specific mechanisms that influence the remodeling that occurs in response to stress. It is likely that different combinations of effector proteins, as well as post-translational modifications, influence the rates and sites of mitochondrial division dynamics. In the long term we wish to understand how ER and mitochondria coordinate their behavior in healthy and pathologic conditions to regulate ER and mitochondrial function, distribution, and the induction of apoptosis in order to understand the impact of these processes on cellular health. Understanding the fundamental mechanism of mitochondrial division and its regulation is directly relevant to understanding the basis of an increasing number of diseases associated with ER stress and dysregulated mitochondrial division, such as Alzheimer's, Parkinson's and diabetes, heart disease and stroke, and will potentially allow for the development of therapies targeting mitochondrial division to treat a wide variety of human diseases.

描述（由申请人提供）：我们建议对调节线粒体分裂动力学的机制进行详细分析。 Drp1 是核心线粒体裂变调节因子，是一种动力相关 GTP 酶，在内在细胞凋亡中也发挥着重要的调节作用。 Drp1在线粒体膜上组装以执行分裂；然而，调节这些事件的机制仍然很大程度上未知。我们正在努力识别和表征 Drp1 效应子，以确定这些蛋白质如何介导 Drp1 招募和组装以执行线粒体分裂（目标 1）。最近观察到内质网（ER）与线粒体接触，而这些接触是线粒体收缩和分裂的部位，这表明内质网强烈影响线粒体动力学。然而，协调这一过程的机制尚不清楚。我们将使用质谱和蛋白质组学方法鉴定 ER 线粒体束缚复合物的成分，并确定这些位点如何影响线粒体分裂动力学（目标 2）。我们将使用已建立的微观和基于细胞的方法以及 siRNA 方法来研究效应器如何在细胞线粒体分裂途径中发挥作用，并使用体外测定来专门解决效应器影响 Drp1 组装和 GTP 水解的机制，并使用负染色电子显微镜确定组装的 Drp1-效应器复合物的结构。我们将使用定位于内质网线粒体接触的候选蛋白来识别新的束缚成分，并表征它们对线粒体分裂动力学的影响。鉴于内质网影响线粒体分裂，我们将研究 Drp1 分裂效应子和内质网线粒体束缚复合物之间的关系，以了解调节线粒体分裂位点和执行的机制。内质网和线粒体在应激条件下都会经历动态重塑；我们将应用我们在健康条件下调节线粒体分裂的知识来了解影响应激反应中发生的重塑的具体机制。效应蛋白的不同组合以及翻译后修饰可能会影响线粒体分裂动力学的速率和位点。从长远来看，我们希望了解内质网和线粒体在健康和病理条件下如何协调它们的行为，以调节内质网和线粒体的功能、分布和诱导细胞凋亡，从而了解这些过程对细胞健康的影响。了解线粒体分裂的基本机制及其调节与了解越来越多与内质网应激和线粒体分裂失调相关的疾病（如阿尔茨海默病、帕金森病、糖尿病、心脏病和中风）的基础直接相关，并且有可能开发针对线粒体分裂的疗法来治疗多种人类疾病。