Regulation of OXPHOS Assembly in Skeletal Muscles

骨骼肌中 OXPHOS 组装的调节

• 批准号: 10660712
• 负责人: Edward Owusu-Ansah
• 金额: $ 47.36万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10660712
• 关键词: Accounting; Address; Adhesions; Animals; Apoptosis Regulation Gene; Apoptotic; Behavioral; Bioenergetics; Biogenesis; Biological Assay; Body Weight; Cell Death; Cellular biology; Chest; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Crista ampullaris; Defect; Down-Regulation; Drosophila genus; Drosophila inturned protein; Drug Metabolic Detoxication; Electrons; Functional disorder; Gel; Generations; Genes; Genetic; Human; Immunoblot Analysis; Inhibition of Apoptosis; Insecta; Leg; Link; Longevity; Measurement; Mediating; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Molecular Chaperones; Muscle; Muscle Contraction; Muscular Atrophy; Mutation; Myopathy; Nature; Nuclear; Organ; Oxidation-Reduction; Oxidative Phosphorylation; Oxidative Regulation; Oxidative Stress Induction; Oxidoreductase; Pathogenicity; Pathologic; Peptide Hydrolases; Phenotype; Physiology; Polyacrylamide Gel Electrophoresis; Process; Property; Proteins; Proteomics; RNA Interference; Reactive Oxygen Species; Reiterated Genes; Research; Resolution; Role; Signal Transduction; Site; Skeletal Muscle; Sorting; System; Testing; Therapeutic; Time; Transgenic Organisms; Transmission Electron Microscopy; Variant; Western Blotting; apoptosis inducing factor; experimental study; genetic analysis; insight; knock-down; model organism; myogenesis; novel; novel therapeutics; proteostasis; respiratory; response; skeletal; therapeutic development; tool; transcriptome sequencing; transmission process

PROJECT SUMMARY Regulation of OXPHOS assembly in skeletal muscles The skeletal musculature is by far the largest organ in animals, accounting for about half the body weight of humans and up to 75% of the body mass of insects. In order to provide the energy required for contraction of muscles, skeletal muscles tend to be highly enriched with mitochondria. Accordingly, mitochondrial disorders frequently present with myopathy as a prominent clinical feature. While the factors responsible for increasing overall mitochondrial mass during myogenesis have been well-characterized, relatively little is known about the specific factors that assist with assembling the oxidative phosphorylation (OXPHOS) complexes in muscles. The broad and long-term objective of my research group is to discover and elucidate the mechanism(s) by which various proteins regulate OXPHOS assembly in skeletal muscles. Apoptosis Inducing Factor (AIF) is a nuclear- encoded oxidoreductase that is largely localized to the mitochondrial intermembrane space. Mutations in AIF cause major alterations in the OXPHOS system and is associated with muscle atrophy in humans. However, the precise mechanism by which AIF exerts its bioenergetics functions has not been resolved. The rising number of pathogenic AIF variants underscores the importance of AIF in human pathophysiology and has made seeking therapeutic options difficult, as it is a major reason for the highly pleiotropic nature of AIF mutations. Therefore, elucidating the mechanism by which AIF regulates OXPHOS assembly in muscles is significant, and is a crucial unmet need, as it will allow the development of therapeutic strategies that exploit various functional properties of AIF to treat specific pathological mutations of the protein in muscles. Accordingly, we have established a genetically tractable system for studying AIF’s function in Drosophila flight muscles. Based on our findings discussed elsewhere in this proposal, we have formulated the following central hypothesis to be tested: AIF is a key signaling hub that regulates OXPHOS assembly through its effect on stabilizing the mitochondrial intermembrane space bridging (MIB) supercomplex, reactive oxygen species (ROS) formation and interaction with other proteins. We will test our hypothesis via three specific aims. First, we will dissect the mechanism by which AIF regulates OXPHOS biogenesis via the MIB supercomplex (Aim 1) and elucidate how ROS signaling impinges on the AIF bioenergetics phenotypes (Aim 2). Finally, we will define and functionally characterize the AIF interactome (Aim 3). We will be using blue native polyacrylamide gel electrophoresis (BN-PAGE), in-gel OXPHOS activity assays, Western blots, RNA-seq, genetics, transmission electron microscopy, and a range of physiology and cell biology assays to address these questions. Altogether, we envisage that the ease of isolating copious amounts of mitochondria from Drosophila flight muscles, extensive arsenal of tools for genetic analyses, relatively short generation time, and limited gene redundancy in Drosophila are assets that should make it feasible to elucidate the mechanism by which AIF regulates OXPHOS assembly.

项目概要 骨骼肌中 OXPHOS 组装的调节 骨骼肌系统是迄今为止动物中最大的器官，约占动物体重的一半 人类和高达 75% 的昆虫体重。为了提供收缩所需的能量 肌肉、骨骼肌往往富含线粒体。因此，线粒体疾病 经常以肌病作为突出的临床特征。虽然导致增加的因素 肌生成过程中线粒体的总体质量已得到很好的表征，但人们对肌生成过程中线粒体质量的了解相对较少。 有助于在肌肉中组装氧化磷酸化 (OXPHOS) 复合物的特定因素。这 我的研究小组的广泛和长期目标是发现和阐明其机制 多种蛋白质调节骨骼肌中 OXPHOS 的组装。细胞凋亡诱导因子（AIF）是一种细胞凋亡诱导因子 编码的氧化还原酶主要位于线粒体膜间隙。 AIF 突变 引起 OXPHOS 系统的重大改变，并与人类肌肉萎缩有关。然而， AIF发挥其生物能学功能的确切机制尚未解决。数量不断增加 致病性 AIF 变异强调了 AIF 在人类病理生理学中的重要性，并促使人们寻求 治疗选择很困难，因为它是 AIF 突变高度多效性的主要原因。所以， 阐明 AIF 调节肌肉中 OXPHOS 组装的机制具有重要意义，也是至关重要的 未满足的需求，因为它将允许开发利用各种功能特性的治疗策略 AIF 治疗肌肉中蛋白质的特定病理突变。据此，我们设立了一个 用于研究果蝇飞行肌肉中 AIF 功能的遗传易处理系统。根据我们的调查结果 在本提案的其他部分讨论过的情况下，我们制定了以下待测试的中心假设：AIF 是一个 通过稳定线粒体的作用来调节 OXPHOS 组装的关键信号中枢 膜间空间桥接 (MIB) 超复合物、活性氧 (ROS) 形成和相互作用 与其他蛋白质。我们将通过三个具体目标来检验我们的假设。首先，我们将剖析该机制 AIF 通过 MIB 超级复合物调节 OXPHOS 生物发生（目标 1）并阐明 ROS 信号传导机制 影响 AIF 生物能学表型（目标 2）。最后，我们将定义并在功能上表征 AIF 相互作用组（目标 3）。我们将使用蓝色天然聚丙烯酰胺凝胶电泳 (BN-PAGE)、凝胶内 OXPHOS 活性测定、蛋白质印迹、RNA-seq、遗传学、透射电子显微镜以及一系列 生理学和细胞生物学测定可以解决这些问题。总而言之，我们预计隔离的容易程度 来自果蝇飞行肌肉的大量线粒体、用于遗传分析的大量工具、 相对较短的世代时间和果蝇有限的基因冗余是使其成为可能的资产 阐明 AIF 调节 OXPHOS 组装的机制是可行的。