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.

项目摘要 在骨骼肌中的Oxphos组装调节 骨骼肌肉是迄今为止动物中最大的器官，约占体重的一半 人类和多达75％的绝缘体重。为了提供收缩所需的能量 肌肉，骨骼肌倾向于高度富集线粒体。根据线粒体疾病 经常出现肌病作为突出的临床特征。而负责增加的因素 在肌发生过程中的总体线粒体质量已经充分表征，对这种情况相对较少了解 有助于组装肌肉中氧化物磷酸化（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该机制 各种蛋白质调节骨骼肌中的Oxphos组装。凋亡诱导因子（AIF）是一种核 编码的氧化氧化还原酶在很大程度上定位于线粒体膜间空间。 AIF中的突变 引起OXPHOS系统的重大改变，并与人类的肌肉萎缩有关。但是， AIF发挥其生物能功能的精确机制尚未解决。数量上升 致病性AIF变体强调了AIF在人类病理生理学中的重要性，并已寻求 治疗方案很困难，因为这是AIF突变高度效应性质的主要原因。所以， 阐明AIF调节肌肉中的Oxphos组装的机制很重要，并且是至关重要的 未满足的需求，因为它将允许开发利用各种功能属性的理论策略 AIF治疗肌肉中蛋白质的特定病理突变。根据，我们已经建立了 可用于研究AIF在果蝇飞行肌肉中的功能的遗传处理系统。根据我们的发现 在本提案中的其他地方讨论了，我们提出了要测试的以下中心假设：AIF是一个 关键信号集线器通过稳定线粒体来调节OXPHOS组装的效果 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。我们将通过三个特定目标检验我们的假设。首先，我们将通过 通过MIB超级复合物（AIM 1）调节OXPHOS生物发生，并阐明ROS信号如何 影响AIF生物能表型（AIM 2）。最后，我们将定义并在功能上表征 AIF Interactome（AIM 3）。我们将使用蓝色天然聚丙烯酰胺凝胶电泳（BN-page），凝胶 Oxphos活性评估，蛋白质印迹，RNA序列，遗传学，透射电子显微镜和一系列 生理学和细胞生物学解决这些问题。总之，我们设想隔离的便利性 果蝇飞行肌肉的大量线粒体，广泛的遗传分析工具， 相关的短生成时间和果蝇中的基因冗余有限是应有的资产 可行的，以阐明AIF调节OXPHOS组装的机制。