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 骨骼肌中OXPHOS组装的调节 骨骼肌肉系统是迄今为止动物中最大的器官，约占体重的一半。 人类和高达75%的昆虫体重。为了提供收缩所需的能量， 在肌肉中，骨骼肌往往高度富含线粒体。因此，线粒体疾病 经常以肌病作为突出的临床特征出现。虽然造成人口增长的因素 在肌发生过程中的整体线粒体质量已经得到了很好的表征，但对线粒体的结构和功能知之甚少。 特定的因子，有助于组装氧化磷酸化（OXPHOS）复合物在肌肉中。的 我的研究小组的一个广泛和长期的目标是发现和阐明机制， 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- 编码的氧化还原酶主要定位于线粒体膜间隙。AIF突变 引起OXPHOS系统的重大改变，并与人类肌肉萎缩有关。但 AIF发挥其生物能量学功能的精确机制尚未解决。越来越多的 致病性AIF变体强调了AIF在人类病理生理学中的重要性， 治疗选择困难，因为这是AIF突变高度多效性的主要原因。因此，我们认为， 阐明AIF调节肌肉中OXPHOS组装的机制是重要的，并且是一个关键的 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。因此，我们建立了一个 遗传学上易于处理的系统，用于研究AIF在果蝇飞行肌肉中的功能。根据我们的调查结果 在本建议的其他地方讨论，我们制定了以下中心假设进行测试：AIF是一个 通过其对稳定线粒体的作用调节OXPHOS组装的关键信号中枢 膜间空间桥（MIB）超复合物，活性氧（ROS）的形成和相互作用 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超复合物（Aim 1）调节OXPHOS生物合成，并阐明ROS信号转导 影响AIF生物能量表型（Aim 2）。最后，我们将定义和功能特性， AIF相互作用组（Aim 3）。我们将使用蓝色非变性聚丙烯酰胺凝胶电泳（BN-PAGE）， OXPHOS活性测定、蛋白质印迹、RNA-seq、遗传学、透射电子显微镜和一系列的免疫组织化学方法。 生理学和细胞生物学分析来解决这些问题。总而言之，我们设想， 大量的果蝇飞行肌线粒体，大量的遗传分析工具， 果蝇相对较短的世代时间和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。