Regulation of OXPHOS Assembly in Skeletal Muscles

项目总结 骨骼肌中OXPHOS组装的调控 骨骼肌肉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%的昆虫的身体质量。为了提供收缩所需的能量 肌肉、骨骼肌往往高度富含线粒体。相应地，线粒体疾病 常以肌病为主要临床表现。而导致增长的因素 整体上，线粒体质量在肌肉发生过程中已经有了很好的表征，相对较少的人知道 帮助组装肌肉中氧化磷酸化(OXPHOS)复合体的特定因素。这个 我的研究组的广泛和长期的目标是发现和阐明(S) 不同的蛋白质调节骨骼肌中OXPHOS的组装。细胞凋亡诱导因子(AIF)是一种核转录因子。 编码的氧化还原酶，主要定位于线粒体膜间间隙。AIF中的突变 会导致OXPHOS系统的重大变化，并与人类肌肉萎缩有关。然而， AIF发挥其生物能量学功能的确切机制尚未解决。越来越多的人 致病的AIF变异体强调了AIF在人类病理生理学中的重要性，并提出了寻求 治疗选择困难，因为这是AIF突变具有高度多效性的主要原因。因此， 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 未得到满足的需求，因为它将允许开发利用各种功能特性的治疗策略 AIF用于治疗肌肉中蛋白质的特定病理突变。因此，我们建立了一个 研究果蝇飞行肌肉中AIF功能的遗传易操作系统。根据我们的发现 在本提案的其他部分讨论过，我们提出了以下要检验的中心假设：AIF是一个 通过稳定线粒体来调节OXPHOS组装的关键信号枢纽 膜间空间桥接(MIB)超复合体、活性氧(ROS)的形成和相互作用 与其他蛋白质结合。我们将通过三个具体目标来检验我们的假设。首先，我们将通过以下方式剖析这一机制 哪种AIF通过MIB超复合体(AIM 1)调节OXPHOS的生物发生，并阐明ROS信号转导 对AIF生物能量表型的影响(目标2)。最后，我们将定义和描述 AIF互动组(AIM 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活性分析、Western blotts、RNA-Seq、遗传学、透射电子显微镜和一系列 生理学和细胞生物学分析可以解决这些问题。总而言之，我们设想隔离的简便性 来自果蝇飞行肌肉的大量线粒体，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它的优势所在 说明AIF调控OXPHOS组装的机制是可行的。

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.