Identifying Remote Regulators of Complex I Biogenesis in Drosophila

果蝇复合体 I 生物发生的远程调节因子的鉴定

• 批准号: 9381113
• 负责人: Edward Owusu-Ansah
• 金额: $ 40万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-08 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9381113
• 关键词: Biogenesis; Biological Models; Candidate Disease Gene; Cardiovascular Diseases; Cell Culture Techniques; Cell Line; Cells; Chronic; Complex; Degenerative Disorder; Development; Disease; Drosophila genus; Enzymes; Flavin Mononucleotide; Generations; Genetic; Human; Impairment; Link; Mammalian Cell; Metabolic Diseases; Mitochondria; Morphologic artifacts; Muscle; Mutation; Neurospora crassa; Organism; Orthologous Gene; Post-Translational Protein Processing; Process; Proteins; Proteomics; Reiterated Genes; Signal Transduction; Skeletal Muscle; System; Testing; Therapeutic; Time; combat; genetic analysis; in vivo; novel; sarcopenia; tool

PROJECT SUMMARY Human Mitochondrial Complex I (CI) is composed of 44 distinct subunits that are assembled together with eight Fe-S clusters and a single flavin mononucleotide, to form a functioning enzyme. Ancillary proteins referred to as assembly factors assist with the assembly process; and a dozen or so bona fide CI assembly factors (CIAFs) have been characterized. However, about half of CI disorders cannot be traced to mutations in any of the 44 CI subunits or known assembly factors, which suggests that additional regulators of CI biogenesis remain to be characterized. Some regulators of CI assembly may not directly interact with any of the 44 CI subunits, but rather interact with CIAFs to regulate CI assembly indirectly. For instance, they may regulate the stability, subcellular localization, degree of post-translational modification, extent of activation, etc. of a CIAF. We refer to this class of regulators as remote regulators (RRs) of CI assembly. We hypothesize that at least some CI disorders may be attributed to mutations in RRs, many of which have not yet been discovered. The ideal model system for discovering RRs of CI assembly will have to satisfy at least 4 criteria: (i) the mechanism of CI assembly should closely mimic that of the human enzyme, (ii) it should be highly enriched with mitochondria to enable the examination of the effects of 1000s of candidate genes on CI assembly rather easily, (iii) the genetic tool kit in such an organism should be significantly advanced to the point where the effects of 1000s of candidate genes on CI assembly can rapidly be tested, and finally (iv) it should be possible to analyze CI assembly in vivo where it is subject to both developmental and environmental signals, and not prone to cell culture artifacts. None of the current model systems for studying CI assembly (in Neurospora crassa and various mammalian cell lines) satisfy all 4 criteria. To facilitate the discovery of RRs of CI assembly, we are using the mitochondria-enriched flight muscles in Drosophila as a novel system to study CI assembly as it satisfies all four criteria. We find that CI biogenesis in Drosophila skeletal muscles proceeds via the formation of ~315-, ~370-, ~550-, and ~815 kDa CI assembly intermediates as has been described in mammalian systems; and Drosophila CI has a comparable number of subunits as the human enzyme. Importantly, mutations in Drosophila orthologs of CIAFs described in humans, also impair CI assembly in Drosophila, further showing that the mechanism of CI assembly is conserved between humans and Drosophila. Here, we propose to use a genetic and proteomic approach to identify novel RRs of CI assembly in this system; and test our candidate regulators in both Drosophila and human cells. The ease of isolating copious amounts of mitochondria from flight muscles, extensive arsenal of tools for genetic analyses, relatively short generation time, and limited gene redundancy in Drosophila are assets that should facilitate the discovery of RRs of CI assembly.

项目摘要 人线粒体复合物I（CI）由44个不同的亚基组装在一起组成 八个Fe-S簇和一个黄素单核苷酸，形成一个功能酶。辅助 被称为组装因子的蛋白质有助于组装过程;还有十几种真正的CI 组装因子（CIAF）的特征。然而，大约一半的CI疾病无法追踪 与44个C1亚基或已知组装因子中的任何一个突变有关，这表明额外的C1亚基或已知组装因子可能与C1亚基的突变有关。 CI生物发生的调节剂仍有待表征。CI组件的一些调节器可能不直接 与44个C1亚基中的任何一个相互作用，而是与CIAF相互作用以间接调节C1组装。 例如，它们可以调节蛋白质的稳定性、亚细胞定位、翻译后表达的程度等。 CIAF的修饰、活化程度等。我们将这类调节器称为远程调节器 (RRs)CI大会。我们假设至少有一些CI障碍可能归因于基因突变， RRs，其中许多尚未被发现。发现竞争情报资源的理想模型系统 组装将必须满足至少4个标准：（i）CI组装的机制应密切模仿 （ii）它应该高度富集线粒体以使检查成为可能 1000个候选基因对CI组装的影响相当容易，（iii）在这样一个 生物体应该是显着先进的点，1000的候选基因的影响， 可以快速测试CI组装，并且最后（iv）应该可以在体内分析CI组装 在那里它受到发育和环境信号的影响，并且不倾向于细胞培养 藏物目前用于研究CI组装的模型系统（在粗糙脉孢菌（Neurospora crassa）和各种 哺乳动物细胞系）满足所有4个标准。为了便于发现CI组装的RR，我们 使用果蝇中富含神经元的飞行肌肉作为一种新的系统来研究CI组装， 满足所有四个标准。我们发现果蝇骨骼肌中的CI生物发生是通过 形成~315-、~370-、~550-和~815 kDa Cl组装中间体，如在 哺乳动物系统;果蝇CI具有与人类酶相当数量的亚基。 重要的是，在人类中描述的CIAF的果蝇直系同源物中的突变也损害了CI组装 在果蝇中，进一步表明CI组装的机制在人类和 果蝇在这里，我们建议使用遗传学和蛋白质组学的方法来确定新的RR CI 在这个系统中组装;并在果蝇和人类细胞中测试我们的候选调节子。的容易程度 从飞行肌肉中分离出大量的线粒体，这是遗传学研究的广泛工具。 分析，相对较短的世代时间和有限的基因冗余果蝇的资产， 应该有助于发现CI组装的RR。