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: 9978888
• 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/9978888
• 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.

项目总结 人类线粒体复合体I(CI)由44个不同的亚基组成，它们组装在一起 由八个铁-S簇和一个黄素单核苷酸组成一个功能酶。辅助的 被称为组装因子的蛋白质帮助组装过程；以及十几个真正的CI 对组装因子(CIAF)进行了表征。然而，大约一半的CI障碍是无法追踪到的 与44个CI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 CI生物发生的调控因素仍有待研究。CI组装的一些监管机构可能不会直接 与44个CI亚基中的任何一个相互作用，而不是与CIAF相互作用，间接调节CI组装。 例如，它们可以调节稳定性、亚细胞定位、翻译后程度 CIAF的修改、激活程度等。我们将这类监管者称为远程监管者 CI组件的(RR)。我们推测，至少一些CI障碍可能归因于 RRS，其中许多尚未被发现。发现竞争情报RRS的理想模型系统 组装必须至少满足4个标准：(I)CI组装的机制应紧密模仿 人类的酶，(Ii)应高度富含线粒体，以便进行检查 1000个候选基因对CI组装的影响是相当容易的，(Iii)这样一种 生物体应该显著地进步到这样一个点，即数千个候选基因对 CI组装可以快速测试，最后(Iv)应该可以在体内分析CI组装 它同时受到发育和环境信号的影响，不容易进行细胞培养 手工艺品。目前还没有研究CI组装的模型系统(在粗糙脉孢菌和各种 哺乳动物细胞系)满足所有4个标准。为了便于发现CI组件的RR，我们 利用果蝇线粒体丰富的飞行肌肉作为研究CI组装的新系统 满足所有四个标准。我们发现果蝇骨骼肌中CI的生物发生是通过 ~315-、~370-、~550-和~815 kDa CI组装中间体的形成 而果蝇CI的亚基数量与人类酶的亚基数量相当。 重要的是，在人类中描述的CIAF的果蝇同源基因突变也损害了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鉴定CI的新的RR 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器。轻松自在 从飞行肌肉中分离大量的线粒体，广泛的遗传工具武器库 分析表明，果蝇相对较短的世代时间和有限的基因冗余是 应有助于发现CI组装的RR。