Identifying Novel Regulators of Mitochondrial Complex I Biogenesis

鉴定线粒体复合物 I 生物发生的新型调节因子

• 批准号: 9401087
• 负责人: Christian Joel Garcia
• 金额: $ 4.4万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9401087
• 关键词: Address; Biochemistry; Biogenesis; Biological Assay; Biological Models; Candidate Disease Gene; Cardiovascular Diseases; Catalysis; Cattle; Cell Culture Techniques; Cell Line; Cells; Chronic; Communities; Complex; Degenerative Disorder; Development; Disease; Drosophila genus; Electron Transport; Embryo; Enzymes; Flavin Mononucleotide; Gel; Generations; Genetic; Goals; Heart Mitochondria; Human; Hydrophobicity; Impairment; Knowledge; Leigh Disease; Link; Mammalian Cell; Mass Spectrum Analysis; Membrane; Membrane Potentials; Metabolic Diseases; Mitochondria; Mitochondrial Matrix; Mitochondrial Proteins; Modeling; Molecular; Molecular Chaperones; Morphologic artifacts; Muscle; Mutation; NADH; Neurospora crassa; Nuclear; Optic Atrophy; Organism; Orthologous Gene; Patients; Physiology; Play; Polyacrylamide Gel Electrophoresis; Process; Proteins; Proteomics; Reiterated Genes; Role; Signal Transduction; Silver Staining; Skeletal Muscle; Structure; System; Testing; Therapeutic; Time; Ubiquinone; Validation; Western Blotting; arm; combat; enzyme activity; genetic analysis; hydrophilicity; improved; in vivo; innovation; insight; kidney cell; novel; protein function; sarcopenia; tool

PROJECT SUMMARY 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. The ideal model system for discovering new regulators 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 novel regulators 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. Because NDUFS3 (one of the 44 CI subunits) has a central role in CI biogenesis we hypothesize that some regulators of CI assembly may directly interact with NDUFS3. Here, we propose to use a genetic and proteomic approach to identify novel regulators of CI assembly that interact with NDUFS3; 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 new regulators of CI assembly.

项目摘要 线粒体复合物I（mitochondrialComplex I，CI）由44个不同的亚基组成，它们与8个Fe-S结合在一起 簇和单一的黄素单核苷酸，形成一个功能酶。辅助蛋白质被称为 组装因素有助于组装过程;十几个真正的CI组装因素（CIAF） 被定性。然而，大约一半的CI障碍不能追溯到44个CI亚基中的任何一个中的突变。 或已知的组装因子，这表明CI生物发生的其他调节因子仍有待表征。 用于发现CI组件的新调节剂的理想模型系统将必须满足至少4个标准： CI组装的机制应该紧密地模仿人酶的机制，（ii）它应该高度富集有 线粒体，以使得能够相当容易地检查1000个候选基因对CI组装的影响，（iii） 这种生物体中的遗传工具包应该大大先进到这样一个程度，即1000个 可以快速测试CI组装上的候选基因，并且最后（iv）应该可以分析CI组装 在体内，它受到发育和环境信号的影响，并且不易于产生细胞培养物的假象。 目前用于研究CI组装（在粗糙脉孢菌和各种哺乳动物细胞中）的模型系统都没有 线）满足所有4个标准。为了促进发现CI组装的新调节子，我们使用了 果蝇中富含维生素A的飞行肌肉作为研究CI组装的新系统，因为它满足所有四个 的搜索.我们发现，果蝇骨骼肌中的CI生物合成通过~315-，~370-， ~550-和~815 kDa CI组装中间体，如哺乳动物系统中所述;和果蝇CI 与人类酶的亚基数量相当。重要的是，果蝇直系同源基因中的突变 在人类中描述的CIAF也损害果蝇中的CI组装，进一步表明CI的机制是不稳定的。 组装在人类和果蝇之间是保守的。因为NDUFS 3（44个CI亚基之一）具有一个 在CI生物发生中的中心作用，我们假设CI组装的一些调节因子可能直接与 NDUFS 3.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调控CI组装 与NDUFS 3相互作用;并在果蝇和人类细胞中测试我们的候选调节因子。的容易性 从飞行肌肉中分离出大量的线粒体，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促进基因组学的资产。 发现CI组装的新调节剂。