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: 10213091
• 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/10213091
• 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; 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）由组装在一起的44个不同的亚基组成 具有八个Fe-S簇和一个单黄素单核苷酸，形成功能性酶。辅助 蛋白质称为组装因子有助于组装过程；还有十几个善意的CI 组装因子（CIAF）已被表征。但是，大约一半的CI疾病无法追踪 在44个CI亚基或已知组装因子中的任何一个中的突变，这表明额外 CI生物发生的调节剂仍有待表征。 CI组装的某些调节器可能不会直接 与44个CI亚基相互作用，而是与CIAF相互作用以间接调节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。我们将此类别的调节器称为远程监管机构 CI组装的（RRS）。我们假设至少某些CI疾病可能归因于 RR，其中许多尚未发现。发现CI RR的理想模型系统 组装将必须满足至少4个标准：（i）CI组装的机制应紧密模仿 人类酶的酶，（ii）应高度富集线粒体以实现检查 1000年代的候选基因对CI组装的影响很容易（iii）这种遗传工具试剂盒 有机体应显着前进到1000候选基因对 CI组装可以迅速进行测试，最后（IV）应该可以在体内分析CI组装 它受发展和环境信号的约束，不容易受到细胞培养 文物。目前没有用于研究CI组装的模型系统（在Neurospora Crassa和各种 哺乳动物细胞系）满足所有4个标准。为了促进发现CI组装RR，我们是 使用果蝇中富含线粒体的飞行肌肉作为研究CI组装的新型系统 满足所有四个标准。我们发现果蝇骨骼肌中的CI生物发生通过 如在 哺乳动物系统；果蝇CI具有相当数量的亚基与人类酶。 重要的是，人类描述的CIAF果蝇直系同源物的突变也损害了CI组装 在果蝇中，进一步表明CI组装的机理是人类和 果蝇。在这里，我们建议使用一种遗传和蛋白质组学方法来识别CI的新RR 在此系统中组装；并在果蝇和人类细胞中测试我们的候选调节剂。轻松 将大量的线粒体与飞行肌肉隔离，遗传的广泛工具 分析，相对较短的生成时间和果蝇中的基因冗余有限是资产 应该促进发现CI组装的RR。

项目成果

Mitochondria in epithelial ovarian carcinoma exhibit abnormal phenotypes and blunted associations with biobehavioral factors.

• DOI: 10.1038/s41598-021-89934-6
• 发表时间: 2021-06-02
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Bindra S;McGill MA;Triplett MK;Tyagi A;Thaker PH;Dahmoush L;Goodheart MJ;Ogden RT;Owusu-Ansah E;R Karan K;Cole S;Sood AK;Lutgendorf SK;Picard M
• 通讯作者: Picard M

The mitochondrial calcium uniporter of pulmonary type 2 cells determines severity of acute lung injury.

• DOI: 10.1038/s41467-022-33543-y
• 发表时间: 2022-10-03
• 期刊: Nature communications
• 影响因子: 16.6

Analyzing the integrity of oxidative phosphorylation complexes in Drosophila flight muscles.

• DOI: 10.1016/j.xpro.2021.101021
• 发表时间: 2021-12-17
• 期刊: STAR protocols
• 作者: Murari A;Owusu-Ansah E
• 通讯作者: Owusu-Ansah E

Dissecting the concordant and disparate roles of NDUFAF3 and NDUFAF4 in mitochondrial complex I biogenesis.

• DOI: 10.1016/j.isci.2021.102869
• 发表时间: 2021-08-20
• 期刊: iScience
• 影响因子: 5.8
• 作者: Murari A;Rhooms SK;Garcia C;Liu T;Li H;Mishra B;Deshong C;Owusu-Ansah E
• 通讯作者: Owusu-Ansah E

Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples.

• DOI: 10.1016/j.jbc.2021.101204
• 发表时间: 2021-10
• 期刊: The Journal of biological chemistry
• 作者: Ansari F;Yoval-Sánchez B;Niatsetskaya Z;Sosunov S;Stepanova A;Garcia C;Owusu-Ansah E;Ten V;Wittig I;Galkin A
• 通讯作者: Galkin A