Understanding the Mechanisms of Respiratory Supercomplexes and mitochondrial Complex I

了解呼吸超级复合物和线粒体复合物 I 的机制

• 批准号: 10405545
• 负责人: James Anthony Letts
• 金额: $ 35.85万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10405545
• 关键词: 10 year old; Address; Basic Science; Biochemical; Bioenergetics; Biological Models; Cell Culture Techniques; Complex; Cryoelectron Microscopy; Defect; Diagnosis; Disease; Electron Transport; Enzymes; Event; Family suidae; Foundations; Future; Genetic; Goals; Heart; Heart Mitochondria; Hela Cells; Human; Individual; Life; Medical; Membrane Proteins; Metabolic; Metabolic Diseases; Metabolic Pathway; Metabolism; Mitochondria; Molecular; Neurospora crassa; Organism; Oxidative Phosphorylation; Paracoccus denitrificans; Patients; Pharmacotherapy; Physiological; Production; Proteobacteria; Regulation; Research; Resolution; Respiration; Role; Structure; System; Testing; Tissues; Work; drug development; effective therapy; insight; particle; protein complex; respiratory; stem; therapeutic development; treatment strategy

PROJECT SUMMARY/ABSTRACT The mitochondrial oxidative phosphorylation electron transport chain (ETC) is composed of five large membrane protein complexes (CI, CII, CIII2, CIV and CV) and is responsible for the production of the majority of cellular ATP. Consequently, the ETC is essential to bioenergetic metabolism. ETC defects are one of the most commonly diagnosed congenital metabolic defects, with CI deficiencies representing roughly a third of these diagnoses. Although ~50% of patients with CI deficiencies die within the first 2 years of life and only ~25% reach 10 years of age, CI remains the least well mechanistically understood of all the ETC complexes. Furthermore, despite the large medical need, there are currently no effective treatments for CI or other ETC deficiencies. This discrepancy stems in part from an incomplete understanding of the molecular mechanisms of the individual complexes and their higher-order assemblies into supercomplexes (SCs). In mammalian heart mitochondria the majority of CI is found in association with CIII2 and CIV (SC I+III2+IV, the respirasome) or in association with CIII2 (SC I+III2). Recent biochemical and structural work has produced the first atomic- resolution structures of mammalian mitochondrial CI and defined the arrangement of the individual complexes within the respirasome and SC I+III2. However, significant questions remain regarding the function, mechanism and regulation of the ETC complexes and SCs. To address these gaps in our understanding and to develop the basic science that will underpin potential treatment strategies of ETC defects, we will establish two major research directions in my lab. Using detailed biochemical and enzymatic analyses together with single particle cryo-electron microscopy structural characterizations, we will elucidate the mechanisms, functions and regulation of 1) isolated CI and 2) respiratory SCs. To achieve this, we propose to perform systematic functional and structural comparisons of respiratory CI and SCs purified from mammalian mitochondria (from both HeLa cell culture and porcine heart tissue), the a-proteobacteria Paracoccus denitrificans and the fungal model system Neurospora crassa. P. denitrificans is one of the closest living organisms to the ancestral a- proteobacteria that originated mitochondria after the endosymbiotic event. N. crassa is an established, powerful genetic and biochemical system for bioenergetics, for which nonetheless no high-resolution ETC structures are available. Comparing the CI and SCs from these divergent and genetically tractable organisms to their mammalian counterparts will allow us to test several key mechanistic hypotheses in the field and to identify the conserved features of CI and SC mechanism and regulation. This will provide deep insights into the energy-converting mechanism of CI and the physiological roles of SC formation, which will define the scientific foundation needed for the development of therapeutic strategies against CI and further ETC deficiencies.

项目概要/摘要 线粒体氧化磷酸化电子传递链（ETC）由五个大链组成 膜蛋白复合物（CI、CII、CIII2、CIV 和 CV），负责大部分的产生 细胞 ATP。因此，ETC 对于生物能量代谢至关重要。 ETC缺陷是其中之一 最常见的先天性代谢缺陷，其中 CI 缺陷约占三分之一 这些诊断。尽管约 50% 的 CI 缺陷患者会在生命的头 2 年内死亡，而且仅 约 25% 的人年满 10 岁，CI 仍然是所有 ETC 复合体中机制理解最差的。 此外，尽管医疗需求巨大，但目前尚无针对 CI 或其他 ETC 的有效治疗方法 的不足。这种差异部分源于对分子机制的不完全理解。 单个复合物及其高阶组装成超级复合物（SC）。在哺乳动物的心脏中 线粒体 大多数 CI 与 CIII2 和 CIV（SC I+III2+IV，呼吸体）相关或在 与 CIII2 (SC I+III2) 相关。最近的生物化学和结构工作产生了第一个原子 哺乳动物线粒体 CI 的解析结构并定义了各个复合物的排列 在呼吸体和 SC I+III2 内。然而，关于功能、机制仍然存在重大问题。 ETC 综合体和 SC 的监管。为了解决我们理解上的这些差距并发展 作为支持 ETC 缺陷潜在治疗策略的基础科学，我们将建立两个主要的 我实验室的研究方向。将详细的生化和酶分析与单颗粒一起使用 冷冻电子显微镜结构表征，我们将阐明其机制、功能和 1) 孤立 CI 和 2) 呼吸 SC 的调节。为了实现这一目标，我们建议进行系统的 从哺乳动物线粒体（来自 HeLa 细胞培养物和猪心脏组织）、a-变形杆菌脱氮副球菌和真菌 粗糙脉孢菌模型系统。脱氮假单胞菌是最接近祖先的生物体之一- 在内共生事件后起源于线粒体的变形菌。粗糙猪笼草是一种已建立的、 强大的生物能遗传和生化系统，但目前还没有高分辨率的 ETC 结构可用。比较这些不同且遗传上易于处理的生物体的 CI 和 SC 与哺乳动物对应物的研究将使我们能够测试该领域的几个关键机制假设，并 识别 CI 和 SC 机制和调节的保守特征。这将为您提供深入的见解 CI 的能量转换机制和 SC 形成的生理作用，这将定义科学 开发针对 CI 和进一步 ETC 缺陷的治疗策略所需的基础。