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.

项目摘要/摘要 线粒体氧化磷酸化电子传输链（ETC）由五个大型 膜蛋白复合物（CI，CII，CIII2，CIV和CV），负责大多数 细胞ATP。因此，ETC对于生物能代谢至关重要。等缺陷是 最常见的先天性代谢缺陷，CI缺陷约占三分之一 这些诊断。尽管约有50％的CI缺陷患者在生命的前两年死亡，仅在 〜25％达到10岁，CI仍然是所有ETC络合物中最低的机械理解。 此外，尽管医疗需求很大，但目前尚无针对CI或其他等有效的治疗方法 缺陷。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件成超级复合物（SC）。在哺乳动物的心中 线粒体大多数CI都与CIII2和CIV（SC I+III2+IV，呼吸症）或IN相关 与CIII2（SC I+III2）关联。最近的生化和结构工作产生了第一个原子 - 哺乳动物线粒体CI的分辨率结构，并定义了单个复合物的排列 在呼吸系统和SC I+III2中。但是，关于功能，机制仍然存在重大问题 以及ETC复合物和SC的调节。在我们的理解中解决这些差距并发展 基础科学将是基础的潜在治疗策略等缺陷的基础科学，我们将建立两个主要的 我实验室的研究指示。使用详细的生化和酶促分析与单个粒子一起进行 冷冻电子显微镜结构表征，我们将阐明机制，功能和 调节1）分离的CI和2）呼吸SC。为了实现这一目标，我们建议执行系统 从哺乳动物线粒体纯化的呼吸道CI和SC的功能和结构比较（来自 HeLa细胞培养和猪心组织），a蛋白核细菌denitrificans和真菌 模型系统Neurospora Crassa。 P. denitrificans是距祖先A-的生物最接近的生物之一 内共生事件后起源于线粒体的蛋白质细菌。 N. Crassa是一个已建立的， 强大的生物能源遗传和生化系统，但没有高分辨率等 结构可用。比较这些不同的和遗传性的生物的CI和SC 与他们的哺乳动物对应物将使我们能够在现场测试几个关键的机械假设，并测试 确定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 制定针对CI和进一步缺陷的治疗策略所需的基础。