The Role of Cardiolipin in Assembly and Function of the Mitochondrial Respirasome

心磷脂在线粒体呼吸体组装和功能中的作用

• 批准号: 9392919
• 负责人: WILLIAM DOWHAN
• 金额: $ 36.77万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-02 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9392919
• 关键词: 3-Methylglutaconic aciduria type 2; Address; Affect; Affinity; Aging; Area; Binding; Binding Sites; Biological Assay; Biological Models; Cardiolipins; Cell Death; Cell physiology; Cells; Cessation of life; Chemicals; Complex; Coupled; Cryoelectron Microscopy; Data; Dependence; Dimensions; Disease; Electron Transport Complex III; Electrons; Equilibrium; Fatty Acids; Future; Glues; Growth; Head; Heart; Heart failure; Hereditary Disease; Image; In Vitro; Individual; Infection; Ions; Ischemia; Life; Lipids; Location; Malignant Neoplasms; Mammalian Cell; Maps; Mass Spectrum Analysis; Medicine; Membrane; Mitochondria; Modeling; Molecular; Muscle function; Myocardium; Neurodegenerative Disorders; Organelles; Oxidative Stress; Oxides; Pathologic; Peripheral; Phenotype; Phospholipids; Physiological; Play; Positioning Attribute; Predisposition; Property; Proteins; Recovery; Reperfusion Therapy; Reporting; Resolution; Respiratory Chain; Respiratory physiology; Role; Saccharomyces cerevisiae; Site; Site-Directed Mutagenesis; Skeletal Muscle; Structure; Surface; System; Tissues; Yeasts; analog; chemical property; complex IV; cytochrome c; cytochrome c oxidase; density; detector; genetic manipulation; improved; innovation; mitochondrial dysfunction; mutant; nanometer resolution; novel; oxidation; particle; premature; reconstitution; respiratory; simulation; symposium

Individual respiratory complexes of mitochondria are in dynamic equilibrium with higher order supercomplex organizations that compose the respirasome. Physiological and pathological perturbations in the mitochondrial cardiolipin (CL) pool directly affect and regulate this equilibrium. Aging, neurodegenerative diseases, heart failure, ischemia/reperfusion, cancer and Barth Syndrome are associated with abnormal CL pools. Saccharomyces cerevisiae mutants lacking CL display similar phenotypes to mammalian cells with reduced CL levels making yeast an excellent model system. We were the first to report that formation of the yeast tetrameric respiratory supercomplex (SC III2IV2) and cytochrome c (cyt c) channeling between complexes III (CIII, bc1 complex) and IV (CIV, cyt c oxidase) within the SC are directly dependent on CL. Using our recently acquired K2 Summit direct electron detector for single particle electron cryo-microscopy, we have significantly improved the quality of EM images. We achieved a higher resolution 3D density map of the tetrameric SC than we previously reported. We are now in a position to attain 3D density maps of SCs at an unprecedented sub- nanometer resolution. We propose an innovative combination of high resolution structural determinations, functional assays, genetic manipulation of yeast cells and novel lipid-dependent reconstitution studies to establish the molecular basis for CL-dependent formation and function of respiratory SCs. Aim 1: A) obtain a sub-nanometer resolution 3D density map for the tetrameric SC to establish the precise dimensions of the lipid- filled gaps and the interface between CIII and CIV; B) determine the location(s) of bound cyt c in the tetrameric SC to decipher how SC formation makes cyt c channeling possible; C) resolve the structure of the III2IV1 trimer to understand how structural differences with the tetramer results in CL-independent formation and lack of cyt c channeling in the trimer; D) perform structural studies of the SC tetramer lacking subunit Qcr6 of CIII coupled with lipid analysis (under Aim 2) to determine whether Qcr6p maintains the lipid-filled gap between CIII and CIV, which makes SC formation sensitive to CL levels. Aim 2: A) integrate structural with quantitative analysis of CL and other lipids present in the above SCs; B) determine the features of CL that support tetrameric SC formation and function using an innovative in vitro SC-reconstitution system employing structural analogs of CL; C) mimic pathological conditions resulting in CL pool alterations to understand how CL levels and CL oxidation affect SC structure and function. Aim 3: A) determine CIII and CIV surface exposed CL-binding sites potentially responsible for SC formation using a photoactivatible CL analog; B) use Molecular Dynamic Simulations to predict surface exposed CL-binding sites; C) employ site-directed mutagenesis at chemically modified and predicted CL-binding sites that lie at the CIII-CIV interface for verification of their involvement in SC formation. This innovative and integrated approach will establish the molecular mechanism of CL- dependent SC formation and function. In the future similar studies will be extended mammalian respirasomes.

线粒体的各个呼吸复合物与高阶超复合物处于动态平衡 组成呼吸体的组织。线粒体的生理和病理扰动 心磷脂（CL）池直接影响和调节这种平衡。衰老、神经退行性疾病、心脏 衰竭、缺血/再灌注、癌症和巴斯综合症与异常的 CL 池有关。 缺乏 CL 的酿酒酵母突变体表现出与 CL 减少的哺乳动物细胞相似的表型 水平使酵母成为一个优秀的模型系统。我们是第一个报告酵母形成的人 四聚体呼吸超复合物 (SC III2IV2) 和复合物 III 之间的细胞色素 c (cyt c) 通道 SC 内的（CIII，bc1 复合物）和 IV（CIV，细胞色素 c 氧化酶）直接依赖于 CL。使用我们最近 购买了用于单粒子电子冷冻显微镜的 K2 Summit 直接电子探测器，我们显着 提高了 EM 图像的质量。我们获得了比之前更高分辨率的四聚体 SC 3D 密度图 我们之前报道过。我们现在能够以前所未有的速度获得 SC 的 3D 密度图 纳米分辨率。我们提出了高分辨率结构测定的创新组合， 功能测定、酵母细胞的基因操作和新型脂质依赖性重组研究 建立呼吸 SC 依赖于 CL 的形成和功能的分子基础。目标 1：A) 获得 四聚体 SC 的亚纳米分辨率 3D 密度图，以确定脂质的精确尺寸 填补了CIII和CIV之间的空白和接口； B) 确定四聚体中结合的细胞色素 c 的位置 SC 破译 SC 的形成如何使 cyt c 通道成为可能； C) 解析 III2IV1 三聚体的结构 了解四聚体的结构差异如何导致 CL 独立形成和细胞色素 c 缺乏 三聚体中的通道； D) 对缺乏 CIII 偶联亚基 Qcr6 的 SC 四聚体进行结构研究 通过脂质分析（目标 2）确定 Qcr6p 是否维持 CIII 和 CIII 之间的脂质填充间隙 CIV，使 SC 的形成对 CL 水平敏感。目标 2：A) 将结构分析与定量分析相结合 上述 SC 中存在的 CL 和其他脂质； B) 确定支持四聚体SC的CL的特征 使用创新的体外 SC 重建系统进行形成和功能，该系统采用结构类似物 CL; C) 模拟导致 CL 池改变的病理条件，以了解 CL 水平和 CL 的变化 氧化影响SC结构和功能。目标 3：A) 确定 CIII 和 CIV 表面暴露的 CL 结合位点 使用可光活化的 CL 类似物可能导致 SC 形成； B) 使用分子动力学 模拟预测表面暴露的 CL 结合位点； C) 采用化学定点诱变 修改和预测位于 CIII-CIV 界面的 CL 结合位点，以验证它们参与 SC的形成。这种创新的综合方法将建立 CL- 的分子机制。 依赖 SC 的形成和功能。未来类似的研究将扩展到哺乳动物呼吸体。