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.

线粒体的单个呼吸复合体与高阶超复合体处于动态平衡 组织组成的共同体。线粒体中的生理和病理扰动 心磷脂（CL）库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Barth综合征与异常CL池相关。 缺乏CL的酿酒酵母突变体显示出与CL降低的哺乳动物细胞相似的表型 水平使酵母一个很好的模型系统。我们是第一个报道酵母的形成 四聚体呼吸超复合物（SC III 2 IV 2）和复合物III之间的细胞色素c（cyt c）通道 (CIII SC内的IV（CIV，cyt c氧化酶）直接依赖于CL。利用我们最近 获得K2 Summit直接电子探测器用于单粒子电子冷冻显微镜，我们有显着 提高了EM图像的质量。我们获得了四聚体SC的分辨率高于 我们之前报道过。我们现在能够在前所未有的亚空间获得SC的3D密度图， 纳米分辨率我们提出了一种创新的高分辨率结构测定组合， 功能测定、酵母细胞的遗传操作和新的脂质依赖性重建研究， 为CL依赖的呼吸系统SC的形成和功能奠定分子基础。目标1：A）获得 四聚体SC的亚纳米分辨率3D密度图，以建立脂质的精确尺寸。 填充的间隙以及CIII和CIV之间的界面; B）确定结合的cyt c在四聚体中的位置 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-独立形成和缺乏细胞色素c D）对缺乏CIII偶联的亚基Qcr6的SC四聚体进行结构研究 脂质分析（目标2），以确定Qcr6p是否维持CIII和 CIV，这使得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 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，采用 CL; C）模拟导致CL池改变的病理条件，以了解CL水平和CL 氧化影响SC的结构和功能。目的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类似物可能负责SC形成; B）使用分子动力学 模拟以预测表面暴露的CL结合位点; C）在化学修饰处采用定点诱变。 位于CIII-CIV界面的修饰和预测的CL-结合位点，用于验证它们参与 SC编队。这种创新和综合的方法将建立CL的分子机制- 依赖SC的形成和功能。在未来，类似的研究将扩展到哺乳动物体内。