Phospholipids and Mitochondrial Function

磷脂和线粒体功能

• 批准号: 7873382
• 负责人: WILLIAM DOWHAN
• 金额: $ 20.42万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-03 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7873382
• 关键词: 3-Dimensional; 3-Methylglutaconic aciduria type 2; Aging; Apoptosis; Binding; Biochemical; Biochemical Genetics; Biological Models; Cardiolipins; Cell Nucleus; Cell physiology; Communication; Complex; Coupled; Cryoelectron Microscopy; Cytochrome bc1 Complex; Cytoplasm; Data; Detergents; Disease; Docking; Electron Microscopy; Electron Transport; Electron Transport Complex III; Energy Metabolism; Eukaryotic Cell; Functional disorder; Funding; Future; Genes; Genetic; Genetic Translation; Glues; Glycogen Branching Enzyme; Green Fluorescent Proteins; Heart failure; High Pressure Liquid Chromatography; Individual; Inner mitochondrial membrane; Ischemia; Light; Lipids; Location; Mammalian Cell; Messenger RNA; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Molecular; Molecular Genetics; Myocardial dysfunction; Negative Staining; Nuclear; Pathway interactions; Phase; Phenotype; Phosphatidyl glycerol; Phosphatidylglycerols; Phospholipids; Physiological; Play; Polyacrylamide Gel Electrophoresis; Property; Regulation; Reperfusion Therapy; Reporting; Research; Resolution; Role; Saccharomyces cerevisiae; Sampling; Signal Pathway; Site; Staging; Stress; Structure; System; Translation Initiation; Translations; Untranslated Regions; Yeasts; analog; base; biological adaptation to stress; complex IV; cytochrome c oxidase; dodecyl maltoside; essential phospholipids; mutant; novel; oligomycin sensitivity-conferring protein; particle; reconstitution; research study; response; stoichiometry; three dimensional structure

DESCRIPTION (provided by applicant): Cardiolipin (CL) is an essential phospholipid for normal mitochondrial energy metabolism so that physiological and pathological perturbations in its levels result in alterations in the structure, function and assembly of mitochondria. CL is an integral part of most of the components of the mitochondrial energy transducing system. Myocardial dysfunction and apoptosis are associated with abnormal CL levels in Barth Syndrome, aging, ischemia/reperfusion, and heart failure. Saccharomyces cerevisiae mutants (crd1?) lacking CL, but containing its precursor phosphatidylglycerol (PG), or mutants (pgs1?) lacking both PG and CL display similar phenotypes to mammalian cells with reduced PG and CL levels or the inability to make PG and CL, respectively. Therefore, yeast is an excellent model system to study their role of these lipids in mitochondrial function. Studies of yeast pgs1? and crd1? mutants led to two findings that are the basis for the proposed studies. (1) Translation repressors that bind 5' of the translation initiation site on mRNA for subunit 4 (Cox4p) of the mitochondrial electron transport chain component cytochrome c oxidase (Complex IV) are specifically activated or synthesized in response to altered mitochondrial phospholipid composition in pgs1? mutants. Studies are proposed to genetically and biochemically define the components and mechanism of this novel mitochondrial stress response pathway. (2) Complex IV and Complex III (cytochrome bc1) form a III2IV2 supercomplex that kinetically behaves as a single unit "respirasome", which was established by using a crd1? mutant to be specifically dependent on CL. Experiments are proposed to determine the phospholipid stoichiometry in the individual Complexes III and IV and in the III2IV2 supercomplex and the location of CL within the latter required for formation and stabilization of the supercomplex. A 3-D structure of the III2IV2 supercomplex has been determined by negative stain-electron microscopy and single particle analysis, which will be refined using cryoelectron microscopy to establish how Complex III and IV are organized in the supercomplex. Phospholipid analysis and structural data will be integrated to establish how CL is involved in "gluing" together the supercomplex components. Defining the roles PG and CL play in normal mitochondrial function will shed light on the molecular basis for cellular dysfunction in physiological and pathological states where these lipids are reduced.

描述（由申请人提供）：心磷脂（CL）是正常线粒体能量代谢所必需的磷脂，因此其水平的生理和病理扰动会导致线粒体结构、功能和组装的改变。CL是线粒体能量传导系统中大部分组成部分的组成部分。在Barth综合征、衰老、缺血/再灌注和心力衰竭中，心肌功能障碍和细胞凋亡与CL水平异常有关。缺乏CL但含有其前体磷脂酰甘油（PG）的酿酒酵母突变体（crd1?）或既缺乏PG又缺乏CL的突变体（pgs1?）表现出与哺乳动物细胞相似的表型，分别是PG和CL水平降低或不能产生PG和CL。因此，酵母是研究这些脂质在线粒体功能中的作用的一个很好的模型系统。酵母pgs1？和crd1吗?突变体导致了两项发现，这两项发现是拟议研究的基础。(1)结合线粒体电子传递链组分细胞色素c氧化酶（复合体IV）亚基4 (Cox4p) mRNA上翻译起始位点5′的翻译抑制因子被特异性激活或合成以响应pgs1？突变体。研究提出了遗传和生物化学定义的组成部分和机制，这一新的线粒体应激反应途径。(2)复合体IV和复合体III（细胞色素bc1）形成一个III2IV2超复合体，其动力学行为作为一个单一的单位“呼吸酶体”，这是通过使用crd1？特异依赖于CL的突变体。提出了实验来确定单个配合物III和IV以及III2IV2超配合物中的磷脂化学计量，以及CL在后者中形成和稳定超配合物所需的位置。通过负染色电镜和单颗粒分析确定了III2IV2超配合物的三维结构，将使用低温电镜对其进行细化，以确定超配合物III和IV是如何组织的。磷脂分析和结构数据将被整合，以确定CL是如何参与“粘合”在一起的超复杂成分。确定PG和CL在正常线粒体功能中的作用，将揭示这些脂质减少的生理和病理状态下细胞功能障碍的分子基础。