Cytochrome c mechanism of ROS signaling in apoptosis

细胞色素c在细胞凋亡中ROS信号传导的机制

• 批准号: 7126261
• 负责人: Valerian E Kagan
• 金额: $ 3.87万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7126261
• 关键词: Address; Affect; Antioxidants; Apoptosis; Apoptotic; Binding; Biochemical; Biological Assay; Biological Models; Cardiolipins; Caspase; Cell Death; Cell membrane; Cells; Characteristics; Chemicals; Collaborations; Complex; Condition; Cytosol; DNA Fragmentation; Data; Electron Spin Resonance Spectroscopy; Electrospray Ionization; Embryonic Development; Epithelial Cells; Fluorescence; Generations; Goals; Grant; High Pressure Liquid Chromatography; Homeostasis; Hydrogen Peroxide; Inflammation; Ischemia; Joints; Link; Lung; Mass Spectrum Analysis; Medicine; Membrane; Mitochondria; Molecular Conformation; Moscow; Nuclear; Pathway interactions; Permeability; Peroxidase; Peroxidases; Peroxides; Phagocytosis; Phase; Phosphatidylserines; Phospholipids; Physical condensation; Play; Production; Proteins; Rate; Reaction; Reactive Oxygen Species; Research; Research Institute; Research Project Grants; Role; Signal Pathway; Signal Transduction; Stress; TNFRSF6 gene; Testing; Tissues; Type II Epithelial Receptor Cell; United States National Institutes of Health; base; cytochrome c; design; improved; macrophage; membrane activity; mitochondrial membrane; oxidation; parent grant; peroxidation; programs; research study

DESCRIPTION (provided by applicant): Apoptosis plays an essential role in embryonic development, tissue homeostasis and various pathological conditions including inflammation and ischemia. Our long term goal is to elucidate mechanisms of reactive oxygen species (ROS) signaling at the early phase of mitochondria-dependent apoptosis. The specific hypothesis is that cytochrome c (cyt c), when attached to inner membrane of mitochondria, can catalytically oxidize cardiolipin upon reaction with hydrogen peroxide. Accumulation of cardiolipin hydroperoxides induces detachments of cyt c from membrane and its subsequent release from mitochondria that inevitably initiates apoptosis. This hypothesis is based on the observations that 1) cyt c in mitochondria exist predominantly in membrane-bound form; 2) membrane-bound cyt c possess significant peroxides-like activity; 3) accelerated rate of ROS production is associated with apoptosis; 4) oxidation of cardiolipin is associated with cyt c release. This joint research project is focused on the catalytic mechanism of ROS-dependent cardiolipin oxidation and on the role of peroxidized cardiolipin in cyt c release. The specific aims are to: 1) identify binding characteristics of cyt c associated with its pro-oxidant activity and determine conditions that amplify or inhibit this activity; 2) establish the catalytic mechanism of cyt c pro-oxidant activity and determine its preferential substrates; 3) determine signaling role of the cyt c pro-oxidant activity in mitochondria during apoptosis. Using biophysical and biochemical approaches including HPLC, ESI mass spectroscopy, EPR spectroscopy and array of quantitative fluorescence and spectroscopic assays we plan to estimate peroxidase activity of membrane-bound cyt c in mitochondria, to characterize products of cyt c-dependent phospholipid oxidation, and, finally, to establish a link between these oxidative reactions and cyt c release from mitochondria. Thus, these experiments will improve our understanding of the signaling role of ROS and products of phospholipid oxidation in apoptosis. This research will be done primarily in Moscow (Russian Federation) at the Research Institute of Physico-Chemical Medicine in collaboration with Dr. Grigory Borisenko as an extension of NIH grant # R01 HL070755 entitled "Pulmonary Phosphatidylserine Oxidation in Apoptosis".

描述（由申请人提供）：细胞凋亡在胚胎发育、组织稳态和各种病理条件（包括炎症和缺血）中起着重要作用。我们的长期目标是阐明活性氧（ROS）信号在线粒体依赖性凋亡的早期阶段的机制。具体假设是细胞色素c （cyt c）附着在线粒体内膜上，与过氧化氢反应后，可催化氧化心磷脂。心磷脂氢过氧化物的积累诱导细胞c脱离细胞膜，随后从线粒体释放，不可避免地引发细胞凋亡。这一假设是基于以下观察：1)线粒体中的cyt c主要以膜结合形式存在；2)膜结合细胞c具有显著的过氧化物样活性；3) ROS生成速度加快与细胞凋亡有关；4)心磷脂的氧化与cyt c的释放有关。本联合研究项目主要研究ros依赖性心磷脂氧化的催化机制，以及过氧化心磷脂在细胞释放中的作用。具体目标是：1)确定与促氧化活性相关的cyt - c的结合特性，并确定放大或抑制该活性的条件；2)建立cyt - c促氧化活性的催化机制，确定其优先底物；3)确定细胞凋亡过程中线粒体细胞c促氧化活性的信号作用。利用生物物理和生化方法，包括高效液相色谱、ESI质谱、EPR光谱和一系列定量荧光和光谱分析，我们计划估计线粒体中膜结合的cyt - c过氧化物酶活性，表征cyt - c依赖的磷脂氧化产物，并最终建立这些氧化反应与线粒体中cyt - c释放之间的联系。因此，这些实验将提高我们对ROS和磷脂氧化产物在细胞凋亡中的信号作用的理解。这项研究将主要在莫斯科（俄罗斯联邦）物理化学医学研究所与Grigory Borisenko博士合作进行，作为NIH拨款# R01 HL070755题为“肺磷脂酰丝氨酸氧化凋亡”的延伸。