Cytochrome c mechanism of ROS signaling in apoptosis

描述（由申请人提供）：细胞凋亡在胚胎发育、组织稳态和包括炎症和缺血在内的各种病理状况中起重要作用。我们的长期目标是阐明活性氧（ROS）信号转导在早期阶段的细胞凋亡依赖。具体的假设是，细胞色素c（cyt c），当连接到线粒体的内膜，可以催化氧化心磷脂与过氧化氢反应。心磷脂过氧化氢的积累诱导细胞色素c从细胞膜上脱落，随后从线粒体上释放，不可避免地引发细胞凋亡。这一假说基于以下观察结果：1）线粒体中的cyt c主要以膜结合形式存在; 2）膜结合的cyt c具有显著的过氧化物样活性; 3）ROS产生的加速速率与凋亡相关; 4）心磷脂的氧化与cyt c的释放相关。该合作研究项目的重点是ROS依赖性心磷脂氧化的催化机制和过氧化心磷脂在细胞色素c释放中的作用。具体目标是：1）鉴定与其促氧化活性相关的CytC的结合特征，并确定放大或抑制该活性的条件; 2）建立CytC促氧化活性的催化机制，并确定其优先底物; 3）确定细胞凋亡期间线粒体中CytC促氧化活性的信号传导作用。使用生物物理和生物化学方法，包括HPLC，ESI质谱，EPR光谱和阵列的定量荧光和光谱分析，我们计划估计过氧化物酶活性的膜结合细胞色素c在线粒体中，表征产品的细胞色素c依赖性磷脂氧化，并最终，建立这些氧化反应和细胞色素c从线粒体释放之间的联系。因此，这些实验将提高我们对细胞凋亡中ROS和磷脂氧化产物的信号作用的理解。本研究将主要在莫斯科（俄罗斯联邦）理化医学研究所与Grigory Borisenko博士合作进行，作为NIH资助#R 01 HL 070755“细胞凋亡中的肺磷脂酰丝氨酸氧化”的扩展。

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".