MITOCHONDRIA AND PULMONARY ENDOTHELIAL CELL DEATH

线粒体和肺内皮细胞死亡

• 批准号: 6390108
• 负责人: JAMES PETERSON
• 金额: $ 20.62万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6390108
• 关键词: NAD(P)H dehydrogenase; NADPH cytochrome c2 reductase; apoptosis; cell death; circular magnetic dichroism; cytochrome oxidase; electron spin resonance spectroscopy; enzyme activity; enzyme inhibitors; free radical oxygen; hydrogen peroxide; iron compounds; membrane potentials; mitochondria; molecular dynamics; nitric oxide; peroxynitrites; respiratory epithelium; tissue /cell culture; NAD(P)H dehydrogenase; NADPH cytochrome c2 reductase; apoptosis; cell death; circular magnetic dichroism; cytochrome oxidase; electron spin resonance spectroscopy; enzyme activity; enzyme inhibitors; free radical oxygen; hydrogen peroxide; iron compounds; membrane potentials; mitochondria; molecular dynamics; nitric oxide; peroxynitrites; respiratory epithelium; tissue /cell culture

DESCRIPTION (Applicant's abstract): Mitochondria are critical loci of cellular respiration, biosynthesis and metabolism of partially reduced oxygen species and participate in intracellular signaling including calcium homeostasis, oxygen sensing and the initiation of apoptosis. Nitric oxide (NO) is unique amongst signaling molecules in that it affects its target sites by both redox chemistry (S-nitrosylation of thiols) and coordination (especially to Fe2+). Consequently biomolecules affected by NO (and peroxynitrite) tend to have critical metalloregulatory and cysteine-enriched active sites. Accordingly, it has been well known since the discovery of the L-arginine biosynthetic pathway that NO can inhibit mitochondrial respiration. Nonetheless neither the precise targets nor the mechanism by which NO affects mitochondrial function are apparent. We hypothesize that NADH dehydrogenase (complex I) with it's multiple non-heme iron-sulfur (Fe/S) centers and cytochrome c reductase (complex III), an hemoprotein, are targets for NO and peroxynitrite. We propose a series of biophysical and biochemical studies that will reveal the molecular mechanism by which NO, partially reduced oxygen species (PROS) and ONO2 affect mitochondrial respiratory chain function. Aim 1: Investigate the molecular mechanism by which peroxynitrite (ONO2) and other oxidants irreversibly inhibit complex I and complex III. Aim 2: Investigate the mechanism by which complex IV (cytochrome c oxidase) catalyses the detoxification of ONO2 and H2O2. Aim 3: Identify the molecular targets of peroxynitrite and ferrous iron in Keilin-Hartee (K-H, submitochondrial) particles and intact mitochondria. Aim 4: describe the oxidant (NO, superoxide anion, ONO2, etc) induced changes (mitochondrial membrane potential) that occur in mitochondria of intact cultured endothelial cells. These studies, using a combination of low temperature electron paramagnetic resonance spectroscopy and magnetic circular and linear dichroism in isolated bovine heart mitochondria and voltage sensitive dyes in intact bovine pulmonary artery endothelial cells, will provide mechanistic information to support a role for NO induced mitochondrial changes in health and disease.

描述（申请人的摘要）：线粒体是细胞的关键基因座 部分减少氧的呼吸，生物合成和代谢 并参与细胞内信号传导，包括钙稳态， 氧气传感和凋亡的启动。一氧化氮（NO）是唯一的 在信号分子中，它影响了两个氧化还原的目标位点 化学（硫醇的S-亚硝基化）和配位（尤其是Fe2+）。 因此，受NO（和过氧亚硝酸盐）影响的生物分子倾向于 临界金属调节和半胱氨酸富集的活性位点。因此，它 自发现L-精氨酸生物合成途径以来，就众所周知 不能抑制线粒体呼吸。尽管如此， 目标或不影响线粒体功能的机制是 显而易见。我们假设NADH脱氢酶（复杂I）与它是多个 非血红素铁硫（Fe/S）中心和细胞色素C还原酶（复合物III），， 血蛋白是NO和过氧亚硝酸盐的靶标。我们提出了一系列 生物物理和生化研究将揭示通过 没有部分减少的氧（PROS）和ono2会影响线粒体 呼吸链功能。目标1：研究分子机制 过氧亚硝酸盐（ono2）和其他氧化剂不可逆地抑制复合物I和 复合体III。 AIM 2：研究复合IV（细胞色素c）的机制 氧化酶）催化Ono2和H2O2的排毒。目标3：确定 keilin-hartee中过氧亚硝酸盐和铁铁的分子靶标（K-H， sbirmochochondrial）颗粒和完整的线粒体。目标4：描述 氧化剂（NO，超氧化阴离子，ono2等）诱导变化（线粒体 在完整培养的内皮的线粒体中发生的膜电位） 细胞。这些研究，结合了低温电子 顺磁共振光谱以及磁循环和线性二色性 在孤立的牛心脏线粒体和电压敏感染料完整中 牛肺动脉内皮细胞将提供机械信息 支持没有诱导的健康和疾病的线粒体变化的角色。