The Physiology of Oxidative Stress in Escherichia coli

大肠杆菌氧化应激的生理学

• 批准号: 10297291
• 负责人: JAMES A. IMLAY
• 金额: $ 54.83万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10297291
• 关键词: Acetates; Aerobic; Affect; Alcohol dehydrogenase; Anaerobic Bacteria; Anoxia; Appearance; Binding Sites; Biochemical; Cell physiology; Cells; Cellular Stress; Chemistry; Cleaved cell; Collaborations; Cysteine; DNA Damage; Data; Disease; Electrons; Environment; Enzymes; Escherichia coli; Event; Family; Fermentation; Genes; Glucose; Goals; Grant; Growth; Grx1 protein; Hydrogen Peroxide; Hydroxyl Radical; Impairment; In Vitro; Injury; Iron; Knowledge; Literature; Lyase; Mass Spectrum Analysis; Measurement; Mediating; Metabolic; Metabolism; Metal Binding Site; Metals; Methionine; Microbe; Minor; Modeling; Modification; Molecular; Mononuclear; Names; Natural regeneration; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Oxidoreductase; Oxygen; Peroxides; Pharmaceutical Preparations; Phenotype; Physiological; Physiology; Plant Roots; Play; Price; Proteins; Proteome; Pyruvate; Pyruvate Metabolism Pathway; Reaction; Reactive Oxygen Species; Regulon; Role; Site; Solvents; Source; Stress; Sulfate; Sulfhydryl Compounds; Sulfur; Sulfur Metabolism Pathway; Superoxides; System; TXN gene; Testing; Thioredoxin-2; Toxic effect; Work; Zinc; biological adaptation to stress; cell growth; dithiol; fitness; formate acetyltransferase activating enzyme; glutaredoxin; in vivo evaluation; iron oxidation; iron oxide; member; metalloenzyme; methionine sulfoxide; microbial; oxidation; reconstruction; redoxin; repaired; response; side effect; transcriptome sequencing; transcriptomics; wasting; weapons

We have learned that most oxidative toxicity arises when oxygen species attack enzymic iron centers and that cellular defenses work by blocking, reversing, or by-passing the resultant injuries. Yet key observations remain unexplained. In Aim 1 we will investigate why superoxide stress precludes the use of sulfate as a sulfur source, and we will examine why thioredoxins and glutaredoxins are strongly induced as part of the cellular reaction to hydrogen peroxide. Extensive work has led us to the proposal that intracellular cysteine and redoxins help to repair damaged metalloenzyme centers. This model would identify a key connection between sulfur redox state and ROS. Two enzymes dedicated to anaerobic metabolism—pyruvate:formate lyase activating enzyme and alcohol dehydrogenase—have been suggested to be inactivated by iron-centered oxidation events when cells are aerated. This would comprise a clever exploitation of reaction types that are usually harmful. The goal of Aim 2 is to test this striking idea. This hypothesis leads to notions of how the cell might seamlessly restore anaerobic metabolism when anoxia is restored. Protein carbonylation (Aim 3) has long been used as a convenient marker of oxidative stress—but the underlying events and physiological impact are unclear. Our data indicate that carbonylation is focused upon relatively few proteins rather than the full proteome, and we suspect that these proteins are mononuclear Fe(II) enzymes. Global mass spectrometry will identify them by name. We will also test the idea that methionine sulfoxide is a disproportionate Fenton product that reductases can repair. The novelty is that methionine may be oxidized by a secondary electron-hopping event, rather than by direct attack. Finally, in Aim 4 we will take a transcriptomic approach to fully define the OxyR peroxide response. We hope to explain our discovery that OxyR activation per se compromises cells fitness, to the point of prohibiting growth on acetate. It is not surprising that a stress response should exert a price, but we do not yet recognize why any OxyR-driven adaptation would have such a profound effect. The emergent theme of oxidative stress is the tendency of oxygen species to react with iron centers, and of cells to respond with layers of defensive tactics. Our four Aims will build upon this knowledge by tackling persistent questions, with the overall goal of assembling a picture of oxidative stress that is detailed, quantitative, and unified.

我们了解到，大多数氧化毒性是在氧物质攻击酶铁中心时产生的 细胞防御通过阻止、逆转或绕过由此产生的损伤来发挥作用。然而关键 观察结果仍无法解释。在目标 1 中，我们将研究为什么超氧化物应激会妨碍使用 硫酸盐作为硫源，我们将研究为什么硫氧还蛋白和谷氧还蛋白被强烈诱导为 细胞对过氧化氢反应的一部分。大量的工作使我们提出了细胞内 半胱氨酸和氧化还原素有助于修复受损的金属酶中心。该模型将识别一个关键 硫氧化还原态与ROS之间的联系。 两种专门用于无氧代谢的酶——丙酮酸：甲酸裂解酶激活酶和 乙醇脱氢酶——已被认为会因细胞内以铁为中心的氧化事件而失活 被充气。这将包括巧妙地利用通常有害的反应类型。目标是 目标 2 是测试这个引人注目的想法。这一假设引出了细胞如何无缝恢复的概念 缺氧恢复后进行无氧代谢。 蛋白质羰基化（目标 3）长期以来一直被用作氧化应激的便捷标记，但 潜在事件和生理影响尚不清楚。我们的数据表明羰基化作用集中于 相对较少的蛋白质而不是完整的蛋白质组，我们怀疑这些蛋白质是单核 Fe(II) 酶。全局质谱分析将通过名称识别它们。我们还将测试蛋氨酸的想法 亚砜是还原酶可以修复的不成比例的芬顿产物。新颖之处在于蛋氨酸可能 通过二次电子跳跃事件被氧化，而不是通过直接攻击。 最后，在目标 4 中，我们将采用转录组学方法来全面定义 OxyR 过氧化物反应。我们 希望解释我们的发现，即 OxyR 激活本身会损害细胞的适应性，甚至达到禁止的程度 在醋酸盐上生长。压力反应会产生代价并不奇怪，但我们还没有认识到 为什么任何 OxyR 驱动的适应都会产生如此深远的影响。 氧化应激的新主题是氧物质与铁中心发生反应的趋势， 以及细胞以多层防御策略做出反应。我们的四个目标将建立在这些知识的基础上 解决持续存在的问题，总体目标是构建详细的氧化应激图景， 数量化、统一化。