The Physiology of Oxidative Stress in Escherichia coli

大肠杆菌氧化应激的生理学

基本信息

批准号：
10458048
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
项目类别：
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/10458048
关键词：
Acetates Aerobic Affect Alcohol dehydrogenase Anaerobic Bacteria Anoxia Appearance Binding Sites Biochemical Cell physiology Cells Cellular Stress Chemistry 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

项目摘要

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。在目标 1 中，我们将研究为什么超氧化物应激阻碍了使用。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使我们提出了细胞内反应的建议。半胱氨酸和氧化还原素有助于修复受损的金属酶中心，该模型将确定一个关键。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这将包括巧妙地利用通常有害的反应类型。目标 2 是测试这个引人注目的想法。这个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，但我们的数据表明，潜在的事件和生理影响尚不清楚。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。全局质谱法将通过名称来识别它们。我们还将测试蛋氨酸的想法。亚砜是还原酶可以修复的芬顿歧化产物，新颖之处在于蛋氨酸可以修复。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。希望解释我们的发现，即 OxyR 激活本身会损害细胞健康，甚至禁止压力反应会产生代价并不奇怪，但我们尚未认识到。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反应的趋势，我们的四个目标将建立在这些知识的基础上破坏长期存在的问题，总体目标是构建一幅详细的氧化应激图景，数量化、统一化。