Oxidative stress and the cellular thiol status of Escherichia coli

大肠杆菌的氧化应激和细胞硫醇状态

• 批准号: 9238154
• 负责人: JAMES A. IMLAY
• 金额: $ 30.13万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9238154
• 关键词: Adverse effects; Affect; Affinity; Alkaline Phosphatase; Antioxidants; Bacteria; Behavior; Binding; Biochemical Genetics; Cells; Cellular Stress; Chemistry; Consequentialism; Copper; Cysteine; Cystine; Data; Devices; Diagnosis; Dissociation; Disulfides; Ecology; Electron Transport; Enzymes; Escherichia coli; Failure; Genetic; Goals; Growth; Human Pathology; Hydrogen Peroxide; Immune system; Iron; Ligands; Measures; Membrane; Metabolic Pathway; Metals; Microbe; Modeling; Molecular; Mononuclear; Nature; Outcome; Oxidants; Oxidative Stress; Oxides; Oxygen; Pathway interactions; Peroxidases; Phenotype; Physiological; Process; Proteins; Reactive Oxygen Species; Resolution; Role; Route; Saccharomyces cerevisiae; Severities; Site; Source; Specificity; Stress; Structure; Substrate Specificity; Sulfhydryl Compounds; Sulfur; Sum; Superoxides; Supplementation; System; TXN gene; Testing; Transcriptional Regulation; Unspecified or Sulfate Ion Sulfates; Vesicle; Work; Zinc; antioxidant therapy; base; cell injury; cohort; disulfide bond; disulfide bond reduction; enzyme activity; functional restoration; genetic analysis; glutaredoxin; iron metabolism; iron oxide; microbial community; microbicide; mutant; oxidation; redoxin; repair enzyme; repaired; theories; uptake

Summary. Oxidative stress has been implicated in a wide range of human pathologies, and it is also determinative in the ecology of microbial communities and the microbicidal actions of the cell-based immune system. However, its molecular nature is incompletely understood. In discussions of oxidative stress, the presence of reactive oxygen species (ROS) is often conflated with the accumulation of protein disulfide bonds. The model bacterium Escherichia coli provides circumstantial evidence to support this view. E. coli responds to H2O2 exposure by inducing several redoxins devoted to the reduction of disulfide bonds, and its growth during superoxide stress is possible only if a rapidly imported cysteine precursor is provided. Yet we and others have shown that the primary ROS—superoxide and hydrogen peroxide—do not oxidize typical cysteine residues at an impactful rate. In this proposal we seek to identify the connection between the presence of these oxidants and the disruption of cellular thiol status. We have begun by delineating the processes that control the intracellular level of cysteine. We have already identified the routes and extremely high rate of cystine entry into E. coli, and in Aim 1 we propose to do the same for cysteine. We will then examine the devices that help to curb cysteine over-accumulation: its efflux via AlaE, and its degradation by YhaM. This approach provides necessary context for interpreting the effects of cysteine supplementation upon the phenotypes of oxidative stress. In Aim 2 we seek to identify the major mechanisms by which oxidative stress can generate disulfide bonds. We will test three widely suspected sources of general disulfide-bond formation: iron-catalyzed thiol oxidation, copper-catalyzed thiol oxidation, and the inadvertent dissemination of disulfide bonds by the primary peroxidase, AhpCF. We will then examine site-specific thiol oxidation that we have observed when H2O2 attacks iron-based enzymes. In particular, we will test whether the induction of redoxins is an important step in the repair of these enzymes. In Aim 3, we seek to explain why superoxide-stressed cells can only grow if they contain high levels of cysteine. Superoxide disrupts enzymic iron-sulfur clusters and triggers the mismetallation of mononuclear iron enzymes. We have shown that the slow step in cluster repair is the delivery of iron, and our recent work suggests that intracellular cysteine mobilizes iron to the enzymes that need it. Further, cysteine is uniquely effective at extracting zinc from mismetallated iron enzymes, which is the rate-limiting step in their repair. Thus our analysis of superoxide stress has revealed opportunities for cysteine to restore the functions of pathways that superoxide otherwise blocks. In aggregate, the successful completion of these aims will reveal how thiol status is connected to oxidative stress. This would fill a large, perplexing hole in our view of how oxidants damage cells and how cells defend themselves against them. It will also finally provide a rational framework for considering the use of thiol antioxidants to diagnose or suppress oxidative stress.

摘要 氧化应激与多种人类病理学有关，并且也是决定性的 微生物群落的生态学和基于细胞的免疫系统的杀微生物作用。 然而，它的分子性质并不完全了解。在氧化应激的讨论中， 活性氧物质（ROS）经常与蛋白质二硫键的积累相混淆。模型 大肠杆菌提供了支持这一观点的间接证据。E.大肠杆菌对H2 O2的反应 暴露通过诱导几个氧化还原蛋白致力于二硫键的还原，和它的增长， 只有当提供快速输入的半胱氨酸前体时，超氧化物应激才是可能的。然而我们和其他人却 表明，初级ROS-超氧化物和过氧化氢-不氧化典型的半胱氨酸残基， 有影响力的利率。在这个建议中，我们试图确定这些氧化剂的存在之间的联系， 以及细胞巯基状态的破坏。我们已经开始描绘的过程，控制 细胞内半胱氨酸水平。我们已经确定了胱氨酸进入的途径和极高的速率 到大肠在目标1中，我们建议对半胱氨酸做同样的事情。然后我们将研究有助于 抑制半胱氨酸过度积累：其通过AlaE流出，以及其通过YhaM降解。这种方法提供 解释补充半胱氨酸对氧化应激表型影响的必要背景 应力在目标2中，我们试图确定氧化应激产生二硫化物的主要机制 债券我们将测试三个被广泛怀疑的一般二硫键形成的来源：铁催化的硫醇 氧化，铜催化的硫醇氧化，和二硫键的无意传播的主要 过氧化物酶，AhpCF。然后，我们将研究我们观察到的位点特异性硫醇氧化，当H2 O2 攻击铁基酶特别是，我们将测试氧化还原蛋白的诱导是否是一个重要的步骤， 这些酶的修复。在目标3中，我们试图解释为什么超氧化物应激细胞只能在以下情况下生长： 含有大量的半胱氨酸。超氧化物破坏酶的铁硫簇，并触发 单核铁酶的错配。我们已经证明，集群修复的缓慢步骤是 我们最近的工作表明，细胞内半胱氨酸将铁动员到酶， 此外，半胱氨酸在从金属错合铁酶中提取锌方面是独特有效的， 限速步骤在他们的修复。因此，我们对超氧化物应激的分析揭示了半胱氨酸的机会， 以恢复超氧化物所阻断的途径的功能。总的来说， 这些目标将揭示硫醇状态如何与氧化应激相关。这将填补一个巨大的，令人困惑的 我们对氧化剂如何破坏细胞以及细胞如何防御氧化剂的看法存在着一个漏洞。它还将 最后提供一个合理的框架，考虑使用硫醇抗氧化剂，以诊断或抑制 氧化应激