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.

概括。 氧化应激已被隐含在广泛的人类病理中，并且也被确定 在微生物群落的生态学和基于细胞的免疫系统的微生物作用中。 但是，其分子性质尚不完全理解。在讨论氧化应激时的存在 活性氧（ROS）通常与蛋白质二硫键的积累混合在一起。模型 细菌大肠杆菌提供了间接证据来支持这一观点。大肠杆菌对H2O2的反应 通过诱导的几种致力于减少二硫键的氧化还原蛋白的接触，其在 仅当提供快速进口的半胱氨酸前体时，超氧化物应激才有可能。但是我们和其他人有 表明原发性ROS（杀菌剂和过氧化氢）典型的半胱氨酸不保留在 有影响力的速度。在此提案中，我们试图确定这些氧化剂的存在之间的联系 以及细胞硫醇状况的破坏。我们已经开始描述控制该过程的过程 半胱氨酸的细胞内水平。我们已经确定了胱氨酸进入的路线和极高的速度 进入大肠杆菌，在目标1中，我们建议对半胱氨酸做同样的事情。然后，我们将检查帮助的设备 遏制半胱氨酸过度蓄积：其通过Alae的外排，Yham的降解。这种方法提供 解释补充半胱氨酸对氧化表型的影响的必要背景 压力。在AIM 2中，我们试图确定氧化物胁迫可以产生二硫化的主要机制 债券。我们将测试三个广泛怀疑的一般二硫键形成来源：铁催化的硫醇 氧化，铜催化的硫醇氧化以及原发性二硫键的无意传播 过氧化物酶，AHPCF。然后，我们将检查当H2O2时观察到的位点特异性氧化物 攻击基于铁的酶。特别是，我们将测试诱导氧化氧蛋白是否是重要的一步 这些酶的修复。在AIM 3中，我们试图解释为什么超氧化物应激细胞只有在它们才能生长 含有高水平的半胱氨酸。超氧化物破坏酶铁硫簇，并触发 单核铁酶的不易裂。我们已经表明，集群维修的缓慢步骤是 铁的递送，我们最近的工作表明，细胞内半胱氨酸动员了铁的酶 需要它。此外，半胱氨酸在从不种类的铁酶中提取锌具有独特的有效性，这是 维修中的限速步骤。我们对超氧化压力的分析揭示了半胱氨酸的机会 恢复超氧化物否则会阻止的途径的功能。总共成功完成 这些目的将揭示硫醇状态如何与氧化应激相关。这会充满大而令人困惑的 我们认为氧化剂如何损害细胞以及细胞如何防御它们的看法。它也会 最终提供了一个合理的框架，以考虑使用硫醇抗氧化剂诊断或抑制 氧化应激。