权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

基本信息

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
项目类别：
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

项目摘要

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.

总结。