Oxidative stress and the cellular thiol status of Escherichia coli

总结。 氧化应激与广泛的人类病理有关，而且也是决定性的。 在微生物群落的生态和基于细胞的免疫系统的杀菌作用中。 然而，人们对其分子性质的了解还不完全。在关于氧化应激的讨论中， 活性氧物种(ROS)经常与蛋白质二硫键的积累混为一谈。模型 大肠杆菌为这一观点提供了间接证据。大肠杆菌对过氧化氢的反应 通过诱导几种致力于还原二硫键的氧化还蛋白来暴露，并在 只有在提供快速进口的半胱氨酸前体的情况下，才有可能发生超氧化物应激。然而，我们和其他人 表明初级ROS--超氧化物歧化和过氧化氢--不会氧化典型的半胱氨酸残基 有影响力的比率。在这项建议中，我们试图确定这些氧化剂的存在之间的联系 以及细胞内硫醇状态的破坏。我们已经开始描述控制 细胞内半胱氨酸水平。我们已经确定了进入的路线和极高的胱氨酸率 转化到大肠杆菌中，在目标1中，我们建议对半胱氨酸做同样的事情。然后，我们将检查有助于 抑制半胱氨酸的过度积累：半胱氨酸通过丙氨酸的外排和Yham的降解。这种方法提供了 解释补充半胱氨酸对氧化表型影响的必要背景 压力。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。我们将测试三个普遍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，以及初级分子不经意间扩散的二硫键 过氧化物酶，AhpCF.然后，我们将检查我们观察到的特定部位的硫醇氧化，当过氧化氢 攻击铁基酵素。特别是，我们将测试氧化还蛋白的诱导是否是 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有在它们 含有高水平的半胱氨酸。超氧化物破坏酶铁硫团簇并触发 单核铁酶的非金属化。我们已经证明，集群修复中最慢的一步是 铁的传递，我们最近的工作表明，细胞内的半胱氨酸将铁动员到 我需要它。此外，半胱氨酸在从非金属化的铁酶中提取锌是唯一有效的，这是 限速维修步骤。因此，我们对过氧化应激的分析揭示了半胱氨酸的机会 以恢复原本被超氧化物阻断的通路的功能。总体而言，成功完成 这些目的将揭示硫醇状态是如何与氧化应激联系起来的。这将填补一个巨大的，令人困惑的 我们对氧化剂如何损害细胞以及细胞如何防御它们的看法存在漏洞。它还将 最后为考虑使用硫醇抗氧化剂进行诊断或抑制提供一个合理的框架 氧化应激。

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.