Soft Metal, Disulfide, and Cysteine Stresses in Escherichia coli

大肠杆菌中的软金属、二硫化物和半胱氨酸应力

• 批准号: 8461150
• 负责人: JAMES A. IMLAY
• 金额: $ 23.9万
• 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 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8461150
• 关键词: Address; Aerobic; Affinity; Antibiotics; Back; Bacteria; Binding; Biochemistry; Biological; Biota; Cadmium; Catheters; Cells; Chemicals; Chemistry; Complex; Coupled; Coupling; Cysteine; Cystine; Cytoplasm; Cytoplasmic Protein; DNA; Data; Disulfides; Drug Metabolic Detoxication; Ensure; Environment; Enzymes; Escherichia coli; Event; Face; Glutathione Disulfide; Goals; Grant; Habitats; Human Pathology; Hydro-Lyases; Hydroxyl Radical; Immune; In Vitro; Investigation; Iron; Knowledge; Link; Mercury; Metal exposure; Metals; Modeling; Mononuclear; Nature; Organism; Outcome; Oxidation-Reduction; Oxidative Stress; Physiological; Plant Roots; Production; Proteins; Publishing; Pump; Reactive Oxygen Species; Recording of previous events; Risk; Silver; Solvents; Stress; Sulfhydryl Compounds; Sulfides; Sulfur; System; Testing; Toxic effect; Work; Zinc; cell injury; cofactor; coping; copper poisoning; disulfide bond; disulfide compound; fitness; in vivo; interest; metal poisoning; research study; respiratory enzyme; stem

DESCRIPTION (provided by applicant): The goals of this study are to investigate the mechanisms by which soft metals, cystine import, and cysteine accumulation can perturb the fitness of Escherichia coli. These three distinct stresses are connected by the central involvement of cysteine chemistry. Soft metals are present in many habitats, where they comprise a threat to bacteria and higher organisms alike. Their toxicity is underscored by the distribution throughout the biota of dedicated detoxification systems that bind and export them. Some of these metals- notably silver and mercury-have a long history of being used as antibiotics. Nevertheless, we have little knowledge of how they actually damage cells. In recent published work we demonstrated that copper poisons E. coli primarily by inactivating Fe/S-dependent dehydratases. It does so by binding the cysteine residues that coordinate the catalytic iron-sulfur clusters of these enzymes, thereby displacing the iron atoms. Preliminary data demonstrate that silver, mercury, cadmium, and zinc have this effect in vitro, too. This study (Aim 1) will test whether these soft metals exert their toxicity through this mechanism in vivo. It will also determine whether soft metals similarly displace iron from mononuclear enzymes, which employ coordinating groups-often including cysteine-that prefer metals other than iron. Aim 2 focuses upon a separate sulfur problem: the risk of disulfide stress when cystine is imported from aerobic environments. Disulfide stress has conventionally been thought to arise from reactive oxygen species, but recent data do not support this idea. However, disulfide stress is a real risk when cells rapidly import disulfide compounds, such as cystine. In principle such an event would seem likely to propagate disulfide bonds to cytoplasmic proteins. Our study of the high-flux transporter suggests that cystine import is linked to reduction, a tacti that would avoid the release of this disulfide into the cytoplasm. In Aim 2 this model will be rigorously tested. Aim 3 addresses the consequence of rapid cystine import: the excessive accumulation of cysteine, which is toxic in its own right. Our data reveals that E. coli deals with this problem by pumping the cysteine back out of the cell. This investigation will test three plausible mechanisms of cysteine toxicity, and it will identify the exporter(s) that averts it. Collectively this work will illuminate chemical problems that arise from the redox activity and metal affinity of cysteine, as well as the strategies that cells have acquired to protect themselve from it. All three of these sulfur-focused stresses-soft-metal exposure, disulfide import, and cysteine accumulation-occur under conditions that are likely to exist in natural habitats.

描述（由申请人提供）：这项研究的目标是研究软金属，半胱氨酸进口和半胱氨酸积累的机制，可以使大肠杆菌的适应性扰动。这三种不同的应力通过半胱氨酸化学的中心参与而连接。软金属存在于许多栖息地中，在那里它们构成了对细菌和更高生物体的威胁。它们的毒性是由绑定和导出它们的专用排毒系统的整个生物群体中的分布强调的。这些金属中的一些 - 尤其是银和汞的悠久历史，被用作抗生素。然而，我们对它们实际损害细胞的实际损害知识知之甚少。在最近发表的工作中，我们证明了铜毒物大肠杆菌主要是通过失活的脱水酶来灭活。它通过结合坐标这些酶的催化铁硫簇的半胱氨酸残基来做到这一点，从而取代铁原子。初步数据表明，银，汞，镉和锌在体外也具有这种作用。这项研究（AIM 1）将测试这些软金属是否通过这种机制在体内施加毒性。它还将确定软金属是否同样从单核酶中取代铁，这些酶采用了通常的组成组，包括半胱氨酸，而不是铁而不是铁。 AIM 2专注于一个单独的硫问题：从有氧环境中进口胱氨酸时，二硫应激的风险。通常认为二硫应激是由活性氧引起的，但最近的数据不支持这一想法。但是，当细胞快速进口二硫化物（例如胱氨酸）时，二硫应激是一种真正的风险。原则上，这种情况似乎很可能会传播到细胞质蛋白的二硫键。我们对高速升级转运蛋白的研究表明，Cystine的进口与还原有关，该策略可以避免该二硫化物释放到细胞质中。在AIM 2中，该模型将经过严格测试。 AIM 3解决了快速进口的后果：半胱氨酸的过度积累，这本身就是有毒的。我们的数据表明，大肠杆菌处理 通过将半胱氨酸从细胞中抽出来，这一问题。这项研究将测试半胱氨酸毒性的三种合理机制，并将确定避免它的出口商。这项工作总的来说，这将阐明由半胱氨酸的氧化还原活性和金属亲和力引起的化学问题，以及细胞保护它们从中获得的策略。所有这三个以硫的应力均在可能存在于自然栖息地中可能存在的条件下，这三个以硫磺为发的金属暴露，二硫化的进口和半胱氨酸的积累 - 占领。