Gene Regulation by Oxidative Stress in Bacillus subtilis

本项目研究了两种细菌调节蛋白对活性氧的反应：枯草杆菌Perr和OhrR的生物学作用和机制。过氧化调节蛋白(Perr)是金属依赖的铁摄取调节蛋白(Fur)家族中的一员。PERR通过与过氧化氢反应产生羟基自由基，从而修饰组氨酸残基，从而协调结合的Fe2辅因子，从而起到过氧化应激传感器的作用。由此产生的氧化蛋白对DNA结合不起作用。OhrR蛋白含有一个高活性的半胱氨酸残基，它与有机过氧化物反应生成半胱氨酸-磺酸，然后与低分子质量的硫醇反应生成混合二硫化物。氧化的OhrR还形成亚磺酰胺衍生物。这些蛋白质修饰使阻遏物失活，导致有机过氧化氢抗性的表达。该项目阐述了蛋白质氧化调节这两种蛋白质活性的详细机制。这项研究将确定氧化的PERR是被降解的，还是新的蛋白质修复途径的主题。将研究蛋白质氧化对OhrR的影响，并定义有助于OhrR重新激活的蛋白质。相关的调节蛋白存在于各种细菌中，包括重要的植物和动物病原体，在这些细菌中，它们的功能是调节对毒力重要的基因。这项研究将涉及蛋白质化学和氧化的基本方面。蛋白质氧化在所有细胞的生命中都很重要，被认为是导致衰老和许多与年龄相关的疾病的原因。该项目将对所有级别的学生进行遗传和蛋白质化学技术培训。

This project addresses the biological roles and mechanisms of two bacterial regulatory proteins that respond to reactive oxygen species: Bacillus subtilis PerR and OhrR. The peroxide regulon (PerR) repressor is a member of the Fur (ferric uptake regulator) family of metal-dependent repressor proteins. PerR functions as a sensor of peroxide stress by reacting with hydrogen peroxide to generate hydroxyl radical, which modifies histidine residues that coordinate the bound Fe2+ cofactor. The resulting oxidized protein is inactive for DNA-binding. The OhrR protein contains a highly reactive cysteine residue that reacts with organic peroxides to generate a cysteine-sulfenic acid that then reacts with low molecular mass thiols to generate mixed-disulfides. Oxidized OhrR also forms a sulfenyl-amide derivative. These protein modifications inactivate the repressor leading to expression of organic hydroperoxide resistance. This project addresses the detailed mechanisms by which protein oxidation regulates the activity of these two proteins. This research will determine whether oxidized PerR is degraded, or the subject of a novel protein repair pathway. The effects of protein oxidation on OhrR will be investigated, and proteins that contribute to reactivation of OhrR will be defined. Related regulatory proteins are present in diverse bacteria, including important plant and animal pathogens where they function to regulate genes important for virulence. This research will address fundamental aspects of protein chemistry and oxidation. Protein oxidation is important in the life of all cells and is thought to contribute to ageing and numerous age-related diseases. The project will train students at all levels in genetic and protein chemistry techniques.