Regulation of thiol-specific electrophile resistance mechanisms in Bacillus subtilis

新的marr型抑制因子YodB， YvaP和MhqR通过控制旁系的硫醇依赖性双加氧酶和氧化还原酶，赋予枯草芽孢杆菌对亲电醌和二胺的抗性。YodB和YvaP是氧化还原敏感的MarR/DUF24家族调节因子，通过保守的n端Cys残基感知类醌亲电试剂和二胺。先前的数据表明YodB抑制因子在体外响应醌时通过巯基-(S)-烷基化调节。蛋白质组学和巯基氧化还原蛋白质组学研究表明，醌类在体内不可逆地烷基化和聚集含巯基的蛋白质。相反，二胺处理导致体内可逆的二硫键形成。本项目将对YodB和YvaP在体外和体内对醌和二胺的感知机制进行详细的研究。此外，本项目还将进一步关注与MhqR抑制相关的调控机制。分析了新型巯基双加氧酶和还原酶在醌和二胺解毒中的作用。除了YodB和YvaP外，枯草芽孢杆菌基因组中还编码了其他6个YodB类似物（YdeP、YybR、YkvN、YdzF、YcdF和HxlR），这些类似物将通过全基因组分析和详细的生化和遗传方法进行表征。除了醌类，羰基化合物（如甲醛或甲基乙二醛）是天然的亲电试剂，它们很可能通过硫醇-(S)-烷基化化学修饰含硫醇的蛋白质，消耗细胞中的硫醇池。新的MerR/ nmlr家族调节因子AdhR （YraB）被鉴定为醛特异性传感器，并且在体内激活AdhR需要保守的Cys52。AdhR控制巯基依赖性甲醛脱氢酶（AdhA）和半胱氨酸蛋白酶YraA，两者都保护细胞免受FA毒性。鉴定甲醛和甲基乙二醛在AdhR和细胞蛋白硫酸盐中引起的翻译后修饰是本项目的另一个重点。AdhA和YraA在醛解毒和修复或降解受损含硫醇蛋白中的功能将被研究。

The novel MarR-type repressors YodB, YvaP and MhqR confer resistance to electrophilic quinones and diamide in Bacillus subtilis via the control of paralogous thiol-dependent dioxygenases and oxidoreductases. YodB and YvaP are redox-sensitive MarR/DUF24 family regulators that sense quinone-like electrophiles and diamide via the conserved N-terminal Cys residue. Previous data suggest that the YodB repressor is regulated via thiol-(S)-alkylation in response to quinones in vitro. Proteomic and thiol-redox proteomic studies revealed that quinones irreversibly alkylate and aggregate thiol-containing proteins in vivo. In contrast, diamide treatment leads to reversible disulfide bond formation in vivo. In this project, the detailed mechanisms of quinone and diamide sensing by YodB and YvaP will be investigated in vitro and in vivo. In addition, the regulatory mechanisms that are involved in derepression of MhqR are further focused in this project. The functions of the novel thiol-dependent dioxygenases and reductases in quinone and diamide detoxification will be analyzed. Besides YodB and YvaP, six other YodB paralogs (YdeP, YybR, YkvN, YdzF, YcdF and HxlR) are encoded in the genome of B. subtilis which will be characterized using genome-wide analyses and detailed biochemical and genetic approaches. Besides quinones, carbonyl compounds (e.g. formaldehyde or methylglyoxal) are natural electrophiles, that most likely modify thiol-containing proteins via the thiol-(S)-alkylation chemistry, depleting the cellular thiol pool. The novel MerR/NmlR-family regulator AdhR (YraB) was identified as aldehyde-specific sensor and activation of AdhR required the conserved Cys52 in vivo. AdhR controls a thiol-dependent formaldehyde dehydrogenase (AdhA) and the cysteine proteinase YraA, both of which protect cells against FA toxicity. The identification of post-translational modifications caused by formaldehyde and methylglyoxal in AdhR and cellular protein thiolates is another focus of this project. The functions of AdhA and YraA in detoxification of aldehydes and repair or degradation of damaged thiol-containing proteins will be investigated.