Regulation of thiol-specific electrophile resistance mechanisms in Bacillus subtilis

新型Marr型阻遏蛋白YodB、YvaP和MhqR通过控制同源的硫醇依赖的双加氧酶和氧化还原酶使枯草芽孢杆菌对亲电性的苯醌和联胺产生抗性。YodB和YvaP是氧化还原敏感型MARR/DUF24家族调节剂，它们通过保守的N-末端半胱氨酸残基来感知类似苯醌的亲电体和联胺。先前的数据表明，YodB抑制物在体外是通过对苯二酚的硫醇(S)烷基化反应来调节的。蛋白质组学和硫醇氧化还原蛋白质组学研究表明，在体内，醌类化合物不可逆地烷基化并聚集含有硫醇的蛋白质。相反，联胺处理会导致体内可逆的二硫键的形成。在本项目中，我们将在体外和体内研究YodB和YvaP对苯二酚和联胺的详细传感机制。此外，参与MhqR去抑制的调控机制也是本项目进一步关注的重点。将分析新型的硫醇依赖的双加氧酶和还原酶在苯醌和联胺解毒中的作用。除YodB和YvaP外，在枯草芽孢杆菌基因组中还编码了另外6个YodB同源基因(YdeP、YbbR、YkvN、YdzF、YcdF和HxlR)，这些同源基因将通过全基因组分析和详细的生化和遗传学方法进行表征。除了苯二酚，羰基化合物(如甲醛或乙二醛)是天然的亲电性物质，最有可能通过硫醇-(S)-烷基化化学修饰含有硫醇的蛋白质，耗尽细胞内的硫醇库。新的MerR/NmlR家族调节子AdhR(YraB)被鉴定为醛特异性感受器，而AdhR的激活需要体内保守的Cys52。AdhR控制着一种硫醇依赖的甲醛脱氢酶(ADHA)和半胱氨酸蛋白酶YraA，这两种酶都可以保护细胞免受FA的毒性。鉴定AdhR和细胞蛋白硫醇酸盐中甲醛和甲基乙二醛引起的翻译后修饰是该项目的另一个重点。ADHA和YraA在醛的解毒和受损的含硫蛋白的修复或降解中的作用将被研究。

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.