Fluorescence thiol modification assay:: oxidatively modified proteins in Bacillus subtilis
Fluorescence thiol modification assay:: oxidatively modified proteins in Bacillus subtilis
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DOI：
10.1111/j.1365-2958.2005.04845.x
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发表时间：
2005-10-01
影响因子：
3.6
通讯作者：
Hecker, M
中科院分区：
文献类型：
--
作者：
Hochgräfe, F;Mostertz, J;Hecker, M
已知氧化修饰的半胱氨酸残基的硫醇基团可以调节越来越多的蛋白质的活性。在本研究中，我们建立了一种基于荧光的硫醇修饰实验，并将其与双向凝胶电泳法和质谱法相结合，以监测细胞质蛋白体内的硫醇状态。对于革兰氏阳性模式生物枯草芽孢杆菌，我们的结果表明生长细胞中的蛋白质硫醇主要以还原状态存在。只有几种蛋白质被发现是硫醇修饰的，例如在其催化循环中包含氧化的硫醇的酶。为了检测对氧化应激特别敏感的蛋白质，我们将正在生长的枯草杆菌细胞暴露在联胺、过氧化氢或超氧化物生成剂百草枯中。联胺能显著增加多种代谢酶中氧化硫醇的含量，而百草枯处理只影响少数蛋白质。暴露在过氧化氢下，尤其是具有半胱氨酸活性部位的蛋白质，如基于半胱氨酸的过氧化物酶和谷氨酰胺氨基转移酶样蛋白的氧化。此外，观察到高水平的过氧化氢影响了这一组蛋白质的等电点，表明产生了不可逆氧化的硫醇。从一组重叠的氧化修饰蛋白质中，还鉴定了蛋氨酸生物合成所必需的酶，例如钴胺非依赖性蛋氨酸合成酶。生长实验表明，在联胺和过氧化氢胁迫后，蛋氨酸受到限制，这表明蛋氨酸的失活依赖于硫醇的氧化。最后，有证据表明，抗生素呋喃妥因在枯草杆菌中参与了氧化硫醇的形成。
Oxidatively modified thiol groups of cysteine residues are known to modulate the activity of a growing number of proteins. In this study, we developed a fluorescence-based thiol modification assay and combined it with two-dimensional gel electrophoresis and mass spectrometry to monitor the in vivo thiol state of cytoplasmic proteins. For the Gram-positive model organism Bacillus subtilis our results show that protein thiols of growing cells are mainly present in the reduced state. Only a few proteins were found to be thiol-modified, e.g. enzymes that include oxidized thiols in their catalytic cycle. To detect proteins that are particularly sensitive to oxidative stress we exposed growing B. subtilis cells to diamide, hydrogen peroxide or to the superoxide generating agent paraquat. Diamide mediated a significant increase of oxidized thiols in a variety of metabolic enzymes, whereas treatment with paraquat affected only a few proteins. Exposure to hydrogen peroxide forced the oxidation especially of proteins with active site cysteines, e.g. of cysteine-based peroxidases and glutamine amidotransferase-like proteins. Moreover, high levels of hydrogen peroxide were observed to influence the isoelectric point of proteins of this group indicating the generation of irreversibly oxidated thiols. From the overlapping set of oxidatively modified proteins, also enzymes necessary for methionine biosynthesis were identified, e.g. cobalamin-independent methionine synthase MetE. Growth experiments revealed a methionine limitation after diamide and hydrogen peroxide stress, which suggests a thiol-oxidation-dependent inactivation of MetE. Finally, evidence is presented that the antibiotic nitrofurantoin mediates the formation of oxidized thiols in B. subtilis.