Patterns of protein carbonylation following oxidative stress in wild-type and sigB Bacillus subtilis cells
Patterns of protein carbonylation following oxidative stress in wild-type and sigB Bacillus subtilis cells
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DOI：
10.1007/s00438-003-0877-4
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发表时间：
2003-08-01
影响因子：
3.1
通讯作者：
Hecker, M
中科院分区：
文献类型：
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作者：
Mostertz, J;Hecker, M
氧化应激导致核酸、膜脂和蛋白质的损伤。一个显著的效应是金属催化的蛋白质的位点特异性羰基化。在革兰氏阳性土壤细菌枯草芽孢杆菌中，PerR依赖的特定应激反应和sigma（B）依赖的一般应激反应共同作用，使细胞对氧化应激更具抗性。在这项研究中，我们分析了羰基化的细胞质蛋白质在响应过氧化氢应激B。枯草杆菌。此外，我们还询问了对氧化应激的sigma（B）依赖性反应是否也赋予了对蛋白质羰基化的保护。为了监测蛋白质损伤的量和特异性，用2，4-二硝基苯肼衍生羰基，并通过一维或二维凝胶电泳分离的蛋白质的免疫分析来检测所得稳定的腙。蛋白质羰基化的总体水平在用过氧化氢处理的细胞中强烈增加。几种蛋白质，包括延伸因子EF-G，TufA和EF-Ts，被发现高度羰基化。在暴露于其他致死水平的过氧化物之前，通过亚致死过氧化物浓度处理诱导过氧化物特异性应激反应，显著降低了蛋白质羰基化的程度。与指数生长的细胞相比，葡萄糖饥饿的细胞也仅显示少量的过氧化物介导的蛋白质羰基化。我们不能检测到野生型和DeltasigB细胞之间的任何差异饥饿的葡萄糖或预适应热处理的蛋白质损伤的量或特异性后，随后暴露于过氧化应激。然而，当细胞随后经受氧化应激时，用通常由sigma（B）依赖性机制诱导的蛋白质进行人工预加载导致较低水平的蛋白质羰基化。
Oxidative stress causes damage to nucleic acids, membrane lipids and proteins. One striking effect is the metal-catalyzed, site-specific carbonylation of proteins. In the gram-positive soil bacterium Bacillus subtilis, the PerR-dependent specific stress response and the sigma(B)-dependent general stress response act together to make cells more resistant to oxidative stress. In this study, we analyzed the carbonylation of cytoplasmic proteins in response to hydrogen peroxide stress in B. subtilis. Furthermore, we asked whether the sigma(B)-dependent response to oxidative stress also confers protection against protein carbonylation. To monitor the amount and specificity of protein damage, carbonyls were derivatized with 2,4-dinitrophenylhydrazine, and the resulting stable hydrazones were detected by immunoanalysis of proteins separated by one- or two-dimensional gel electrophoresis. The overall level of protein carbonylation increased strongly in cells treated with hydrogen peroxide. Several proteins, including the elongation factors EF-G, TufA and EF-Ts, were found to be highly carbonylated. Induction of the peroxide specific stress response by treatment with sub-lethal peroxide concentrations, prior to exposure to otherwise lethal levels of peroxide, markedly reduced the degree of protein carbonylation. Cells starved for glucose also showed only minor amounts of peroxide-mediated protein carbonylation compared to exponentially growing cells. We could not detect any differences between wild-type and DeltasigB cells starved for glucose or preadapted by heat treatment with respect to the amount or specificity of protein damage incurred upon subsequent exposure to peroxide stress. However, artificial preloading with proteins that are normally induced by sigma(B)-dependent mechanisms resulted in a lower level of protein carbonylation when cells were later subjected to oxidative stress.