McDonagh Brian, Sheehan David
Proteomics Research Group, Department of Biochemistry and Environmental Research Institute, University College Cork, Cork, Ireland.
Proteomics. 2007 Sep;7(18):3395-403. doi: 10.1002/pmic.200700241.
Protein thiols are targets of oxidative stress. Their modification was analysed in gill extracts of the mussel Mytilus edulis, exposed to menadione. Diagonal gel electrophoresis revealed two clusters of carbonylated proteins involved in interchain disulphide linkages. Immunoblotting identified these as being associated with protein disulphide isomerase (PDI) and actin and this was confirmed by immunoprecipitation. Protein free thiols (-SH) were identified in 2-DE separations by labelling with 5-iodoacetamidofluorescein (IAF). Cysteines involved in disulphide bridges were identified by blocking free -SH with N-ethylmaleimide, reducing disulphides with DTT and IAF labelling. Several protein spots containing free thiols disappeared on exposure to menadione. Conversely, new protein spots containing disulphides appeared in response to menadione which may be protective against oxidative stress. In-gel tryptic digestion followed by LC/MS-MS and database searching identified some of the free thiol targets: PDI; hsp gp96; calreticulin; heavy metal binding protein. Tubulin, PDI, enolase and gelsolin contained new disulphide bridges in response to menadione. Our findings indicate a protein level response to oxidative stress principally involving PDI, chaperone-like and cytoskeletal proteins. Since many environmental pollutants cause oxidative stress, studies on PDI and structural proteins may be particularly relevant to understanding toxicity in this popular sentinel species.
蛋白质硫醇是氧化应激的靶点。在暴露于甲萘醌的紫贻贝鳃提取物中分析了它们的修饰情况。对角线凝胶电泳显示了参与链间二硫键连接的两组羰基化蛋白质。免疫印迹鉴定这些蛋白质与蛋白质二硫键异构酶（PDI）和肌动蛋白有关，免疫沉淀证实了这一点。通过用5-碘乙酰氨基荧光素（IAF）标记在二维电泳分离中鉴定蛋白质游离硫醇（-SH）。通过用N-乙基马来酰亚胺封闭游离-SH、用二硫苏糖醇（DTT）还原二硫键并进行IAF标记来鉴定参与二硫键的半胱氨酸。暴露于甲萘醌后，几个含有游离硫醇的蛋白质斑点消失。相反，响应甲萘醌出现了含有二硫键的新蛋白质斑点，这可能对氧化应激具有保护作用。凝胶内胰蛋白酶消化，随后进行液相色谱/串联质谱分析和数据库搜索，鉴定了一些游离硫醇靶点：PDI；热休克蛋白gp96；钙网蛋白；重金属结合蛋白。微管蛋白、PDI、烯醇化酶和凝溶胶蛋白响应甲萘醌含有新的二硫键。我们的研究结果表明，蛋白质水平对氧化应激的反应主要涉及PDI、伴侣样蛋白和细胞骨架蛋白。由于许多环境污染物会导致氧化应激，对PDI和结构蛋白的研究可能与理解这种常见指示物种的毒性特别相关。
