Free Radical Research Group, Department of Pathology and the National Research Centre for Growth and Development, University of Otago Christchurch, P.O. Box 4345, Christchurch, New Zealand.
Arch Biochem Biophys. 2012 Nov 1;527(1):45-54. doi: 10.1016/j.abb.2012.07.011. Epub 2012 Jul 31.
Neutrophil oxidants, including the myeloperoxidase products, HOCl and chloramines, have been linked to endothelial dysfunction in inflammatory diseases such as atherosclerosis. As they react preferentially with sulfur centers, thiol proteins are likely to be cellular targets. Our objectives were to establish whether there is selective protein oxidation in vascular endothelial cells treated with HOCl or chloramines, and to identify sensitive proteins. Cells were treated with HOCl, glycine chloramine and monochloramine, reversibly oxidized cysteines were labeled and separated by 1D or 2D SDS-PAGE, and proteins were characterized by mass spectrometry. Selective protein oxidation was observed, with chloramines and HOCl causing more changes than H(2)O(2). Cyclophilin A was one of the most sensitive targets, particularly with glycine chloramine. Cyclophilin A was also oxidized in Jurkat T cells where its identity was confirmed using a knockout cell line. The product was a mixed disulfide with glutathione, with glutathionylation at Cys-161. Glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxins and cofilin were also highly sensitive to HOCl/chloramines. Cyclophilins are becoming recognized as redox regulatory proteins, and glutathionylation is an important mechanism for redox regulation. Cells lacking Cyclophilin A showed more glutathionylation of other proteins than wild-type cells, suggesting that cyclophilin-regulated deglutathionylation could contribute to redox changes in HOCl/chloramine-exposed cells.
中性粒细胞氧化剂，包括髓过氧化物酶产物次氯酸 (HOCl) 和氯胺，与动脉粥样硬化等炎症性疾病中的内皮功能障碍有关。由于它们优先与硫中心反应，因此巯基蛋白很可能是细胞靶标。我们的目标是确定用 HOCl 或氯胺处理的血管内皮细胞中是否存在选择性蛋白氧化，并确定敏感蛋白。用 HOCl、甘氨酸氯胺和单氯胺处理细胞，用 1D 或 2D SDS-PAGE 标记并分离可逆氧化的半胱氨酸，然后通过质谱法对蛋白质进行表征。观察到选择性蛋白氧化，氯胺和 HOCl 引起的变化比 H2O2 多。亲环蛋白 A 是最敏感的靶标之一，尤其是用甘氨酸氯胺处理时。亲环蛋白 A 在 Jurkat T 细胞中也被氧化，在使用敲除细胞系的情况下证实了其身份。产物是与谷胱甘肽的混合二硫键，在 Cys-161 处发生谷氨酰化。甘油醛-3-磷酸脱氢酶、过氧化物酶和丝切蛋白也对 HOCl/氯胺高度敏感。亲环蛋白正在被认为是氧化还原调节蛋白，谷氨酰化是氧化还原调节的重要机制。缺乏亲环蛋白 A 的细胞比野生型细胞表现出更多的其他蛋白质的谷氨酰化，这表明亲环蛋白调节的去谷氨酰化可能有助于 HOCl/氯胺暴露细胞中的氧化还原变化。
