Free radical-operated proteotoxic stress in macrophages primed with lipopolysaccharide.
Free radical-operated proteotoxic stress in macrophages primed with lipopolysaccharide.
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DOI：
10.1016/j.freeradbiomed.2012.04.023
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发表时间：
2012-07-01
影响因子：
7.4
通讯作者：
Ramirez, Dario C.
中科院分区：
文献类型：
--
作者：
Zhai, Zili;Gomez-Mejiba, Sandra E.;Gimenez, Maria S.;Deterding, Leesa J.;Tomer, Kenneth B.;Mason, Ronald P.;Ashby, Michael T.;Ramirez, Dario C.
炎症部位活化巨噬细胞的自由基操作死亡机制尚不清楚，但重要的是对其进行定义，以便找到防止进一步组织功能障碍的靶点。炎症部位巨噬细胞激活的明确模型是用脂多糖 (LPS) 处理 RAW 264.7 细胞，从而产生活性氧 (ROS)。 ROS 和其他自由基可以用硝酮自旋陷阱 5,5-二甲基-1-吡咯啉 N-氧化物 (DMPO) 捕获，这是一种具有抗氧化特性的细胞渗透性探针，从而干扰自由基操作的氧化过程。在这里，我们使用免疫自旋捕获来研究自由基操作的蛋白质氧化在 LPS 诱导的巨噬细胞细胞毒性中的作用。用 LPS 处理 RAW 264.7 细胞会导致 ROS 产生增加、蛋白质氧化、细胞形态变化和细胞毒性增加。研究发现 DMPO 可以捕获蛋白质自由基，形成蛋白质-DMPO 硝酮加合物，减少蛋白质羰基，并阻止 LPS 诱导的细胞死亡。 N-乙酰半胱氨酸（还原型谷胱甘肽的来源）、二亚苯基碘鎓（NADPH 氧化酶的抑制剂）和 2,2'-联吡啶（Fe2+ 螯合剂）可防止 LPS 诱导的氧化应激和细胞死亡，并减少 DMPO-硝酮加合物的形成，表明 ROS、金属和蛋白质自由基的形成在 LPS 诱导的细胞毒性中发挥着关键作用。我们还确定了蛋白质-DMPO 硝酮加合物的亚细胞定位，并通过 LC-MS/MS 鉴定了一些用于 DMPO 附着的候选蛋白质。 LC-MS/MS 数据与 3-磷酸甘油醛脱氢酶一致，甘油醛是细胞中最丰富、最敏感且普遍存在的蛋白质之一，当细胞用 LPS 引发时，它会被 DMPO 标记。这些信息将有助于找到治疗炎症相关组织功能障碍的策略，重点关注预防自由基操作的蛋白毒性应激和巨噬细胞死亡。
The free-radical-operated mechanism of death of activated macrophages at sites of inflammation is unclear, but it is important to define it in order to find targets to prevent further tissue dysfunction. A well-defined model of macrophage activation at sites of inflammation is the treatment of RAW 264.7 cells with lipopolysaccharide (LPS), with the resulting production of reactive oxygen species (ROS). ROS and other free radicals can be trapped with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), a cell-permeable probe with antioxidant properties, which thus interferes with free-radical-operated oxidation processes. Here we have used immuno-spin trapping to investigate the role of free-radical-operated protein oxidation in LPS-induced cytotoxicity in macrophages. Treatment of RAW 264.7 cells with LPS resulted in increased ROS production, oxidation of proteins, cell morphological changes and cytotoxicity. DMPO was found to trap protein radicals to form protein–DMPO nitrone adducts, to reduce protein carbonyls, and to block LPS-induced cell death. N-Acetylcysteine (a source of reduced glutathione), diphenyleneiodonium (an inhibitor of NADPH oxidase), and 2,2′-dipyridyl (a chelator of Fe2+) prevented LPS-induced oxidative stress and cell death and reduced DMPO–nitrone adduct formation, suggesting a critical role of ROS, metals, and protein-radical formation in LPS-induced cell cytotoxicity. We also determined the subcellular localization of protein–DMPO nitrone adducts and identified some candidate proteins for DMPO attachment by LC-MS/MS. The LC-MS/MS data are consistent with glyceraldehyde-3-phosphate dehydrogenase, one of the most abundant, sensitive, and ubiquitous proteins in the cell, becoming labeled with DMPO when the cell is primed with LPS. This information will help find strategies to treat inflammation-associated tissue dysfunction by focusing on preventing free radical-operated proteotoxic stress and death of macrophages.
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影响因子：
4.8
作者：
Gomez-Mejiba, Sandra E.;Zhai, Zili;Ramirez, Dario C.
通讯作者：
Ramirez, Dario C.
影响因子：
5.3
作者：
Cassina, Patricia;Cassina, Adriana;Radi, Rafael
通讯作者：
Radi, Rafael
影响因子：
7.4
作者：
Domazou, Anastasia S.;Koppenol, Willem H.;Gebicki, Janusz M.
通讯作者：
Gebicki, Janusz M.
DOI：
10.1038/nri3071
发表时间：
2011-10-10
期刊：
Nature reviews. Immunology
影响因子：
--
作者：
通讯作者：
--
DOI：
10.1111/j.1582-4934.2006.tb00407.x
发表时间：
2006-04
影响因子：
5.3
作者：
Dalle-Donne I;Aldini G;Carini M;Colombo R;Rossi R;Milzani A
通讯作者：
Milzani A