The Heart Research Institute, Newtown, Sydney, NSW 2042, Australia.
Free Radic Biol Med. 2010 Apr 15;48(8):1071-8. doi: 10.1016/j.freeradbiomed.2010.01.025. Epub 2010 Jan 25.
Hydroperoxides are major products of the reactions of radicals and singlet oxygen with amino acids, peptides, and proteins. These species can generate radicals in the presence of metal ions and oxidize thiols via nonradical reactions, but the effects of these materials on cells are poorly understood. In this study the exposure of murine macrophage-like cells to preformed peptide or protein hydroperoxides is shown to result in hydroperoxide consumption and cellular thiol oxidation; these effects precede loss of cell viability. N-acetyltryptophan methyl ester hydroperoxides, but not the decomposed species, decreased total cellular thiols and GSH, with the latter occurring more rapidly. Time-dependent inhibition of lysosomal cathepsins B and L was also observed, together with diminished caspase 3/7 activity. A number of other cytosolic thiol- and non-thiol-dependent enzymes were not affected significantly. Hydroperoxides formed on BSA did not deplete total thiols or GSH within cells, although such reactions are rapid in model systems. In contrast, selective inhibition of cathepsins B and L (but not cathepsin D or arylsulfatase) of the endosomal-lysosomal system was detected, consistent with localization within these compartments. Decomposed BSA hydroperoxides did not induce these effects, indicating a requirement for the hydroperoxide group. The differences between these hydroperoxides are ascribed to their mechanisms of penetration into cells. Overall these studies provide valuable data on the initial cellular events arising from exposure to exogenous protein and amino acid peroxides and indicate that cellular thiols are a major target. This selective oxidation may modulate cellular redox balance and subsequent cell behavior.
氢过氧化物是自由基和单线态氧与氨基酸、肽和蛋白质反应的主要产物。这些物质在金属离子存在下可以产生自由基，并通过非自由基反应氧化硫醇，但这些物质对细胞的影响还知之甚少。在这项研究中，暴露于预先形成的肽或蛋白质氢过氧化物的鼠巨噬细胞样细胞导致氢过氧化物消耗和细胞硫醇氧化；这些效应先于细胞活力丧失。N-乙酰色氨酸甲酯氢过氧化物，但不是分解产物，降低了总细胞硫醇和 GSH，后者发生得更快。还观察到溶酶体组织蛋白酶 B 和 L 的时间依赖性抑制，以及 caspase 3/7 活性的降低。许多其他细胞溶质硫醇和非硫醇依赖性酶没有受到显著影响。BSA 形成的氢过氧化物不会使细胞内总硫醇或 GSH 耗尽，尽管在模型系统中这些反应很快。相比之下，仅抑制溶酶体-内体系统中的组织蛋白酶 B 和 L（而不是组织蛋白酶 D 或芳基硫酸酯酶）被检测到，这与这些隔室中的定位一致。分解的 BSA 氢过氧化物不会诱导这些效应，表明需要氢过氧化物基团。这些氢过氧化物之间的差异归因于它们进入细胞的机制。总的来说，这些研究提供了关于暴露于外源性蛋白质和氨基酸过氧化物引起的初始细胞事件的有价值的数据，并表明细胞硫醇是主要靶标。这种选择性氧化可能调节细胞氧化还原平衡和随后的细胞行为。
