Sierra-Rivera E, Meredith M J, Voorhees G J, Oberley L W, Eisert D R, Freeman M L
Vanderbilt Center for Radiation Oncology, Vanderbilt University School of Medicine, Nashville, TN 37232.
Int J Hyperthermia. 1994 Jul-Aug;10(4):573-86. doi: 10.3109/02656739409009359.
The relationship between glutathione metabolism, menadione sodium bisulphite oxidation of protein thiols, and the synthesis of hsc70 was investigated using CHO cells. A 30-min/37 degrees C exposure to menadione, a compound which redox cycles to produce superoxide anion radicals and hydrogen peroxide, resulted in rapid accumulation of hsc70 mRNA. PAGE and Western blot analysis indicated increased synthesis such that accumulation of hsc70 occurred. These changes were preceded by rapid oxidation of GSH to GSSG, followed by GSH depletion, and subsequent protein thiol oxidation. As a test of whether a correlation existed between GSH oxidation and depletion, protein thiol oxidation and hsp synthesis, cells were exposed to menadione in the absence and presence of glucose. Synthesis of hsc70 was increased in cells exposed to menadione in the absence of glucose compared with its presence. As a further test, cells were exposed to BSO/DEM in order to deplete GSH and then exposed to menadione. The synthesis of hsc70 following exposure to menadione was greatly increased in GSH-depleted cells compared with GSH-replete cells. Experiments were conducted to determine if electroporation of cells in GSSG containing buffer affected hsp synthesis. Electroporation in glucose-free buffer containing 3 mM GSSG did not affect hsp synthesis. We interpret these results to indicate that the inability to maintain glutathione in a reduced form during menadione redox cycling resulted in protein thiol oxidation. This, in turn, resulted in accumulation of hsc70 mRNA with a subsequent increase in the synthesis of hsc70.
利用CHO细胞研究了谷胱甘肽代谢、甲萘醌亚硫酸氢钠对蛋白质硫醇的氧化作用以及热休克蛋白70（hsc70）的合成之间的关系。将细胞暴露于甲萘醌30分钟/37℃，甲萘醌这种化合物通过氧化还原循环产生超氧阴离子自由基和过氧化氢，导致hsc70 mRNA迅速积累。聚丙烯酰胺凝胶电泳（PAGE）和蛋白质免疫印迹分析表明合成增加，从而出现hsc70的积累。这些变化之前是谷胱甘肽（GSH）迅速氧化为氧化型谷胱甘肽（GSSG），随后GSH耗竭，接着是蛋白质硫醇氧化。作为对GSH氧化与耗竭、蛋白质硫醇氧化和热休克蛋白（hsp）合成之间是否存在相关性的测试，细胞在有无葡萄糖的情况下暴露于甲萘醌。与有葡萄糖存在时相比，在无葡萄糖情况下暴露于甲萘醌的细胞中hsc70的合成增加。作为进一步的测试，细胞先暴露于丁硫氨酸亚砜胺/二乙胺基二硫代甲酸钠（BSO/DEM）以耗尽GSH，然后再暴露于甲萘醌。与GSH充足的细胞相比，GSH耗尽的细胞在暴露于甲萘醌后hsc70的合成大大增加。进行实验以确定在含有GSSG的缓冲液中对细胞进行电穿孔是否会影响hsp合成。在含有3 mM GSSG的无葡萄糖缓冲液中进行电穿孔不影响hsp合成。我们对这些结果的解释是，在甲萘醌氧化还原循环过程中无法将谷胱甘肽维持在还原形式导致蛋白质硫醇氧化。这进而导致hsc70 mRNA积累，随后hsc70的合成增加。
