Hydrogen Peroxide as Intracellular Messenger

去年，我们发现某些真核生物的过氧化还蛋白(PRX)酶活性部位的半胱氨酸残基在催化过程中会发生可逆氧化为亚磺酸(Cys？SOOH)。一种名为硫氧还蛋白(SRX)的酶已被确定为逆转酵母中所产生的酶失活。我们现在已经确定了酵母SRX的哺乳动物同源物的特征，该方法基于使用针对亚磺酸状态的抗体的免疫印迹分析来监测亚磺酸PRX的还原。哺乳动物SRX的亚磺酸还原是一个缓慢的过程(kcat=0.18/min)，需要ATP水解酶。可以有效地用GTP、dATP或dGTP取代ATP，但不能用CTP、UTP、dCTP或dTTP取代。谷胱甘肽和硫氧还蛋白都是SRX反应的潜在生理电子供体，因为它们的Km值(分别为1.8 mM和1.2微米)在其胞内浓度范围内，而两种还原剂得到的Vmax值相似。虽然SRX的pKa相对较低(~7.3)，但即使当大多数PRX分子的活性中心半胱氨酸被氧化时，SRX的活性中心半胱氨酸仍保持还原状态。最后，通过RNA干扰去除人SRX表明，SRX在很大程度上减少了A549人细胞中PRX的Cys？SOOH。 各种促凋亡刺激可增加线粒体产生超氧化物歧化酶和过氧化氢。虽然超氧化物歧化损伤线粒体功能，并被依赖于Mn2+的超氧化物歧化酶清除，但线粒体过氧化氢22在细胞凋亡中的作用和代谢尚不清楚。现已通过RNA干扰在HeLa细胞中研究了线粒体特异的过氧化氢清除酶--过氧化还蛋白(PRX)III的缺失对细胞凋亡信号的影响。PRX III的缺失导致细胞内过氧化氢水平增加，并使细胞对星形孢子素或肿瘤坏死因子-α诱导的细胞凋亡敏感。在Prx III？耗竭的细胞中，线粒体膜电位崩溃、细胞色素c释放和caspase激活的速率增加，这些作用可被Prx III或线粒体靶向过氧化氢酶的异位表达所逆转。PRX III的缺失也加剧了促凋亡刺激对线粒体大分子的损伤。我们的结果表明，Prx III是线粒体H_2O_2丰度的关键调节因子，线粒体H_2O_2本身与其他凋亡信号调节因子协同促进细胞凋亡。

Last year we showed that the cysteine residue in the active site of certain eukaryotic peroxiredoxin (Prx) enzymes undergoes reversible oxidation to sulfinic acid (Cys?SOOH) during catalysis. An enzyme named sulfiredoxin (Srx) has been identified as responsible for reversal of the resulting enzyme inactivation in yeast. We have now characterized mammalian orthologs of yeast Srx with an assay based on monitoring of the reduction of sulfinic Prx by immunoblot analysis with antibodies specific for the sulfinic state. Sulfinic reduction by mammalian Srx was found to be a slow process (kcat = 0.18/min) that requires ATP hydrolysis. ATP could be efficiently replaced by GTP, dATP, or dGTP but not by CTP, UTP, dCTP, or dTTP. Both glutathione and thioredoxin are potential physiological electron donors for the Srx reaction, given that their Km values (1.8 mM and 1.2 microM, respectively) are in the range of their intracellular concentrations and the Vmax values obtained with the two reductants were similar. Although its pKa is relatively low (~7.3), the active site cysteine of Srx remained reduced even when the active site cysteine of most Prx molecules became oxidized. Finally, depletion of human Srx by RNA interference suggested that Srx is largely responsible for reduction of the Cys?SOOH of Prx in A549 human cells. Various proapoptotic stimuli increase the production of superoxide and H2O2 by mitochondria. Whereas superoxide impairs mitochondrial function and is removed by Mn2+-dependent superoxide dismutase, the role and metabolism of mitochondrial H2O22 during apoptosis have remained unclear. The effects on apoptotic signaling of depletion of peroxiredoxin (Prx) III, a mitochondrion-specific H2O2-scavenging enzyme, have now been investigated by RNA interference in HeLa cells. Depletion of Prx III resulted in increased intracellular levels of H2O2 and sensitized cells to induction of apoptosis by staurosporine or TNF-alpha. The rates of mitochondrial membrane potential collapse, cytochrome c release, and caspase activation were increased in Prx III?depleted cells, and these effects were reversed by ectopic expression of Prx III or mitochondrion-targeted catalase. Depletion of Prx III also exacerbated damage to mitochondrial macromolecules induced by the proapoptotic stimuli. Our results suggest that Prx III is a critical regulator of the abundance of mitochondrial H2O2, which itself promotes apoptosis in cooperation with other mediators of apoptotic signaling.