Mutation in G6PD gene leads to loss of cellular control of protein glutathionylation: Mechanism and implication
Mutation in G6PD gene leads to loss of cellular control of protein glutathionylation: Mechanism and implication
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DOI：
10.1002/jcb.21394
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发表时间：
2008-01-01
影响因子：
4
通讯作者：
Koch, Cameron J.
中科院分区：
文献类型：
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作者：
Ayene, Iraimoudi S.;Biaglow, John E.;Koch, Cameron J.
由于葡萄糖-6-磷酸脱氢酶(G6PD)缺乏，超过4亿人容易受到氧化应激的影响。蛋白质谷胱甘肽基化被认为是氧化应激过程中蛋白质功能和/或细胞信号丧失的原因。为了阐明G6PD缺乏对蛋白质谷胱甘肽基化的细胞控制的影响，我们使用了羟乙基二硫化物(HEDS)，这是一种与现有硫醇进行二硫键交换的氧化剂。与野生型(K1)细胞相比，经HEDS处理的G6PD缺陷型(E89)细胞的蛋白质谷胱甘肽基化显著增加。为了确定HEDS是否导致一种重要蛋白质的功能丧失，我们利用一种新的ELISA法比较了HEDS对全球蛋白质谷胱甘肽基化的影响和对多功能DNA修复蛋白Ku蛋白功能的影响。经HEDS处理的E89细胞显示Ku蛋白与DNA的结合显著丧失。HEDS可降低E89细胞的细胞蛋白硫醇和谷胱甘肽，而对K1细胞无明显影响。与K1细胞相比，E89细胞对HEDS的解毒作用较低，即将二硫化物HEDS转化为游离的巯基乙醇(ME)。在HEDS存在的情况下，K1细胞的NADH水平保持不变，而E89细胞的NADH水平在类似的暴露后下降了10倍。NADPH是一种维持硫醇还原形式所需的辅因子，在E89细胞中比K1细胞中减少得更多。通过将G6PD基因重新导入到表型正常的E89(A1A)细胞中，进一步证明了G6PD在控制这种全局蛋白谷胱甘肽基化和Ku功能中的特殊作用。
More than 400 million people are susceptible to oxidative stress due to glucose-6-phosphate dehydrogenase (G6PD) deficiency. Protein glutathionylation is believed to be responsible for loss of protein function and/or cellular signaling during oxidative stress. To elucidate the implications of G6PD deficiency specifically in cellular control of protein glutathionylation, we used hydroxyethyldisulfide (HEDS), an oxidant which undergoes disulfide exchange with existing thiols. G6PD deficient (E89) cells treated with HEDS showed a significant increase in protein glutathionylation compared to wild-type (K1) cells. In order to determine whether increase in global protein glutathionylation by HEDS leads to loss of function of an important protein, we compared the effect of HEDS on global protein glutathionylation with that of Ku protein function, a multifunctional DNA repair protein, using a novel ELISA. E89 cells treated with HEDS showed a significant loss of Ku protein binding to DNA. Cellular protein thiol and GSH, whose disulfide is involved in protein glutathionylation, were decreased by HEDS in E89 cells with no significant effect in K1 cells. E89 cells showed lower detoxification of HEDS, that is, conversion of disulfide HEDS to free sulfhydryl mercaptoethanol (ME), compared to K1 cells. K1 cells maintained their NADH level in the presence of HEDS but that of E89 cells decreased by tenfold following a similar exposure. NADPH, a cofactor required to maintain reduced form of the thiols, was decreased more in E89 than K1 cells. The specific role of G6PD in the control of such global protein glutathionylation and Ku function was further demonstrated by reintroducing the G6PD gene into E89 (A1A) cells, which showed a normal phenotype.