Thioredoxin-like domains required for glucose regulatory protein 58-mediated reductive activation of mitomycin C leading to DNA cross-linking
Thioredoxin-like domains required for glucose regulatory protein 58-mediated reductive activation of mitomycin C leading to DNA cross-linking
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DOI：
10.1158/1535-7163.mct-07-0160
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发表时间：
2007-10-01
影响因子：
5.7
通讯作者：
Jaiswal, Anil K.
中科院分区：
文献类型：
--
作者：
Adikesavan, Anbu Karani;Jaiswal, Anil K.
已知葡萄糖调节蛋白（GRP 58）介导丝裂霉素C（MMC）诱导的DNA交联。然而，这一机制仍然难以捉摸。我们推测，硫氧还蛋白样结构域，一个在NH 2末端和另一个在COOH末端，需要GRP 58介导的MMC还原激活导致DNA交联。定点诱变将硫氧还蛋白结构域中的半胱氨酸突变为丝氨酸。将野生型（WT）和突变型GRP 58克隆到pcDNA中以在哺乳动物细胞中转染时产生GRP 58 V5标记的WT和突变蛋白。分析了瞬时表达WT和突变型GRP 58的人结肠癌（HCT 116）细胞和稳定表达WT和突变型GRP 58的中国仓鼠卵巢细胞的MMC诱导的DNA交联。WT GRP 58在MMC诱导的DNA交联中是高效的。然而，NH 2-和COOH-末端硫氧还蛋白突变体均显示出DNA交联中MMC的显著减少。GRP 58与硫氧还蛋白还原酶1的共表达和/或用NADPH处理细胞增加了MMC诱导的WT GRP 58的DNA交联。在类似的实验中，硫氧还蛋白还原酶1的siRNA抑制导致MMC诱导的DNA交联减少。进一步的实验表明，硫氧还蛋白结构域的突变导致MMC的代谢还原活化显著降低。这些结果得出结论，GRP 58通过其两个硫氧还蛋白样结构域，作为还原酶发挥功能，导致生物还原药物MMC活化和DNA交联。
Glucose regulatory protein (GRP58) is known to mediate mitomycin C (MMC)-induced DNA cross-linking. However, the mechanism remains elusive. We hypothesized that thioredoxin-like domains, one at NH2 terminus and another at COOH terminus, are required for GRP58-mediated MMC reductive activation leading to DNA cross-linking. Site-directed mutagenesis mutated cysteines in thioredoxin domains to serines. Wild-type (WT) and mutant GRP58 were cloned in pcDNA to produce GRP58 V5-tagged WT and mutant proteins on transfection in mammalian cells. Human colon carcinoma (HCT116) cells transiently expressing and Chinese hamster ovary cells stably expressing WT and mutant GRP58 were analyzed for MMC-induced DNA cross-linking. WT GRP58 was highly efficient in MMC-induced DNA cross-linking. However, both NH2- and COOH-terminal thioredoxin mutants showed significant reduction in MMC-in DNA cross-linking. The coexpression of GRP58 with thioredoxin reductase 1 and/or treatment of cells with NADPH increased MMC-induced DNA crosslinking from the WT GRP58. In similar experiments, siRNA inhibition of thioredoxin reductase 1 led to decreased MMC-induced DNA cross-linking. Further experiments revealed that mutations in thioredoxin domains led to significant decrease in metabolic reductive activation of MMC. These results led to conclusion that GRP58, through its two thioredoxin-like domains, functions as a reductase leading to bioreductive drug MMC activation and DNA cross-linking.