Site-directed mutagenesis of cysteine to serine in the DNA binding region of Nrf2 decreases its capacity to upregulate antioxidant response element-mediated expression and antioxidant induction of NAD(P)H:quinone oxidoreductase1 gene
Site-directed mutagenesis of cysteine to serine in the DNA binding region of Nrf2 decreases its capacity to upregulate antioxidant response element-mediated expression and antioxidant induction of NAD(P)H:quinone oxidoreductase1 gene
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DOI：
10.1038/sj.onc.1205288
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发表时间：
2002-03-28
期刊：
影响因子：
8
通讯作者：
Jaiswal, AK
中科院分区：
文献类型：
--
作者：
Bloom, D;Dhakshinamoorthy, S;Jaiswal, AK
NF-E2相关因子2（Nrf 2）是CNC/b-zip蛋白，其调节解毒酶基因（包括NAD（P）H：醌氧化还原酶1（NQO 1））的抗氧化反应元件（ARE）介导的表达和抗氧化诱导。来自不同物种的Nrf 2以及与其他b-zip蛋白的比较揭示了Nrf 2的DNA结合结构域中的位置506处存在高度保守的半胱氨酸残基。使用定点诱变将该半胱氨酸突变为丝氨酸。在人肝母细胞瘤（Hep-G2）细胞中的转染/过表达实验表明，含有C506 S突变的突变体Nrf 2（mNrf 2）在激活ARE介导的基因表达和响应于叔丁基对苯二酚（t-BHQ）的诱导方面的效率显著低于野生型Nrf 2。N-乙基马来酰亚胺（NEM），一种巯基交联剂，抑制Nrf 2，但不抑制mNrf 2C 506 S介导的NQO 1表达。这进一步暗示了在ARE介导的基因表达的Nrf 2调节中的位置506处的半胱氨酸。核定位实验表明，C506 S突变并不影响Nrf 2的保留INrf 2/Keap 1在胞质溶胶中，或其释放响应抗氧化剂。然而，条带和超位移分析显示，与野生型Nrf 2相比，mNrf 2C 506 S与NQO 1基因ARE的结合显著减少。因此，Nrf 2中的C506 S突变降低了其对ARE的亲和力，导致NQO 1的表达和抗氧化诱导降低。
NF-E2 related factor 2 (Nrf2) is a CNC/b-zip protein that regulates antioxidant response element (ARE)mediated expression, and antioxidant induction, of detoxifying enzyme genes, including NAD(P)H:quinone oxidoreductasel (NQO1). A comparison of Nrf2 from different species, and with other b-zip proteins, revealed the presence of a highly conserved cysteine residue at position 506 in the DNA binding domain of Nrf2. Site-directed mutagenesis was used to mutate this cysteine, to serine. Transfection/over expression experiments in human hepatoblastoma (Hep-G2) cells demonstrated that mutant Nrf2 (mNrf2), containing the C506S mutation, was significantly less efficient in activating ARE-mediated gene expression, and induction in response to tert-butyl hydroquinone (t-BHQ), as copmpared with wild-type Nrf2. N-ethyl malemide (NEM), a sulfhydryl cross- linker, inhibited Nrf2 but not mNrf2C506S-mediated expression of NQO1. This further implicated the cysteine at position 506 in Nrf2 regulation of ARE-mediated gene expression. Nuclear localization experiments revealed that C506S mutation did not affect the retention of Nrf2 by INrf2/Keap1 in the cytosol, or its release in response to antioxidants. However, band and supershift assays showed a significant reduction in the binding of mNrf2C506S to the NQO1 gene ARE as compared with wild-type Nrf2. Therefore, the C506S mutation in Nrf2 lowered its affinity for the ARE, leading to decreased expression, and antioxidant induction, of NQO1.