期刊
ONCOGENE
卷 21, 期 14, 页码 2191-2200出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/sj.onc.1205288
关键词
NQO1; ARE; detoxifying genes; Nrf2; cysteine; redox regulation
资金
- NIGMS NIH HHS [GM47466] Funding Source: Medline
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.
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