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.
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 oxidoreductase1 (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.
Bloom
David
D
Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, TX 77030, USA.
Dhakshinamoorthy
Saravanakumar
S
Jaiswal
Anil K
AK
D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005033:Ethylmaleimide / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D051259:MafG Transcription Factor; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D051267:NF-E2-Related Factor 2; D012097:Repressor Proteins / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000975:Antioxidants; D004268:DNA-Binding Proteins; C103944:MAFG protein, human; D051259:MafG Transcription Factor; C491247:Mafg protein, mouse; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; C495636:Nfe2l2 protein, mouse; D012097:Repressor Proteins; D015534:Trans-Activators; D012694:Serine; D009247:NADH, NADPH Oxidoreductases; C072396:NAD(P)H plastoquinone oxidoreductase; D003545:Cysteine; D005033:Ethylmaleimide