Li W, Yu SW, Kong ANNrf2 possesses a redox-sensitive nuclear exporting signal in the Neh5 transactivation domain. J Biol Chem 281: 27251-27263
Department of Pharmaceutics, Ernest-Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854, USA. Journal of Biological Chemistry
(Impact Factor: 4.57).
10/2006; 281(37):27251-63. DOI: 10.1074/jbc.M602746200
NF-E2-related factor 2 (Nrf2) is the key transcription factor regulating the antioxidant response. Previous studies identified a nuclear localization signal (NLS) in the basic region and a nuclear exporting signal (NES) in the leucine zipper domain of Nrf2. In this study, we characterize a new functional NES (175LLSIPELQCLNI186) in the transactivation (TA) domain of Nrf2. A green fluorescence protein (GFP)-tagged Nrf2 segment (amino acids162-295) called GFP-NESTA exhibited a cytosolic distribution that could be disrupted by L184A mutation or leptomycin B treatment. Chimeric expression of this NESTA with a nuclear protein GAL4DBD could expel GAL4DBD into the cytoplasm. A variety of oxidants, including sulforaphane, tert-butylhydroquinone, and H2O2, could effectively induce nuclear translocation of GFP-NESTA. Mutational studies showed that cysteine 183 may mediate the redox response of NESTA. The discovery of multiple NLS/NES motifs in Nrf2 and the redox sensitivity of NESTA imply Nrf2 may be self-sufficient to sense and transduce oxidative signals into the nucleus, consequently initiating antioxidant gene transcription.
Available from: Francesco Galli
- "Deciphering the functional properties of these diselenides may help in developing novel pharmacological modulators of GSTP signaling with selective effects on hormetic genes, which include a broad spectrum of drug metabolizing proteins involved in the detoxification and antioxidant response   . "
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ABSTRACT: Glutathione S-transferase π (GSTP), a phase II gene downstream of the Nrf2-ARE/EpRE transcription pathway, plays a key role in both the signaling and detoxification response to Se-organic compounds with thiol peroxidase activity. We here investigated the role of GSTP on the Nrf2 activation response of cells challenged with a new class of diselenides derived from the basic structure of (PhSe)2. These diselenides, and particularly DSBA, behave as mild thiol peroxidases leading to a moderate generation of H2O2 and NOx, and signaling of stress-activated and survival-promoting MAPKs, which ultimately control the mitochondrial pathway of apoptosis. Used in murine embryonic fibroblasts (MEFs) and HepG2 human hepatocarcinoma cells to produce sub-maximal conditions of stress, the diselenide compounds stimulated Nrf2 nuclear translocation and the transcription of the same Nrf2 gene as well as of GSTP and other phase II genes. This resulted in a higher degree of protection against H2O2 cytotoxicity (hormetic effect). Diselenide toxicity increased in GSTP knockout MEFs by a higher generation of NOx and SAPK-JNK activation. A lowered hormetic potential of these cells was observed in association with an abnormal expression and nuclear translocation of Nrf2 protein. Immunoprecipitation and affinity purification experiments revealed the existence of a Nrf2/GSTP complex in MEFs and HepG2 cells. Covalent oligomers of GSTP subunits were observed in DSBA treated cells. In conclusion, GSTP gene expression influences the Nrf2-dependent response to hormetic diselenides. Mechanistic interpretation for this GSTP-dependent effect may include a direct and redox-sensitive interaction of GSTP with Nrf2 protein.
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Available from: PubMed Central
- "This raises an interesting possibility that direct modification of Nrf2 amino acid residue(s) by ARE inducers constitutes an alternative mechanism for ARE activation . In another study, Li et al. have identified a potential nuclear export sequence (NES) motif in the Neh5 transactivation domain of Nrf2 protein and observed that mutating cysteine residue at 183 position into alanine (C183A) abrogated the NES function of Nrf2, rendering a nuclear accumulation of Nrf2 . In addition, several potential NES sequence motifs together with putative nuclear localization sequence (NLS) motifs were identified in the Nrf2 protein sequence . "
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ABSTRACT: Isothiocyanates (ITCs) are abundantly found in cruciferous vegetables. Epidemiological studies suggest that chronic consumption of cruciferous vegetables can lower the overall risk of cancer. Natural ITCs are key chemopreventive ingredients of cruciferous vegetables, and one of the prime chemopreventive mechanisms of natural isothiocyanates is the induction of Nrf2/ARE-dependent gene expression that plays a critical role in cellular defense against electrophiles and reactive oxygen species. In the present review, we first discuss the underlying mechanisms how natural ITCs affect the intracellular signaling kinase cascades to regulate the Keap1/Nrf2 activities, thereby inducing phase II cytoprotective and detoxifying enzymes. We also discuss the potential cellular protein targets to which natural ITCs are directly conjugated and how these events aid in the chemopreventive effects of natural ITCs. Finally, we discuss the posttranslational modifications of Keap1 and nucleocytoplasmic trafficking of Nrf2 in response to electrophiles and oxidants.
Available from: Rathnam Chaguturu
- "Some active compounds may lead to ubiquitination of Keap1 instead of Nrf2, and facilitate Nrf2 accumulation . Some active compounds may cause an inactivation of the Nrf2 export signal, increasing Nrf2 accumulation in the nucleus . Some active compounds may increase nuclear export of Bach1, which competes with Nrf2 for small Maf binding . "
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ABSTRACT: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces a battery of cytoprotective genes involved in antioxidant defense through binding to Antioxidant Response Elements (ARE) located in the promoter regions of these genes. To identify Nrf2 activators for the treatment of oxidative/electrophilic stress-induced diseases, the present study developed a high-throughput assay to evaluate Nrf2 activation using AREc32 cells that contain a luciferase gene under the control of ARE promoters. Of the 47,000 compounds screened, 238 (top 0.5% hits) of the chemicals increased the luminescent signal more than 14.4-fold and were re-tested at eleven concentrations in a range of 0.01-30 µM. Of these 238 compounds, 231 (96%) increased the luminescence signal in a concentration-dependent manner. Chemical structure relationship analysis of these 231 compounds indicated enrichment of four chemical scaffolds (diaryl amides and diaryl ureas, oxazoles and thiazoles, pyranones and thiapyranones, and pyridinones and pyridazinones). In addition, 30 of these 231 compounds were highly effective and/or potent in activating Nrf2, with a greater than 80-fold increase in luminescence, or an EC50 lower than 1.6 µM. These top 30 compounds were also screened in Hepa1c1c7 cells for an increase in Nqo1 mRNA, the prototypical Nrf2-target gene. Of these 30 compounds, 17 increased Nqo1 mRNA in a concentration-dependent manner. In conclusion, the present study documents the development, implementation, and validation of a high-throughput screen to identify activators of the Keap1-Nrf2-ARE pathway. Results from this screening identified Nrf2 activators, and provide novel insights into chemical scaffolds that might prevent oxidative/electrophilic stress-induced toxicity and carcinogenesis.
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