Nrf2 possesses a redox-sensitive nuclear exporting signal in the Neh5 transactivation domain.
ABSTRACT 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.
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ABSTRACT: Nrf2/ARE pathway plays an important role in adapt to oxidative stress caused by pro-oxidants and electrophiles through up-regulating phase II detoxifying enzymes. Our previous study has demonstrated that PCB quinone exposure causes severe cellular oxidative stress (Toxicology in Vitro 26 (2012) 841-848). There are no reports describing the ability of PCB quinone on Nrf2/ARE activation. In the present study, we found that exposure to PCB29-pQ resulted in a significant increase in Nrf2 and Keap1 expression in total protein, as well as the Nrf2 targeting genes, including NQO1 and HO-1. Next, immunocytochemistry analysis identified the accumulation of Nrf2 in nucleus subsequent to PCB29-pQ treatment. The increased Nrf2 and constant Keap1 expression in nucleus suggested the dissociation of Nrf1/Keap1 complex. Similarly, mRNA level of Nrf2 was elevated significantly with PCB29-pQ treatment, but not Keap1. Additionally, PCB29-pQ treatment led to significant up-regulation of the mRNA level of antioxidant enzymes, NQO1 and HO-1, in a concentration-dependent manner. Electrophoretic mobility shift assay and luciferase reporter assay further confirmed the formation of Nrf2-ARE complex. PCB29-pQ treatment has no effect on mitogen-activated protein kinase signaling, however, phospho-AKT was up-regulated and GSK-3β was down-regulated. Pretreatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), suppressed the phosphorylation of AKT and inhibited PCB29-pQ induced Nrf2/HO-1 activation, meanwhile, GSK-3β expression was increased accordingly. At last, reactive oxygen species (ROS) scavengers inhibited PCB29-pQ induced Nrf2 activation partly. These results suggested that Nrf2 activation by PCB29-pQ in HepG2 cells is associated with ROS and AKT pathway but not MAPK signaling, the activation of Nrf2/ARE may be an adaptive response to oxidative stress.Chemico-biological interactions 12/2013; · 2.46 Impact Factor
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ABSTRACT: Orphan receptor Nurr1 participates in the acquisition and maintenance of the dopaminergic cell phenotype, modulation of inflammation and cytoprotection, but little is known about its regulation. In this study, we report that Nurr1 contains a bipartite nuclear localization signal (NLS) within its DNA binding domain and two leucine-rich nuclear export signals (NES) in its ligand binding domain. Together, these signals regulate Nurr1 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nurr1 is mostly nuclear. A Nurr1 mutant lacking the NLS failed to enter the nucleus. The Nurr1 NLS sequence, when fused to green fluorescence protein, led to nuclear accumulation of this chimeric protein, indicating that this sequence was sufficient to direct nuclear localization of Nurr1. Furthermore, two NES were characterized in the ligand binding domain, whose deletion caused Nurr1 to accumulate predominantly in the nucleus. The Nurr1 NES was sensitive to CRM1 and could function as an independent export signal when fused to enhanced green fluorescence protein (EGFP). Sodium arsenite, an agent that induces oxidative stress, promoted nuclear export of ectopically expressed Nurr1 in HEK293T cells, and the antioxidant N-acetyl cysteine. Similarly, in dopaminergic MN9D cells, arsenite induced the export of endogenous Nurr1, resulting in the loss of expression of Nurr1-dependent genes. This study illustrates that Nurr1 shuttling between cytosol and nucleus is controlled by specific nuclear import and export signals, and that oxidative stress can unbalance the distribution of Nurr1 to favor its cytosolic accumulation.Journal of Biological Chemistry 01/2013; · 4.65 Impact Factor
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ABSTRACT: The regulatory mechanisms by which hydrogen peroxide (H2O2) modulates the activity of transcription factors in bacteria (OxyR and PerR), lower eukaryotes (Yap1, Maf1, Hsf1 and Msn2/4) and mammalian cells (AP-1, NRF2, CREB, HSF1, HIF-1, TP53, NF-κB, NOTCH, SP1 and SCREB-1) are reviewed. The complexity of regulatory networks increases throughout the phylogenetic tree, reaching a high level of complexity in mammalians. Multiple H2O2 sensors and pathways are triggered converging in the regulation of transcription factors at several levels: (1) synthesis of the transcription factor by upregulating transcription or increasing both mRNA stability and translation; (ii) stability of the transcription factor by decreasing its association with the ubiquitin E3 ligase complex or by inhibiting this complex; (iii) cytoplasm-nuclear traffic by exposing/masking nuclear localization signals, or by releasing the transcription factor from partners or from membrane anchors; and (iv) DNA binding and nuclear transactivation by modulating transcription factor affinity towards DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes. We also discuss how H2O2 biological specificity results from diverse thiol protein sensors, with different reactivity of their sulfhydryl groups towards H2O2, being activated by different concentrations and times of exposure to H2O2. The specific regulation of local H2O2 concentrations is also crucial and results from H2O2 localized production and removal controlled by signals. Finally, we formulate equations to extract from typical experiments quantitative data concerning H2O2 reactivity with sensor molecules. Rate constants of 140 M(-1) s(-1) and ≥1.3 × 10(3) M(-1) s(-1) were estimated, respectively, for the reaction of H2O2 with KEAP1 and with an unknown target that mediates NRF2 protein synthesis. In conclusion, the multitude of H2O2 targets and mechanisms provides an opportunity for highly specific effects on gene regulation that depend on the cell type and on signals received from the cellular microenvironment.Redox biology. 01/2014; 2:535-562.