Expression of antioxidant and phase 2 xenobiotic metabolizing enzyme genes is regulated through cis-acting sequences known as antioxidant response elements. Transcriptional activation through the antioxidant response elements involves members of the CNC (Cap 'n' Collar) family of basic leucine zipper proteins including Nrf1 and Nrf2. Nrf2 activity is regulated by Keap1-mediated compartmentalization in the cell. Given the structural similarities between Nrf1 and Nrf2, we sought to investigate whether Nrf1 activity is regulated similarly to Nrf2. Nrf1 also resides normally in the cytoplasm of cells. Cytoplasmic localization however, is independent of Keap1. Colocalization analysis using green fluorescent protein-tagged Nrf1 and subcellular fractionation of endogenous Nrf1 and fusion proteins indicate that Nrf1 is primarily a membrane-bound protein localized in the endoplasmic reticulum. Membrane targeting is mediated by the N terminus of the Nrf1 protein that contains a predicted transmembrane domain, and deletion of this domain resulted in a predominantly nuclear localization of Nrf1 that significantly increased the activation of reporter gene expression. Treatment with tunicamycin, an endoplasmic reticulum stress inducer, caused an accumulation of a smaller form of Nrf1 that correlated with detection of Nrf1 in the nucleus by biochemical fractionation and immunofluorescent analysis. These results suggest that Nrf1 is normally targeted to the endoplasmic reticulum membrane and that endoplasmic reticulum stress may play a role in modulating Nrf1 function as a transcriptional activator.
"In contrast to Nrf2, the N-terminal domain (NTD) of Nrf1 (TCF11), which anchors Nrf1 to the endoplasmic reticulum (ER) membrane and the nuclear membrane, regulates Nrf1 activation and its translocation to the nucleus –. Furthermore, the human Nrf1 gene can generate both full length 120 kDa Nrf1 (p120-Nrf1) and several truncated (36, 55, 65, and 95 kDa) isoforms of Nrf1 , . Of these smaller Nrf1 isoforms, the N-terminal-truncated 65 kDa isoform (p65-Nrf1) has been shown to possess significant regulatory effects with regard to Nrf2 mediated transcription. "
[Show abstract][Hide abstract] ABSTRACT: Despite androgen deprivation therapy (ADT), persistent androgen receptor (AR) signaling enables outgrowth of castration resistant prostate cancer (CRPC). In prostate cancer (PCa) cells, ADT may enhance AR activity through induction of oxidative stress. Herein, we investigated the roles of Nrf1 and Nrf2, transcription factors that regulate antioxidant gene expression, on hormone-mediated AR transactivation using a syngeneic in vitro model of androgen dependent (LNCaP) and castration resistant (C4-2B) PCa cells. Dihydrotestosterone (DHT) stimulated transactivation of the androgen response element (ARE) was significantly greater in C4-2B cells than in LNCaP cells. DHT-induced AR transactivation was coupled with higher nuclear translocation of p65-Nrf1 in C4-2B cells, as compared to LNCaP cells. Conversely, DHT stimulation suppressed total Nrf2 levels in C4-2B cells but elevated total Nrf2 levels in LNCaP cells. Interestingly, siRNA mediated silencing of Nrf1 attenuated AR transactivation while p65-Nrf1 overexpression enhanced AR transactivation. Subsequent studies showed that Nrf1 physically interacts with AR and enhances AR's DNA-binding activity, suggesting that the p65-Nrf1 isoform is a potential AR coactivator. In contrast, Nrf2 suppressed AR-mediated transactivation by stimulating the nuclear accumulation of the p120-Nrf1 which suppressed AR transactivation. Quantitative RT-PCR studies further validated the inductive effects of p65-Nrf1 isoform on the androgen regulated genes, PSA and TMPRSS2. Therefore, our findings implicate differential roles of Nrf1 and Nrf2 in regulating AR transactivation in PCa cells. Our findings also indicate that the DHT-stimulated increase in p65-Nrf1 and the simultaneous suppression of both Nrf2 and p120-Nrf1 ultimately facilitates AR transactivation in CRPC cells.
PLoS ONE 01/2014; 9(1):e87204. DOI:10.1371/journal.pone.0087204 · 3.23 Impact Factor
"The full-length Nrf1 is first synthesized as a 95-kDa non-glycosylated protein [i.e. the term non-glycosylation is defined that the primordial portion of Nrf1 residing on the cytoplasmic side of membranes has actually never been glycosylated at the consensus asparagines (Asn-X-Ser/Thr, in which X represents any of other amino acids rather than proline) in its Asn/Ser/Thr-rich (NST) domain, because it is clear that the N-linked glycosylation reaction catalyzed by oligosaccharyltransferases (OST) occurs in the lumen of the endoplasmic reticulum (ER)3435]. Subsequently, the non-glycosylated Nrf1 protein is co-translationally targeted to the ER through its N-terminal homology box 1 (NHB1) signal sequence363738. The NHB1-associated transmembrane region (called TM1, aa 7–26) determines the membrane-topology of Nrf1 (refs. 38,39,40), which is highly conserved with those of TCF11 (ref. "
[Show abstract][Hide abstract] ABSTRACT: The integral membrane-bound Nrf1 transcription factor fulfils important functions in maintaining cellular homeostasis and organ integrity, but how it is controlled vectorially is unknown. Herein, creative use of Gal4-based reporter assays with protease protection assays (GRAPPA), and double fluorescence protease protection (dFPP), reveals that the membrane-topogenic vectorial behaviour of Nrf1 dictates its post-translational modification and transactivation activity. Nrf1 is integrated within endoplasmic reticulum (ER) membranes through its NHB1-associated TM1 in cooperation with other semihydrophobic amphipathic regions. The transactivation domains (TADs) of Nrf1, including its Asn/Ser/Thr-rich (NST) glycodomain, are transiently translocated into the ER lumen, where it is glycosylated in the presence of glucose to become a 120-kDa isoform. Thereafter, the NST-adjoining TADs are partially repartitioned out of membranes into the cyto/nucleoplasmic side, where Nrf1 is subject to deglycosylation and/or proteolysis to generate 95-kDa and 85-kDa isoforms. Therefore, the vectorial process of Nrf1 controls its target gene expression.
"It was previously shown that Nrf1a shares structural similarities with Nrf2, which includes the Neh2 (Nrf2-ECH homology 2) domain that serves to destabilize Nrf2 through interaction with the KEAP1 protein. Although Nrf1a interacts with KEAP1, the location and function of Nrf1a is not regulated by KEAP1 . Since Nrf1b possesses the Neh2 domain, it is reasonable to speculate that the cellular localization and stability of Nrf1b may be regulated by KEAP1. "
[Show abstract][Hide abstract] ABSTRACT: Nuclear factor E2-related factor 1 (Nrf1) is a basic leucine zipper transcription factor that plays an important role in the activation of cytoprotective genes through the antioxidant response elements. The previously characterized long isoform of Nrf1 (Nrf1a) is targeted to the endoplasmic reticulum and accumulates in the nucleus in response to activating signals. Here we characterized a novel Nrf1 protein isoform (Nrf1b) generated through an alternative promoter and first exon that lacks the ER targeting domain of Nrf1a. The 5'-flanking region of Nrf1b directed high levels of luciferase reporter expression in cells. RT-PCR and Western blotting showed Nrf1b is widely expressed in various cell lines and mouse tissues. Immunoblot analysis of subcellular fractions and imaging of green fluorescence protein (GFP)-tagged Nrf1b demonstrate Nrf1b is constitutively localized to the nucleus. Nrf1b can activate GAL4-dependent transcription when fused to the heterologous GAL4 DNA-binding domain. Gel-shift and coimmunoprecipitation experiments demonstrate that Nrf1b forms a complex with MafG, and expression of Nrf1b activates the expression of antioxidant response element containing reporters and genes in cells. These results suggest Nrf1b is targeted to the nucleus where it activates ARE-driven genes and may play a role in modulating antioxidant response elements.
PLoS ONE 10/2012; 7(10):e48404. DOI:10.1371/journal.pone.0048404 · 3.23 Impact Factor
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