The Experts below are selected from a list of 102003 Experts worldwide ranked by ideXlab platform
Jefferson Y Chan - One of the best experts on this subject based on the ideXlab platform.
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the deubiquitinating enzyme usp7 regulates the transcription factor NRF1 by modulating its stability in response to toxic metal exposure
Journal of Biological Chemistry, 2021Co-Authors: John Jw Han, Hyun M Kim, Jun Y Lee, Yerin S Jeon, Jefferson Y ChanAbstract:The Nuclear factor E2-related factor 1 (NRF1) transcription factor performs a critical role in regulating cellular homeostasis as part of the cellular stress response, and drives the expression of antioxidants and detoxification enzymes among many other functions. Ubiquitination plays an important role in controlling the abundance and thus nuclear accumulation of NRF1 proteins, but the regulatory enzymes that act on NRF1 are not fully defined. Here, we identified ubiquitin specific protease 7 (USP7), a deubiquitinating enzyme, as a novel regulator of NRF1 activity. We found that USP7 interacts with NRF1a and TCF11-the two long protein isoforms of NRF1. Expression of wild type USP7, but not its catalytically defective mutant, resulted in decreased ubiquitination of TCF11 and NRF1a, leading to their increased stability, and increased transactivation of reporter gene expression by TCF11 and NRF1a. In contrast, knockdown or pharmacologic inhibition of USP7 dramatically increased ubiquitination of TCF11 and NRF1a, and reduction of their steady state levels. Loss of USP7 function attenuated the induction of NRF1 protein expression in response to treatment with arsenic and other toxic metals, and inhibition of USP7 activity significantly sensitized cells to arsenic treatment. Collectively, these findings suggest that USP7 may act to modulate abundance of NRF1 protein to induce gene expression in response to toxic metal exposure.
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nuclear factor erythroid 2 related transcription factor 1 NRF1 is regulated by o glcnac transferase
Free Radical Biology and Medicine, 2017Co-Authors: Jeong Woo Han, Joshua Valdez, Candy Lee, Hyunmin Kim, Xiaorong Wang, Lan Huang, Jefferson Y ChanAbstract:The NRF1 (Nuclear factor E2-related factor 1) transcription factor performs a critical role in regulating cellular homeostasis. Using a proteomic approach, we identified Host Cell Factor-1 (HCF1), a co-regulator of transcription, and O-GlcNAc transferase (OGT), the enzyme that mediates protein O-GlcNAcylation, as cellular partners of NRF1a, an isoform of NRF1. NRF1a directly interacts with HCF1 through the HCF1 binding motif (HBM), while interaction with OGT is mediated through HCF1. Overexpression of HCF1 and OGT leads to increased NRF1a protein stability. Addition of O-GlcNAc decreases ubiquitination and degradation of NRF1a. Transcriptional activation by NRF1a is increased by OGT overexpression and treatment with PUGNAc. Together, these data suggest that OGT can act as a regulator of NRF1a.
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Induction of Herpud1 expression by ER stress is regulated by NRF1
FEBS letters, 2015Co-Authors: Jefferson Y ChanAbstract:Herpud1 is an ER-localized protein that contributes to endoplasmic reticulum (ER) homeostasis by participating in the ER-associated protein degradation pathway. The NRF1 transcription factor is important in cellular stress pathways. We show that loss of NRF1 function results in decreased Herpud1 expression in cells and liver tissues. Expression of Herpud1 increases in response to ER stress, but not in NRF1 knockout cells. Transactivation studies show that NRF1 acts through antioxidant response elements located in the Herpud1 promoter, and chromatin immunoprecipitation demonstrates that Herpud1 is a direct NRF1 target gene. These results indicate that NRF1 is a transcriptional activator of Herpud1 expression during ER stress, and they suggest NRF1 is a key player in the regulation of the ER stress response in cells.
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Glycogen synthase kinase 3 regulates expression of nuclear factor-erythroid-2 related transcription factor-1 (NRF1) and inhibits pro-survival function of NRF1.
Experimental cell research, 2013Co-Authors: Madhurima Biswas, Erick K. Kwong, Eujean Park, Parminder Nagra, Jefferson Y ChanAbstract:Abstract Nuclear factor E2-related factor-1 (NRF1) is a basic leucine zipper transcription factor that is known to regulate antioxidant and cytoprotective gene expression. It was recently shown that NRF1 is regulated by SCF–Fbw7 ubiquitin ligase. However our knowledge of upstream signals that targets NRF1 for degradation by the UPS is not known. We report here that NRF1 expression is negatively regulated by glycogen synthase kinase 3 (GSK3) in Fbw7-dependent manner. We show that GSK3 interacts with NRF1 and phosphorylates the Cdc4 phosphodegron domain (CPD) in NRF1. Mutation of serine residue in the CPD of NRF1 to alanine (S350A), blocks NRF1 from phosphorylation by GSK3, and stabilizes NRF1. Knockdown of NRF1 and expression of a constitutively active form of GSK3 results in increased apoptosis in neuronal cells in response to ER stress, while expression of the GSK3 phosphorylation resistant S350A–NRF1 attenuates apoptotic cell death. Together these data suggest that GSK3 regulates NRF1 expression and cell survival function in response to stress activation.
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deficiency in the nuclear related factor erythroid 2 transcription factor NRF1 leads to genetic instability
FEBS Journal, 2012Co-Authors: Diamanda Rigas, Ara Cho, Jefferson Y ChanAbstract:Nuclear factor erythroid-derived 2-related factor 1 (NRF1) regulates cellular stress response genes, and has also been suggested to play a role in other cellular processes. We previously demonstrated that hepatocyte-specific deletion of NRF1 in mice resulted in spontaneous apoptosis, inflammation, and development of liver tumors. Here, we showed that both fibroblasts derived from NRF1 null mouse embryos and fibroblasts expressing a conditional NRF1 allele showed increased micronuclei and formation of abnormal nuclei. Lentiviral shRNA-mediated knockdown of NRF1 in SAOS–2 cells also resulted in increased micronuclei, abnormal mitosis and multi-nucleated cells. Metaphase analyses showed increased aneuploidy in NRF1−/− embryonic fibroblasts. Nuclear defects in NRF1-deficient cells were associated with decreased expression of various genes encoding kinetochore and mitotic checkpoint proteins. Our findings suggest that NRF1 may play a role in maintaining genomic integrity, and that NRF1 dysregulation may induce tumorigenesis.
Yiguo Zhang - One of the best experts on this subject based on the ideXlab platform.
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Activation of the membrane-bound NRF1 transcription factor by USP19, a tail-anchored ubiquitin-specific protease in the endoplasmic reticulum
2020Co-Authors: Hu Shaofan, Xiang Yuancai, Wang Meng, Yiguo ZhangAbstract:The membrane-bound transcription factor NRF1 (i.e., encoded by Nfe2l1) is activated by sensing glucose deprivation, cholesterol excess, proteasomal inhibition and oxidative stress, and then mediates distinct signaling responses in order to maintain cellular homeostasis. Here, we found that NRF1 stability and transactivity are enhanced by USP19, a tail-anchored ubiquitin-specific protease in the endoplasmic reticulum (ER). Further experiments revealed that USP19 directly interacts with NRF1 in proximity to the ER and acts as a deubiquitinating enzyme to remove ubiquitin moieties from this protein and hence circumvent potential proteasomal degradation. Such USP19-mediated effect takes place only after NRF1 is retrotranslocated by p97 out of ER membranes. Conversely, knockout of USP19 causes significant decreases in NRF1 abundance and its active isoform entering the nucleus, resulting in down-regulation of its target proteasomal subunits. This led to a modest reduction of USP19-/--derived tumor growth in xenograft mice, when compared with wild-type controls. Altogether, these demonstrate that USP19 serves as a novel mechanistic modulator of NRF1, but not Nrf2. In turn, our additional evidence has also unraveled that transcriptional expression of endogenous USP19 and its promoter-driven reporter genes is regulated by Nrf2, as well by NRF1, at distinct layers within a complex hierarchical regulatory network.
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A unity of opposites in between NRF1- and Nrf2-mediated responses to the endoplasmic reticulum stressor tunicamycin
2019Co-Authors: Yu-ping Zhu, Ze Zheng, Zhuo Fan, Lu Qiu, Yiguo ZhangAbstract:The water-soluble Nrf2 is accepted as a master regulator of antioxidant responses to cellular stress, it was also identified as a direct target of the endoplasmic reticulum (ER)-anchored PERK. However, the membrane-bound NRF1 response to ER stress remains elusive. Herein, we report a unity of opposites in both NRF1- and Nrf2-coordinated responses to the ER stressor tunicamycin (TU). The TU-inducible transcription of NRF1 and Nrf2, as well as GCLM and HO-1, was accompanied by activation of ER stress signaling networks. The unfolded protein response (UPR) mediated by ATF6, IRE1 and PERK was significantly suppressed by NRF1-specific knockout, but hyper-expression of Nrf2, GCLM and HO-1 was retained in NRF1-/- cells. By contrast, Nrf2-/-{Delta}TA cells with a genomic deletion of its transactivation domain resulted in significant decreases of GCLM, HO-1 and NRF1; this was accompanied by partial decreases of IRE1 and ATF6, but not PERK, along with an obvious increase of ATF4. Notably, NRF1 glycosylation and its trans-activity to mediate transcriptional expression of 26S proteasomal subunits were repressed by TU. This inhibitory effect was enhanced by NRF1-/- and Nrf2-/-{Delta}TA, but not by a constitutive activator caNrf2{Delta}N (that increased abundances of non-glycosylated and processed NRF1). Furthermore, caNrf2{Delta}N also enhanced induction of PERK and IRE1 by TU, but reduced expression of ATF4 and HO-1. Such distinct roles of NRF1 and Nrf2 are unified to maintain cell homeostasis by a series of coordinated ER-to-nuclear signaling responses to TU. Overall, NRF1 acts in a cell-autonomous manner to determine transcription of most of UPR-target genes, albeit Nrf2 is also partially involved in this process.
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Topovectorial mechanisms control the juxtamembrane proteolytic processing of NRF1 to remove its N-terminal polypeptides during maturation of the CNC-bZIP factor
Toxicology and applied pharmacology, 2018Co-Authors: Yuancai Xiang, Zhuo Fan, Lu Qiu, Josefin Halin, Meng Wang, Zhengwen Zhang, Peter Mattjus, Yiguo ZhangAbstract:The topobiological behaviour of NRF1 dictates its post-translational modification and its ability to transactivate target genes. Here, we have elucidated that topovectorial mechanisms control the juxtamembrane processing of NRF1 on the cyto/nucleoplasmic side of endoplasmic reticulum (ER), whereupon it is cleaved and degraded to remove various lengths of its N-terminal domain (NTD, also refolded into a UBL module) and acidic domain-1 (AD1) to yield multiple isoforms. Notably, an N-terminal ~12.5-kDa polypeptide of NRF1 arises from selective cleavage at an NHB2-adjoining region within NTD, whilst other longer UBL-containing isoforms may arise from proteolytic processing of the protein within AD1 around PEST1 and Neh2L degrons. The susceptibility of NRF1 to proteolysis is determined by dynamic repositioning of potential UBL-adjacent degrons and cleavage sites from the ER lumen through p97-driven retrotranslocation and -independent pathways into the cyto/nucleoplasm. These repositioned degrons and cleavage sites within NTD and AD1 of NRF1 are coming into their bona fide functionality, thereby enabling it to be selectively processed by cytosolic DDI-1/2 proteases and also partiality degraded via 26S proteasomes. The resultant proteolytic processing of NRF1 gives rise to a mature ~85-kDa CNC-bZIP transcription factor, which regulates transcriptional expression of cognate target genes. Furthermore, putative ubiquitination of NRF1 is not a prerequisite necessary for involvement of p97 in the client processing. Overall, the regulated juxtamembrane proteolysis (RJP) of NRF1, though occurring in close proximity to the ER, is distinctive from the mechanism that regulates the intramembrane proteolytic (RIP) processing of ATF6 and SREBP1.
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NRF1 is paved as a new strategic avenue to prevent and treat cancer, neurodegenerative and other diseases.
Toxicology and applied pharmacology, 2018Co-Authors: Jianxin Yuan, Shuwei Zhang, Yiguo ZhangAbstract:Abstract Transcription factor NRF1 acts as a unique vital player in maintaining cellular homeostasis and organ integrity during normal development and growth throughout the life process. Loss-of-function of NRF1 results in severe oxidative stress, genomic instability, embryonic lethality, developmental disorders, and adult diseases such as non-alcoholic steatohepatitis, hepatocellular carcinoma, diabetes and neurogenerative diseases. Thereby, NRF1 is critically implicated in a variety of important physio-pathological processes by governing robust target genes in order to reinforce antioxidant, detoxification and cytoprotective responses to cellular stress. Notably, there also exists a proteasomal ‘bounce-back’ response mediated by NRF1, insofar as to enhance the drug resistance to proteasomal inhibitors in clinical treatment of neuroblastoma, multiple myeloma and triple-negative breast cancers. Recently, several drugs or chemicals are found or re-found in new ways to block the proteasomal compensatory process through inhibiting the multistep processing of NRF1. Conversely, activation of NRF1 induced by some drugs or chemicals leads to cytoprotection from cell apoptosis and promotes cell viability. This is the start of constructive and meaningful studies, approaching to explore the mechanism(s) by which NRF1 is activated to protect neurons and other cells from malignant and degenerative diseases. Overall, NRF1 has appealed attentions as a new attractive therapeutic strategy for human diseases including cancers
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Topovectorial mechanisms control the juxtamembrane proteolytic processing of NRF1 to remove its N-terminal polypeptides during maturation of the CNC-bZIP factor
2018Co-Authors: Yuancai Xiang, Zhuo Fan, Lu Qiu, Josefin Halin, Meng Wang, Zhengwen Zhang, Peter Mattjus, Yiguo ZhangAbstract:The topobiological behaviour of NRF1 dictates its post-translational modification and its ability to transactivate target genes. Here, we have elucidated that topovectorial mechanisms control the juxtamembrane processing of NRF1 on the cyto/nucleoplasmic side of endoplasmic reticulum (ER), whereupon it is cleaved and degraded to remove various lengths of its N-terminal domain (NTD, also refold into a UBL module) and acidic domain-1 (AD1) to yield multiple isoforms. Notably, an N-terminal ~12.5-kDa polypeptide of NRF1 arises from selective cleavage at an NHB2-adjoining region within NTD, whilst other longer UBL-containing isoforms may arise from proteolytic processing of the protein within AD1 around PEST1 and Neh2L degrons. The susceptibility of NRF1 to proteolysis is determined by dynamic repositioning of potential UBL-adjacent degrons and cleavage sites from the ER lumen through p97-driven retrotranslocation and -independent pathways into the cyto/nucleoplasm. These repositioned degrons and cleavage sites within NTD and AD1 of NRF1 are coming into their bona fide functionality, thereby enabling it to be selectively processed by cytosolic DDI-1/2 proteases and also degraded via 26S proteasomes. The resultant proteolytic processing of NRF1 gives rise to a mature ~85-kDa CNC-bZIP transcription factor, which regulates transcriptional expression of cognate target genes. Furthermore, putative ubiquitination of NRF1 is not a prerequisite necessary for involvement of p97 in the client processing. Overall, the regulated juxtamembrane proteolysis (RJP) of NRF1, though occurring in close proximity to the ER, is distinctive from the mechanism that regulates the intramembrane proteolytic (RIP) processing of ATF6 and SREBP1.
John D Hayes - One of the best experts on this subject based on the ideXlab platform.
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The selective post-translational processing of transcription factor NRF1 yields distinct isoforms that dictate its ability to differentially regulate gene expression.
Scientific reports, 2015Co-Authors: Yiguo Zhang, Lu Qiu, Yuancai Xiang, Huakan Zhao, John D HayesAbstract:Upon translation, the N-terminal homology box 1 (NHB1) signal anchor sequence of NRF1 integrates it within the endoplasmic reticulum (ER) whilst its transactivation domains [TADs, including acidic domain 1 (AD1), the flanking Asn/Ser/Thr-rich (NST) domain and AD2] are transiently translocated into the ER lumen, whereupon the NST domain is glycosylated to yield an inactive 120-kDa glycoprotein. Subsequently, these TADs are retrotranslocated into extra-luminal subcellular compartments, where NRF1 is deglycosylated to yield an active 95-kDa isoform. Herein, we report that AD1 and AD2 are required for the stability of the 120-kDa NRF1 glycoprotein, but not that of the non-glycosylated/de-glycosylated 95-kDa isoform. Degrons within AD1 do not promote proteolytic degradation of the 120-kDa NRF1 glycoprotein. However, repositioning of AD2-adjoining degrons (i.e. DSGLS-containing SDS1 and PEST2 sequences) into the cyto/nucleoplasm enables selective topovectorial processing of NRF1 by the proteasome and/or calpains to generate a cleaved active 85-kDa NRF1 or a dominant-negative 36-kDa NRF1γ. Production of NRF1γ is abolished by removal of SDS1 or PEST2 degrons, whereas production of the cleaved 85-kDa NRF1 is blocked by deletion of the ER luminal-anchoring NHB2 sequence (aa 81–106). Importantly, NRF1 activity is positively and/or negatively regulated by distinct doses of proteasome and calpain inhibitors.
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Transcription Factor NRF1 Negatively Regulates the Cystine/Glutamate Transporter and Lipid-Metabolizing Enzymes
Molecular and cellular biology, 2014Co-Authors: Tadayuki Tsujita, Liam Baird, Masayuki Yamamoto, Vivian Peirce, Yuka Matsuyama, Misaki Takaku, Shawn V Walsh, Julian L Griffin, Akira Uruno, John D HayesAbstract:Liver-specific NRF1 (NF-E2-p45-related factor 1) knockout mice develop nonalcoholic steatohepatitis. To identify postnatal mechanisms responsible for this phenotype, we generated an inducible liver-specific NRF1 knockout mouse line using animals harboring an NRF1(flox) allele and a rat CYP1A1-Cre transgene (NRF1(flox/flox)::CYP1A1-Cre mice). Administration of 3-methylcholanthrene (3-MC) to these mice (NRF1(flox/flox)::CYP1A1-Cre+3MC mice) resulted in loss of hepatic NRF1 expression. The livers of mice lacking NRF1 accumulated lipid, and the hepatic fatty acid (FA) composition in such animals differed significantly from that in the NRF1(flox/flox)::CYP1A1-Cre control. This change was provoked by upregulation of several FA metabolism genes. Unexpectedly, we also found that the level of glutathione was increased dramatically in livers of NRF1(flox/flox)::CYP1A1-Cre+3MC mice. While expression of glutathione biosynthetic enzymes was unchanged, xCT, a component of the cystine/glutamate antiporter system x(c)(-), was significantly upregulated in livers of NRF1(flox/flox)::CYP1A1-Cre+3MC mice, suggesting that NRF1 normally suppresses xCT. Thus, stress-inducible expression of xCT is a two-step process: under homeostatic conditions, NRF1 effectively suppresses nonspecific transactivation of xCT, but when cells encounter severe oxidative/electrophilic stress, NRF1 is displaced from an antioxidant response element (ARE) in the gene promoter while Nrf2 is recruited to the ARE. Thus, NRF1 controls both the FA and the cystine/cysteine content of hepatocytes by participating in an elaborate regulatory network.
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Transcription factor NRF1 Is topologically repartitioned across membranes to enable target gene transactivation through Its acidic glucose-responsive domains
PloS one, 2014Co-Authors: Yiguo Zhang, Yonggang Ren, John D HayesAbstract:The membrane-bound NRF1 transcription factor regulates critical homeostatic and developmental genes. The conserved N-terminal homology box 1 (NHB1) sequence in NRF1 targets the cap‘n’collar (CNC) basic basic-region leucine zipper (bZIP) factor to the endoplasmic reticulum (ER), but it is unknown how its activity is controlled topologically within membranes. Herein, we report a hitherto unknown mechanism by which the transactivation activity of NRF1 is controlled through its membrane-topology. Thus after NRF1 is anchored within ER membranes, its acidic transactivation domains (TADs), including the Asn/Ser/Thr-rich (NST) glycodomain situated between acidic domain 1 (AD1) and AD2, are transiently translocated into the lumen of the ER, where NST is glycosylated in the presence of glucose to yield an inactive 120-kDa NRF1 glycoprotein. Subsequently, portions of the TADs partially repartition across membranes into the cyto/nucleoplasmic compartments, whereupon an active 95-kDa form of NRF1 accumulates, a process that is more obvious in glucose-deprived cells and may involve deglycosylation. The repartitioning of NRF1 out of membranes is monitored within this protein by its acidic-hydrophobic amphipathic glucose-responsive domains, particularly the Neh5L subdomain within AD1. Therefore, the membrane-topological organization of NRF1 dictates its post-translational modifications (i.e. glycosylation, the putative deglycosylation and selective proteolysis), which together control its ability to transactivate target genes.
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Identification of topological determinants in the N-terminal domain of transcription factor NRF1 that control its orientation in the endoplasmic reticulum membrane
Biochemical Journal, 2010Co-Authors: Yiguo Zhang, John D HayesAbstract:NF-E2-related factor 1 (NRF1) is a cap‘n'collar (CNC) basic-region leucine zipper (bZIP) transcription factor that is tethered to endoplasmic reticulum (ER) and nuclear envelope membranes through its N-terminal signal peptide (residues 1-30). Besides the signal peptide, amino acids 31-90 of NRF1 also negatively regulate the CNC-bZIP factor. In this paper we have tested the hypothesis that amino acids 31-90 of NRF1, and the overlapping N-terminal homology box 2 (NHB2, residues 82-106), inhibit NRF1 because they control its topology within membranes. This region contains three amphipathic a-helical regions comprising amino acids 31-50 [called the signal peptide-associated sequence (SAS)], 55-82 [called the cholesterol recognition amino acid consensus sequences (CRACs)], and 89-106 (part of NHB2). We present experimental data showing that the signal peptide of NRF1 contains a transmembrane 1 region (TM1, residues 7-24) that is orientated across the ER membrane in an Ncyt/Clum fashion with its N-terminus facing the cytoplasm and its C-terminus positioned in the lumen of the ER. Once NRF1 is anchored to the ER membrane through TM1, the remaining portion of the N-terminal domain (residues 1-124) is transiently translocated into the ER lumen. Thereafter, NRF1 adopts a topology in which the SAS is inserted into the membrane, the CRACs are probably repartitioned to the cytoplasmic side of the ER membrane, and NHB2 may serve as an anchor switch, either lying on the luminal surface of the ER or traversing the membrane with an Ncyt/Clum orientation. Thus, NRF1 can adopt several topologies within membranes that are determined by its N-terminal domain.
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Identification of topological determinants in the N-terminal domain of transcription factor NRF1 that control its orientation in the endoplasmic reticulum membrane.
The Biochemical journal, 2010Co-Authors: Yiguo Zhang, John D HayesAbstract:NRF1 [NF-E2 (nuclear factor-erythroid 2)-related factor 1] is a CNC (cap'n'collar) bZIP (basic-region leucine zipper) transcription factor that is tethered to ER (endoplasmic reticulum) and nuclear envelope membranes through its N-terminal signal peptide (residues 1-30). Besides the signal peptide, amino acids 31-90 of NRF1 also negatively regulate the CNC-bZIP factor. In the present study we have tested the hypothesis that amino acids 31-90 of NRF1, and the overlapping NHB2 (N-terminal homology box 2; residues 82-106), inhibit NRF1 because they control its topology within membranes. This region contains three amphipathic alpha-helical regions comprising amino acids 31-50 [called the SAS (signal peptide-associated sequence)], 55-82 [called the CRACs (cholesterol-recognition amino acid consensus sequences)] and 89-106 (part of NHB2). We present experimental data showing that the signal peptide of NRF1 contains a TM1 (transmembrane 1) region (residues 7-24) that is orientated across the ER membrane in an N(cyt)/C(lum) fashion with its N-terminus facing the cytoplasm and its C-terminus positioned in the lumen of the ER. Once NRF1 is anchored to the ER membrane through TM1, the remaining portion of the N-terminal domain (NTD, residues 1-124) is transiently translocated into the ER lumen. Thereafter, NRF1 adopts a topology in which the SAS is inserted into the membrane, the CRACs are probably repartitioned to the cytoplasmic side of the ER membrane, and NHB2 may serve as an anchor switch, either lying on the luminal surface of the ER or traversing the membrane with an N(cyt)/C(lum) orientation. Thus NRF1 can adopt several topologies within membranes that are determined by its NTD.
Masayuki Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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discovery of an NRF1 specific inducer from a large scale chemical library using a direct NRF1 protein monitoring system
Genes to Cells, 2015Co-Authors: Tadayuki Tsujita, Liam Baird, Yuki Furusawa, Satomi Gotoh, Shinichi Kawaguchi, Fumiki Katsuoka, Masayuki YamamotoAbstract:: NRF1 (NF-E2-p45-related factor 1) plays an important role in the regulation of genes encoding proteasome subunits, a cystine transporter, and lipid-metabolizing enzymes. Global and tissue-specific disruptions of the NRF1 gene in mice result in embryonic lethality and spontaneous development of severe tissue defects, respectively, suggesting NRF1 plays a critical role in vivo. Mechanistically, the continuous degradation of the NRF1 protein by the proteasome is regarded as a major regulatory nexus of NRF1 activity. To develop NRF1-specific inducers that act to overcome the phenotypes related to the lack of NRF1 activity, we constructed a novel NRF1ΔC-Luc fusion protein reporter and developed cell lines that stably express the reporter in Hepa1c1c7 cells for use in high-throughput screening. In screening of a chemical library with this reporter system, we identified two hit compounds that significantly induced luciferase activity. Through an examination of a series of derivatives of one of the hit compounds, we identified T1-20, which induced a 70-fold increase in luciferase activity. T1-20 significantly increased the level of NRF1 protein in the mouse liver, indicating that the compound is also functional in vivo. Thus, these results show the successful identification of the first small chemical compounds which specifically and significantly induce NRF1.
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Discovery of an NRF1‐specific inducer from a large‐scale chemical library using a direct NRF1‐protein monitoring system
Genes to cells : devoted to molecular & cellular mechanisms, 2015Co-Authors: Tadayuki Tsujita, Liam Baird, Yuki Furusawa, Satomi Gotoh, Shinichi Kawaguchi, Fumiki Katsuoka, Yoshika Hou, Masayuki YamamotoAbstract:NRF1 (NF-E2-p45-related factor 1) plays an important role in the regulation of genes encoding proteasome subunits, a cystine transporter, and lipid-metabolizing enzymes. Global and tissue-specific disruptions of the NRF1 gene in mice result in embryonic lethality and spontaneous development of severe tissue defects, respectively, suggesting NRF1 plays a critical role in vivo. Mechanistically, the continuous degradation of the NRF1 protein by the proteasome is regarded as a major regulatory nexus of NRF1 activity. To develop NRF1-specific inducers that act to overcome the phenotypes related to the lack of NRF1 activity, we constructed a novel NRF1ΔC-Luc fusion protein reporter and developed cell lines that stably express the reporter in Hepa1c1c7 cells for use in high-throughput screening. In screening of a chemical library with this reporter system, we identified two hit compounds that significantly induced luciferase activity. Through an examination of a series of derivatives of one of the hit compounds, we identified T1-20, which induced a 70-fold increase in luciferase activity. T1-20 significantly increased the level of NRF1 protein in the mouse liver, indicating that the compound is also functional in vivo. Thus, these results show the successful identification of the first small chemical compounds which specifically and significantly induce NRF1.
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CNC-bZIP Protein NRF1-Dependent Regulation of Glucose-Stimulated Insulin Secretion
Antioxidants & redox signaling, 2015Co-Authors: Hongzhi Zheng, Masayuki Yamamoto, Rui Zhao, Peng Xue, Jian Dong, Dianxin Liu, Qingchun Tong, Weiping TengAbstract:Abstract Aims: The inability of pancreatic β-cells to secrete sufficient insulin in response to glucose stimulation is a major contributing factor to the development of type 2 diabetes (T2D). We investigated both the in vitro and in vivo effects of deficiency of nuclear factor-erythroid 2-related factor 1 (NRF1) in β-cells on β-cell function and glucose homeostasis. Results: Silencing of NRF1 in β-cells leads to a pre-T2D phenotype with disrupted glucose metabolism and impaired insulin secretion. Specifically, MIN6 β-cells with stable knockdown of NRF1 (NRF1-KD) and isolated islets from β-cell-specific NRF1-knockout [NRF1(b)-KO] mice displayed impaired glucose responsiveness, including elevated basal insulin release and decreased glucose-stimulated insulin secretion (GSIS). NRF1(b)-KO mice exhibited severe fasting hyperinsulinemia, reduced GSIS, and glucose intolerance. Silencing of NRF1 in MIN6 cells resulted in oxidative stress and altered glucose metabolism, with increases in both glucose uptake and ae...
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transcription factor NRF1 negatively regulates the cystine glutamate transporter and lipid metabolizing enzymes
Molecular and Cellular Biology, 2014Co-Authors: Tadayuki Tsujita, Liam Baird, Vivian Peirce, Yuka Matsuyama, Misaki Takaku, Shawn V Walsh, Julian L Griffin, Akira Uruno, Masayuki YamamotoAbstract:Liver-specific NRF1 (NF-E2-p45-related factor 1) knockout mice develop nonalcoholic steatohepatitis. To identify postnatal mechanisms responsible for this phenotype, we generated an inducible liver-specific NRF1 knockout mouse line using animals harboring an NRF1(flox) allele and a rat CYP1A1-Cre transgene (NRF1(flox/flox)::CYP1A1-Cre mice). Administration of 3-methylcholanthrene (3-MC) to these mice (NRF1(flox/flox)::CYP1A1-Cre+3MC mice) resulted in loss of hepatic NRF1 expression. The livers of mice lacking NRF1 accumulated lipid, and the hepatic fatty acid (FA) composition in such animals differed significantly from that in the NRF1(flox/flox)::CYP1A1-Cre control. This change was provoked by upregulation of several FA metabolism genes. Unexpectedly, we also found that the level of glutathione was increased dramatically in livers of NRF1(flox/flox)::CYP1A1-Cre+3MC mice. While expression of glutathione biosynthetic enzymes was unchanged, xCT, a component of the cystine/glutamate antiporter system x(c)(-), was significantly upregulated in livers of NRF1(flox/flox)::CYP1A1-Cre+3MC mice, suggesting that NRF1 normally suppresses xCT. Thus, stress-inducible expression of xCT is a two-step process: under homeostatic conditions, NRF1 effectively suppresses nonspecific transactivation of xCT, but when cells encounter severe oxidative/electrophilic stress, NRF1 is displaced from an antioxidant response element (ARE) in the gene promoter while Nrf2 is recruited to the ARE. Thus, NRF1 controls both the FA and the cystine/cysteine content of hepatocytes by participating in an elaborate regulatory network.
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Transcription Factor NRF1 Negatively Regulates the Cystine/Glutamate Transporter and Lipid-Metabolizing Enzymes
Molecular and cellular biology, 2014Co-Authors: Tadayuki Tsujita, Liam Baird, Masayuki Yamamoto, Vivian Peirce, Yuka Matsuyama, Misaki Takaku, Shawn V Walsh, Julian L Griffin, Akira Uruno, John D HayesAbstract:Liver-specific NRF1 (NF-E2-p45-related factor 1) knockout mice develop nonalcoholic steatohepatitis. To identify postnatal mechanisms responsible for this phenotype, we generated an inducible liver-specific NRF1 knockout mouse line using animals harboring an NRF1(flox) allele and a rat CYP1A1-Cre transgene (NRF1(flox/flox)::CYP1A1-Cre mice). Administration of 3-methylcholanthrene (3-MC) to these mice (NRF1(flox/flox)::CYP1A1-Cre+3MC mice) resulted in loss of hepatic NRF1 expression. The livers of mice lacking NRF1 accumulated lipid, and the hepatic fatty acid (FA) composition in such animals differed significantly from that in the NRF1(flox/flox)::CYP1A1-Cre control. This change was provoked by upregulation of several FA metabolism genes. Unexpectedly, we also found that the level of glutathione was increased dramatically in livers of NRF1(flox/flox)::CYP1A1-Cre+3MC mice. While expression of glutathione biosynthetic enzymes was unchanged, xCT, a component of the cystine/glutamate antiporter system x(c)(-), was significantly upregulated in livers of NRF1(flox/flox)::CYP1A1-Cre+3MC mice, suggesting that NRF1 normally suppresses xCT. Thus, stress-inducible expression of xCT is a two-step process: under homeostatic conditions, NRF1 effectively suppresses nonspecific transactivation of xCT, but when cells encounter severe oxidative/electrophilic stress, NRF1 is displaced from an antioxidant response element (ARE) in the gene promoter while Nrf2 is recruited to the ARE. Thus, NRF1 controls both the FA and the cystine/cysteine content of hepatocytes by participating in an elaborate regulatory network.
Senthil K. Radhakrishnan - One of the best experts on this subject based on the ideXlab platform.
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NRF1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex
The Journal of biological chemistry, 2018Co-Authors: Janakiram R. Vangala, Senthil K. RadhakrishnanAbstract:Inhibition of the proteasome leads to proteotoxic stress, which is characterized by the buildup of ubiquitinated proteins that cannot be degraded properly. The transcription factor NRF1 (also called NFE2L1) counteracts proteotoxic stress by inducing transcription of proteasome subunit genes, resulting in the restoration of proteasome activity. Further understanding of the NRF1 pathway is therefore of interest in both neurodegeneration, where proteasome activity could be enhanced, and cancer, where suppression of this pathway could potentiate the cell-killing effect mediated by proteasome inhibitor drugs. Here, to identify novel regulators of NRF1, we performed an RNAi screen in an engineered cell line, reporting on NRF1 transcriptional activity. In addition to validating known regulators, we discovered that the AAA+ ATPase RUVBL1 is necessary for NRF1's transcriptional activity. Given that RUVBL1 is part of different multisubunit complexes that play key roles in transcription, we dissected this phenomenon further and found that the TIP60 chromatin-regulatory complex is essential for NRF1-dependent transcription of proteasome genes. Consistent with these observations, NRF1, RUVBL1, and TIP60 proteins were co-recruited to the promoter regions of proteasome genes after proteasome inhibitor treatments. More importantly, depletion of RUVBL1 or TIP60 in various cancer cells sensitized them to cell death induced by proteasome inhibition. Overall, our study provides a framework for manipulating the TIP60-NRF1 axis to alter proteasome function in various human diseases, including cancer.
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Proteotoxic stress-induced NRF1 transcriptional program requires a functional TIP60 complex
2018Co-Authors: Janakiram R. Vangala, Senthil K. RadhakrishnanAbstract:In response to inhibition of the cellular proteasome, the transcription factor NRF1 (also called NFE2L1) induces transcription of proteasome subunit genes resulting in the restoration of proteasome activity and thus enabling the cells to mitigate the proteotoxic stress. To identify novel regulators of NRF1, we performed an RNA interference screen and discovered that the AAA+ ATPase RUVBL1 is necessary for its transcriptional activity. Given that RUVBL1 is part of different multi-subunit complexes that play key roles in transcription, we dissected this phenomenon further and found that the TIP60 chromatin regulatory complex is essential for NRF1-dependent transcription of proteasome genes. Consistent with these observations, NRF1, RUVBL1, and TIP60 proteins were co-recruited to the promoter regions of proteasome genes after proteasome inhibitor treatments. More importantly, depletion of RUVBL1 or TIP60 in various cancer cells sensitized them to cell death induced by proteasome inhibition. Our study provides a framework for manipulating the NRF1-TIP60 axis to alter proteasome function in various human diseases including cancer.
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NRF1 can be processed and activated in a proteasome-independent manner
Current biology : CB, 2016Co-Authors: Janakiram R. Vangala, Elke Krüger, Franziska Sotzny, Raymond J. Deshaies, Senthil K. RadhakrishnanAbstract:In response to proteasome inhibition, the transcription factor NRF1 facilitates de novo synthesis of proteasomes by inducing proteasome subunit (PSM) genes 1 and 2. Previously, we showed that activation of the p120 form of NRF1, a membrane-bound protein in the endoplasmic reticulum (ER) with the bulk of its polypeptide in the lumen, involves its retrotranslocation into the cytosol in a manner that depends on the AAA-ATPase p97/VCP [3]. This is followed by proteolytic processing and mobilization of the transcriptionally active p110 form of NRF1 to the nucleus. A subsequent study suggested that site-specific proteolytic processing of NRF1 by the proteasome yields an active 75 kDa fragment [4]. We show here that under conditions where all three active sites of the proteasome are completely blocked, p120 NRF1 can still be proteolytically cleaved to the p110 form, which is translocated to the nucleus to activate transcription of PSM genes. Thus, our results indicate that a proteasome-independent pathway can promote the release of active p110 NRF1 from the ER membrane.
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p97-dependent retrotranslocation and proteolytic processing govern formation of active NRF1 upon proteasome inhibition
eLife, 2014Co-Authors: Senthil K. Radhakrishnan, Willem Den Besten, Raymond J. DeshaiesAbstract:Cells exposed to high temperatures, infections and other forms of stress often produce oxygen ions and peroxide molecules that can cause damage to proteins and DNA. Cells therefore rely on molecular machines called proteasomes to eliminate damaged proteins, before they cause too much harm. Two related transcription factors—proteins that interact with DNA to ‘switch on’ the expression of genes—are involved in a cell’s responses to stress, but in different ways. Nrf2 switches on genes that limit the damage caused by oxygen ions and peroxide molecules, while NRF1 switches on the genes that encode the components of the proteasome. As such, NRF1 helps to restart proteasome activity if it has been shut off—a phenomenon known as ‘bounce-back’. Within a cell, NRF1 is known to start off embedded within the membranes of a structure called the endoplasmic reticulum. However, it is not clear how activated NRF1 leaves this membrane and enters the nucleus to interact with the cell’s DNA. Now, Radhakrishnan et al. show that when NRF1 is produced, most of its length is found inside the endoplasmic reticulum, with only a small piece being anchored in the surrounding membrane. This is unlike previously described transcription factors that associate with the endoplasmic reticulum, which are stuck to the outside of this structure. Radhakrishnan et al. also discovered that the activation of NRF1 depends on an enzyme called p97 or VCP. This enzyme helps to flip NRF1 from the inside of the endoplasmic reticulum to its outside surface. In most cells, the proteasome then breaks down this part of NRF1. However, if the proteasome is inhibited, an unknown enzyme cuts NRF1 free from the endoplasmic reticulum, allowing it to migrate to the nucleus and promote the production of more proteasome components to counteract the inhibition. Interestingly, drugs that inhibit the proteasome are used to combat cancer because the build-up of damaged proteins is toxic to the cancer cells. By showing that p97 promotes the ‘bounce-back’ of the proteasome, the work of Radhakrishnan et al. suggests that combining existing proteasome inhibitors with drugs that inhibit p97 could eventually lead to new, more effective, therapies for cancer or other diseases.
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p97-dependent retrotranslocation and proteolytic processing govern formation of active NRF1 upon proteasome inhibition
2013Co-Authors: Senthil K. Radhakrishnan, Willem Den Besten, Raymond J. DeshaiesAbstract:Proteasome inhibition elicits an evolutionarily conserved response wherein proteasome subunit mRNAs are upregulated, resulting in recovery (i.e. 'bounce-back') of proteasome activity. We previously demonstrated that the transcription factor NRF1/NFE2L1 mediates this homeostatic response in mammalian cells. We show here that NRF1 is initially translocated into the lumen of the ER, but is rapidly and efficiently retrotranslocated to the cytosolic side of the membrane in a manner that depends on p97/VCP. Normally, retrotranslocated NRF1 is degraded promptly by the proteasome and active species do not accumulate. However, in cells with compromised proteasomes, retrotranslocated NRF1 escapes degradation and is cleaved N-terminal to Leu-104 to yield a fragment that is no longer tethered to the ER membrane. Importantly, this cleavage event is essential for NRF1-dependent activation of proteasome gene expression upon proteasome inhibition. Our data uncover an unexpected role for p97 in activation of a transcription factor by relocalizing it from the ER lumen to the cytosol. Article now published as eLife 2014;3:e01856 http://dx.doi.org/10.7554/eLife.01856
