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Transcription Factor Nrf1 Is Negatively Regulated by Its O-Glcnacylation Status

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Transcription Factor Nrf1 Is Negatively Regulated by Its O-Glcnacylation Status FEBS Letters 589 (2015) 2347–2358 journal homepage: www.FEBSLetters.org Transcription factor Nrf1 is negatively regulated by its O-GlcNAcylation status Jiayu Chen a,b,c,1, Xiping Liu b,c,1, Fenglin Lü a, Xinping Liu c,YiRuc, Yonggang Ren a, Libo Yao c,2, ⇑ Yiguo Zhang a, ,2 a Laboratory of Cell Biochemistry and Gene Regulation, College of Medical Bioengineering and Faculty of Life Sciences, University of Chongqing, No. 174 Shazheng Street, Shapingba District, Chongqing 400044, China b Department of Biochemistry and Molecular Biology, Zunyi Medical College, Zunyi 563000, China c State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi’an 710032, China article info abstract Article history: O-Linked N-acetylglucosamine transferase (OGT) was identified as an Nrf1-interacting protein. Received 4 April 2015 Herein, we show that Nrf1 enables interaction with OGT and their co-immunoprecipitates are Revised 19 July 2015 O-GlcNAcylated by the enzyme. The putative O-GlcNAcylation negatively regulates Nrf1/TCF11 to Accepted 20 July 2015 reduce both its protein stability and transactivation activity of target gene expression. The turnover Available online 29 July 2015 of Nrf1 is enhanced upon overexpression of OGT, which promotes ubiquitination of the CNC-bZIP Edited by Ivan Sadowski protein. Furthermore, the serine/theorine-rich sequence of PEST2 degron within Nrf1 is identified to be involved in the protein O-GlcNAcylation by OGT. Overall, Nrf1 is negatively regulated by its O-GlcNAcylation status that depends on the glucose concentrations. Keywords: Nuclear factor erythroid 2-related factor Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. (Nrf1) O-GlcNAcylation O-Linked N-acetylglucosamine transferase (OGT) Transcriptional regulation Post-translational modification 1. Introduction Glycosylation and deglycosylation are a pair of ubiquitous rever- Abbreviations: AD1, acidic domain 1; ARE, antioxidant response element; bZIP, basic region-leucine zipper; CHX, cycloheximide; CNC, cap’n’collar; EGFP, enhanced sible modifications of proteins contributing to numerous biological green fluorescent protein; ER, endoplasmic reticulum; Glc, glucose; GCLM, gluta- functions in all systems of life [1–3]. A glycomic estimate suggests mate cysteine ligase modifier subunit; GlcNAc, b-D-N-acetylglucosamine; GlcNH2, that over 50% of all proteins are glycosylated by the covalent attach- glucosamine; GST, glutathione S-transferase; HCF1, host cell factor 1; IP, immuno- ment of P7,000 glycans to polypeptide structures through amide precipitation; IB, immunoblotting; Nrf1, nuclear factor erythroid 2-related factor; NST, Asn/Ser/Thr-rich region; OGT, O-linked N-acetylglucosamine transferase; OGA, linkages (i.e. N-glycosylation) to asparagine side chains, through O-GlcNAcase; PEST2, proline-glutamate-serine-threonine-rich sequence 2; siOGT, glycosidic linkages (i.e. O-glycosylation) to side chains of serine siRNA targeting against OGT; siNC, a scrambled siRNA as a negative control; TCF11, and/or threonine, hydroxylysine (e.g. collagen) or tyrosine residues transcription factor 11; Ub, ubiquitin (e.g. glycogenin), or through C–C linkages (i.e. C-glycosylation) to the Author contributions: Jiayu Chen and Xiping Liu performed all the experiments, C2 position of tryptophan [4]. This estimate is also thought to be far collected the data, made the figures and statistical analysis, and wrote the too low, because it does not consider that many, if not most, pro- manuscript draft. Fenglin Lv and Xinpin Liu participated in the revision of the manuscript for intellectual content and study supervision. Yi Ru and Yonggang Ren teins within the nucleus and cytoplasm are dynamically modified analyzed the data and provided technical support; Yiguo Zhang and Libo Yao by the attachment of b-D-N-acetylglucosamine (GlcNAc) moieties supervised the study and provided the funding support. Yiguo Zhang conceptual- to the hydroxyl group of serine and/or threonine residues (i.e. ized the project, designed the experiments, analyzed the data, wrote and revised O-GlcNAcylation) [5]. The unique O-GlcNAcylation of such soluble the paper. ⇑ Corresponding author. nucleocytoplasmic proteins, as well as secreted or membrane glu- E-mail addresses: [email protected], [email protected] (Y. Zhang). coproteins, occurs in response to cell biological or environmental 1 Contributed equally to this work. cues, including growth factors, signaling molecules, glucose and 2 Co-corresponding author for the paper. http://dx.doi.org/10.1016/j.febslet.2015.07.030 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 2348 J. Chen et al. / FEBS Letters 589 (2015) 2347–2358 other nutrient fluxes, and various stresses [6]. The capability of variety of critical homeostatic and developmental pathways O-GlcNAcylation is of paramount importance in the regulation of through regulating the expression of antioxidant response element cellular metabolism, signaling, transcription, and other processes (ARE)-driven genes, encoding antioxidant proteins, detoxification [6,7]. Conversely, dysregulation of O-GlcNAcylation contributes to enzymes, metabolic enzymes and 26S proteosomal subunits [21– the etiology of cancer, diabetes, neurodegenerative and inflamma- 23]. The CNC-bZIP family comprises the Drosophila Cnc protein, tory diseases [8–11]. the Caenorhabditis elegans skinhead-1 (Skn-1), the vertebrate acti- The O-GlcNAcylation reaction is catalyzed by O-linked vator NF-E2 p45 and related Nrf1 (including TCF11 and its short N-acetylglucosamine transferase (OGT, encoded by the Ogt/Sxc form LCR-F1/Nrf1b), Nrf2 and Nrf3, as well as the transcription gene residing in close proximity to the XIST locus [12,13]), which repressors Bach1 (BTB and CNC homolog 1) and Bach2. In mam- enables the transfer of a single O-GlcNAc moiety from uridine dis- mals, Nrf1 and Nrf2 are two principal CNC-bZIP factors to regulate phosphate N-acetylglucosamine (UDP-GlcNAc) to a serine or thre- ARE-driven cytoprotective genes against cellular stress [24,25]. onine residue of protein substrates, whilst removal of the However, a sharp distinction between Nrf1 and Nrf2 is defined O-GlcNAc (i.e. de-GlcNAcylation) is catalyzed by O-GlcNAcase by the findings that the soluble Nrf2 is dispensable for develop- (OGA, encoded by the single Oga/MGEA5 gene) [14,15]. The ment [26], whereas the membrane-bound Nrf1 is essential for O-GlcNAc cycling is mediated by OGT (with three alternatively maintaining cellular homeostasis and organ integrity during spliced isoforms of 106kDa, 103kDa and 74.5kDa) and OGA (with development and growth. The latter conclusion is drawn from two spliced isoforms of 130kDa and 75kDa); both are per se gene-targeting experiments showing that global knockout of Nrf1 modified by O-GlcNAcylation in metazoan animals [16,17]. The (also called nfe2l1) in mice causes embryonic lethality and severe enzymatic activity of OGT and its substrate specificity are oxidative stress [27–29] and specifically conditional knockout of dependent on concentrations of UDP-GlcNAc, which is controlled the gene in the liver and brain results in non-alcoholic steatohep- by several major metabolic pathways in response to stimuli and atitis, hepatoma [30,31] and neurodegeneration [32,33]. nutrient availability (Fig. 1A). Generally, 2–5% of glucose that The unique biological function of Nrf1 is dictated by its enters the cell is via the glycolytic pathway to produce membrane-topovectorial processing within and around endoplas- fructose-6-phosphate (Fruc-6-p) committed to the hexosamine mic reticulum (ER) to produce several isoforms through different biosynthetic pathway (HBP) to make UDP-GlcNAc as an end prod- post-translational modifications [34–39]. Upon translation of uct. The HBP flux is also integrated with metabolisms of amino Nrf1, its uncleavable N-terminal signal sequence (called NHB1) acids, fatty acids and nucleotides. Glutamine (Gln) serves as the enables the nascent protein to be integrated in a specific topology nitrogen donor to form glucosamine-6-phosphate (GlcNH2–6-p), within and around the ER. After Nrf1 is anchored within the ER the latter acetylation reaction occurs by catalytically trans- membrane through the NHB1-associated region, its connecting ferring the acetyl group from the donor acetyl-CoA to yield transactivation domains (TADs, including AD1, NST and AD2) are N-acetylglucosamine-6-phosphate (GlcNAc-6-p), and subsequent transiently translocated into the lumen, where Nrf1 is cascade reactions with the uridine trisphosphate (UTP) promotes N-glycosylated by the covalent attachment of the glycan biosynthesis of UDP-GlcNAc. Collectively, UDP-GlcNAc is well Glc3Man9GlcNAc2 through amide linkages to asparagine residues placed at a major nexus between cellular metabolic networks in the central Asn/Ser/Thr-rich (NST) domain, enabling Nrf1 to and signaling responsive pathways. represent an inactive 120kDa glycoprotein. Subsequently, glycosy- O-GlcNAcylation is abolished by disruption of Ogt, leading to lated NST-containing TADs are allowed for dynamic repartitioning the lethality of XY stem cells from male mice and resulting embry- out of the ER and repositioning into the cyto/nucloplasm, onic lethality at 5.5 days post-coitus [18]. Loss of the essential whereupon Nrf1 is deglycosylated to yield an active 95kDa factor function of OGT for O-GlcNAcylation in early development of ver- because
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