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

FEBS Letters 589 (2015) 2347–2358

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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 its TADs can gain access to the general transcriptional tebrate is also accompanied by a series of dramatic effects on cel- machinery, enabling target gene transactivation. The membrane- lular metabolism, signaling, transcription, gene expression, and topovectorial organization of Nrf1 also dictates selective adaptation in response to various pathophysiological cues proteolytic processing of the CNC-bZIP protein to generate distinct [6,7,17]. It is well defined that the nutrient-sensitive OGT is isoforms of between 85kDa and 25kDa that differentially regulate allowed for gene regulation by interacting and modifying key ARE-battery genes [37,39–41]. In the present study, we have transcriptional factors (e.g. c-Myc, p53, FoxO1, CREB, CRTC2, investigated whether and how the protein stability of Nrf1 is HCF1, PPARc,ERb, OCT4, NANOG, SOX2, SIN3A and ZFP281), and influenced by OGT-mediated O-GlcNAcylation. The resultant epigenetic regulators (e.g., PcG, TrxG, HDAC and NCOR2), as well evidence that has been presented here demonstrates that the as C-terminal domain of the RNA polymerase II, to promote the putative O-GlcNAcylation of Nrf1 negatively regulates the assembly of distinct functional coding machineries. Interestingly, constitutive expression of the CNC-bZIP factor, its protein stability amongst over 1,000 substrates of OGT, only an evolutionary and its transactivation activity to down-regulate ARE-driven target conserved transcriptional co-activator called host cell factor 1 genes. (HCF1) undergoes an unusual proteolytic cleavage to produce both its N-terminal and C-terminal subunits (i.e. HCF1N and 2. Materials and methods HCF1C, that are involved in control of the cell cycle at G1 and M1 phases, respectively), in the process whereby OGT is 2.1. Chemicals, antibodies and other regents considered to act as a novel protease involving the O-GlcNAcylation reaction [19]. All chemicals were of the highest quality commercially avail- Recently, the O-GlcNAcylation enzyme OGT, as well as its sub- able, of which Alloxan (as an OGT-specific inhibitor), PUGNAc (as strates HCF1 and casein kinase 2 (CK2), had also been detected an OGA-specific inhibitor), MG132 (as a pan proteasomal inhibitor) as one of nuclear factor-erythroid 2 (NF-E2) p45-related factor 1 and cycloheximide (CHX, which blocks nascent polypeptide (Nrf1)-interacting proteins by liquid chromatography-mass spec- biosynthesis) were purchased from Sigma–Aldrich (St Louis, MO, trometry (LC–MS)/MS (see Supplemental data from [20]). USA). Two antibodies against the glycan O-GlcNAc epitope However, it is unknown whether OGT mediates O-GlcNAcylation (CTD110.6) and TCF11/Nrf1 (D5B10) were obtained from Cell of Nrf1 and its long isoforms TCF11 (transcription factor 11). The Signaling Technology (Bedford, MA, USA). Antibodies against OGT latter two proteins belong to the family of cap’n’collar (CNC) basic (H-300) were from Santa Cruz Biotechnology (Santa Cruz, CA, region-leucine zipper (bZIP) transcription factors that control a USA). Antibodies against each of the Flag, V5, HA and GST epitopes, J. Chen et al. / FEBS Letters 589 (2015) 2347–2358 2349

A Glc Gln GlcNH2 Fatty acids

Uridine

metabolism Nucleotide Glc Gln GlcNH CoA metabolism Glu 2 Acetyl-CoA GlcNAc-6-p Glc-6-p Fruc-6-p GlcNH -6-p ets hsht pathway phosphate Pentose 2 GlcNAc-1-p UTP Hexosamine biosynthetic pathway Pi UDP-GlcNAc Glycolysis Glycogen UDP Alloxan GlcNAc-O OH OGT Substrate Substrate Proteins (Nrf1) Proteins (Nrf1) OGA H2O O-GlcNAcylation PUGNAc GlcNAc

B 5 mM Glc medium C 25 mM Glc medium + Alloxan + PUGNAc - (100 μM) - (50 μM)

O-GlcNAc O-GlcNAc

1.00 0.85 1.00 1.21

140-kDa 130-kDa TCF11 140-kDa 120-kDa /hNrf1 130-kDa TCF11 100-kDa 120-kDa /hNrf1 100-kDa

1.00 1.42 1.00 0.75

Tubulin Tubulin

Fig. 1. Expression of human Nrf1/TCF11 is influenced by the intracellular status of O-GlcNAc that depends on the glucose (Glc) concentrations. (A) Schematic representation of the hexosamine biosynthetic pathway (HBP) to yield UDP-GlcNAc and subsequent cycling of O-GlcNAc. The cycling enzymes OGT and OGA control a pair of reversible modifications: O-GlcNAcylation and ensuing de-GlcNAcylation, respectively. Of importance is that O-GlcNAcylation has been considered as a nutrient sensor because the donor sugar, UDP-GlcNAc, integrates inputs from multiple metabolic pathways. The OGT activity to transfer the monosaccharide GlcNAc to serine/threonine residues depends upon UDP-GlcNAc concentrations. The enzymatic activity of OGT can be inhibited by Alloxan, whilst PUGNAc is an inhibitor of the OGA that hydrolyses the sugar O-GlcNAc to be removed from substrates. Upon addition of GlcNH2, it is enabled to directly enter the HBP flux. All abbreviations were defined in the main text. (B) HEK293T cells were allowed to be grown in a hypoglucose medium (containing 5 mmol/L glucose) and also treated with 100 lmol/L Alloxan for 6 h. The total cell lysates were subjected to separation of proteins by SDS–PAGE containing 8% polyacrylamide, followed by Western blotting with antibodies against O-GlcNAc and Nrf1/TCF11. In addition, tubulin served as the internal control for loading proteins. The intensity of blots was quantified by using the ImageJ software and normalized to the control value. (C) HEK293T cells were allowed to be grown in a hyperglucose medium (containing 25 mmol/L glucose) and treated with 50 lmol/L PUGNAc for 6 h. Subsequently, the total cell lysates were subjected to separation of proteins by SDS–PAGE containing 10% polyacrylamide, followed by Western blotting and quantification of the blot intensity by densitometry as described above. along with other antibodies against tubulin, b-actin and GADPH, 2.2. Plasmids and siRNAs were from Signalway Antibody (College Park). The GST-based Protein Interaction Pull-Down Kit was purchased from Thermo sci- Two expression constructs for GST-OGT fusion protein entific. Both SYBR Green universal master mix and Multiscript RT (pGEX-OGT) and its mutant [pcDNA-OGTD(TPR1-6)] [42], together were from TaKaRa Biotechnology Co., Ltd (Dalian, China). with the free-GST expression plasmid pGEX-4T, were a kind gift 2350 J. Chen et al. / FEBS Letters 589 (2015) 2347–2358 provided by Professor Xiaoyong Yang (at School of Medicine, Yale overnight at 4 °C. Thereafter, the antibody blots were University). The plasmid pcDNA-Flag-OGT (#29760) and the cross-reacted for 1 h with the corresponding species-specific sec- pRL-CMV reporter were purchased from Addgene and Promega, ondary antibodies, followed by visualization by using either the respectively. Additional expression constructs for Nrf1 and its enhanced chemiluminescence or the Odyssey Imaging System mutant that had been created on the base of pcDNA3.1/V5His, as (Li-Cor Biosciences). The intensity of immunoblotted protein bands well as pcDNA-3 HA-Ub and the ARE-driven reporter gene was quantified by using the ImageJ software, and normalized to

PSV406 ARE-GSTA2-Luc, were developed in our laboratory distinct protein-loading and other controls as described in figure [34–39]. All these constructs were verified by DNA sequencing. legends. Amongst the primary antibodies, O-GlcNAc-specific anti- Two sequences of small interfering RNAs (siRNAs): 50-GGAGCCUU body (CTD110.6) was used to detect the O-GlcNAcylation status GCAGUGUUAUA-30 and 50-GAGGCAGUUCGCUUGUAUCGU-30, of protein substrates of OGT [42]. target against the human OGT mRNA coding region so as to knockdown its expression. In addition, a scrambled siRNA 2.6. Co-immunoprecipitation and ubiquitination assays sequence (5’-UUCUCCGAACGUGUCACG-3’) that has no homology with any genes was used as an internal negative control. HEK293T cells had been grown on 10-cm dishes, and transfected for 48 h with each (20 lg of DNA) of expression constructs 2.3. Cell culture and transfection for OGT, Nrf1 and their mutants. The cells were treated with 10 lM MG132 for an additional 4 h before being harvested in lysis Human embryonic kidney (HEK293T) cell lines were main- buffer: 50 mM Tris, pH 8.0, 20% glycerol, 500 mM NaCl, 0.5% NP-40, tained in the State Key Laboratory of Cancer Biology, the Fourth 5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 protease Military Medical University. The cells were grown in Dulbecco’s inhibitors (from Roche). The supernatants collected from centrifu- Modified Eagle Medium (DMEM, from Gibco BRL, Grand Island, gation were pre-clarified by the protein A/G PLUS-agarose (from NY, USA) supplemented with 10% fetal bovine serum (FBS, from Santa Cruz Biotechnology) for 40 min at 4 °C, followed by immuno- Gibco BRL), 100 U/ml penicillin and 100 lg/ml streptomycin (both precipitation with antibodies against the V5 epitope or the normal from Invitrogen, Carlsbad, CA, USA) in a 37 °C incubator with 5% mouse IgG overnight at 4 °C. The precipitates were re-incubated

CO2 in the humidified air. These cells were subjected to with the protein A/G PLUS-agarose for 6 h and then washed five transfection experiments that had been conducted by using times with the buffer: 50 mM Tris, pH 7.5, 10% glycerol, 100 mM TurboFect Transfection Reagent (from Thermo Scientific) following NaCl, 0.1% NP-40, 1 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, before the manufacturer’s instructions. being subjected to protein resolution by SDS–PAGE, followed by Western blotting analysis. For in vivo ubiquitination assay, the cells 2.4. Quantitative real-time PCR analysis were co-transfected with HA-tagged ubiquitin (Ub) and/or each of the indicated expression constructs for 48 h, before being treated HEK293T cells that had been transfected with the indicated with 10 lM MG132 for 4 h. The cell lysates were subjected to siRNAs were subjected to isolation of total RNAs, which were co-immunoprecipitation with a V5 antibody, and immunoblotting carried out by using the RNAiso kit (from Takara) according to with antibodies against the HA epitope or the O-GlcNAc glycan to the manufacturer’s instructions. Subsequently, 500 ng of total detect the status of ubiquitination [43] or O-GlcNAcylation [42]. RNAs was added in retrotranscription reaction with the PrimeScript RT Master Mix (from TaKaRa) to generate the first 2.7. GST-based pull-down assay strand of cDNA, which served as the template for qRT-PCR that was performed in the following conditions: activation at 95 °C Expression of GST or its fusion protein with OGT (i.e. GST-OGT) for 30 s, followed by 40 cycles of 10 s at 95 °C, and 30 s at 58 °C. was induced by 0.2 mmol/L IPTG in Escherichia coli BL21 (DE3, At last, the melting curve was added to examine the amplification pLys), before they were immobilized on glutathione-agarose by quality. Expression of mRNA for b-actin was used as an internal using the GST Protein Interaction Pull-Down Kit (from Thermo sci- control standard. Two pairs of primers used were as follows: entific), which was carried out according to the manufacturer’s OGT with 50-GGCAGTTCGCTTGTATCGT-30 (forward) and 50-GATG instructions. HEK293T cells were transfected with expression con- GCACGCGTATAACAC-30 (reverse) sequences, and b-actin with structs for Nrf1, before being incubated with equal amounts of 50-AATCTGGCACCACAC CTT CTACAA-30 (forward) and 50-TAGCAC immobilized GST or its fusion proteins for 16 h at 4 °C. The AGCCTGGATAGCAACG-30 (reverse) sequences. GST-bound agarose beads were washed five times with a wash solu- tion enclosed in the kit, before GST-bound proteins were eluted, 2.5. Western blotting with O-GlcNAcylation analysis separated by SDS–PAGE, and then visualized by Western blotting.

Experimental cells were either treated with the indicated chem- 2.8. Luciferase reporter assay icals or transfected with distinct expression constructs before being harvested and homogenized in RIPA lysis buffer (50 mM Cells were co-transfected with 200 ng of ARE-driven luciferase

Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, reporter (PSV406 ARE-GSTA2-Luc) and 20 ng of renilla luciferase 0.1% SDS) containing 2 lg/mL protease inhibitor cocktail (from reporter (pRL-CMV), along with siRNA or other effecter plasmids Roche, Germany). The clariﬁed supernatants were collected and as required. At 24 h after transfection, the reporter activity was then the protein concentrations were determined by using a BCA measured by using the dual luciferase reporter assay system protein assay kit (from Pierce Biotechnology, Rockford, IL, USA). (E1910 from Promega), according to the manufacturer’s instruc- Equal amounts (20 lg) of protein extracts were subjected to elec- tion, and then was normalized to the value of corresponding renilla tropherotic separation by SDS–PAGE containing 6% to 10% poly- luciferase activity. Subsequently, differences in their transcrip- acrylamide. Subsequently, the resolved proteins were transferred tional activity were subjected to statistical analyses. onto the Amersham’s nitrocellulose membranes, followed by Western blotting with distinct primary and secondary antibodies. 2.9. Cycloheximide chase experiment The protein-transferred membranes were blocked by incubation with 5% bovine serum albumin at room temperature for 1 h and HEK293T cells had been transfected with the indicated siRNAs, was then re-incubated with each of the primary antibodies for and 24 h latter were subjected to cycloheximide (CHX, 100 lg/ml) J. Chen et al. / FEBS Letters 589 (2015) 2347–2358 2351 chase experiment, which was performed for the indicated times (0, expressed as another major 130kDa and 100kDa proteins (cf. 0.5, 1, 2 and 4 h). Subsequently, the cell lysates were examined by Fig. 1C, middle panel, with Fig. 1B, middle panel). Intriguingly, the Western blotting experiments to assess the protein stability of Nrf1 enhancement of O-GlcNAcylation by PUGNAc appeared to enable and OGT. a 25% reduction in the overall expression of TCF11/hNrf1, with particularly 120kDa isoform to be blocked (Fig. 1C, middle panel, 2.10. Statistical analysis it should be here noted that the 120kDa of TCF11/hNrf1 was separated by the pH 8.9 Laemmli SDS–PAGE containing 10% The statistical signiﬁcance of changes in the ARE-driven repor- polyacrylamide, which are proposed to be a processed and/or non ter activity and in the gene expression was determined using the glycosylated/deglycosylated isoform that is sharply distinctive Student’s t test or Multiple Analysis of Variations (MANOVA). The from the full-length glycoprotein of 120kDa Nrf1 that has been data are shown as a fold change (mean ± S.D), each of which previously shown to be isolated by the pH 7.0 LDS/NuPAGE gel represents at least 3 independent experiments that were each containing 4–12% polyacrylamide [36–38]). Collectively, these data performed triplicate. suggest that changes in the putative O-GlcNAc status (of proteins such as TCF11/hNrf1 and its regulators) monitors the abundance 3. Results and discussion of the CNC-bZIP protein (and/or its stability).

3.1. The abundance of human Nrf1/TCF11 is monitored by its putative 3.2. Interaction of OGT with Nrf1 enables O-GlcNAcylation of the CNC- O-GlcNAc status bZIP protein

Molecular cloning of human Nrf1 (hNrf1) and its long form An amino acid sequence alignment of hNrf1, TCF11 and mouse TCF11 reveals both polypeptides comprise 742 and 772 aa, respec- Nrf1 (mNrf1) was made by using an programme called T-Coffee tively; hNrf1 arises from alternatively splicing of the full-length (http://www.ebi.ac.uk/Tools/msa/tcoffee/), revealing that mNrf1 mRNA transcript to delete Exon 4 encoding aa 242–271 within has 97.432% sequence identity with hNrf1 and its long form acidic domain 1 (AD1) of TCF11 [44,45]; this region called Neh4L TCF11, with the only variable region being presented (Fig. 2A). It is highly conserved with the Neh4 transactivation domain of should also be noted that hNrf1-mediated reporter gene activity Nrf2 [39,46]. Almost equal amounts of hNrf1 and TCF11 are appears to be unaffected by lack of aa 242–271 within AD1 of expressed in normal human cell lines, whereas human cancer cell TCF11, albeit this region contains a nuclear export signal [47,54]. lines predominantly express hNrf1 rather than TCF11 (data unpub- This is consistent with the fact that not a similar form to TCF11 lished in our group). However, these two isoforms have similar exists in the mouse and thus mNrf1 is essential for exerting its transactivation activity mediated primarily by AD1 [47], though unique functions to maintain cell homeostasis against various the TAD region of Nrf1 lacks the Neh4 subdomain that is present stresses [21–23]. Thus, an expression construct for mNrf1 has been in TCF11 (which is absent in the mouse [48,49]). employed in transfection experiments with HEK293 cells, in order Herein, we investigated whether the abundance of endogenous to keep a comparison with our previous work focused on the spe- TCF 11/hNrf1 is influenced by the O-GlcNAc status. Western blot- cies CNC-bZIP factor. ting of total lysates with a specific antibody against the O-GlcNAc To address the question of what effects the putative O-GlcNAc epitope revealed that treatment of HEK293T cells (that had been status controlled by OGT has on expression of Nrf1 and/or TCF 11, grown in a 5 mmol/L glucose-containing medium enabling the de we next examined whether the CNC-bZIP protein interacts with novo glycosylation cascade to slow down under the hypoglucose OGT enabling it to be O-GlcNAcylated. Firstly, a mixture of either conditions so that those such as non-glycosylated and/or glutathione S-transferase (GST) or its fusion protein GST-OGT (puri- de-glycosylated 100kDa Nrf1 were expressed to relative higher fied from IPTG-induced E. coli strain, Fig. 2B, left panel) with total extents, as described previously [37,38]) with 100 lmol/L of lysates of HEK293T cells that had been transfected with an expres- Alloxan (which acts as an OGT-specific inhibitor to block sion construct for mNrf1 tagged with the V5 epitope (mNrf1-V5) O-GlcNAcylation [50,51]) caused a decrease in the O-GlcNAc levels was respectively subjected to pull-down experiments from to 85% of the control value measured from untreated cells glutathione-agarose beads. Subsequently, three fractions of elution (Fig. 1B, upper panel). Interestingly, the modest inhibition of by reduced glutathione were determined by immunoblotting with O-GlcNAcylation by Alloxan was accompanied by a resulting antibodies against either GST (Fig. 2B, right upper panel) or V5 epi- 1.42-fold increase in the abundance of TCF 11/hNrf1 between topes (right lower panel). The results revealed that a minor 120kDa 140kDa and 100kDa, in particular both 130kDa and 100kDa pro- glycoprotein of mNrf1-V5 and its major 95kDa and 85kDa iso- teins (Fig. 1B, middle panel). It should be herein noted that distinct forms are recovered in the first fractional elutant of the GST-OGT TCF11/hNrf1 isoforms were separated in the pH 8.9 Laemmli fusion protein rather than the GST moiety, suggesting a possibility SDS–PAGE containing 8% polyacrylamide, and therefore their that Nrf1 has a physical interaction with OGT in vitro. Herein, it is electrophoretic mobility on the pH 8.9 SDS–PAGE gel is obviously notable that the in vitro non-denatured lysate conditions should different from the equivalent mobility exhibited on the pH 7.0 enable the 120kDa mNrf1-V5 deglycosylation to become instead NuPAGE containing 4–12% polyacrylamide [36–38]). of a major 95kDa deglycoprotein, followed by an unidentified pro- Conversely, 50 lmol/L of PUGNAc (which is the potent inhibitor cessing to yield an N-terminally-truncated 85kDa isoform. of OGA to block de-GlcNAcylation [52,53]) was allowed for treat- Secondly, to examine whether the putative interaction of Nrf1 ment of HEK293T cells (that had been grown in a 25 mmol/L with OGT occurs in vivo, total lysates of HEK293T cells that had glucose-containing medium facilitating the de novo glycosylation been grown in 25 mmol/L glucose-medium and co-transfected reaction to be promoted under the hyperglucose condition as with expression constructs for mNrf1-V5 and OGT-Flag (Fig. 2C, described previously [37,38]), leading to increased levels of left panel) were subjected to co-immunoprecipitation (CO-IP) O-GlcNAc by 21% higher than the control level obtained from with antibody against the V5 epitope. The results indicate untreated cells (Fig. 1C, upper panel). This was accompanied by ele- co-immunoprecipitates of mNrf1-V5 with OGT-Flag, but not a sim- vated expression of TCF11/hNrf1 as both major 140kDa and ilar immunoprecipitate with DOGT [which is an OGT-truncated 120kDa proteins, relative to their equivalent abundances under mutant lacking the tetraticopeptide repeats 1 (TPR1) to 6 motifs] the hypoglucose conditions so that TCF 11/hNrf1 was principally was observed (Fig. 2B, right panel). In addition, although that the 2352 J. Chen et al. / FEBS Letters 589 (2015) 2347–2358

A aa 242--271 in TCF11 but not in Nrf1 mNrf1 208 252 hNrf1 208 251 TCF11 208 281

B Total cell lysates GST pull-down experiments pGEX-4T pGEX-OGT

pGEX-4TpGEX-OGT 123123Fraction GST-OGT

Anti-GST

free GST

120-kDa 95-kDa mNrf1-V5 85-kDa

C Input IP: IgG Anti-V5 + - OGT-Flag + + - OGT-Flag + + mNrf1-V5 + ΔOGT G -- Anti-Flag + + + mNrf1-V5 F G F Anti-Flag mNrf1-V5 120-kDa IB

G Anti-IgG F Anti-OGT

D IP: Anti-V5 IP: Anti-V5 GlcNH (5μM) ++ 2 ++- + - OGT-Flag + + +- +- mNrf1-V5 + +- +-

G Anti-OGT F

O-GlcNAc mNrf1-V5 (Nrf1-interacting 120-kDa proteins) 95-kDa 85-kDa

Fig. 2. Interaction of OGT with Nrf1 occurs both in vitro and in vivo. (A) A multiple sequence alignment of hNrf1, TCF11 and mNrf1 was made by using the T-Coffee programme. Amongst them, only one variable region was presented herein. The symbol (⁄) indicates identical amino acids, whereas other amino acids with complete similarity or semi-similarity are represented by other symbols (: and ), respectively. In addition, aa 242–271 within AD1 of TCF11 are lost in either hNrf1 or mNrf1. (B) Lysates of HEK293T cells expressing mouse V5-tagged Nrf1 (i.e. mNrf1-V5), as the source of prey proteins, were incubated with either free GST moiety or its fusion protein GST-OGT (puriﬁed from IPTG-induced E. coli lysates) as bait proteins that were immobilized on glutathione-agarose beads for the pull-down experiments. The bait-prey elutant was then collected in order, and was thereafter loaded on lanes 1, 2, 3 of SDS–PAGE gels, followed by Western blotting analysis with antibodies against GST and the V5 epitope. (C) HEK293T cells were co-transfected with expression constructs for mNrf1-V5, together with either OGT-Flag or its mutant DOGT (lacking its TPR1 to 6 motifs). Immunoprecipitation of mNrf1-V5 was carried out with anti-V5 antibody. The resultant precipitates were immunoblotted with antibodies against the Flag epitope or IgG (as an internal control). It is noted that each of the ’input’ samples represents 5% of the whole-cell lysates used for the above immunoprecipitation. (D) HEK293T cells co- expressing mNrf1-V5 and OGT-Flag were allowed for being grown in a hypoglucose (5 mmol/L) medium containing 5 mmol/L GlcNH2. Subsequently, the immunoprecipitates of mNrf1 with anti-V5 antibody were identiﬁed by immunoblotting with antibodies against O-GlcNAc, OGT or Nrf1. In addition, the putative O-GlcNAcylated OGT isoform (C) and (D) is indicated by the letter G, whereas the letter F represents the intact full-length non-glycosylated and/or deglycosylated OGT protein.

hyperglucose condition had been showed to allow mNrf1 to be co-immunoprecipitates with the V5 antibody were visualized by expressed as a major 120kDa glycoprotein [37,38], herein the Western blotting with additional three different antibodies present expression of 120kDa mNrf1 was repressed by (Fig. 2D). These results revealed that the co-immunoprecipitates over-expression of ectopic OGT-Flag (Fig. 2C, left middle panel). of mNrf1 and its interacting proteins (e.g. OGT itself) were obvi-

Thirdly, whether co-immunoprecipitates of mNrf1-V5 is ously modiﬁed through GlcNH2-derived O-GlcNAcylation reaction O-GlcNAcylated by OGT was determined next. The HEK293T with ectopic OGT-Flag and/or endogenous OGT (Fig. 2D, left and cells co-expressing mNrf1-V5 and OGT-Flag had been allowed for right panels); one of two major electrophoretic bands of OGT pro- being grown in the hypoglucose medium (i.e. 5 mmol/L tein is proposed to be a putative O-GlcNAcylation isoform (indi- glucose enabling de nevo glycosylation to shut down), before cated by G, right upper panel), which was enriched within the being added by 5 lmol/L glucosamine (GlcNH2), which serves immunoprecipitates more than that visualized by general as an additional original material of the HBP to recover the Western blotting of total lysates elsewhere. In addition, it is noted intracellular O-GlcNAcylation catalyzed by OGT). The resultant that the hypoglucose conditions enabled mNrf1-V5 to express as a J. Chen et al. / FEBS Letters 589 (2015) 2347–2358 2353

A Real-time PCR D 100 Luciferase reporter assay p < 0.01 p < 0.05 $ 80

** 20

Relative expression ** siNC siOGT-1 siOGT-2 0 Relative luciferase activity siNC siOGT-1

siNC siOGT-1 B siNC siOGT-1 E TCF11/hNrf1 140-kDa G e1 130-kDa 120-kDa F OGT 100-kDa 1.00 1.75 1.00 0.64 Tubulin e2 Lipin1 1.00 2.13 C siNC siOGT-1 e3 GCLM 1.00 1.73

e4 Tubulin

F O-GlcNAc 2.4 siNC $$ siOGT-1 $ $ 1.6

0.8 1.00 0.59 Relative expression 0.0 Tubulin

TCF11 Lipin1 GCLM /hNrf1

Fig. 3. Knockdown of OGT causes an increase in the abundance of hNrf1/TCF11 and its activity to up-regulate its target gene expression. (A) Total RNAs were isolated from HEK293T cells that had been transfected with two siRNA sequences targeting against OGT (i.e. siOGT-1 and siOGT-2) or a scrambled control siNC, and were then reversely transcribed into the first strand of cDNA. Subsequently, the mRNA levels of OGT were measured by quantitative real-time PCR (qRT-PCR). The results were calculated as a fold change (mean ± S.D) and siOGT-1 causes a significant decrease (**P < 0.01, n = 9), relative to the basal OGT expression measured from siNC-transfected cells. (B and C) Total lysates of the above transfected cells were subjected to Western blotting with antibodies against OGT, O-GlcNAc and tubulin. (D) HEK293T cells had been co-transfected with siOGT-1 or siNC, together with two reporter genes PSV40GSTA2-6 ARE-Luc and pRL-CMV. At 24 h after transfection, the ARE-driven activity was measured by using double luciferase assay kit and then normalized by the corresponding value of the Renialla activity. The results were calculated as a fold change (mean ± S.D) of reporter gene transactivation (i.e. mediated by endogenous Nrf1/TCF11 and/or Nrf2) upon siOGT knockdown. A significant increase ($P < 0.05, n = 9) is relative to the basal activity measured from siNC-transfected cells. (E and F) Total lysates of the above-transfected cells were examined by Western blotting with antibodies against hNrf1/TCF11, Lipin1, GCLM and tubulin. Subsequently, the intensity of blots was quantified by densitometry using the ImageJ software and normalized to the siNC value. These data shown here are a representative of at least three independent experiments undertaken on separate occasions, and significant increases ($P < 0.05 and $$P < 0.01, n = 6) are indicated. major 95kDa non-glycosylated and/or de-glycosylated protein in and Western blotting. The result obtained from qRT-PCR showed COS-1 cells [37,38]. Herein, the abundance of the 95kDa mNrf1 that the expression of OGT mRNA was significantly knocked down was suppressed by either ectopic OGT-Flag or endogenous OGT by either siOGT-1 or siOGT-2 to approximately 10–25% of the con- isoenzymes (Fig. 2D, left lower panel). Taken together with trol value measured obtained from the cells that had been trans- LC-MS/MS data (showing that OGT is an Nrf1-interacting protein fected with scrambled siRNA as an internal negative control (i.e. [20]), these results indicate that a physical interaction of Nrf1 with siNC, with its value of 1 being set) (Fig. 3A). Western blotting OGT allows the CNC-bZIP protein to be O-GlcNAcylated, resulting revealed that siOGT-1 knockdown also caused a 36% decrease in its destabilization. in the endogenous OGT protein (Fig. 3B), as accompanied by an additional 41% reduction in the entire O-GlcNAcylated proteins 3.3. Knockdown of OGT increases expression of Nrf1 and its target (Fig. 3C), when compared with the control siNC levels. genes However, relative gene expression of the ARE-driven reporter

PSV40GSTA2-6 ARE-Luciferase was signiﬁcantly increased to HEK293T cells were transfected with each OGT-targeting siRNA 2-fold changes by knockdown of siOGT-1 (Fig. 3D). Further exam- oligonucleotides (i.e. siOGT-1 and siOGT-2) and subsequently the inations of TCF11/hNrf1 indicated that siOGT-1 knockdown expression levels of the O-GlcNAcycling enzyme were examined appeared to cause stabilization of the endogenous CNC-bZIP pro- by quantitative real-time polymerase chain reaction (qRT-PCR) teins (particularly 130/140kDa isoforms) such that it was 2354 J. Chen et al. / FEBS Letters 589 (2015) 2347–2358

A B D Control Control(EGFP)OGT-Flag (EGFP) OGT-Flag G 140-kDa OGT 130-kDa TCF11 Luciferase reporter assay F 120-kDa /hNrf1 100-kDa 70 p < 0.01 1.00 4.83 1.00 0.57 60 Tubulin 50

C 40 6 Control OGT-Flag $$ O-GlcNAc 30 4 $ 20 ** 2 10 Relative luciferase activity (fold)

Relative expression * 0 Control OGT-Flag 1.00 3.14 0 OGT O-GlcNAc TCF11/ (EGFP) hNrf1 Tubulin

Fig. 4. Over-expression of OGT causes a reduction in expression of hNrf1/TCF11 and its transcriptional activity. (A and B) OGT was allowed for over-expression in HEK293T cells that had been transfected with an expression construct for OGT-Flag, when compared with an EGFP-expressing control plasmid. Then total cell lysates were analyzed by Western blotting with antibodies against OGT, O-GlcNAc and Nrf1/TCF11. In addition, tubulin served as an internal loading control. (C) The intensity of the above immunoblots was quantified by densitometry and normalized to the control values measured from the EGFP-transfected cells. These data that were graphically shown herein are a representative of at least three independent experiments undertaken on separate occasions, with significant increases ($P < 0.05 and $$P < 0.01, n = 6) and significant decrease (*P < 0.05, n = 6) being indicated. (D) HEK293T cells had been co-transfected with an expression construct for OGT-Flag or EGFP (as a control), together with two reporter genes PSV40GSTA2-6 ARE-Luc and pRL-CMV. At 24 h after transfection, ARE-driven luciferase activity was measured as described for Fig. 3D, and were calculated as a fold change (mean ± S.D) of reporter gene transactivation (i.e. mediated by endogenous Nrf1/TCF11 and/or Nrf2) upon over-expression of OGT. A significant decrease (**P < 0.01, n = 9) is relative to the basal activity measured from EGFP-transfected cells. increased by 75%, with a slightly slower electrophoretic mobility OGT-Flag over-expression (Fig. 4D). Overall, these data indicate being exhibited on SDS–PAGE gels (Fig. 3, E1 and F). Moreover, that forced expression of OGT results in marked decreases in the endogenous expression of TCF11/Nrf1-target gene products abundance of endogenous hNrf1/TCF11 and its transactivation Lipin1 [55] and GCLM [29,56] was markedly increased by activity to mediate ARE-driven gene expression. siOGT-1 knockdown (Fig. 3, E2, E3 and F). Collectively, these results suggest that knockdown of OGT appears to promote stabilization of 3.5. The turnover of Nrf1 is regulated by OGT-directed O- hNrf1/TCF11, leading to an increase in expression of its GlcNAcylation downstream genes. The above-described results raise a question of why the poten- 3.4. Over-expression of OGT reduces the abundance of Nrf1 and its tial O-GlcNAcylation of Nrf1/TCF11 by OGT causes a decrease in the transactivation activity amount of the CNC-bZIP factor and its transcriptional activity. Although it is clear that the stability of Nrf1/TCF11 is regulated To further determine the putative effect of OGT on Nrf1/TCF11, through different ubiquitin (Ub) ligases Hrd1, SCFb-TrCP and we transfected an expression construct for either the C-terminally SCFFbw7 targeting the CNC-bZIP protein to distinct degradation flagged OGT (i.e. OGT-Flag) or enhanced green fluorescent protein pathways [22,57,58], it is unknown of whether ubiquitination of (EGFP, as an internal control) into HEK293T cells. Western blotting Nrf1/TCF 11 is regulated by its O-GlcNAcylation status. Herein, in of the cell lysates showed that over-expression of ectopic OGT-Flag order to address whether or how O-GlcNAcylation of Nrf1/TCF11 protein caused a 3.14-fold increase in the entire O-GlcNAcylation monitors the protein stability through the ubiquitin-proteasomal mediated by this enzyme, when compared with the control value degradation pathway, three expression constructs for both mNrf1 of 1.00 that mediated by the endogenous OGT enzyme (Fig. 4A, and Ub-HA (i.e. Ub tagged by HA C-terminally) together with either upper and middle panels and 4C). By comparison with OGT-Flag or EGFP (as an internal control) were co-transfected into siOGT-mediated blockage of O-GlcNAcylation, the pattern of HEK293T cells (Fig. 5A). Western blotting of the total cell lysates O-GlcNAcylated proteins decreased by siOGT-1 (Fig. 3C) is mark- showed that over-expression of ectopic OGT-Flag and Ub-HA did edly distinctive from that of the OGT-elevated O-GlcNAcylated pro- cause an obvious increase in the amount of total ubiquitinated proteins (including TCF11/hNrf1 and OGT itself that were deduced as teins, including the enzyme OGT itself (Fig. 5A, upper and lower major specific glycosylated proteins) (Fig. 4A, middle panel). panels); this was, however, accompanied by decreased expression Intriguingly, the ectopic OGT-Flag over-expression led to an level of the 120kDa mNrf1 glycoprotein (middle panel). Taken obvious reduction in the abundance of endogenous TCF11/hNrf1 together with other MG132-based experimental evidence showing proteins (particularly 130kDa and 140kDa) to 57% of the control that the turnover of Nrf1/TCF11 occurs via Hrd1-, SCFb-TrCP- and/or levels measured from the cells that had been transfected with an SCFFBW7-directed ubiquitin-mediated protein degradation path- EGFP-expression construct (Fig. 4B, upper panel and 4C). Further, ways [22,57–59], it is therefore postulated that O-GlcNAcylation luciferase reporter assays also revealed that a significant decrease of mNrf1 by OGT enables destabilization of the CNC-bZIP protein, in transcriptional expression of the PSV40GSTA2-6 ARE-Luc possibly via the ubiquitin-proteasomal degradation pathway, reporter gene from 60- to 10-fold changes was resulted from albeit detailed mechanistic studies are required. J. Chen et al. / FEBS Letters 589 (2015) 2347–2358 2355

A Control B (EGFP) OGT-Flag Glc: 25 mM 5 mM ++mNrf1-V5 ++- + mNrf1-V5 ++Ub-HA - +++ OGT-Flag 120-kDa mNrf1-V5 (TCF11/ Anti-Flag 95-kDa hNrf1) Input 1.00 0.05 0.26 1.87

Anti-V5 OGT-Flag (120-kDa mNrf1) 1.00 7.11 8.61 9.24

β-Actin IP: Anti-V5 IB: Anti-HA 10% SDS-PAGE

C ΔOGT mNrf1+OGT mNrf1+ 120-kDa Ubiquitinated mNrf1-V5 proteins 95-kDa 1.00 1.69 Anti-OGT full-length OGT Glyco- ΔOGT truncated ΔOGT

Tubulin

6% SDS-PAGE

D siNC siOGT E 00.5124M00.512 4hafterCHX siOGT_TCF11/hNrf1 siNC_TCF11/hNrf1 TCF11 /hNrf1

Y = -0.3794x + 0.408 R2 = 0.8563 1.00 0.79 0.69 0.49 0.35 1.60 1.64 0.74 0.50 0.39

Y = -0.2421x - 0.0997 R2 = 0.9463

OGT of TCF11/Nrf1_siNC) 0 /[A] 1.00 1.21 1.17 1.09 1.07 0.59 0.55 0.51 0.49 0.32 t

β-Actin Abundance of TCF11/hNrf1 Ln( [ A ]

Time after CHX treatment (h)

Fig. 5. The O-GlcNAc promotes the turnover of Nrf1/TCF11 proteins via the ubiquitin-mediated degradation pathway. (A) HEK293T cells had been co-transfected with OGT- Flag (or EGFP as a control) plasmids, together with additional two expression constructs for mouse Nrf1 (i.e. mNrf1-V5) and HA-tagged ubiquitin (Ub-HA), before being treated with the proteasomal inhibitor MG132. Subsequently, an in vivo assay was employed to assess ubiquitination of mNrf1-V5 immunoprecipitates, whilst the ‘Input’ samples were subjected to Western blotting with antibodies against the Flag or V5 epitopes. (B) The indicated expression constructs were co-transfected into HEK293T cells that were allowed for growth in a complete medium containing 5 or 25 mmol/L glucose. The total cell lysates were then subjected to separation of proteins by SDS–PAGE containing 10% polyacrylamide, followed by Western blotting with antibodies against OGT, hNrf1/TCF11 and b-actin (as an internal loading control). (C) Total lysates of HEK293T cells that were allowed for over-expression of mNrf1-V5, together with either OGT or its mutant DOGT (lacking its TPR1–6 motifs), were subjected to separation of proteins by SDS–PAGE containing 6% polyacrylamide, followed by immunoblotting with antibodies against the V5 epitope, OGT or tubulin (as an internal loading control). (D) HEK293T cells that had been transfected with siOGT or siNC were treated with 100 lg/ml cycloheximide (CHX) for the indicated times (i.e. 0, 0.5, 1, 2 and 4 h). The CHX chase experiments were employed to assess the protein stability of hNrf1/TCF11 and OGT by Western blotting. Subsequently, the intensity of all blots was quantiﬁed by densitometry and normalized to the control values measured from siNC-transfected cells. These data shown herein are a representative of at least three independent experiments undertaken on separate occasions. (E) The relative abundance of hNrf1/TCF11 expression in siOGT- and siNC-transfected cells was calculated and the turnover of the CNC-bZIP proteins was shown graphically.

Next, we examined whether the role of OGT in the negative reg- hNrf1/TCF11 (of between 140kDa and 100kDa) (Fig. 5B, two ulation of Nrf1 is inﬂuenced by the concentration of glucose. When central lanes in upper and middle panels). Conversely, expression HEK293T cells had been grown in a 25-mmol/L hyperglucose med- of these CNC-bZIP proteins (particularly of 95kDa) appeared to ium enabling de novo metabolism to product a certain amount of be markedly increased to 1.87-fold, even though ectopic the major substrate UDP-GlcNAc required for the OGT-catalyzed OGT-Flag expression was retained in the HEK293T cells that had O-GlcNAcylation, over-expression of OGT-Flag caused signiﬁcant been grown in a 5-mmol/L hypoglucose medium, in which the decreases in the abundances of the major ectopic mNrf1-V5 pro- yield of UDP-GlcNAc was reduced leading to elimination of the teins (between 120kDa and 95kDa) and the minor endogenous OGT-catalyzed O-GlcNAcylation. Further examinations revealed 2356 J. Chen et al. / FEBS Letters 589 (2015) 2347–2358

mNrf1 Δ2-80 Δ1-291 Δ299-400Δ403-440Δ441-455 Δ456-519Δ454-488Δ489-580Δ403-506 - + - + - + - + - + - + - + - + - + - + OGT-Flag + + + + + + + + + + Control mNrf1 ------(EGFP) 120-kDa 95-kDa * * 85-kDa 90-kDa § 55-kDa * 80-kDa 46-kDa * * 36-kDa

lanes #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 120-kDa 85-kDa 55-kDa 85-kDa 110-kDa 115-kDa 115-kDa 120-kDa 90-kDa 90-kDa 80-kDa Ratios 1.00/0.79 0.43/0.37 0.43/0.53 1.02/0.38 1.00/0.74 1.06/0.81 0.61/0.81 0.54/0.73 1.15/0.96 0.31/1.14 0.69/1.49 (1.00/0.87) (1.00/1.24) (1.00/0.37) (1.00/0.76) (1.00/1.32) (1.00/1.35) (1.00/0.84) (1.00/3.68) (1.00/2.15)

OGT OGT

GADPH GADPH

Fig. 6. Two regions within Nrf1 enable it to be associated with its putative O-GlcNAcylation directed by OGT. HEK293T cells had been co-transfected with an expression construct for wild-type mNrf1-V5 or its mutants (lacking various lengths of discrete regions within mNrf1), together with (+) or without () OGT-Flag or EGFP (as an internal control) plasmids. Subsequently, the total cell lysates were subjected to separation of proteins by SDS–PAGE containing 6% polyacrylamide, followed by Western blotting with antibodies against the V5 epitope, OGT and GADPH (as an internal loading control). Subsequently, the intensity of all blots was quantified by densitometry and normalized to the wild-type Nrf1 protein expressed in OGT-transfected cells (its value of 1). The ratio of the major longer isoforms of mNrf1 or its mutant protein in OGT/Nrf1-transfected cells to their corresponding yields from EGFP/Nrf1-transfected cells was shown on the bottom of the upper panel. The images shown herein are a representative of at least three independent experiments undertaken on separate occasions. The position of electrophoretic migration of the V5-tagged mNrf1 polypeptides was estimated to be 120, 95, 85, 55, 46, and 36kDa. (⁄) indicates a recovery of the relatively longer isoform of each mutants from the inhibitory effect of OGT on Nrf1 in the co-transfected cells, whereas ($) represents an enhanced effect of OGT on the Nrf1 mutant when compared with its wild-type Nrf1 expression in both co-transfected cells. that inhibition of mNrf1-V5 by OGT-Flag was obviously recovered siNC control levels. Collectively, these data indicate that OGT to 1.69-fold expression in the cells that had been co-transfected has a negative capability to decrease basal expression of with a construct for the DOGT mutant (lacking its TPR1–6 motifs) hNrf1/TCF11, but the detailed mechanism by which the process (Fig. 5C, upper panel), and the recovery was not associated with the of the proteolytic degradation of the existing CNC-bZIP proteins abundance of DOGT expressed as a major truncated protein, is regulated by the O-GlcNAcylation enzyme is not well under- together with a relative minor and slower glycosylated isoform, stood. Intriguingly, siOGT knockdown rendered the enhanced being displayed on the pH 8.9 SDS–PAGE gel containing 6% poly- hNrf1/TCF11 protein to exhibit a slower mobility during acrylamide (Fig. 5C, middle panel). Taken together with the above electrophoresis than the corresponding control case of siNC data (shown in Figs. 2–4), these results suggest that the TPR1–6 (Fig. 5D, cf. right 5 lanes with left 5 lanes). Together with OGT region of OGT enables the O-GlcNAc enzyme to interact with over-expression data, this finding leads us to surmise that OGT Nrf1/TCF11, allowing the latter CNC-bZIP protein to be modified could act as an indirect regulator to promote ubiquitin-mediated by O-GlcNAcylation. degradation, and might also serve as a direct functional protease Subsequently, the inhibitory effect of OGT on the stability of as described for the processing of HCF1 [19], although these events Nrf1/TCF 11 and its turnover was further determined by cyclohex- require further investigation. imide (CHX, that inhibits biosynthesis of nascent polypeptides) chase experiments of HEK293T cells that had been transfected 3.6. O-GlcNAcylation of Nrf1 is associated with its serine-rich portion with siOGT or siNC before being treated with the chemical of the PEST2 sequence (100 lg/ml) in medium containing 25 mM glucose. After knockdown of OGT, its residual protein became fainter as the chase time The C-terminal enzymatic activity of OGT to catalyze addition of was extended to 4 h after treatment with CHX (Fig. 5D, middle O-GlcNAc to cognate substrate polypeptides is regulated primarily panel). By contrast, the endogenous hNrf1/TCF11 expression was by its N-terminal TPR domain, that dictates different features of all increased by siOGT knockdown, when compared with the control three spliced isoforms of 116-, 103- and 74.5kDa and can also pro- siNC levels (upper panels). Further observations revealed that the vide a scaffold structure enabling the enzyme to physically interact abundance of the longer hNrf1/TCF 11 proteins was significantly with its substrate protein [16,17]. As described above, a potential decreased following 1 h-treatment of siNC-transfected cells with interaction of OGT with Nrf1 is likely to be dictated by its TPR CHX, and the chemical treatment of siOGT-transfected cells only domain, allowing the CNC-bZIP protein to be O-GlcNAcylated caused the turnover of hNrf1/TCF11 to be slightly prolonged to (Fig. 2). In turn, O-GlcNAcylation of hNrf1/TCF11 by OGT enables 2h (Fig. 5D, left upper panel). However, the half-life (estimated down-regulation of its basal expression and/or its protein stability, 50 min) of the existing major longer CNC-bZIP proteins after whilst removal of the TPR1–6 motifs causes a recovery from treatment with CHX appeared to be unaltered by knockdown of the inhibitory effects of OGT on Nrf1 (Figs. 3–5). Herein, to siOGT (Fig. 5E), although the stoichiometric graphs and lines provide a better understanding of which regions within Nrf1 are presented a different tendency to reveal the turnover of engaged in association with OGT-directed O-GlcNAcylation, we hNrf1/TCF11 after knockdown of siOGT when compared with the performed further experiments of HEK293T cells that had been J. Chen et al. / FEBS Letters 589 (2015) 2347–2358 2357 co-transfected with expression constructs for each of wild-type how the putative O-GlcNAcylation of Nrf1 monitors the selective mNrf1 and its mutants lacking various lengths of the protein, processing of the CNC-bZIP factor to yield distinct isoforms and/or together with OGT or the EGFP control, followed by Western blot- its topovectorial repositioning to dislocate from the ER to the ting to examine which mutants enable mNrf1 to be recovered from nucleus, enabling it to fine-tune ARE-driven gene expression. the inhibitory effect of OGT on the CNC-bZIP factor. By comparison of the intensity ratios of Western blots for Nrf1 or Acknowledgements its mutant proteins in OGT + Nrf1-expressing cells to their corresponding yields from EGFP + Nrf1-expressing cells (Fig. 6,onthe The authors have no conflict of interests to disclose. We grate- bottom of the upper panels), the results revealed that deletion of fully acknowledge the kind help of Dr. Xiaoyong Yang (at School of the serine-repeats (SR) domain (aa 454–488, lanes #8 with Medicine, Yale University, New Haven) for providing pGEX-4T, 1.00/1.35), the SR-adjoining PEST2 sequence (aa 456–519, lanes #7 pGEX-OGT and pcDNA-OGTA(TPR1 -6). We are thankful to Dr. with 1.00/1.32) or the PEST2-containing large region (aa 403–506, Xia Li and Mr. Jian Zhang for their helpful discussion. This work lanes #10 with 1.00/3.68) from mNrf1, but not removal of its was supported by the National Natural Science Foundation of Neh6L domain (aa 489–580, lanes #9), caused a marked China (NSFC, key program 91129703, 91429305 and project recovery from the putative inhibitory effect of OGT-directed 31270879) awarded to Prof. Yiguo Zhang (University of O-GlcNAcylation on the protein, leading to an increase in the abun- Chongqing, China) and other NSFC Grants (Nos. 31270879 and dance of the major longer isoform of each of Nrf1 mutants (as indi- 31470803) awarded to Prof. Libo Yao, on the base of the Open fund cated⁄), when compared to the wild-type protein with the 1.00:0.79 from the State Key Laboratory of Cancer Biology at Fourth Military ratio of its expression with OGT to its production in the absence of Medical University (CBSKL201312 and CBSK201203), but these the enzyme (Fig. 6, lanes #1). This finding suggests that the SR por- funding sources had no direct involvement in the research work. tion of the PEST2 degron appears to be associated with the putative O-GlcNAcylation of wild-type mNrf1 by OGT, although the detailed References mechanism remains to be explored. In addition, a modest recovery of mNrf1 from OGT-mediated inhibition was observed upon dele- [1] Helenius, A. and Aebi, M. (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu. Rev. Biochem. 73, 1019–1049. tion of the N-terminal 191-aa region covering both NTD and AD1 [2] Ohtsubo, K. and Marth, J.D. (2006) Glycosylation in cellular mechanisms of of mNrf1 (Fig. 6, lanes #3 with a ratio of 1.00/1.24), but not removal health and disease. Cell 126, 855–867. of its N-terminal 80-aa small region (lanes #2), hence implying that [3] Moremen, K.W., Tiemeyer, M. and Nairn, A.V. (2012) Vertebrate protein glycosylation: diversity, synthesis and function. Nat. Rev. Mol. 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