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• W2011181458 abstract "Selenocysteine is inserted into selenoproteins via the translational recoding of a UGA codon, normally used as a stop signal. This process depends on the nature of the selenocysteine insertion sequence element located in the 3′ UTR of selenoprotein mRNAs, selenium bioavailability, and, possibly, exogenous stimuli. To further understand the function and regulation of selenoproteins in antioxidant defense and redox homeostasis, we investigated how oxidative stress influences selenoprotein expression as a function of different selenium concentrations. We found that selenium supplementation of the culture media, which resulted in a hierarchical up-regulation of selenoproteins, protected HEK293 cells from reactive oxygen species formation. Furthermore, in response to oxidative stress, we identified a selective up-regulation of several selenoproteins involved in antioxidant defense (Gpx1, Gpx4, TR1, SelS, SelK, and Sps2). Interestingly, the response was more efficient when selenium was limiting. Although a modest change in mRNA levels was noted, we identified a novel translational control mechanism stimulated by oxidative stress that is characterized by up-regulation of UGA-selenocysteine recoding efficiency and relocalization of SBP2, selenocysteine-specific elongation factor, and L30 recoding factors from the cytoplasm to the nucleus. Selenocysteine is inserted into selenoproteins via the translational recoding of a UGA codon, normally used as a stop signal. This process depends on the nature of the selenocysteine insertion sequence element located in the 3′ UTR of selenoprotein mRNAs, selenium bioavailability, and, possibly, exogenous stimuli. To further understand the function and regulation of selenoproteins in antioxidant defense and redox homeostasis, we investigated how oxidative stress influences selenoprotein expression as a function of different selenium concentrations. We found that selenium supplementation of the culture media, which resulted in a hierarchical up-regulation of selenoproteins, protected HEK293 cells from reactive oxygen species formation. Furthermore, in response to oxidative stress, we identified a selective up-regulation of several selenoproteins involved in antioxidant defense (Gpx1, Gpx4, TR1, SelS, SelK, and Sps2). Interestingly, the response was more efficient when selenium was limiting. Although a modest change in mRNA levels was noted, we identified a novel translational control mechanism stimulated by oxidative stress that is characterized by up-regulation of UGA-selenocysteine recoding efficiency and relocalization of SBP2, selenocysteine-specific elongation factor, and L30 recoding factors from the cytoplasm to the nucleus. Selenium is an essential trace element implicated in muscular, thyroid, immune, and brain functions (1Rayman M.P. Selenium and human health.Lancet. 2012; 379: 1256-1268Abstract Full Text Full Text PDF PubMed Scopus (2084) Google Scholar, 2Papp L.V. Holmgren A. Khanna K.K. Selenium and selenoproteins in health and disease.Antioxid. Redox Signal. 2010; 12: 793-795Crossref PubMed Scopus (187) Google Scholar). Numerous epidemiological and intervention studies have recognized the link between low levels of selenium in human body fluids and an increased risk of cancers, including prostate, lung, and colon cancer. Selenium is incorporated as selenocysteine, a rare amino acid, into at least 25 human selenoproteins, an essential family of redox enzymes (3Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigó R. Gladyshev V.N. Characterization of mammalian selenoproteomes.Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1847) Google Scholar). Many selenoproteins are involved in antioxidant defense, redox homeostasis, and redox signaling through the action of glutathione peroxidase (Gpx1-Gpx6), methionine sulfoxide reductase (MsrB1 or SelX), thioredoxin reductases (TR1-TR3), and endoplasmic reticulum (ER) 3The abbreviations used are: ERendoplasmic reticulumSECISselenocysteine insertion sequenceROSreactive oxygen speciesGpxglutathione peroxidaseTRthioredoxin reductaseCtrlcontrolSupsupplementedt-BHPter-butyl hydroperoxideLucluciferaseCu-OOHcumene hydroperoxide. selenoproteins (Sel15, SelS, SelK, and SelM). Nevertheless, about half of the selenoproteins are without a known function, and the regulation of the selenoproteome in response to oxidative stress remains uncharacterized. endoplasmic reticulum selenocysteine insertion sequence reactive oxygen species glutathione peroxidase thioredoxin reductase control supplemented ter-butyl hydroperoxide luciferase cumene hydroperoxide. Selenocysteine is cotranslationally inserted by the ribosome using an UGA codon, normally read as a stop signal. The 3′ UTRs of selenoprotein mRNAs contain a secondary structure called the selenocysteine insertion sequence (SECIS) element. The SECIS controls the faithful recoding of the UGA codon as selenocysteine, thereby preventing the formation of truncated proteins (for reviews, see Refs. 2Papp L.V. Holmgren A. Khanna K.K. Selenium and selenoproteins in health and disease.Antioxid. Redox Signal. 2010; 12: 793-795Crossref PubMed Scopus (187) Google Scholar, 4Allmang C. Wurth L. Krol A. The selenium to selenoprotein pathway in eukaryotes: more molecular partners than anticipated.Biochim. Biophys. Acta. 2009; 1790: 1415-1423Crossref PubMed Scopus (141) Google Scholar, 5Berry M.J. Tujebajeva R.M. Copeland P.R. Xu X.M. Carlson B.A. Martin 3rd, G.W. Low S.C. Mansell J.B. Grundner-Culemann E. Harney J.W. Driscoll D.M. Hatfield D.L. Selenocysteine incorporation directed from the 3′UTR: characterization of eukaryotic EFsec and mechanistic implications.Biofactors. 2001; 14: 17-24Crossref PubMed Scopus (56) Google Scholar, 6Driscoll D.M. Copeland P.R. Mechanism and regulation of selenoprotein synthesis.Annu. Rev. Nutr. 2003; 23: 17-40Crossref PubMed Scopus (317) Google Scholar, 7Hatfield D.L. Carlson B.A. Xu X.M. Mix H. Gladyshev V.N. Selenocysteine incorporation machinery and the role of selenoproteins in development and health.Prog. Nucleic Acid Res. Mol. Biol. 2006; 81: 97-142Crossref PubMed Scopus (152) Google Scholar). Two categories of recoding factors have been characterized as components of the selenocysteine insertion machinery, including tRNASec binding proteins (selenocysteine-specific elongation factor (EFsec), SECp43, O-phosphoseryl-tRNA(Sec) kinase (PSTK), and selenocysteine synthase (8Fagegaltier D. Hubert N. Yamada K. Mizutani T. Carbon P. Krol A. Characterization of mSelB, a novel mammalian elongation factor for selenoprotein translation.EMBO J. 2000; 19: 4796-4805Crossref PubMed Scopus (243) Google Scholar, 9Tujebajeva R.M. Copeland P.R. Xu X.M. Carlson B.A. Harney J.W. Driscoll D.M. Hatfield D.L. Berry M.J. Decoding apparatus for eukaryotic selenocysteine insertion.EMBO Rep. 2000; 1: 158-163Crossref PubMed Scopus (243) Google Scholar, 10Ding F. Grabowski P.J. Identification of a protein component of a mammalian tRNA(Sec) complex implicated in the decoding of UGA as selenocysteine.RNA. 1999; 5: 1561-1569Crossref PubMed Scopus (39) Google Scholar, 11Xu X.M. Mix H. Carlson B.A. Grabowski P.J. Gladyshev V.N. Berry M.J. Hatfield D.L. Evidence for direct roles of two additional factors, SECp43 and soluble liver antigen, in the selenoprotein synthesis machinery.J. Biol. Chem. 2005; 280: 41568-41575Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 12Small-Howard A. Morozova N. Stoytcheva Z. Forry E.P. Mansell J.B. Harney J.W. Carlson B.A. Xu X.M. Hatfield D.L. Berry M.J. Supramolecular complexes mediate selenocysteine incorporation in vivo.Mol. Cell Biol. 2006; 26: 2337-2346Crossref PubMed Scopus (124) Google Scholar, 13Carlson B.A. Xu X.M. Kryukov G.V. Rao M. Berry M.J. Gladyshev V.N. Hatfield D.L. Identification and characterization of phosphoseryl-tRNA[Ser]Sec kinase.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 12848-12853Crossref PubMed Scopus (142) Google Scholar)) and SECIS binding proteins (SECIS binding protein 2 (SBP2), ribosomal protein L30 (rpL30), translation initiation factor 4A3 (eIF4A3), and nucleolin (14Copeland P.R. Fletcher J.E. Carlson B.A. Hatfield D.L. Driscoll D.M. A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs.EMBO J. 2000; 19: 306-314Crossref PubMed Scopus (312) Google Scholar, 15Chavatte L. Brown B.A. Driscoll D.M. Ribosomal protein L30 is a component of the UGA-selenocysteine recoding machinery in eukaryotes.Nat. Struct. Mol. Biol. 2005; 12: 408-416Crossref PubMed Scopus (147) Google Scholar, 16Budiman M.E. Bubenik J.L. Miniard A.C. Middleton L.M. Gerber C.A. Cash A. Driscoll D.M. Eukaryotic initiation factor 4a3 is a selenium-regulated RNA-binding protein that selectively inhibits selenocysteine incorporation.Mol. Cell. 2009; 35: 479-489Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 17Miniard A.C. Middleton L.M. Budiman M.E. Gerber C.A. Driscoll D.M. Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression.Nucleic Acids Res. 2010; 38: 4807-4820Crossref PubMed Scopus (65) Google Scholar)). However, despite much effort, the precise mechanism of selenoprotein synthesis and regulation remains elusive. Many studies have shown that selenium levels differentially control the expression of the selenoproteome in mammals and cultured cells. This regulation occurs moderately at the level of mRNA transcription/stability, but mostly during translation, to maintain essential selenoproteins at the expense of the others (18Bermano G. Arthur J.R. Hesketh J.E. Selective control of cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase mRNA stability by selenium supply.FEBS Lett. 1996; 387: 157-160Crossref PubMed Scopus (72) Google Scholar, 19Bermano G. Nicol F. Dyer J.A. Sunde R.A. Beckett G.J. Arthur J.R. Hesketh J.E. Tissue-specific regulation of selenoenzyme gene expression during selenium deficiency in rats.Biochem. J. 1995; 311: 425-430Crossref PubMed Scopus (261) Google Scholar, 20Weiss Sachdev S. Sunde R.A. Selenium regulation of transcript abundance and translational efficiency of glutathione peroxidase-1 and -4 in rat liver.Biochem. J. 2001; 357: 851-858Crossref PubMed Scopus (117) Google Scholar, 21Wingler K. Böcher M. Flohé L. Kollmus H. Brigelius-Flohé R. mRNA stability and selenocysteine insertion sequence efficiency rank gastrointestinal glutathione peroxidase high in the hierarchy of selenoproteins.Eur. J. Biochem. 1999; 259: 149-157Crossref PubMed Scopus (170) Google Scholar). This has been particularly described for Gpx1 and Gpx4, two members of the Gpx family that are ubiquitously expressed. Gpx1 expression was more affected than Gpx4 by selenium availability (22Lei X.G. Evenson J.K. Thompson K.M. Sunde R.A. Glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are differentially regulated in rats by dietary selenium.J. Nutr. 1995; 125: 1438-1446Abstract Full Text PDF PubMed Google Scholar), essentially via a translational control mechanism (23Latrèche L. Duhieu S. Touat-Hamici Z. Jean-Jean O. Chavatte L. The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.RNA Biol. 2012; 9: 681-690Crossref PubMed Scopus (34) Google Scholar). We established HEK293 cell lines stably expressing luciferase-SECIS constructs to quantitatively evaluate the translational regulation of selenoprotein expression (23Latrèche L. Duhieu S. Touat-Hamici Z. Jean-Jean O. Chavatte L. The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.RNA Biol. 2012; 9: 681-690Crossref PubMed Scopus (34) Google Scholar, 24Latrèche L. Jean-Jean O. Driscoll D.M. Chavatte L. Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine.Nucleic Acids Res. 2009; 37: 5868-5880Crossref PubMed Scopus (76) Google Scholar). Indeed, we demonstrated that, in response to selenium level variations, selenocysteine insertion was driven by the nature of the SECIS element. To date, no stimulus other than selenium has been shown to stimulate selenoprotein expression. Reactive oxygen species (ROS) are generated either endogenously during cellular metabolism, mostly by mitochondria, or exogenously from environmental stress. ROS are also emerging as essential signaling molecules at low concentrations that transduce signals from the mitochondrial compartment to other compartments of the cell. To maintain ROS homeostasis, cells possess enzymatic antioxidant systems such as Gpxs, TRs, catalase, superoxide dismutase, and non-enzymatic antioxidant systems, including glutathione (GSH), vitamin E, and ascorbate. Among the selenoproteins, the Gpxs are implicated in the elimination of peroxides, whereas the TRs are critical for many cellular functions involving thiol-dependent redox mechanisms. Gpxs reduce hydrogen and lipid peroxides using GSH as a cofactor, which is subsequently recycled by glutathione reductases (25Brigelius-Flohé R. Maiorino M. Glutathione peroxidases.Biochim. Biophys. Acta. 2013; 1830: 3289-3303Crossref PubMed Scopus (1166) Google Scholar). TRs catalyze the NADPH-dependent reduction of oxidized thioredoxin and, therefore, participate in the defense against oxidative stress during DNA synthesis and in redox signaling (26Arnér E.S. Focus on mammalian thioredoxin reductases: important selenoproteins with versatile functions.Biochim. Biophys. Acta. 2009; 1790: 495-526Crossref PubMed Scopus (519) Google Scholar). In addition to thioredoxin, a vast range of small molecules can also be reduced by TRs and participate in its antioxidant function. In parallel, the ER-resident selenoproteins, which include SelS, SelK, Sel15, and SelM, have important functions in protein folding and the ER stress response (27Shchedrina V.A. Zhang Y. Labunskyy V.M. Hatfield D.L. Gladyshev V.N. Structure-function relations, physiological roles, and evolution of mammalian ER-resident selenoproteins.Antioxid. Redox Signal. 2010; 12: 839-849Crossref PubMed Scopus (155) Google Scholar). ROS can severely damage a variety of macromolecules, including proteins, lipids, and nucleic acids. Depending on the nature of oxidative damage, these oxidized molecules can be repaired by various cellular machineries, several of which involve selenoproteins such as Gpxs, SelX, or TRs. When ROS production is overwhelming, oxidized macromolecules accumulate, hamper biological functions, and lead to pathological conditions. Down-regulation of selenoprotein expression is expected to increase ROS production and ER stress, which would lead to accumulation of oxidized molecules and DNA damage, and to impair cellular homeostasis and cell cycle progression. Accumulation of ROS-induced cellular damage is associated with cancer, neurological disorders, atherosclerosis, inflammation, and aging. ROS have been proposed to link the selenium activity in mammals with these pathologies (28Steinbrenner H. Sies H. Protection against reactive oxygen species by selenoproteins.Biochim. Biophys. Acta. 2009; 1790: 1478-1485Crossref PubMed Scopus (395) Google Scholar). The transcriptional regulation of the antioxidant defense has been well described in response to oxidative stress. ROS induce a rapid activation of the NRF2 and NF-κB transcription factors (29Buelna-Chontal M. Zazueta C. Redox activation of Nrf2 and NF-κB: a double end sword?.Cell. Signal. 2013; 25: 2548-2557Crossref PubMed Scopus (195) Google Scholar). Their targets include a multitude of genes involved in the stress response, antioxidant activity, the anti-inflammatory response, DNA repair, molecular chaperones, proteasome systems, and two selenoproteins, Gpx2 (mostly expressed in the intestine) and TR1 (an essential cytoplasmic enzyme). The degree of expression of these genes involves other transcription factors (30Brigelius-Flohé R. Müller M. Lippmann D. Kipp A.P. The yin and yang of nrf2-regulated selenoproteins in carcinogenesis.Int. J. Cell Biol. 2012; 2012: 486147Crossref PubMed Scopus (59) Google Scholar, 31Müller M. Banning A. Brigelius-Flohé R. Kipp A. Nrf2 target genes are induced under marginal selenium-deficiency.Genes Nutr. 2010; 5: 297-307Crossref PubMed Scopus (74) Google Scholar). A link between selenium, selenoprotein expression, and the antioxidant response has been established via NRF2, which is induced in certain cell lines during severe selenium deficiency or complete loss of selenoproteins obtained by tRNASec gene inactivation (32Suzuki T. Kelly V.P. Motohashi H. Nakajima O. Takahashi S. Nishimura S. Yamamoto M. Deletion of the selenocysteine tRNA gene in macrophages and liver results in compensatory gene induction of cytoprotective enzymes by Nrf2.J. Biol. Chem. 2008; 283: 2021-2030Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The remarkable feature of selenoprotein expression lies in the many levels of control. Indeed, the complex network of regulation during transcription, translation, and mRNA stabilization allows a hierarchical expression of these antioxidant components (6Driscoll D.M. Copeland P.R. Mechanism and regulation of selenoprotein synthesis.Annu. Rev. Nutr. 2003; 23: 17-40Crossref PubMed Scopus (317) Google Scholar). Whether and how the selenoproteins that are involved in antioxidant defense are regulated by oxidative stress remains to be studied. In this work, we investigated the regulation of selenoprotein expression in the context of H2O2-induced oxidative stress as a function of selenium concentration in the culture media, defined as control (Ctrl) and supplemented (Sup) conditions. We used HEK293 cells in our study because selenoproteins are expressed at significant levels in this cell line (23Latrèche L. Duhieu S. Touat-Hamici Z. Jean-Jean O. Chavatte L. The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.RNA Biol. 2012; 9: 681-690Crossref PubMed Scopus (34) Google Scholar, 24Latrèche L. Jean-Jean O. Driscoll D.M. Chavatte L. Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine.Nucleic Acids Res. 2009; 37: 5868-5880Crossref PubMed Scopus (76) Google Scholar) and also because we developed cellular tools to quantitatively study the recoding of UGA as selenocysteine in this particular cell line (23Latrèche L. Duhieu S. Touat-Hamici Z. Jean-Jean O. Chavatte L. The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.RNA Biol. 2012; 9: 681-690Crossref PubMed Scopus (34) Google Scholar). We found that down-regulation of the antioxidant defense in the Ctrl condition was linked to a selective reduction of several selenoproteins. Thus, HEK293 cells grown in selenium-limiting conditions produced more ROS, accumulated more oxidized proteins, and were more sensitive to oxidative challenge. Remarkably, in this condition (i.e. Ctrl), several selenoproteins, including Gpx1, Gpx4, TR1, SelS, SelK, and Sps2, were specifically up-regulated by H2O2 treatment. However, only limited changes were observed in the Sup condition. We determined that H2O2-induced up-regulation of selenoproteins is due to a novel translational control mechanism leading to the stimulation of UGA recoding efficiency in selenoprotein mRNAs. Luciferase reporter plasmids and cell lines were generated as in Refs. 23Latrèche L. Duhieu S. Touat-Hamici Z. Jean-Jean O. Chavatte L. The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.RNA Biol. 2012; 9: 681-690Crossref PubMed Scopus (34) Google Scholar, 24Latrèche L. Jean-Jean O. Driscoll D.M. Chavatte L. Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine.Nucleic Acids Res. 2009; 37: 5868-5880Crossref PubMed Scopus (76) Google Scholar). Cell culture media, FCS, and supplements were purchased from Invitrogen. H2O2, t-BHP, Cu-OOH, NADPH, thioredoxin, L-GSH, glutathione reductase, and 5,5′-dithio-bis(2-dinitrobenzoic acid) were purchased from Sigma-Aldrich. Antibodies were purchased from Euromedex (Gpx1, catalog no. 2971; Gpx4, catalog no. 3649-1; TR1, catalog no. LF-MA0015; Sel15, catalog no. 3364; and SelP, catalog no. 3718), Sigma-Aldrich (SelS, catalog no. HPA010025; TR2, catalog no. HPA003323; actin, catalog no. A1978; tubulin, catalog no. T9026), Covalab (EFsec (23Latrèche L. Duhieu S. Touat-Hamici Z. Jean-Jean O. Chavatte L. The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.RNA Biol. 2012; 9: 681-690Crossref PubMed Scopus (34) Google Scholar)), and ProteinTech Group (SBP2, catalog no. 1289-1-AP). HEK293 cells (Invitrogen) were grown and maintained as described in Ref. 23Latrèche L. Duhieu S. Touat-Hamici Z. Jean-Jean O. Chavatte L. The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.RNA Biol. 2012; 9: 681-690Crossref PubMed Scopus (34) Google Scholar, 24Latrèche L. Jean-Jean O. Driscoll D.M. Chavatte L. Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine.Nucleic Acids Res. 2009; 37: 5868-5880Crossref PubMed Scopus (76) Google Scholar. Ctrl and Sup culture media were made according to Refs. 23Latrèche L. Duhieu S. Touat-Hamici Z. Jean-Jean O. Chavatte L. The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.RNA Biol. 2012; 9: 681-690Crossref PubMed Scopus (34) Google Scholar, 24Latrèche L. Jean-Jean O. Driscoll D.M. Chavatte L. Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine.Nucleic Acids Res. 2009; 37: 5868-5880Crossref PubMed Scopus (76) Google Scholar) and the references therein. The same lot number of FCS was used in all experiments because selenium is provided by this source. The selenium concentration in the Ctrl medium was determined by inductively coupled plasma mass spectrometry (ICP-MS) to be 15 nm. Sup medium was made by adding 30 nm sodium selenite to the Ctrl medium, and, therefore, it contained 45 nm selenium. One day prior to H2O2 exposure, cells were plated in 100-mm dishes in either Ctrl or Sup medium. When the cells reached 70% confluency, the medium was changed to a freshly prepared solution of FCS-free DMEM that contained different concentrations of H2O2, Cu-OOH, or t-BHP as indicated. After 30 min of exposure, the media were removed and replaced by the appropriate ones. Cell extracts were harvested 24 h post-treatment in 300 μl of lysis buffer (23Latrèche L. Duhieu S. Touat-Hamici Z. Jean-Jean O. Chavatte L. The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.RNA Biol. 2012; 9: 681-690Crossref PubMed Scopus (34) Google Scholar, 24Latrèche L. Jean-Jean O. Driscoll D.M. Chavatte L. Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine.Nucleic Acids Res. 2009; 37: 5868-5880Crossref PubMed Scopus (76) Google Scholar). Cellular fractionations were performed with the ProteoJET cytoplasmic and nuclear protein extraction kit (Fermentas) following the instructions of the manufacturer. Protein concentrations were measured using the DC kit (Bio-Rad) in microplate assays. HEK293 cells that were grown in Ctrl or Sup medium were incubated with 5 μm MitoSOXTM Red (Invitrogen) for 15 min at 37 °C in 5% CO2. Antimycine A (2.5 μg/ml), an inhibitor of respiratory complex III, was added as indicated. Oxidation of MitoSOXTM Red reagent by superoxide produced red fluorescence that was measured as a function of time on a Partech flow cytometer according to the instructions of the manufacturer. Fluorescence kinetics were determined from three independent experiments. HEK293 cells were seeded in 6-well plates and grown in either Ctrl or Sup medium for 1 day. Cells were treated with 0 or 300 μm H2O2 for 30 min in serum-free medium and then harvested 24 h later. Cells were fixed in 70% ethanol/PBS for 2 h at −20 °C, rehydrated in PBS, and stained for 2 h at 37 °C with propidium iodide (50 μg/ml) and RNase A (250 μg/ml). Cell fluorescence was then analyzed by flow cytometry. Cell cycle analyses were done using Flowmax and Multicycle software. Protein extracts (50 μg) were separated in BisTris NuPAGE Novex Midi gels (Invitrogen). Proteins were transferred onto nitrocellulose membranes that were probed with primary antibodies (as indicated) and HRP-conjugated anti-rabbit or anti-mouse secondary antibodies. Detection of total carbonyl contents in cell extracts was performed with the OxyblotTM protein oxidation detection kit (Millipore). Protein carbonyls from cell extracts (5 μg) were derived using 2,4-dinitrophenylhydrazine, separated on a 10% SDS-PAGE, transferred to a nitrocellulose membrane, and detected with an anti-2,4-dinitrophenol antibody according to the instructions of the manufacturer. The chemiluminescence signal was detected using an ECL Advance Western blotting detection kit (GE Healthcare) and an LAS3000 charge-coupled device (CCD) camera (GE Healthcare). Gpx activity was measured in an enzymatic coupled assay according to methods described previously (19Bermano G. Nicol F. Dyer J.A. Sunde R.A. Beckett G.J. Arthur J.R. Hesketh J.E. Tissue-specific regulation of selenoenzyme gene expression during selenium deficiency in rats.Biochem. J. 1995; 311: 425-430Crossref PubMed Scopus (261) Google Scholar). The reaction mixture was composed of 50 μg of protein extract, 0.25 mm NADPH, 2 mm reduced l-glutathione, and 1.5 IU of glutathione reductase adjusted to a total volume of 250 μl with 50 mm potassium phosphate buffer (pH 7.5). The reaction was started by the addition of 300 nm t-BHP, and consumption of NAPDH was followed at 340 nm with a Fluostar reader. Enzymatic activities were expressed as nanomoles of glutathione per minute per milligram (milliunits per milligram of protein). TR activity was measured as described in Ref. 33Arnér E.S. Recombinant expression of mammalian selenocysteine-containing thioredoxin reductase and other selenoproteins in Escherichia coli.Methods Enzymol. 2002; 347: 226-235Crossref PubMed Scopus (39) Google Scholar. Briefly, the catalytic reduction of oxidized thioredoxin by TR is coupled with the oxidation of one molecule of NADPH. In this work, the recycling of NADP in NADPH is associated with the conversion of 5,5′-dithio-bis(2-dinitrobenzoic acid) to 5-thio-2-nitrobenzoic acid, which is visible at 410 nm. The reaction mixture was composed of 50 μg of protein extract, 0.2 mm NADPH, 10 mm EDTA, and 0.2 mg/ml BSA, adjusted to a total volume of 250 μl with 50 mm potassium phosphate buffer (pH 7.5). The reaction was started by the addition of 25 mm 5,5′-dithio-bis(2-dinitrobenzoic acid), and the production of 5-thio-2-nitrobenzoic acid was followed at 340 nm with a Fluostar reader. Enzymatic activities were expressed as nanomoles of NADPH per minute per milligram (milliunits per milligram of protein). In every enzymatic analysis, the assays were performed in duplicate in three independent experiments. Total RNAs were extracted using the Nucleospin RNA II kit (Macherey Nagel). Synthesis of cDNA was carried out using a Transcriptor high-fidelity cDNA synthesis kit (Roche Applied Science) according to the instructions of the manufacturer. Real-time PCR was performed in triplicate using LightCycler® 480 SYBR Green I Master (Roche Applied Science). The PCR program was 95 °C for 5 min, 45 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s. Data were analyzed using LightCycler® 480 software and normalized relative to the mRNA levels of Hrpt. The primers used are described in Ref. 34Legrain Y. Touat-Hamici Z. Chavatte L. Interplay between selenium levels, selenoprotein expression and replicative senescence in WI-38 human fibroblasts.J. Biol. Chem. 2014; 289: 6299-6310Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar. HEK293 cells that stably express the Luc UGA/Gpx4 or Luc UGA/Gpx1 constructs (23Latrèche L. Duhieu S. Touat-Hamici Z. Jean-Jean O. Chavatte L. The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.RNA Biol. 2012; 9: 681-690Crossref PubMed Scopus (34) Google Scholar) were plated in the different culture media 1 day prior to H2O2, Cu-OOH, or t-BHP exposure. Cells were harvested 24 h later in lysis buffer and analyzed for luciferase activity as described in Refs. 23Latrèche L. Duhieu S. Touat-Hamici Z. Jean-Jean O. Chavatte L. The differential expression of glutathione peroxidase 1 and 4 depends on the nature of the SECIS element.RNA Biol. 2012; 9: 681-690Crossref PubMed Scopus (34) Google Scholar, 24Latrèche L. Jean-Jean O. Driscoll D.M. Chavatte L. Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine.Nucleic Acids Res. 2009; 37: 5868-5880Crossref PubMed Scopus (76) Google Scholar with a Lumistar reader (BMGLabtech). Luciferase activity in arbitrary units was expressed relative to micrograms of protein. Cells were seeded on coverslips in 6-well plates, treated as described above, washed with PBS, fixed in 4% paraformaldehyde/PBS for 15 min at room temperature, permeabilized with 0.5% Triton/PBS for 8 min, and blocked in 1% BSA/PBS for 30 min. Coverslips were incubated with anti-SBP2 antibody at 4 °C overnight, washed with PBS, and incubated with an Alexa Fluor 488 dye-conjugated anti-rabbit secondary antibody. Nuclei were stained with DAPI. Epifluorescence images were captured in 10 z-sections on an AxioOberver Z1 microscope (Zeiss) equipped with an Evolve electron multiplying charge-coupled device (EMCCD) camera (Roper Scientific). Image analysis was performed with National Institutes of Hea" @default.
• W2011181458 created "2016-06-24" @default.
• W2011181458 creator A5008981622 @default.
• W2011181458 creator A5023711162 @default.
• W2011181458 creator A5073646319 @default.
• W2011181458 creator A5088331529 @default.
• W2011181458 date "2014-05-01" @default.
• W2011181458 modified "2023-09-30" @default.
• W2011181458 title "Selective Up-regulation of Human Selenoproteins in Response to Oxidative Stress" @default.
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