ActivationofNRF2byNitrosativeAgentsandH2O2 Involves ... cells to hydrogen peroxide, ... transferred onto a nitrocellulose membrane, ... acetone,thenthepelletsweredissolvedinHENbuffercontain-

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  • Activation of NRF2 by Nitrosative Agents and H2O2 InvolvesKEAP1 Disulfide Formation*SReceived for publication, August 3, 2009, and in revised form, December 29, 2009 Published, JBC Papers in Press, January 8, 2010, DOI 10.1074/jbc.M109.051714

    Simon Fourquet, Raphael Guerois, Denis Biard, and Michel B. Toledano1

    From the Laboratoire Stress Oxydants et Cancer, SB2MS, and CNRS, URA 2096, IBITECS, CEA-Saclay, F-91191 Gif-sur-Yvette, France andCEA-DSV-iRCM/INSERM U935, Institut A. Lwoff-CNRS, BP 8, F-94801 Villejuif Cedex, France

    TheNRF2 transcription factor regulates amajor environmen-tal and oxidative stress response. NRF2 is itself negatively regu-lated by KEAP1, the adaptor of a Cul3-ubiquitin ligase complexthat marks NRF2 for proteasomal degradation by ubiquitina-tion. Electrophilic compounds activate NRF2 primarily byinhibiting KEAP1-dependent NRF2 degradation, through alkyl-ation of specific cysteines. We have examined the impact onKEAP1 of reactive oxygen and nitrogen species, which are alsoNRF2 inducers. We found that in untreated cells, a fraction ofKEAP1 carried a long range disulfide linking Cys226 and Cys613.Exposing cells to hydrogen peroxide, to the nitric oxide donorspermine NONOate, to hypochlorous acid, or to S-nitrosocys-teine further increased this disulfide andpromoted formationofa disulfide linking two KEAP1 molecules via Cys151. None ofthese oxidants, except S-nitrocysteine, caused KEAP1 S-ni-trosylation. A cysteine mutant preventing KEAP1 intermolecu-lar disulfide formation also prevented NRF2 stabilization inresponse to oxidants, whereas those preventing intramoleculardisulfide formation were functionally silent. Further, simulta-neously inactivating the thioredoxin and glutathione pathwaysled both to major constitutive KEAP1 oxidation and NRF2 sta-bilization. We propose that KEAP1 intermolecular disulfideformation via Cys151 underlies the activation of NRF2 by reac-tive oxygen and nitrogen species.

    TheCapncollar bZip transcription factorNRF2 regulates anenvironmental and oxidative stress response of major physio-logical importance in mammals. NRF2 is activated by reactiveoxygen and nitrogen species, electrophilic xenobiotics, andheavymetals and promotes cytoprotection and survival towardthese stresses (for a review, see Refs. 1 and 2). Activation ofNRF2 is intricate, engaging controls at the level of subcellulardistribution, interaction with other proteins, phosphorylation,and protein stability (reviewed in Ref. 2). Among these, proteinstability is a major control determinant, involving KEAP1, theadaptor of a Cul3-ubiquitin ligase complex that ubiquitinatesNRF2 and marks it for proteasomal degradation (36). Stresssignals that activate NRF2, herein named NRF2 inducers, are

    primarily sensed at the level of KEAP1, causing NRF2 proteinstabilization (79) by inhibiting KEAP1-mediated NRF2 ubiq-uitination (10, 11).The large number of NRF2 inducers and their quite different

    chemical nature have raised the question of how they are spe-cifically sensed by KEAP1. Although NRF2 inducers are chem-ically very different, they all have electrophilic properties,which has led to the proposal that they must operate by alkyla-tion and/or oxidation of KEAP1 Cys residues (12). The 624-amino acid-long KEAP1 protein has 25 (mouse) or 27 (human)Cys residues and carries a Broad complex, Tramtrack, Bric-a-Brac (BTB)2 dimerization domain, an intervening region (IVR),and a six-Kelch repeat domain (Kelch) (see Fig. 2). It also bindszincwith a 1:1 stoichiometry, possibly through the IVR residuesCys254, Cys273, Cys288, and Cys293, as suggested by the 100-foldlower zinc affinity of mutants lacking these residues (13).Several laboratories have sought to identify in vitro which of

    the KEAP1 Cys residues are modified by NRF2 inducers, eachidentifying a different set of Cys residues, with most residuesidentified at least once (summarized in Refs. 14 and 15). Still theBTB domain Cys151 and IVR Cys288 came out as the most fre-quently identified and themost reactive residues. In vivo proofsof the modification of KEAP1 at Cys residues have also beenobtained from cells treated with oxidized lipids (1618), a car-nosic acid derivative (19), nitric oxide and 8-nitro-cGMP (20,21), and N-iodoacetyl-N-biotinylhexylenediamine (22), thuscorroborating the hypothesis of a Cys residue-based mecha-nism in KEAP1 regulation. The latter study also mapped mod-ified residues that included IVR cysteines and Cys151 (22).

    A third and very informative approach to the KEAP1 redoxmechanism has been to evaluate the effect of Cys residue sub-stitution on KEAP1 function. In cells expressing KEAP1 mu-tants that lack Cys273 or Cys288, NRF2 is constitutively active(11, 17, 23). Because these residues might contribute to zinccoordination, their substitution (or modification) could alterKEAP1 function through the loss of a structural or a redoxregulatory zinc motif. In cells that express a KEAP1 mutantlackingCys151,NRF2 basal activity is in contrast low and cannotbe induced by tert-butylhydroquinone (t-BHQ) andmany otherNRF2 inducers (6, 11, 2426). Mice transgenic complementa-tion rescue experiments have confirmed the functional impor-tance of KEAP1 Cys273, Cys288, and Cys151 (26). Further, a

    * This work was supported by funds from ARC, ANR, and Region Ile-de-FranceDIM SEnT (to M. B. T.) and by a Programme Toxicologie Nucleaire fellow-ship (to S. F.).

    S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1S4.

    1 Recipient of the fund program Equipe Labellisee Ligue 2009 from La LigueContre le Cancer. To whom correspondence should be addressed: LSOC,IBITECS, CEA-Saclay, Bat. 142, F-91191 Gif-Sur-Yvette, France. Tel.: 33-1-69-08-82-44; Fax: 33-1-69-08-80-46; E-mail: michel.toledano@cea.fr.

    2 The abbreviations used are: BTB, Broad complex, Tramtrack, Bric-a-Brac; Cys-NO, S-nitrocysteine; NEM, N-ethylmaleimide; SpNO, spermine NONOate;t-BHQ, tert-butyl hydroquinone; IVR, intervening region; HA, hemaggluti-nin; shRNA, small hairpin RNA; YFP, yellow fluorescent protein; TrxR1,thioredoxin reductase 1.

    THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 11, pp. 84638471, March 12, 2010 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

    MARCH 12, 2010 VOLUME 285 NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8463

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  • recent systematic study in zebra fish classified NRF2 inducersinto at least two classes, one requiring KEAP1 Cys151 and theother requiring Cys273, thus re-emphasizing the functionalimportance of these residues (27). By concluding that differentNRF2 inducers trigger different Cys-based regulatory mecha-nisms, this study may also explain at least in part why the invitro searches for reactive Cys residues, which have been con-ducted by different laboratories using different inducers, haveyielded different results.In the present study, we have examined the mechanism of

    KEAP1 regulation by H2O2, NO, andHOCl. These compoundsare NRF2 inducers (20, 2832) and are physiologically impor-tant as endogenously produced. A negative NRF2 regulation byH2O2 has been described though (33). Although strongly elec-trophilic, these compounds differ chemically from the NRF2inducers studied so far, because they are oxidants and not alkyl-ating agents and are thus anticipated to modify Cys residue byoxidation. Despite themany studies of the KEAP1 redoxmech-anism, it is not known whether its Cys residues could undergooxidation in vivo. We have thus carefully monitored the effectof H2O2, the NO releasing agent spermine NONOate (SpNO),andHOCl on the KEAP1 redox state and found that these com-pounds similarly oxidize KEAP1 with formation of intra- andintermolecular disulfides. Evaluation of the role of the oxidizedresidues on KEAP1 function suggests that the intermoleculardisulfide is important for activation, whereas the intramolecu-lar disulfide could have a structural role. We also show thatsimultaneous inactivation of the thioredoxin and glutathionesystems leads to constitutive KEAP1 oxidation and strongNRF2 stabilization.

    EXPERIMENTAL PROCEDURES

    Cell Culture and ReagentsHeLa cells were grown at 37 C,5% CO2, in Dulbeccos modified Eagles medium containing1 g/liter of glucose, 110 mg/ml sodium pyruvate, 4 mMGlutaMAX (Invitrogen), complemented with 10% fetal calfserum (Sigma). For plasmid transfection, 5.5 105 cells wereincubated 5 h with 3 g of DNA and Lipofectamine 2000(Invitrogen) following the suppliers recommendations, washed,and incubated in fresh medium. The cells were treated asdescribed in the text 24 h after transfection. For the TRxR1knockdown, 2.5 105 cells were transfected with 3 g of DNA,by the same protocol. After 24 h, hygromycin B was added (250g/ml) to the culturemedium twice every 2 days. The cells werethen expanded and kept in the presence of hygromycin B (125g/ml). The selection was withdrawn before experiments.N-Ethylmaleimide (NEM), t-BHQ, H2O2, and cycloheximidewere purchased from Sigma, and SpNO was from CaymanChemical. S-nitrosocysteine (Cys-NO) was prepared bymixingstoichiometric amounts of L-cysteine and sodium nitrite at pH4, followed by themeasure of its concentration by recording theabsorbance at 334 nm.PlasmidsPlasmid pcDNA3-HA-mKEAP1 was a gift from

    Dr. M. Yamamoto, pCI-HA-mNRF2 from was J. A. Diehl, andpeYFP-N1 was purchased from Clontech. Plasmid pcDNA3-Myc-His-mKEAP1 was constructed by PCR-mediated re-placement of the HA tag sequence of pcDNA3-HA-mKEAP1with three Myc tag sequences followed by a stretch of eight

    His codons. Mutagenesis was done with the StratageneQuikChange multi kit following the manufacturers instruc-tions. For the TrxR1 knockdown, we used TrxR1-specific smallhairpin RNA (shRNA) that targeted the following sequenceswithin the open reading frame: GGATTAAGGCAACA-AATAA (sh1), GCATCAAGCAGCTTTGTTA (sh2), andGCAAGACTCTCGAAATTAT (sh3). shRNA sequences weredesigned with the DSIR program that also operates an exactsimilarity search algorithm for potential off target detection(34). These shRNAwere expressed under the control of the H1promoter from the Epstein-Barr virus-based replicative plas-mid that contains an oriP, the EBNA open reading frame, and ahygromycin B selection cassette (35). Control lines expressed anonfunctional shRNA as reported (35).Redox WesternThe cells were washed on ice with 40 mM

    NEM in phosphate-buffered saline and lysed in lysis buffer (0.1M Tris-HCl, pH 8.0, 120 mM NaCl, 0.2% deoxycholic acid, 5%Nonidet P-40, 0.2mMNaF, 0.2mMEGTA, 0.1mMphenylmeth-ylsulfonyl fluoride, Roche-Complete mini protease inhibitormixture, 40 mM NEM). Centrifuged-cleared lysates weredilutedwith 2 volumes of 3 loading buffer (0.2MTris-HCl, pH6.8, 45% glycerol, 6% SDS, 0.03% bromphenol blue). Half of thesamples were reduced by the addition of -mercaptoethanol(6% v/v). After heat denaturation, the proteins were separatedby SDS-PAGE, transferred onto a nitrocellulose membrane,and immunostained with anti-NRF2 (H300; Santa Cruz),anti-HA (HA11; Covance), anti-Myc (9E10; a kind gift from G.Clement and C. Creminon, France), anti-TrxR1 (a kind giftfrom A Holmgren, Sweden), or anti-YFP (JL8; Clontech) spe-cific antibodies. Detection was performed after chromophore-coupled secondary antibody staining, using the LICOROdysseyinfrared imager.Protein PulldownAssaysFor pulling downHis tag-contain-

    ing polypeptides, the cells (106/sample)werewashed in ice-coldphosphate-buffered saline containing NEM (40 mM) and lysedin precipitation buffer (0.1 M Tris-HCl, pH 8, 1% Nonidet P-40,2% glycerol, 0.3 M NaCl, 0.2 mM phenylmethylsulfonyl fluoride,Roche-Complete mini protease Inhibitor mixture, 40 mMNEM). 10 mM imidazole was then added, and 90% of thesample (250 l) was incubated with 50 l of a nickel-nitrilo-triacetic acid resin (Qiagen) for 1 h. After extensive washingwith wash buffer (0.1 M Tris-HCl, pH 8, 1% Nonidet P-40, 2%glycerol, 0.3 M NaCl, 0.2 mM phenylmethylsulfonyl fluoride,10 mM NEM, 10 mM imidazole), the proteins were elutedwith 2 protein-loading buffer containing 10 mM NEM andseparated by SDS-PAGE.The Biotin-switch TechniqueThe biotin-switch method

    was performed as described in Ref. 36, with the difference thatcells were directly lysed inHEN buffer (250mMHEPES, pH 7.7,1 mM EDTA, 0.1 mM neocuproine) containing SDS (2.5%) andthe thiol-reactive compound S-methyl methanethiosulfonate(0.1%) to block thiol modification during and after the lysis.Briefly, the proteins were precipitated and washed twice withacetone, then the pellets were dissolved in HEN buffer contain-ing SDS (1%), and reduction and labeling of S-nitrosothiolswere achieved by the addition of sodium ascorbate (100 M)and biotin-HPDP (0.25 mg/ml) upon incubation in the dark atroom temperature. For the specific detection of S-nitroso-

    Oxygen and Nitrogen Species Induce KEAP1 Disulfide Formation

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  • KEAP1, the samples were precipitated with acetone, washedwith acetone, dissolved in HEN buffer 0.1 containing SDS(1%). 3 volumes of (v/v) neutralization buffer (25 mM HEPES,pH 7.5, 100 mM NaCl, 1 mM ETDA, 1% Triton X-100) wasadded before incubation with a streptavidin-agarose slurry.Elution was performed in 0.1 HEN buffer containing -mer-captoethanol (1%). Western blots were then performed usinganti-Myc antibodies.

    RESULTS

    Oxidation of KEAP1 inCells Exposed toH2O2Weevaluatedwhether H2O2 could oxidize KEAP1 at Cys residues by analyz-ing the redox state ofHA-KEAP1 ectopically expressed inHeLacells. To prevent Cys residue oxidation, free sulfhydryls wereblocked with NEM during sample preparation. HA-KEAP1from untreated cell lysates that had been reduced by -mer-captoethanol migrated in SDS-PAGE as a single band withan apparent molecular mass of 70 kDa (Fig. 1A, lowerpanel). When reduction was omitted, HA-KEAP1 from thesame lysates still migrated as a major band of samemolecularmass (denoted Red for reduced), but now a second lessintense band of faster mobility was observed (denoted OxIMfor oxidized intramolecular; see below) (Fig. 1A, upperpanel). A 5-min exposure of cells to H2O2 (200 M) furtheraltered migration of KEAP1 under nonreducing conditions

    (Fig. 1A, lane 2); the reduced KEAP1 70-kDa band decreasedin intensity, whereas OxIM increased and two new bands ofslower migration appeared (denoted OxIR1 and OxIR2 foroxidized intermolecular; see below; lane 2). OxIM, OxIR1,and OxIR2 were absent under reducing conditions, indicat-ing that they probably result from disulfide formation. OxIMmight correspond to an intramolecular disulfide (see below),the formation of which is predicted to increase SDS-PAGEmobility because of a decrease in the hydrodynamic radius ofthe SDS-bound polypeptide, especially if its two constitutiveCys residues are far apart in...

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