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Interaction of adenanthin with glutathione and thiol enzymes: Selectivity for thioredoxin reductase and inhibition of peroxiredoxin recycling Marjolein Soethoudt, Alexander V. Peskin, Nina Dickerhof, Louise N. Paton, Paul E. Pace, Christine C. Winterbourn www.elsevier.com/locate/freeradbiomed

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S0891-5849(14)00444-4 http://dx.doi.org/10.1016/j.freeradbiomed.2014.09.025 FRB12162

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Free Radical Biology and Medicine

Received date: 16 July 2014 Revised date: 19 September 2014 Accepted date: 19 September 2014 Cite this article as: Marjolein Soethoudt, Alexander V. Peskin, Nina Dickerhof, Louise N. Paton, Paul E. Pace, Christine C. Winterbourn, Interaction of adenanthin with glutathione and thiol enzymes: Selectivity for thioredoxin reductase and inhibition of peroxiredoxin recycling, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2014.09.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Interactionofadenanthinwithglutathioneandthiolenzymes:selectivityfor thioredoxinreductaseandinhibitionofperoxiredoxinrecycling  MarjoleinSoethoudt1,2,AlexanderVPeskin1,NinaDickerhof1,LouiseNPaton1, PaulEPace1andChristineCWinterbourn1  1

Centre for Free Radical Research, Department of Pathology, University of Otago

Christchurch,ChristchurchNewZealand 2

Present address: BioOrganic Synthesis, Leiden Institute of Chemistry, Leiden University,

Leiden,TheNetherlands  Towhomcorrespondenceshouldbeaddressed: POBox4345,Christchurch8041,NewZealand Phone+6433640564 Email[email protected]  



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ABSTRACT Thediterpenoid,adenanthin,repressestumorgrowthandprolongssurvivalinmousepromyelocytic leukemiamodels(LiuetalNatureChemBiol8,486,2012).Itwasproposedtodothisbyinactivating peroxiredoxins (Prxs) 1 and 2 through forming an adduct specifically on the resolving Cys residue. We confirmed that adenanthin underwent Michael addition to isolated Prx2, thereby inhibiting oxidationtoadisulfidelinkeddimer.However,contrarytotheoriginalreport,boththeperoxidatic and resolving Cys residues could be derivatized. Glutathione also formed an adenanthin adduct, reactingwithasecondorderrateconstantof25+5M1s1.With50μMadenanthin,theperoxidatic and resolvingCysofPrx2 reactedwith half times of7 and 40min respectively, compared with 10 minforGSH.WhenerythrocytesorJurkatTcellsweretreatedwithadenanthin,wesawnoevidence forareactionwithPrxs1or2.Instead,adenanthincausedtimeandconcentrationdependentloss of GSH followed by dimerization of the Prxs. Prxs undergo continuous oxidation in cells and are normallyrecycledbythioredoxinreductaseandthioredoxin.OurresultsindicatethatPrxreduction wasinhibited.Weobservedrapidinhibitionofpurifiedthioredoxinreductase(halftime5minwith2 μMadenanthin)andincells,.thioredoxinreductasewasmuchmoresensitivethanGSHandlossof bothprecededaccumulationofoxidizedPrxs.Thus,adenanthinisnotaspecificPrxinhibitorof,and its reported antitumor and antiinflammatory effects are more likely to involve more general inhibitionofthioredoxinand/orglutathioneredoxpathways.  Keywords Peroxiredoxin, electrophile, thioredoxin reductase, glutaredoxin, adenanthin, glutathione, glutathionereductase   Abbreviations APL, acute promyelocytic leukemia; DTNB, dithiobis2nitrobenzoic acid: DTPA, di ethylenetriaminepentaaceticacid;NEM,Nethylmaleimide;Prx,peroxiredoxin  

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 Introduction Adenanthin (Fig. 1) is a natural diterpenoid that was identified in a drug screen as a promising therapeutic agent for acute promyelocytic leukemia (APL) [1]. It was shown to induce the differentiationofAPLcells,torepresstumorgrowthandprolongsurvivalinmouseleukemiamodels. It has also been reported to delay the development of mouse experimental autoimmune encephalomyelitis[2].WhenLiuetal[1]searchedforbindingpartnersintheAPLcellsusingbiotin adenanthin, their pull down experiments detected a single protein band which they showed to containperoxiredoxins(Prxs)1and2.Adenanthin(Fig.1)hasanelectrophiliccentrethatwouldbe expectedtotargetthiolgroupsandformMichaeladditionproducts.Consistentwiththis,acovalent adduct with the resolving Cys residue of the Prxs was identified [1]. Based on these findings, the authors highlighted the potential of adenanthin as a lead compound for drug development [3]. In addition, if adenanthin were a specific inhibitor of Prxs, it would have widespread application for investigatingtheirmanyphysiologicalandpathologicalfunctions. Peroxiredoxinsarecysteinedependentperoxidaseswithantioxidantpropertiesandareubiquitously distributed in multiple organisms, including humans. They provide cellular protection against oxidative stress, and also modulate redox signaling cascades involved in growth factor responses, cellproliferationandapoptosis[4].KnockdownofindividualPrxsgivesrisetovariousphenotypes, includingincreasedcancerrisk,alteredinflammatoryresponse,anemiaandshortenedlifespan[59]. Furthermore,alteredPrxlevelsareassociatedwithavarietyofpathologies[1013].Mammalshave six Prxs, which subdivide into three major subclasses based on their catalytic mechanism and the numberofcysteinesinvolved.Allhaveacatalyticcysteineresiduethathasextremelyhighreactivity with peroxides [4, 14, 15]. The typical 2Cys Prxs, which include Prxs 1 and 2, function as homodimers and operate by a mechanism in which there is initial oxidation of the active site peroxidativeCysononesubunittoformasulfenicacidthatcondenseswiththeresolvingCysonthe other subunit to give an interchain disulfide. The catalytic cycle is completed by reduction by thioredoxin,whichisregeneratedbythioredoxinreductaseandNADPH.CellularPrx1andPrx2are kept mainly in their reduced forms, but become oxidized on exposure to low concentrations of peroxides.Iftherecyclingmechanismisslow(asinerythrocytes[16])orifitisinhibited,thedisulfide form accumulates and can be detected as a dimer by nonreducing SDSpolyacrylamide electrophoresis (SDSPAGE). Thus, if adenanthin adds to the resolving Cys, it should inhibit dimer formation.

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AlthoughadenanthinappearedtoshowselectivityforPrxs1and2inAPLcells[1],asanelectrophile it might be expected to form adducts with GSH and other cellular thiol compounds. Indeed, the finding that Prxs did not appear to be the target in the encephalitis model [2] implies that adenanthin may not be as selective as initially proposed.  In order to understand more about the biologicalactivityofadenanthin,wehavecompareditsreactivitywithPrxs,GSHandcomponentsof thereductivepathwaysinvolvedinPrxandGSHrecycling.WereportthatpurifiedPrx2hassimilar reactivitytoGSH,andthatinerythrocytesandJurkatcellsPrxs1or2arenotselectivelytargeted. Instead, adenanthindepletes GSH andinhibitsthioredoxin reductase. As a result, reductionof the Prxs is inhibited and they accumulate as disulfides following exposure of the cells to low concentrationsofH2O2.  2.MaterialsandMethods 2.1Materials Adenanthin (MW 490 Da, 99% purity) was purchased from BioBioPha Co. (Kunming, China) and a stocksolution(20mM)waspreparedinDMSO.OtherchemicalswerefromSigmaAldrich(StLouis, MO, USA), Merck (Billerica, MASS) or Roche (Mannheim, Germany). BioRad (Hercules, CA, USA) supplied the electrophoresis and Bradford reagents and enhanced chemiluminescence reagents were from GE Healthcare (Buckinghamshire, UK). Rabbit polyclonal antibodies to Prx1, Prx2 and horseradishperoxidaseconjugatedgoatantirabbitantibodieswerepurchasedfromAbcam,Sigma AldrichandDAKOrespectively. Recombinant Histagged wild type Prx2 andC172S (resolving cysteine to serine) and C51S (peroxidaticcysteinetoserine)mutantswerepreparedasdescribed[17]withafewmodifications. Briefly,cDNAencodinghumanPrx2withanaminoterminalFactorXacleavagesitewasclonedintoa pET28a vector.  Specific oligonucleotides were used to mutate this template byPCR to generateseparatecDNAconstructsencodingtheCystoSermutants.Theproteinswereexpressed in E. coli and purified on His60 Ni Superflow Resin (Clontech) according to the manufacturer’s instructions.HistagswereremovedbyincubationwithFactorXa(Roche)togivePrx2proteinswith noadditionalaminoacids.Eachisolatedproteinranasasinglebandat~22kDaonreducingSDS PAGEandgaveasinglepeakwhenanalyzedbyLC/MS.Immediatelybeforeexperiments,Prx2was reducedby50mMdithiothreitol(DTT),thenpassedthroughaMicroBioSpin 6column(BioRad), whichwasfirstwashedwithwater,thenwith100lofa10mg/mlcatalasesolution,followedby5 ml of 50 mM phosphate buffer, pH 7.4 or 8.0, containing 0.1 mM diethylenetriaminepentaacetic

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acid (DTPA). The phosphate buffer was pretreated with 10 g/ml catalase (to remove any H2O2), thenpassedthroughanAmiconUltra1510Kfilter.ThisprocedurewasadoptedtoensurethatPrx2 wasfullyreducedbeforetreatment.  2.2.ReactionofpurifiedPrx2withadenanthin Wild type or mutant protein (5 μM) was incubated at 37oC with 60 μM adenanthin in phosphate buffer, pH 7.4 containing 0.1 mM DTPA. Aliquots were taken at specified times and immediately analyzedbyLC/MS.ForWesternblotanalysis,5μMwildtypePrx2wasincubatedat37oCeitherwith 50μMadenanthinforspecifiedtimesorwithvariousconcentrationsofadenanthinfor1h.Ateach sampling, two aliquots were taken and one was treated for 5 min with 30 μM H2O2. Both were blockedwithNEM(25mM),andanalyzedbySDSPAGE.  2.3.ReactionofGSHwithadenanthin Forproductanalysis,GSHandadenanthinwerereactedatequimolarconcentrations(40μM)atpH 7.4.After2hat37oC,thereactionwasstoppedbywith400μMNEMandproductswereanalyzedby LC/MS (Section 2.9). For the time course, 5 μM adenanthin was incubated with 50 μM GSH in phosphatebuffer,pH7.4at37°C.Atspecifiedtimepoints,aliquotswereblockedwithNEM(final concentration1mM)andanalyzedbyLC/MS.  2.4. Reactions of purified glutathione reductase, thioredoxin reductase, and glutaredoxin with adenanthin The activity of recombinant rat thioredoxin reductase 1 (TrxR1) obtained from IMCO (Stockholm, Sweden)wasanalyzedbySecystinedependentNADPHoxidationasdescribedbyCunniffetal.[18]. To test for inhibition, 0.2 μM TrxR1 was incubated with adenanthin in the presence of 100 μM NADPH at 37 oC.  Aliquots were diluted 1:24 into the assay mixture after appropriate times and activitywasmeasured.Theactivityofpurifiedglutathionereductase(SigmaAldrich)wasanalyzed by measuring GSSGdependent NADPH oxidation as described by Beutler [19].  The activity of glutaredoxin(IMCOCorporationLtdAB,Stockholm)wasanalyzedbymeasuringoxidationofNADPH in the presence of GSH, glutathione reductase and 2hydroxyethyl disulfide [20]. To test for inhibition,theglutathionereductase(1:100dilutionofthecommercialsuspension)orglutaredoxin (5μM)waspreincubatedfor30minat37 oCwitheither50μMNADPHor30μMGSHrespectively, and10160Madenanthin,inphosphatebuffer,pH7.4containing0.1mMDTPA.Afterincubation, the enzymes were placed on ice, then diluted 50 fold into the assay mixture. The control rate of

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NADPH oxidation was 123 + 1 μM/min for glutathione reductase and 13.7 + 0.3 μM/min for glutaredoxin.  2.5.Treatmentoferythrocytes Erythrocyteswereobtainedfromhealthyhumanvolunteerswithinformedconsent,asapprovedby theNewZealandSouthernARegionalEthicsCommittee.BloodwascollectedintoheparinorEDTA. Erythrocytes were separated by centrifugation, washed 3 times with 1.5 volumes of cold PBS (10 mM sodium phosphate buffer pH 7.4 in 0.14 M NaCl) then kept on ice for use on the day of collection. Cell counts were made using a hemocytometer. Erythrocytes (107 in 100 μl) in PBS containing 5 mM glucose were treated with adenanthin under conditions described for each experiment. Control cells were treated with an equivalent concentration of DMSO. At the end of eachexperiment,samplesforPrx2analysis(15μl)wereaddedto30μlPBSor30μlof100mMNEM inPBS,stoodonicefor30minandthenaddedtononreducingsamplebuffer(45μl)forSDSPAGE. Theremaining70 μl was usedtomeasure theconcentration ofGSHusingdithiobis2nitrobenzoic acid(DTNB).  2.6.TreatmentandanalysisofJurkatcells Jurkat Tlymphoma cells (originally obtained from AmericanTypeCell Culture, Rockville, MD, USA) were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum and penicillin/streptomycin(Invitrogen,Carlsbad,CA,USA),at37°Cinahumidifiedatmospherewith5% CO2. Cell density was maintained at 0.20.8 × 106 cells/ml. Before treatment, the medium was removed and the cells were resuspended at 5 x 106/ml in PBS. The cells were treated with adenanthin under conditions described in each figure legend. For Prx2 analysis, 500 μl of each samplewasaddedto50lof200mMNEMandincubatedonicefor30min.Thecellswerepelleted and lysed in 100 l extractbuffer (40 mMHEPES, 50 mMNaCl,1mM EDTA, 1 mM EGTA,pH 7.4, containingproteaseinhibitorsand1%CHAPS),thenaddedtosamplebufferforSDSPAGE.  ForGSH analysis,2.5x 106cellswerespundown andextractedinto100μlextractbuffer.Protein was precipitated and GSH was analyzed using the DTNB assay [19]. Thioredoxin reductase activity was measured in 2.5 x 106 cells extracted into 300 μl extract buffer, following centrifugation to removecelldebris.DTNBwasaddedandthedifferenceinrateofabsorbancechangebefore and afterNADPHadditionwasusedtocalculateenzymeactivity[21].Allmeasuredconcentrationswere normalizedtototalproteinconcentration,asanalyzedusingtheBradfordassay.  6 

Todeterminecellviability,thecellswereanalyzed30minaftertreatmentandafterresuspensionin RPMI for 24 h. For the analysis, 105 cells were resuspended in 200 l PBS, propidium iodide (final concentration5g/ml)wasaddedandthepercentageofcellsthattookuppropidiumiodidewas determinedusingaCytomicsFC500MPLflowcytometer(BeckmanCoulter,Brea,CA).  2.7.Polyacrylamidegelelectrophoresis(PAGE)andWesternblotting SDSPAGEwasperformedunderreducingornonreducingconditionsusingaBioRadMiniProteanII or MiniProtean 3 cell apparatus as described previously [16]. Molecular masses were estimated with Precision Plus Dual Color standards (Biorad). A 3% stacking gel and a 12% resolving gel were usedunlessstatedotherwise.Totalproteinloadingwas6ng/laneforpurePrx2,0.40.8μgperlane forerythrocytesamplesand5μgperlaneforJurkatcellextracts.Forimmunoblotanalysis,proteins were transferred to polyvinylidene difluoride membranes.  Incubation with primary antibodies to Prx2 (1:10 000 dilution) or Prx1 (1:1000 dilution) in 5% (w/v) was followed by incubation with horseradishperoxidaseconjugatedgoatantirabbitIgG(1:10000dilution).Bandsweredetectedby enhancedchemiluminescence(ECL,Pierce)andvisualizedwithaChemiDocXRSgeldocumentation system (Uvitec, Cambridge). The relative intensities of the Prx monomer and dimer bands were quantified by densitometric analysis using ImageJ (National Institute of Health), and results are expressedasapercentageofthetotalintensity.Erythrocytesamplesoftengaveabandat~30kDa, whichislikelytorepresentnonspecificpseudoperoxidaseactivityofdimerichemoglobin[16].  2.8.WholeproteinmassspectrometryofPrx2adenanthin Samples containing 500 ng protein were loaded onto an Accucore150C4 (50 x 2.1 mm, 2.6 m, Thermo Scientific, Waltham, MA) column using a Dionex UltiMate 3000 HPLC system (Thermo Scientific).ThiolswerenotderivatizedtoavoidanyconfusionbetweentheadditionofAdn(490Da) with4NEMmolecules(each125Da).Anacetonitrilegradientfrom90%solventA(0.1%formicacid inwater)to80%solventB(0.1%formicacidinacetonitrile)wasrunover4.6min.SolventBwasheld at 80% for 2.5 min followed by column reequilibration for  2.5 min with 90% solvent A. The flow rate was 400 μl/min and the column temperature was 60 °C. The HPLC was coupled inline to an electrospray ionization source of a Velos Pro mass spectrometer (Thermo Scientific) and mass spectraldatawereacquiredbetweenm/z50and2000inpositivemode.Thevoltagewas4kVolts andthenitrogengas flow was20AU.Thetemperatureofthe heatedcapillarywas275°Candthe vaporizertemperaturewas400°C.Spectrawereaveragedoverthefulllengthofeachproteinpeak and deconvoluted using ProMass for Xcalibur (version 2.8; Novatia LLC, Monmouth Junction, NJ). Theaccuracyofthedeconvolutedmasseswas±3Da. 7 

 2.9.DetectionofglutathionespeciesandtheGSHadenanthinadductbymassspectrometry GSH and GSSG were measured by isotope dilution mass spectrometry as described [22]. Samples wereseparatedonaJupiterC18HPLCcolumn(150x2mm,5m,100,Phenomenex,Torrance, CA), using the same LC/MS system as in section 2.9. An acetonitrile gradient from 95% solvent A (0.1%formicacidinwater)to95%solventB(0.1%formicacidinacetonitrile)wasrunover7min. Solvent B was held at 95% for 10 min followed by column reequilibration for 10 min with 95% solvent A. The flow rate was 200 μl/min and the column temperature was 40 °C. For initial characterizationoftheGSHadenanthinadduct,MSdatawereacquiredinnegativemode.Aspecies withthepredictedm/zof796.5foranegativelysinglychargedGSHadenanthinadductwasdetected at11.0min.Toconfirmthestructuralidentityofthisspecies,fullCIDMS/MSspectrarangingfrom m/z 200 to 2000 were acquired for m/z 796.5. The window for precursor selection was 1 Da on eithersideofthem/z;thenormalizedcollisionenergywas35andactivationtime10msec.Thetime course samples were analyzed in positive ion mode and the peak for the GSHadenanthin adduct (m/z798.4)wasintegrated.  3.Results 3.1.ReactionofadenanthinwithisolatedPrx2 Reduced Prx2 (containing a small amount of disulfidebonded dimer) was incubated with adenanthin. This did not alter the proportions of Prx monomer and dimer, but did cause a concentrationdependent (Fig. 2A) and timedependent (Fig. 2B) shift in the monomer band to a slightlyhigherpositionwhenanalyzedbySDSPAGE(lefthandlanes).ExposureofuntreatedPrx2to a slight molar excess of H2O2 resulted in oxidation to the dimer (right hand lanes).  Pretreatment with adenanthin caused concentration and time dependent inhibition of dimerization. These resultsareconsistentwithasmallmobilityshiftofthemonomerduetoadditionofadenanthin(MW 490 Da), and consequent inhibition of dimerization due to blockage of one of the active site Cys residues. AnalysisbymassspectrometryconfirmedformationofPrx2adenanthinadducts.Prx2gaveamajor masspeakat21,893Daplusasmallamountofdimer(Fig.3A).Withadenanthintreatment,there was time dependent addition of first one and then two adenanthin molecules to the Prx. The spectrummeasuredafter2h(Fig.3B)showsPrxmonomerwithtwoadenanthinadductsandasmall amountwiththree.ThePrx2underwentsubstantialdimerizationwiththisprocedure(eitherduring incubation or sample workup) and the dimer also had adenanthins attached. This indicates that

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more than one Cys residue is vulnerable. To simplify analysis and compare reactivity of the Cys residues, a similar experiment was carried out with recombinant Prx2 in which the resolving Cys (172) or peroxidative Cys (51) had been mutated to Ser. Both formed a single adduct with adenanthin (Figs. 3C & D respectively). These results confirm that these two Cys residues are the major sites for adduct formation and imply that binding to other sites is minimal.  Time course experimentsshowedthatadditiontotheperoxidaticCys(intheresolvingmutant)wassubstantially faster than addition to the resolving Cys (Fig. 3E). Incubation for extended periods resulted in additionofafurtheradenanthinmolecule.Thisadduct(whichismoreevidentfortheC127Smutant in Fig. 3D), and the adduct with three adenanthins in Fig. 3B, likely represent conjugation to the third, noncatalytic cysteine. The data for the mutants in Fig. 3E fitted well to exponential curves. AnalysisofthesecurvesgavehalflivesforadductformationontheperoxidaticandresolvingCysof 7 and 40 min, respectively. As these experiments were carried out under pseudofirst order conditionswith50μMadenanthin,correspondingsecondorderrateconstantsofapproximately30 and5M1s1,respectively,canbecalculated. 3.2.ReactionofadenanthinwithGSH LC/MS analysis showed that adenanthin also reacts with GSH. Untreated adenanthin gave a mass spectrum with amajorpeak atm/z 508.3 (correspondingtoawateradduct) and smaller peaks at m/z 981.3 and 998.5 (Fig. 4A). The latter two correspond to adenanthin dimers with and without waterandareconsistentwithadvicefromthesupplierthatadenanthinfliespartiallyasadimerin the mass spectrometer. After incubation with equimolar GSH, analysis by LC/MS after 2h showed that only small amounts of GSH and adenanthin remained (Fig. 4B). A new peak was formed with m/z 796.5 (Fig 4C), consistent with a Michael addition product of adenanthin with GSH (Fig. 4D). Fragmentation of the m/z 796.5 peak resulted in a single species with m/z 306, consistent with negativelychargedGSH(Fig.4Cinset). Fig.4EshowsthetimecourseofformationoftheadenanthinGSHadduct,carriedoutunderpseudo firstorderconditionswithexcessGSH.ThedatainFig.4Efittedwelltoexponentialcurves,givinga halflifes for adduct formation of 20 and 10 min for 25 and 50 μM GSH respectively. Adduct formationwasaccompaniedbyprogressivelossofapproximately1020%oftheGSH(measuredby stableisotopedilutionLC/MS),whichisconsistentwiththeexcessofGSHanda1:1stoichiometry. RatesofGSHlossandadductformationwerecomparable;forexampleinoneexperimentwith25 mMGSH,pseudofirstorderrateconstantswere0.024and0.033min1respectively.Resultsfrom6 analysesofadductformationcarriedoutwith25or50μMGSHgaveasecondorderrateconstant forthereactionof25+6M1s1.

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 3.3Reactionsofadenanthininerythrocytes When erythrocytes were incubated with adenanthin, we did not observe the shift in the electrophoretic mobility of Prx2 expected for adduct formation. Instead, Western blotting of non reducing gels showed progressive accumulation of Prx2 dimer over time (Fig. 5A, left lanes). Also, when the cells were lysed without NEMblocking (a procedure that results in oxidation of Prx2 by peroxidesinthelysisbuffer[23])allthePrx2dimerized(rightlanes).Asdimerizationcannotoccur when adenanthin is bound to either of the active site cysteines, this indicates that little, if any, adducthadbeenformed.DimerizationofPrx2increasedwithincreasingadenanthinconcentration (Fig. 5B), and Prx1 showed similar susceptibility to oxidation as Prx2 (Fig. 5C). The observed accumulation of Prx dimer is analogous to what is seen when erythrocytes are incubated with dinitrochlorobenzene [16], which inhibits thioredoxin reductase and other reductive mechanisms. Therefore, these results imply that adenanthin impairs the regeneration of the reduced Prxs from theirdisulphideforms. Totestthisproposal,effectsofadenanthinoncomponentsoferythrocytereductionpathwayswere investigated. As expected from the results with pure GSH, adenanthin treatment resulted in the progressivelossoferythrocyteGSH(Fig.5D).Ofnote,muchoftheGSHwasdepletedbeforethere wassubstantialPrxoxidation. 3.4.Reactionsofadenanthinwiththioredoxinreductase,glutaredoxinandglutathionereductase We examined effects of adenanthin on isolated thioredoxin reductase, glutathione reductase and glutaredoxin.Allwereprogressivelyinhibitedbypreincubationwithadenanthin(inthepresenceof either GSH or NADPH to ensure that the proteins were reduced). Analysis of the concentration dependencecurvesforglutaredoxinandglutathionereductaseshowed50%inhibitionafter30min incubationwith100150μMadenanthin(datanotshown).IncomparisonwithFig.2B,whichshows about 50% adduct formation and inhibition of dimerization when Prx2 was treated with 50 μM adenanthin for 60120 min, these results suggest that the reactivities of the three proteins are comparable.  Thioredoxinreductasewasmuchmorereactive.Adenanthinat2μMcausedprogressiveinhibition over 20 min (Fig. 6A) and the concentrationdependence curve at 10 min showed up to complete inhibition(Fig.6B).Inhibitionrequiredtheenzymetobereduced,asnonewasseenintheabsence ofNADPH(notshown).Theenzymewasdiluted24foldforanalysisafterexposuretoadenanthin,

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implyingthatinhibitionwasnoteasilyreversible.ThetimecourseinFig.6Afittedpseudofirstorder kineticswithahalflifeof5min.Thiscorrespondstoasecondorderrateconstantof1200M1s1.  3.5.ReactionsofadenanthininJurkatcells We first characterized the effects of adenanthin on Prx2 oxidation and GSH content. As reported previously [24], Western blotting of nonreducing gels showed that most of the Prx2 in untreated Jurkat cells was reduced. As was the case with erythrocytes, treatment with adenanthin gave no detectableshiftinthemonomerband(notshown).Therewas,however,progressivePrx2oxidation with increasing adenanthin concentration and more pronounced depletion of GSH (Fig. 7A). However, the most dramatic effect was on thioredoxin reductase activity. Thioredoxin reductase wasmuchmoresensitivethanGSH,with70%inhibitedafteranhour’sincubationwithonly10μM adenanthin. Adenanthin at concentrations up to 100 μM had no immediate effect on cell viability (Fig. 7B). However,whenexaminedafter24h,cellsexposedto20μMorhigherconcentrationswerealmost alldead.  4.Discussion Adenanthin has been reported to selectively target peroxiredoxins 1 and 2 in leukemic cells by undergoingaMichaeladditionreactionbetweenits,unsaturatedcarbonylandtheirresolvingCys [1].Basedonthesefindings,weexpectedtoseespecifictargetingofadenanthintotheresolvingCys of isolated Prx 2. We also expected adduct formation to occur in erythrocytes and Jurkat cells, thereby preventing the Prxs from forming disulfidelinked dimers when the cells were exposed to H2O2.Ineachcasethiswasnotwhatweobserved. PurePrx2 did reactwithadenanthintogivethe expected inhibitionof dimerization. However, the reaction was not selective for the resolving Cys. Multiple adducts were formed and our kinetic studieswithCysmutantsshowedthattheperoxidaticCysisapproximatelysixtimesmorereactive than the resolving Cys. Liu et al’s evidence for specificity included MS data showing adenanthin attachmenttothetrypticpeptidecontainingtheresolvingCysofPrx1[1].Theydidnotpresentdata showingthattheperoxidativeCyswasunaltered,butifthiswerethecaseitmightbeexplainedby our observation that the adduct did not survive extended incubation or reduction. However, they did not pull down the resolving Cys mutant with biotinadenanthin, and the reason for the

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discrepancy with our results is not clear. Another electrophile that forms an adduct with 2Cys peroxiredoxins,conoidinA,wasfoundtoreactwiththeperoxidaticCys[25]. Not unexpectedly, adenanthin also formed a Michael addition product with GSH. GSH (estimated secondorderrateconstant20M1s1)showedcomparablereactivitytotheactivesitethiolsofPrx2 (rateconstantsof30and5M1s1).Onthisbasis,adenanthinwouldbeexpectedtoreactwithother thiolproteinsanditwouldnotbehighlyselectiveforPrx2incells.Consistentwiththisconclusion, weobservedasimilardegreeofinactivationofisolatedglutaredoxinandglutathionereductaseas seenwithPrx2,andMuchowiczandcoworkers[26]recentlyreportedadenanthinadductformation and inhibition of thioredoxin and protein disulfide isomerase. However, we found thioredoxin reductasetobemoresensitivetoinactivationanditmaybeitsmostpreferredtarget. We observed rapid inhibition of thioredoxin reductase with low micromolar concentrations of adenanthin and determined a rate constant (1200 M1s1) that is almost two orders of magnitude higherthanforGSHandPrx2.WealsosawinactivationinJurkatcellsatlowerconcentrationsthan those required to see GSH loss or Prx2 disulfide accumulation. This high sensitivity is not unexpected,asthioredoxinreductaseisaselenoenzymeandseleniumisastrongernucleophilethan sulfur. It is also inhibited by another diterpenoid, 15oxospiramilactone [27], by 4hydroxynonenal [28] and by electrophilic derivatives of polyphenols [29, 30]. Thioredoxin reductase operates by a complexmechanisminvolvingtworedoxsitesanditisbeyondthescopeofthisstudytocharacterize where adenanthin acts. However, its inhibitory behavior is similar to that of 4hydroxynonenal, whichbindsprimarilyattheCys496Sec497activesite[28]. OurfindingthatadenanthinisnotselectiveforPrxs1and2isinagreementwithotherswhoshowed that  biotinylated 15oxospiramilactone reacted with a number of other cell proteins [27]. Also, althoughPrxsweretheonlyadenanthinbindingproteinsdetectedwithleukemiccells[1]thisgroup observedinteractionwithcomponentsoftheNFNBsignalingpathwayintheirmodelofautoimmune encephalomyelitis[2]. In spite of adenanthin reacting with isolated Prx2, we saw no evidence for adduct formation or inhibitionofPrxdimerizationwhenitwasaddedtoerythrocytesorJurkatcells.Theexactopposite happened.AdenanthinpromotedtheaccumulationofPrxdimers.CellularPrxscontinuallyundergo redoxcyclingduetoendogenousgenerationandremovalofreactiveoxygenspecies.Ifreductionis inhibited,thedimericformsaccumulate[31,32].Prxs1and2arerecycledbyreducedthioredoxin, whichisregeneratedbythioredoxinreductase/NADPH[33].Inhibitionofthisreductivemechanism wouldexplainwhytheoxidizedPrxsaccumulated.Basedonthestronginhibitionseenin isolation

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and in Jurkat cells, thioredoxin reductase is likely to be the prime target, although inactivation of thioredoxin [26]  and loss of GSH and glutaredoxin activity could have contributed. Glutaredoxin/GSHcanreducethioredoxin[34]andprovideanauxiliarymechanismforPrxrecycling whenthioredoxinreductaseactivityislow.ThesubstantiallossofGSHpriortoPrxoxidation,seenin both cell types, could have been due to a combination of adduct formation and inhibition of glutathionereductase. OurresultsshowthatalthoughdirectinhibitionofPrxs1and2byadenanthinisunlikelyincells,Prx metabolismisaffected.Byinactivatingthethioredoxinsystem,aswellasdepletingGSH,Prxcycling isinhibitedandthecellswouldhaveimpairedabilitytocounteractoxidativestress,althoughviaa different mechanism. However, the impact would be much wider, as the thioredoxin reductase/thioredoxinandglutaredoxin/GSHsystemsdriveanextensivearrayofcellularfunctionsin additiontoprotectingagainstoxidativestress[33,3537].Theseincluderibonucleotidereductionfor DNAsynthesis,methioninesulfoxidereduction,aswell ascontrollingtheredoxstateofnumerous transcription factors and other thiol proteins. Consequently, these redox pathways are important regulatorsofcellproliferation,survivalandapoptosis. WeobservedthatwhileadenanthinwasnotimmediatelytoxictoJurkatcells,concentrationsinthe range that inhibited thioredoxin reductase caused loss of viability on longer incubation. Other electrophiles[27,29,30]alsoinduceapoptoticcelldeathinassociationwiththioredoxinreductase inhibition,anditseemslikelythatthismechanismcontributedtothelossofcellviabilitycausedby adenanthin. Likewise, other observed effects of adenanthin (or 15oxospiramilactone) on cell differentiation and tumor growth in APL [1], dampening immune responses and altering NFNB signaling[2]orWnt/cateninsignaling[38],orpromotingcelldeathinhepatocarcinomacells[39] may involve the thioredoxin and GSH redox systems. There is considerable interest in thioredoxin reductase as a target for anticancer therapy [37], as it promotes tumor growth, is upregulated in various cancers [36, 4043], and is associated with drug resistance. With the exception of gold compounds, our results with Jurkat cells suggest that adenanthin is at least as effective as other identifiedinhibitors. Inconclusion,wehaveconfirmedthatadenanthinreactsdirectlywithpurifiedreducedPrx2,butnot specifically with the resolving Cys. In two cell types it preferentially interacted with thioredoxin reductaseandGSH,inhibitingrecyclingofPrxdisulfidesandcausingaccumulationofPrxs1and2as oxidizeddimers.AlthoughadenanthinisnotselectiveforPrxs,itispotentiallyusefulasasensitive inhibitorofcellularthiolreductivesystems.

13 

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[13]KangDH,LeeDJ,KimJ,LeeJY,KimHW,KwonK,etal.Vascularinjuryinvolvestheoveroxidation of peroxiredoxin type II and is recovered by the peroxiredoxin activity mimetic that induces reendothelialization.Circulation.2013;128:83444. [14] Hall A, Karplus PA, Poole LB. Typical 2Cys peroxiredoxinsstructures, mechanisms and functions.FebsJ.2009;276:246977. [15]PeskinAV,LowFM,PatonLN,MaghzalGJ,HamptonMB,WinterbournCC.Thehighreactivityof peroxiredoxin2withH(2)O(2)isnotreflectedinitsreactionwithotheroxidantsandthiolreagents.J BiolChem.2007;282:1188592. [16]LowFM,HamptonMB,PeskinAV,WinterbournCC.Peroxiredoxin2functionsasanoncatalytic scavengeroflowlevelhydrogenperoxideintheerythrocyte.Blood.2007;109:26117. [17] Pace PE, Peskin AV, Han MH, Hampton MB, Winterbourn CC. Hyperoxidized peroxiredoxin 2 interactswiththeproteindisulfideisomeraseERp46.TheBiochemicaljournal.2013;453:47585. [18]CunniffB,SniderGW,FredetteN,HondalRJ,HeintzNH.Adirectandcontinuousassayforthe determinationofthioredoxinreductaseactivityincelllysates.AnalBiochem.2013;443:3440. [19] Beutler E. Red Cell Metabolism; A manual of biochemical methods. 3 ed. Orlando: Grune & Stratton1984. [20] Hashemy SI, Johansson C, Berndt C, Lillig CH, Holmgren A. Oxidation and Snitrosylation of cysteinesinhumancytosolicandmitochondrialglutaredoxins:effectsonstructureandactivity.JBiol Chem.2007;282:1442836. [21] Arner ES, Zhong L, Holmgren A. Preparation and assay of mammalian thioredoxin and thioredoxinreductase.MethodsEnzymol.1999;300:22639. [22] Harwood DT, Kettle AJ, Brennan S, Winterbourn CC. Simultaneous determination of reduced glutathione,glutathionedisulphideandglutathionesulphonamideincellsandphysiologicalfluidsby isotopedilutionliquidchromatographytandemmassspectrometry.JChromatogrBAnalytTechnol BiomedLifeSci.2009;877:33939. [23] Poynton RA, Hampton MB. Peroxiredoxins as biomarkers of oxidative stress. Biochim Biophys Acta.2014;1840:90612. [24]CoxAG,PearsonAG,PullarJM,JonssonTJ,LowtherWT,WinterbournCC,etal.Mitochondrial peroxiredoxin 3 is more resilient to hyperoxidation than cytoplasmic peroxiredoxins. Biochem J. 2009;421:518. [25]HaraldsenJD,LiuG,BottingCH,WaltonJG,StormJ,PhalenTJ,etal.IdentificationofConoidina asaCovalentInhibitorofPeroxiredoxinIi.OrgBiomolChem.2009;7:30408. [26]MuchowiczA,FirczukM,ChlebowskaJ,NowisD,StachuraJ,BarankiewiczJ,etal.Adenanthin targetsproteinsinvolvedintheregulationofdisulphidebonds.BiochemPharmacol.2014;89:2106. [27] Liu J, Mu C, Yue W, Li J, Ma B, Zhao L, et al. A diterpenoid derivate compound targets selenocysteine of thioredoxin reductases and induces Bax/Bakindependent apoptosis. Free Radic BiolMed.2013. [28]FangJ,HolmgrenA.Inhibitionofthioredoxinandthioredoxinreductaseby4hydroxy2nonenal invitroandinvivo.JAmChemSoc.2006;128:187985.

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[29] Fang J, Lu J, Holmgren A. Thioredoxin reductase is irreversibly modified by curcumin: a novel molecularmechanismforitsanticanceractivity.JBiolChem.2005;280:2528490. [30]LuJ,PappLV,FangJ,RodriguezNietoS,ZhivotovskyB,HolmgrenA.InhibitionofMammalian thioredoxin reductase by some flavonoids: implications for myricetin and quercetin anticancer activity.CancerRes.2006;66:44108. [31] Low FM, Hampton MB, Winterbourn CC. Peroxiredoxin 2 and peroxide metabolism in the erythrocyte.AntioxidRedoxSignal.2008;10:162130. [32] Stacey MM, Vissers MC, Winterbourn CC. Oxidation of 2cys peroxiredoxins in human endothelialcellsbyhydrogenperoxide,hypochlorousacid,andchloramines.AntioxidRedoxSignal. 2012;17:41121. [33]LuJ,HolmgrenA.Thethioredoxinantioxidantsystem.FreeRadicBiolMed.2014;66:7587. [34] Du Y, Zhang H, Lu J, Holmgren A. Glutathione and glutaredoxin act as a backup of human thioredoxin reductase 1 to reduce thioredoxin 1 preventing cell death by aurothioglucose. J Biol Chem.2012;287:382109. [35] Allen EM, Mieyal JJ. Proteinthiol oxidation and cell death: regulatory role of glutaredoxins. AntioxidRedoxSignal.2012;17:174863. [36] Lee S, Kim SM, Lee RT. Thioredoxin and thioredoxin target proteins: from molecular mechanismstofunctionalsignificance.AntioxidRedoxSignal.2013;18:1165207. [37]UrigS,BeckerK.Onthepotentialofthioredoxinreductaseinhibitorsforcancertherapy.Semin CancerBiol.2006;16:45265. [38] Wang W, Liu H, Wang S, Hao X, Li L. A diterpenoid derivative 15oxospiramilactone inhibits Wnt/betacateninsignalingandcoloncancercelltumorigenesis.CellRes.2011;21:73040. [39]HouJK,HuangY,HeW,YanZW,FanL,LiuMH,etal.AdenanthintargetsperoxiredoxinI/IItokill hepatocellularcarcinomacells.CellDeathDis.2014;5:e1400. [40] Selenius M, Rundlof AK, Olm E, Fernandes AP, Bjornstedt M. Selenium and the selenoprotein thioredoxinreductaseintheprevention,treatmentanddiagnosticsofcancer.AntioxidRedoxSignal. 2010;12:86780. [41]KakolyrisS,GiatromanolakiA,KoukourakisM,PowisG,SouglakosJ,SivridisE,etal.Thioredoxin expressionisassociatedwithlymphnodestatusandprognosisinearlyoperablenonsmallcelllung cancer.ClinCancerRes.2001;7:308791. [42] Engman L,AlMaharikN,McNaughtonM,BirminghamA,PowisG.Thioredoxinreductase and cancercellgrowthinhibitionbyorganotelluriumantioxidants.AnticancerDrugs.2003;14:15361. [43] Lincoln DT, Ali Emadi EM, Tonissen KF, Clarke FM. The thioredoxinthioredoxin reductase system:overexpressioninhumancancer.AnticancerRes.2003;23:242533.   



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FigureLegends:  Fig.1.Structureofadenanthin  Fig.2.Concentration(A)andtimedependence(B)oftheeffectofadenanthinonpurifiedPrx2.Prx2 (5 μM) was incubated at 37°C, in 50 mM phosphate buffer, pH 8 for 1 h with indicated concentrations of adenanthin (A) or with 50 M adenanthin for varying times at pH 7.4 (B). The samples intheleftfourlaneswereuntreatedandtherightsamplesweretreatedwith7 μMH2O2 before adding 50 μM NEM and separation by nonreducing SDSPAGE and silverstaining. Gels are representative of 3 independent experiments. Arrows shows slower moving monomer band consistentwithadditionofadenanthin.  Fig.3.MassspectralanalysisofPrx2aftertreatmentwithadenanthin.(A)UntreatedwildtypePrx2 (after reduction with DTT); (B) wild type Prx2; (C)  Prx2C51S,  and (D) Prx2C172S, all at 5 μM treated with adenanthin (60 μM) for 1h at pH 7.4. (E) Time course for formation of a single adenanthinadductfortheC51SandC172Smutants.(z)unmodifiedPrx2,(S)Prx2withoneAdn adduct,(U)Prx2withtwoadducts.Aftertreatment,proteinswereimmediatelyseparatedbyLC/MS without further derivatization. Masses of the underivatized proteins are: wildtype 21 892 Da, and C51SorC172S,21876Da.Additionofeachadenanthin(Adn)adds490Da.Exponentialcurvesfitted tothedatainEgavepseudofirstorderrateconstantsforAdnadditionof0.10and0.018min1and halflivesof7and40minfortheC172SandC51Smutantsrespectively.  Fig. 4. Adduct formation between GSH and adenanthin (Adn). A. Mass spectrum of adenanthin (positiveionmode).B.ChromatographicseparationofmixtureafterreactingadenanthinwithGSH (both40μM)for2hat37oCandpH7.4,thenderivatizingexcessGSHwithNEM.C:Massspectrum ofthe11minpeak(negativeionmode).Inset,MS/MSspectrumshowingglutathionefragment.D. ProposedstructureoftheGSHadenanthinadduct.E.TimecourseforadenanthinbindingtoGSH. GSH (25 and 50 μM) was reacted with 5 μM adenanthin and formation of the GSH adduct was followedbyLC/MS.Datafromarepresentativeexperimentareshownandexponentialcurveshave been fitted and used to calculate pseudo first order rate constants of 0.035 and 0.068 min1 respectively.  Fig.5.EffectsofadenanthinontheredoxstateoferythrocytePrxs1and2.(A)TimecourseforPrx2 oxidation in erythrocytes treated with 50 μM adenanthin (Adn)  at 37°C. After stated times, cells were treated with NEM then subjected to nonreducing SDSPAGE and western blotting  for Prx2 17 

(lefthand lanes).Samples on the righthadnoNEMadded (a procedure that allows oxidation and thereforedimerizationofPrx2withfreeactivesitecysteines).(B)Erythrocyteswereincubatedasin (A)withthestatedconcentrationofadenanthinfor1h,thentreatedwithNEMandimmunoblotted forPrx2.(C)Cellsweretreatedasfor(B)andimmunoblottedforPrx1.Dimerandmonomerbands aremarked. Xis anonspecificbandthought to bedimerichemoglobinwhichreactswith the ECL detection system. (D) Time course of Prx2 dimerization and GSH loss. GSH levels were measured usingDTNBandpercentagesofPrx2monomerweredeterminedbydensitometryofblotsasin(A). Results are means and SD from 3 independent experiments. (E) Changes in GSH and GSSG concentrations over time in erythrocyes at 108/ml treated with adenanthin as in (A). Glutathione speciesweremeasuredbymassspectrometry.  Fig. 6. Inhibition of isolated thioredoxin reductase 1 (TrxR1) by adenanthin. (A) Time course of inactivationof0.2μMTrxR1by2μMadenanthin.Resultsaremeansandrange(withinsymbolsize) fromtwoindependentexperiments.Exponentialfit(R2 =0.99)correspondstoapseudofirstorder rateconstant(withexcessadenanthin)of0.14min1.(B)Concentrationdependencofinactivationof 0.2μMTrxR1treatedwithstatedconcentrationsofadenanthinfor10min.  Fig. 7. (A) Oxidation of Prx2 and losses of GSH and thioredoxin reductase activity in Jurkat cells treatedwithadenanthin.Cells(5x106)weretreatedwithvariousconcentrationsofadenanthinfor1 hat37oC.AproportionofeachsamplewastreatedwithNEM(200mM)andlysedfordetectionof Prx2oxidationbyWesternblottingofnonreducingSDSgels(‹)andtheremainderwasanalyzed forGSH(„)andTrxRactivity(z).Prxoxidationisexpressedastheproportionpresentasmonomer andallresultsarenormalizedtocontrolcellsincubatedwithoutadenanthin.Resultsaremeansand SDfor3independentexperiments.(B)ViabilityofJurkatcellsfollowingtreatmentwithadenanthin. Cellsweretreatedasin(A)andviabilityafter30min(z)or24h(„)wasmeasuredusingpropidium iodideandflowcytometry.         18 

 HIGHLIGHTS AdenanthinbindstotheperoxidaticandresolvingCysofperoxiredoxin(Prx)2. ItalsoreactswithGSH,glutaredoxin,andatamuchfasterratewiththioredoxinreductase. AdenanthincausedPrx2dimeraccumulationincellsduetoinhibitionofreductivemechanisms. ItsantitumoreffectsarelikelytoinvolvethiolreductionpathwaysratherthanPrxbinding. 

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Figure 2

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 3E

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 4E

Figure 5A-C

Figure 5D

Figure 5E

Figure 6A

Figure 6B

Figure 7A

Figure 7B

Graphical Abstract (for review)