Ageneralmethodfortheassayofdeubiquitinatingenzymeswasdescribedindetailusing¹²⁵I-labeledubiquitin-fusedαNH-MHISPPEPESEEEEEHYC(referredtoasUb-PESTc)asasubstrate.SincethetyrosineresidueinthePESTcportionofthefusionproteinwasalmostexclusivelyrADIoiodinatedunderamildlabelingcondition,suchasusingIODO-BEADS,theenzymescouldbeassayeddirectlybysimplemeasurementoftheradioactivityreleasedintoacidsolubleproducts.Usingthisassayprotocol,wecouldpurifysixdeubiquitinatingenzymesfromchickskeletalmuscleandyeastandcomparetheirspecificactivities.SincetheextractsofE.colishowedlittleornoactivityagainstthesubstrate,theassayprotocolshouldbeusefulforidentificationandpurificationofeukaryoticdeubiquitinatingenzymesclonedandexpressedinthecells.

Ubiquitin(Ub)isahighlyconserved76aminoacidproteinfoundinalleukaryoticcells(1,2).Ubiscovalentlyligatedtoavarietyofintracellularproteinsthroughanisopeptidelinkage.UbsbythemselvesorthathavealreadybeenconjugatedtoproteinsmayalsobeligatedtoadditionalUbmoleculestoformbranchedpoly-Ubchains.Thisubiquitinationhasbeenimplicatedintheregulationofdiversecellularprocesses,suchasselectiveproteinbreakdown,cellcycleregulation,andstressresponse(3-5).Inaddition,proteinubiquitinationinvivoisadynamic,reversIBLeprocessthatisundercontrolrespondingtoexternalstimuli,suchasheatshockandstarvation(6-8).Therefore,theenzymesthatproteolyticallyremoveUbfromUb-proteinconjugatesshouldbeofimportanceinmaintainingthesteady-statelevelsoffreeUbforitsdiversecellularfunctions.

Ubsareencodedbytwodistinctgeneclasses.Oneisapoly-Ubgenethatencodesapoly-proteinoftandemlyrepeatedUbs(9,10).TheotherencodesafusionproteininwhichasingleUbislinkedtoaribosomalproteinconsistingof52or76-80aminoacids.ThetransientassociationofUbwiththeribosomalproteinshasbeensuggestedtopromotetheirincorporationintoribosomes(11).Therefore,proteolysisatthepeptidebondsbetweenUbandtheextensionproteinsisrequiredforgenerationofribosomalproteinsforribosomebiogenesisaswellasoffreeUbs.

Deubiquitinatingenzymes(DUBs)areknowntoconsistofalargeproteinfamilyineukaryotes.Forexample,thebuddingyeasthas17genesforDUBs(4).Moreover,sofarmorethan60full-lengthDUBsequenceshavebeenidentifiedineukaryotes(12).However,comprehensivesearchesforDUBs,particularlyinmammaliancells,werehamperedduetothelackofrapidandefficientmethodsforassayingtheenzymes.Wehaverecentlyreportedthat¹²⁵I-labeledUb-PESTcservesasanexcellentsubstrateforthesensitiveandquantitativeassayofvariousDUBs(13,14).Usingthisassay,wehavealsoisolatedanumberofnovelDUBsinchickskeletalmuscleandyeast(13,15-18).HerewedescribethedetailedprotocolforassayingDUBsusing¹²⁵I-labeledUb-PESTc.Wealsocomparethesensitivityofthismethodtothatusingafluorogenicpeptidesubstrate,carbobenzoxy-LRGG-7-amido-4-methylcoumarin(Cbz-LRGG-AMC),thathasalsobeenusedasasubstrateforDUBs(19).

Materials

YeastUbhydrolase-1(YUH1)waspurifiedasdescribedpreviously(20).ThepurifiedUb-specificprotease-6(yUBP-6)inyeastandcUBP41,UbC-terminalhydrolase-1(cUCH-1),cUCH-6,andcUCH-8inchickskeletalmusclewerepreparedasdescribed(13,15-18).Ub-PESTcwaspurifiedfromanE.colistrainAR13carryingpNMHUB-PESTcasdescribedbyYooetal.(21).Ub-aldehydewaspreparedasdescribed(13).Cbz-LRGG-AMCwaskindlyprovidedbyDr.K.Tanaka(TokyoMetropolitanInstituteofMedicalScience,Japan).

RadioiodinationofUb-PESTc

Ub-PESTcwasradiolabeledwithNa¹²⁵IusingIODO-BEADS(Pierce)byfollowingtheprocedurerecommendedbythemanufacturerandMarkwell(22).OneIODO-BEADwasincubatedin0.2mlof0.1MTris-HCl(pH7)inamicrofugetubefor5minatroomtemperature.Afterincubation,0.2mgofthepurifiedUb-PESTcand200μCiofNa¹²⁵Iwereaddedtothetubeandfurtherincubatedforthenext15min.TheradioiodinatedUb-PESTc(i.e.,theliquidportion)wasthenremovedandsubjectedtogelfiltrationonaSephadexG-10columnequilibratedwiththeTrisbuffertoremovefreeiodine.Upontheiodinationprocedure,approximately85%of¹²⁵IwasincorporatedintoUb-PESTc.

Assayforhydrolysisof125I-labeledUb-PESTc

Reactionmixtures(final0.1ml)containedproperamountsofthepurifiedDUBs,10-20μgof¹²⁵I-labeledUb-PESTc,0.1MTris-HCl(pH7.8),1mMEDTA,1mMdithiothreitol,and5%(v/v)glycerol.Afterincubationofthemixturesforappropriateperiodsat37°C,thereactionwasterminatedbyadding50μlof40%(v/v)trichloroaceticacidand50μlof1.2%(w/v)bovineserumalbumin.Thesampleswerevortexedandcentrifugedfor10minat10,000xgusingamicrofuge,andaliquots(0.1ml)oftheresultingsupernatantswerecountedforradioactivityusingagamma-counter(13).

AssayforhydrolysisofCbz-LRGG-AMC

Reactionmixtures(final0.1ml)containedappropriateamountsofthepurifiedDUBs,0.2mMCbz-LRGG-AMC,0.1MTris-HCl(pH7.8),1mMEDTA,1mMdithiothreitol,and5%(v/v)glycerol.Afterincubationofthemixturesforvariousperiodsat37°C,thereactionwasterminatedbyadding0.1mlof1%(w/v)SDSand0.8mlofH₂O.ReleaseoffreeAMCbytheenzymereactionwasdeterminedbymeasuringitsfluorescenceat380nm(excitation)and440nm(emission)(23).ProteinswerequantifiedasdescribedbyBradford(24).

Gelelectrophoresis

PolyacrylamidegelelectrophoresisinthepresenceofSDSand2-mercaptoethanolwasperformedusingTris-TricinebufferasdescribedbySchäggerandvonJagow(25).Thediscontinuousslabgelscontained4,10and16%polyacrylamidetoimproveresolutionofsmallproteins.Thesamplebuffercontained150mMTris-HCl(pH6.8),1.5%(w/v)SDS,2%(v/v)2-mercaptoethanol,0.002%(w/v)bromophenolblueand7%(v/v)glycerol.Afterelectrophoresis,thegelswerestainedwithCoomassieblueR-250orsubjectedtoautoradiography.

Rechsteinerandcoworkers(21)haveconstructedUb-αNH-peptideextensionscontaining"PEST"sequences.Ofthese,Ub-PESTccontainsapeptideextensionof18aminoacids,whichcarriesasingletyrosineresiduethatcanberadioiodinatedandisshortenoughtobereleasedasanacid-solubleproductwhenincubatedwithDUBs.Moreover,theyhavereportedthattherecombinantUb-PESTcproteincanbeeasilypurifiedbyheating,suchasat85°C,becausefusionoftheshortpeptidetoUbdoesnotaltertheheatresistanceofUbmolecule.Inaddition,theyhavedemonstratedthatUb-PESTcappearscorrectlyprocessedtoyieldfreeUbuponincubationwiththechromatographicfractionsofrabbitreticulocytes.Therefore,wechoseUb-PESTcforlabelingwith¹²⁵IandhenceforusingthelabeledproteinasasubstratefortheassayofDUBs.OverallprocedurefortheenzymeassayissummarizedinFig.1

Byfollowingtheprotocol,wedeterminedtheactivityofYUH1bymeasuringitsABIlitytoreleaseradioactivePESTcintoanacid-solubleformfrom¹²⁵I-labeledUb-PESTc.ThepurifiedenzymewasincubatedwiththeradioiodinatedsubstrateforvariousperiodsintheabsenceandpresenceofUb-aldehyde,whichisknownasaspecificinhibitorofDUBs(26).Table1showsthattheacid-solubleradioactivityincreasesinanincubationtime-dependentfashionandthisincreasecanbecompletelyblockedbythetreatmentofUb-aldehyde.SinceYUH1aswellasotherDUBsareknowntospecificallycleavethecarboxylsideoftheC-terminalGlyresidueofUb,theacid-solubleproductsshouldrepresentthePESTcpeptidereleasedfromtheUb-peptideextension.

Fig.1:[Enlarge]

SchematicrepresentationforamethodforassayingDUBsusing125I-Ub-PESTc.

Tovalidatefurthertheassaymethod,¹²⁵I-labeledUb-PESTcwasincubatedwithincreasingamountsofYUH1for30minat37°C.Thesampleswerethensubjectedtopolyacrylamidegelelectrophoresisinduplicateunderdenaturingconditions.Afterelectrophoresis,oneofthegelswasstainedwithCoomassieR-250.Fig.2AshowsthattheintensityofaproteinbandcorrespondingtothesizeofUbincreasesuponincubationwithincreasingamountsofYUH1.

Inthisgel,however,wecouldnotfindthebandcorrespondingtothePESTcpeptide,whichmighthavebeendiffusedoutduringthestaininganddestainingprocess.Therefore,theothergelwascoveredwithaSaranwrapanddirectlyexposedonanX-rayfilm.Upontheautoradiography,wewereabletodetectthebandofthePESTcpeptide,whoseintensityalsoincreaseduponincubationwithincreasingamountofYUH1.Inaddition,thisincreaseinthebandintensitywasapproximatelyproportionaltotheincreaseinthereleaseofacid-solubleradioactivity(datanotshown).FurThermore,theaminoacidsequenceoftheacid-solubleproductdeterminedbyEdmandegradationafterseparationfromundigested¹²⁵I-labeledUb-PESTcbygelfiltrationwasshowntobeidenticalwiththeN-terminalsequenceofPESTc(seeFig.8ofref.13).Thus,itisclearthattheacid-solubleradioactivityrepresentsPESTcreleasedfrom¹²⁵I-labeledPESTcbytheactionofYUH1.

Fig.2:[Enlarge]

SDS-polyacrylamidegelelectrophoresisoftheproductsgeneratedbyincubationofYUH1and125I-labeledUb-PESTc.Theradioiodinatedsubstrate(10μg)wasincubatedintheabsence(laneb)andpresenceof0.1μg(lanec),0.2μg(laned),and0.4μg(lanee)ofthepurifiedYUH1for30minat37°C.ThesampleswerethenelectrophoresedinduplicateonadiscontinuousgelscontainingSDSand2-mercaptoethanol.Afterelectrophoresis,oneofthegelswasstainedwithCoomassieR-250(A),andtheotherwasdirectlyexposedonanX-rayfilm(B).Laneacontains10μgofunlabeledUbasacontrol.

Surprisingly,however,thebandintensityofUbupontheautoradiographywasfarlowerthantheothers,despitethefactthatUbitselfhasasingletyrosineresidueatthe59thpositionthatcanalsobelabeledby¹²⁵I.Typically,chloramineTisusedforradioiodinationofUbmolecules.Inourstudies,weusedIODO-BEADS,inwhichchloramineTisimmobilizedonnon-porouspolystyrenebeads.Perhaps,thetyrosineresidueinUbisnotaccessibletoelectrophilicidodinespecies(i.e.,I⁺),whichareproducedbytheimmobilizedchloramineT,duetostructuralbarrier,unlikethatinPESTc,thatisfusedtotheflexibleC-terminalregionofUb.Inanyevent,wecouldobtain¹²⁵I-labeledUb-PESTc,inwhichPESTcwasalmostexclusivelyradioiodinated.ThisfortuitousfindingallowedustodeterminerapidlytheactivityofYUH1aswellasofotherDUBsandtoquantifypreciselycleavageproductsbysimplemeasurementoftheradioactivityreleasedintotheacid-solubleproducts.

Usingtheassaymethod,wehavepreviouslypurifiedseveralnovelDUBsfromchickskeletalmuscleandyeast,includingcUBP41(15),cUCH-1(16),cUCH-6(13),cUCH-8(17),andyUBP-6(18),usingconventionalchromatographicprocedures.TocomparetheactivitiesoftheseDUBsagainst¹²⁵I-Ub-PESTc,thesameamountofeachenzymewasincubatedwiththesubstrateforvariousperiods.Fig.3showsthatthehydrolyticratesdiffermarkedlyfromeachother.AmongtheDUBs,cUCH-6hydrolyzedUb-PESTcmostrapidly.TheK_mvaluesfortheenzymeswerealsodifferent,rangingfrom5to65μM(e.g.,5.1μMforcUCH-6and64.5μMforyUBP-6.)

Ubendswiththeaminoacidsequenceof-RLRGG⁷⁶.BasedontheC-terminalsequence,Steinandcoworkers(19)havesynthesizedavarietyoffluorogenicpeptidesbyconjugatingAMCtotheC-terminiofthepeptidesandusedthemassubstratesfordeterminationofthespecificityofisopeptidaseT,whichexistsinallmammaliancellsandhydrolyzestheisopeptidelinkagesofpoly-Ubchains(27).Ofthese,Cbz-LRGG-AMCwasusedinthepresentstudiestocompareitssensitivitytothepurifiedDUBstothatof¹²⁵I-Ub-PESTc.Table2showsthatalloftheDUBshaveatleastthreeorderhigherspecificactivitiesagainst¹²⁵I-Ub-PESTcthanthoseagainstCbz-LRGG-AMC,indicatingthattheassaymethodusing¹²⁵I-Ub-PESTcismuchmoresensitivefordeterminingtheactivitiesofDUBsthanthatusingthefluorogenicpeptidesubstrate.Inaddition,wehaverecentlyfoundthattheextractsofE.colibythemselvesarecapableofreleasingAMCfromthepeptidesubstrate(datanotshown),implyingthatE.colicontainsaprotease(s),whichspecificallycleavesoffAMCfromthepeptidesubstrate.Ontheotherhand,thesameextractscouldnothydrolyze¹²⁵I-Ub-PESTcatall.Thus,theprotease(s)islikelytointerferewiththeassayforoverproducedeukaryoticDUBsinE.colicells,whenCbz-LRGG-AMCwasusedasasubstrate.Furthermore,extractspreparedfrommostofeukaryoticcellsalsocontainaprotease(s)thatrapidlycleavesCbz-LRGG-AMCbutnot¹²⁵I-Ub-PESTc.Thus,theassaymethodusing¹²⁵I-Ub-PESTcshouldbeappropriateforidentificationofDUBsineukaryoticcellsandtheirclonesexpressedinE.coli.Infact,wewereabletoidentifyandpartiallypurifyatleast10differentDUBsfromtheextractofchickskeletalmuscle(13)andtopurifyseveralnovelchickDUBsclonedandexpressedinE.coli(15,28).

Fig.3:[Enlarge]

Hydrolysisof125I-labeledUb-PESTcbyvariousDUBs.Thesameamount(0.1μg)ofthepurifiedYUH1(opencircle),yUBP6(opentriangle),cUCH-1(solidtriangledown),cUCH-6(solidcircle),cUCH-8(solidtriangleup),orcUBP41(solidsquare)wasincubatedwith20μgof125I-Ub-PESTcat37°Cforvariousperiods.Afterincubation,thereleaseofPESTcwasdeterminedasdescribedunderMaterialsandMethods.

Recently,Steinandcoworkers(29)havedevelopedanewassaymethodforDUBsbasedonthesubstrateUbC-terminalAMC(Ub-AMC).Theyshowedthattherateconstants(kc/K_m)forthehydrolysisofUb-AMCare10⁴-and10⁷-foldoverthoseforthecleavageofCbz-LRGG-AMCforisopeptidaseTandUCH-L3respectively.UCH--L3isa26kDaUbC-terminalhydrolase(30)isolatedfromrabbitreticulocytes.However,ithasnotyetbeentestedwhetherUb-AMCissusceptibletoanyproteaseinbacterialoreukaryoticcellsotherthanDUBs.

WearegratefultoDr.MartinRechsteiner(UniversityofUtah)forprovidingE.colistrainAR13carryingpNMHUB-PESTc,forwhichUb-PESTcwaspurified.ThisworkwassupportedbygrantsfromKoreaScienceandEngineeringfoundationthroughResearchCenterforCellDifferentiation,LotteFoundation,andKoreaMiNISTryofEducation(BSRI-97-4415).

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