mechanism, specificity and structure of the · pdf filemechanism, specificity and structure....

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CHAPTER 6

MECHANISM, SPECIFICITY AND STRUCTURE OF THE DEUBIQUITINASES

David Komander*

Abstract: Removalofubiquitinfrommodifiedproteinsisanimportantprocesstoregulatethe ubiquitin system. Roughly 100 dedicated enzymes for this purpose, thedeubiquitinases,existinhumancellsandareintricatelyinvolvedinawidevarietyof cellular processes, although many enzymes remain unstudied to date. Thedeubiquitinases consist of five enzyme families that containUSP,OTU,UCH,Josephin,orJAMM/MPN+domainsprovidingcatalyticactivity.Wenowunderstandthecatalyticmechanismsofalldeubiquitinasefamiliesfromstructuralworkandmoreimportantly,haveobtainedinsightintoanunanticipatedvarietyofwaystoexercisespecificity.Itemergesthatdeubiquitinasesexploittheentirecomplexityoftheubiquitinsystembyrecognizingtheirsubstrates,particularubiquitinchainlinkagesandeventhepositionwithinaubiquitinchain.Thischapterdescribesthemechanismsofdeubiquitinationandthedifferentlayersofdeubiquitinasespecificity.The individual deubiquitinase families are discussedwith a focus on structure,regulationandspecificityfeaturesforselectedenzymes.

INTRODUCTION

Protein ubiquitination is emerging as one of the most important regulatoryposttranslationalmodifications.Mostprominentandwell‑studiedareitsrolesinproteindegradation,1however,recentyearshaveseenanexplosionofdataonnonproteolyticrolesofubiquitinationincellsignallingprocesses,intracellulartraffickingandtheDNAdamageresponse.2Theversatilitytomodulatesuchdiverseprocessesisachievedbytheabilityofubiquitintoformatleasteightdifferenttypesofpolymers(reviewedinrefs.3,4).Insuchubiquitinchains,isopeptidebondsareformedbetweentheubiquitinC‑terminusandone

*DavidKomander—MedicalResearchCouncilLaboratoryofMolecularBiology,ProteinandNucleicAcidChemistryDivision,HillsRoad,Cambridge,CB20QH,UK. Email:dk@mrc‑lmb.cam.ac.uk

Conjugation and Deconjugation of Ubiquitin Family Modifiers,editedbyMarcusGroettrup. ©2010LandesBioscienceandSpringerScience+BusinessMedia.

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70 CONJUGATION AND DECONJUGATION OF UBIQUITIN FAMILY MODIFIERS

ofsevenlysineresiduesofasecondubiquitin(Lys6,Lys11,Lys27,Lys29,Lys33,Lys48andLys63).Alternatively,alsotheN‑terminalaminogroupcanbeusedforubiquitinlinkages togenerate linearubiquitinchains.5The linkage typeof theubiquitinchaindetermineswhetheraubiquitinationeventwilltriggerproteasomaldegradation(mediatedbyLys48‑andLys11‑linkedchainsandpossiblyotherchaintypes)orsignallingprocessessuchasproteinkinaseactivation,orDNArepairpathways(mediatedbyLys63‑linkedandlinearchains).4

Likeotherposttranslationalmodifications,ubiquitinationisreversible.Thehumangenomeencodes∼98deubiquitinatingenzymes,alsoknownasdeubiquitinasesorDUBs,whichprovidedifferentfunctionalitiesandspecificitiestocarefullyregulateubiquitinationevents.These enzymes cluster in five structurally unrelated families:6,7 the ubiquitin specificproteases (USP,56 individualmembers inhumansplus11additionalgenesfromtheUSP17multigenefamily),8theOvarianTumor(OTU)DUBs(15members),theUbiquitinC‑terminalhydrolases(UCH,4members),theJosephindomainDUBs(4members)andtheJAB1/MPN/MOV34(JAMM/MPN+)DUBs(8members).6,7

An important roleofDUBs is themaintenanceofa freeubiquitinpool incells.UbiquitingenesproducepolyubiquitinprecursorproteinsandspecializedDUBssuchasUSP5/IsoTarerequiredtoprocesstheseprecursorsintomonoubiquitin.9Ubiquitinhasahalf‑lifeofseveraldaysincells,whichisachievedbyrecyclingofubiquitinfromdegradedsubstrates.TheproteasomeitselfharborsthreeDUBs(USP14,UCHL5andPOH1)thathydrolyzethechainspriortodegradingofthesubstrate,hencerecyclingubiquitinforfurtheruse.10TheserolesofDUBsinmaintainingastablepoolofmonoubiquitinareperformedbyahandfulofdedicatedenzymes.

ThemajorityofDUBshoweverdirectlyregulateproteinubiquitinationevents.Mostcommonly,ubiquitinationwillleadtoproteindegradationandhencedeubiquitinationhasastabilizingeffect,activelyincreasingproteinlevelsincells.Deubiquitinationcanalsoinhibitcellularsignallingcascadesthatareactivatedbynondegradativechainstypes.Asproteinhomeostasisaswellascellsignallingoftenrequirestighttemporalandspatialregulation,theDUBsaffectingthesepathwaysarealsoregulatedinmanydifferentways.Furthermore,DUBshavemaintainedremarkablespecificity,withregardtotheselectionofsubstrates,theirpreferenceforparticularchaintypesandeventheirpositioningonaubiquitinchain.

De‑regulationofdeubiquitinationcanleadtoimbalancesofproteinlevelsandhencetodisease.Forexample,thedegradationoftheoncogenec‑mycismediatedbyUSP28,whichretainsMYCinthenucleusandpreventsitfromenteringthenucleolus,whereitisdegraded.11ProliferationofsomecancercelllinesdependsonhighMYClevelsandknock‑downofUSP28inhibitsgrowthofthesecelllines,suggestinganoncogenicroleofUSP28.11However,USP28alsostabilizesseveralimportantmediatorsoftheDNAdamageresponse,includingChk2and53BP1,afterDNAdamagehasoccurred.12Hence,lossofUSP28attenuates the cellular response toDNAdamage, renderingUSP28alikelytumorsuppressorcandidate.Asimilarlycomplexexampleistheregulationofthep53tumorsuppressorbythedeubiquitinaseUSP7.USP7isthoughttodirectlystabilizep53levels,butinaddition,USP7alsostabilizesthelevelsofthep53‑destabilizingE3ubiquitinligase,MDM2incells.13,14ThesetwoexamplesillustratetheimportanceofDUBsinregulatingproteinstability.

Severalcell‑signallingDUBshavefurtherwell‑establishedlinkstocancer.Familialcylindromatosis,ararebenignskincanceraffectinghairfolliclesandsweatglandsofskinandneck,hasitsgeneticcauseintruncationofthecyldtumorsuppressorgene.15



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71MECHANISM, SPECIFICITY AND STRUCTURE OF THE DEUBIQUITINASES

TruncationablatesthefunctionoftheUSPdeubiquitinasedomainofCYLD.16‑19ThisdomainhasspecificityforLys63‑linkedandlinearpolyubiquitinchains19,20andhasbeenimplicatedinregulationofnondegradativesignallingpathwaysleadingtotheactivationoftheNF‑κBtranscriptionfactor.21CYLDalsohasrolesinnumerousotherLys63‑dependentprocessesandmayserveasageneralhousekeepingenzymeregulatingLys63ubiquitinlinkages.22OtherDUBsaffectingprimarilynondegradativeubiquitinationeventsaretheNF‑κBregulatorandtumorsuppressorA20,23,24theTRABIDenzymeinvolvedintheWnt/β‑cateninpathway25andOTUD5involvedintheinterferonresponsefactor(IRF)‑3signallingpathway.26

Hence,DUBshaveestablishedrolesincancer,butalsoininflammationandimmuneresponsesandinneurologicaldisorders.Thishasledtoanincreasinginteresttotargettheseenzymespharmacologically,forwhichadetailedmechanisticandstructuralinsightisessential.Thischapterprovidesanoverviewofthestructuralfeaturesofthedeubiquitinasesanddiscussestheirmechanismandcommonconceptsofspecificityandregulation.Forfurtherinformation,readersarereferredtorecentreviewsonthetopic.7,27,28

MECHANISMS OF DEUBIQUITINATION

DUBs are proteases that hydrolyze the isopeptide bond between the ubiquitinC‑terminusandtheLysε‑aminogroup.FourofthefivehumanDUBfamilies(USP,OTU,UCH,Josephin)areCysproteaseswhiletheJAMM/MPN+DUBsarezincdependentmetalloproteases.

Mechanism of Cys‑Dependent DUBs

TheCys‑dependentdeubiquitinasefamiliescompriseacatalyticdiadortriadandtheirmechanismissimilartothatoftheCysproteasepapain.29,30AcatalyticCysperformsanucleophilicattackontheisopeptidelinkageofaubiquitinatedLysresidue.ThisisfacilitatedbyanearbyHissidechainthatlowersthepKaoftheCys.Athirdresidue,usuallyAsporAsn,alignsandpolarizesthecatalyticHis.ThisisnotalwaysessentialandsomeenzymeslackthethirdresidueandpolarizetheHisbyothermeans.Thismechanismhas two additional features. A negatively charged transient reaction intermediate isstabilizedbyanoxyanionholeformednearbybyhydrogen‑donatingresidues.Amorestableacyl‑intermediateisformedwhenthecarboxyl‑groupiscovalentlyboundtotheenzyme,aftertheaminogrouphasbeenhydrolyzed.Thereactioncycleiscompletedbywater‑mediatedhydrolysisoftheacyl‑Cysintermediate.

ThemechanismofCys‑baseddeubiquitinaseshasbeenexploitedbythegenerationofmodifiedubiquitin‑derivedprobesthathavereactiveC‑termini.31,32 In thesimplestmolecule,ubiquitinaldehyde,theC‑terminalcarboxylgroupofGly76isexchangedtoanaldehydegroup,whichafterbindingtothecatalyticCys,isnothydrolyzedbywater.ThismoleculeactsasapotentandspecificinhibitorofCys‑dependentdeubiquitinases.33 Theseubiquitinprobeshavebeenimprovedsince34andseveralprobesarecommerciallyavailable,includingubiquitinvinyl‑sulfone(Ub‑VS)andubiquitinvinylmethylester(Ub‑VME).Ubiquitinprobeswereinstrumentalinidentifyingnoveldeubiquitinasesincells31andtoobtainthefirstubiquitin‑DUBcomplexesforstructuralcharacterisation.35‑37 However,differentDUBsdisplaydifferentaffinitiesforindividualprobes38andsomeenzymescannotbemodifiedbythesereagents.



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Mechanism of Metalloprotease DUBs

JAMM/MPN+familydeubiquitinasesarezinc‑dependentmetalloproteases.Withintheircatalyticsite,invariantHis,AspandSersidechainscoordinatetwozincions.39 The structureofthefirstJAMM/MPN+domainrevealedsimilaritiestocytidinedeaminase,suggesting that thesefamilieswereevolutionarilyrelated.40Thecatalyticmechanismwasproposedtobesimilarbetweenthesetwohydrolyticenzymes.Thezincioninthecatalyticsiteactivatesawatermoleculetoformahydroxideion,whichisabletoattackthecarboxylcarbonintheisopeptidelink.Thetransienttetrahedralintermediatecollapseswitheliminationoftheε‑aminogroupandreplacementoftheaminewithahydroxylgroupfromtheactivatedwatermolecule.AnearbyinvariantGluresidueactsbothasaprotonacceptoranddonorinthiscatalyticcycle.40ThesepredictionswererecentlysupportedbycrystalstructuresoftheAMSH‑LPJAMM/MPN+domaininisolationandboundtodiubiquitin(seebelow).41

CONSIDERATIONS FOR DEUBIQUITINASE SPECIFICITY

The98humanDUBsareadiversesuperfamilyofenzymes.Aswillbediscussedin detail below, the catalytic domains of the five DUB families share no sequencesimilarityandhavedistinctstructuralfolds.MostDUBshoweverhydrolyzeubiquitinchainsintomonoubiquitin.Hencetheycanbindtotwoubiquitinmoieties,placingtheisopeptidebondtobecleavedacrosstheiractivesite.Inthisarrangement,the‘distal’ubiquitinmoleculepresentsitsC‑terminalGlytothecatalyticcentre,whilethe‘proximal’ubiquitinisboundthroughitsmodifiedLys.AllDUBsanalyzedtodatebindubiquitinthroughasignificantdistalbindingsite,whiletheproximalubiquitinbindingsiteislessextensive.Thecatalyticcentre,boundtotheflexiblelinkerbetweenubiquitinmoieties,rigidifiesthelinkerregionbytightinteractions.WhilethesegeneralprinciplesholdtrueformostDUBs,subtledifferencesinubiquitinbindingcanchangeenzymaticpropertiessignificantlyandcontributetoDUBspecificity.

ItisimportanttocomprehendthecomplexityoftheubiquitinsysteminordertodiscussDUBspecificity.Incontrasttoothermodificationssuchasphosphorylationoracetylation,whereasinglemodifyinggroupisattached,ubiquitinationisfurtherorganizedbyitspolymericnature.Ubiquitinchainsaretheprincipaloutcomeofubiquitinationandhavedifferentstructuralandtopologicalfeatures.Bydealingwithubiquitinchains,DUBsfacemanyadditionallayerswheredecisionsregardingspecificityhavetobemade.Itisnotyetclearwhetherallthewaystoexercisespecificityareemployedinvivo,yetmanyobservationssuggestthatDUBsexploitthesystemtoitsfullpotential.ThefollowingsectionoutlinestheemergingconceptsinDUBspecificity.

Ubiquitin versus Ubiquitin‑Like Protein Cleavage

Ubiquitinisoneof17smallubiquitin‑like(UBL)modifiersinhumanswhichallcontainthecharacteristicubiquitinfold.42SeveralUBLs,includingSUMO,Nedd8,ISG15,FAT10andATG12modifyproteinsusingasimilarmechanismcomparedtoubiquitin.42,43 Theresultisatopologicallysimilarmodification(SUMO,Nedd8andAtg12areroughlythesamesizeandshapeasubiquitin,whileISG15andFAT10resemblediubiquitin)yet



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DUBsareabletodistinguishbetweenubiquitinandUBLs.ThekeytothisselectivityliespartlyintheC‑terminalfourresiduespreceedingtheGly‑Glymotif.SUMO,Atg12andFAT10sharenosequencesimilaritywithubiquitinwithintheseresidues.However,Nedd8hasasimilarsequenceandISG15hasanidenticalsequencecomparedtoubiquitin.ItisthereforenotsurprisingthatbothNedd8andISG15canalsobehydrolyzedbysomecross‑reactiveDUBs(seebelowforexamples).

Isopeptide versus Peptide Bond Cleavage

NotallubiquitinchainsarelinkedtoLysresiduesviaisopeptidebonds,butchainscanalsobelinkedthroughtheα‑aminogroupoftheN‑terminus(linearubiquitinchains).5 ThischaintypehasnonproteolyticrolesinNF‑κBsignaling44andlinearchainsarealsothesourceofmonoubiquitinincellsasubiquitinistranslatedfromlinearpolygenes.ThisrequiresDUBstodealwiththisparticularchaintypeandpeptidebonds.Duetostructuraldifferencesbetween the isopeptide (linked throughanelongated,flexiblesidechain)and thepeptidebond(bulkysidechainofMet1,Ramachandranrestraints),cleavageoflinearchainsrequiresamorespaciousactivesiteenvironment.20RecentdatashowsthatUSPenzymescancleavelinearchains,albeitwithloweractivity.MostotherDUBfamiliesdonothydrolyzethischaintype,althoughenzymesactingonlinearchainsmayexistwithinthesefamilies.20CleavageofpeptidebondsbyUSPsmayalsoallowthemtohydrolyzenon‑ubiquitinsequencesandwassuggestedtobeusedintheobservedUSP1autoproteolysiswithinitsUSPdomain.45

Linkage Specificity within a Ubiquitin Chain

ThemoststrikinglayerofDUBspecificityistheabilityofmanyenzymestoselectbetweendifferentubiquitinchainlinkages.20Importantly,chainlinkagespecificityisnotdeterminedbyDUBfamily.Thisisincontrasttoe.g.,phosphatasesthatutilizedifferentenzymefamiliesforremovalofphosphatesfromTyr,orSer/Thrresidues.46Forexample,OTUandUSPfamilyenzymeshaveevolvedLys48‑andLys63‑specificmembers.20 The JAMM/MPN+familyofDUBsmayhaveintrinsicspecificityforLys63‑linkedchains(seebelow).

Currently, however, only threeubiquitin chain types (Lys63‑,Lys48‑linked andlinear)areavailableforinvitrostudiesofDUBspecificity.Hencetheoverallpictureremainsincompleteandrequiresdevelopmentofnewandbetterreagentsandassays.AshighlyspecificDUBsexist,itispossiblethatevennewDUBfamiliesmaybediscoveredonceproperreagentsareavailable.

Exo‑ vs. Endo Activity within a Ubiquitin Chain

Polymersofubiquitincanbecleavedfromtheend(exo)orwithinachain(endo)andbothmechanismshavebeendescribed.19,47Thismechanisticdifferencehasprofoundconsequences.Anendo‑DUBwouldbeabletoremoveentirechainsfromsubstrates,reversingpolyubiquitinationmostefficiently.ItwouldhoweverresultinfreechainsandfurtherDUBaction(likelybydistinctenzymes)isrequiredtorecyclemonoubiquitinfromthereleasedchains.Incontrast,exo‑DUBactivityseemsinefficientifchainsarelong;suchactivitywouldberequiredthoughforrecycling,e.g.,proteasome‑bound,DUBs.



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Chain Cleavage versus Substrate Deubiquitination

Ubiquitinationcanoftenbedividedintotwoindependentsteps,chaininitiationandchainelongation.Onemechanisticreasonforthisisthatthesequencecontextofthe‘first’ubiquitinonasubstrateLysisdistinctfromthe(alwaysequivalent)ubiquitinsequenceusedforelongatingthechain.DUBsfacethesameproblem.SomeDUBsmayonly targetubiquitin‑ubiquitin linkages,but theiractionmightnotremovetheproximalubiquitin,leavingthesubstratemonoubiquitinated.Infact,itisoftennotclearwhatthephysiologicalendproductofadeubiquitinationreactionis.Ubiquitinchainediting,48i.e.,theswitchfromonechaintype(e.g.,a‘signalling’Lys63‑linkedchain)toanothertype(e.g.,‘degrading’Lys48‑linkedchain)maybenefitfromsubstratesnotfullydeubiquitinated.Insuchscenario,DUBactiononasubstrateleavesaplatform,i.e.,monoubiquitin,forsubsequentubiquitinationwithadifferentchaintype.EnzymesthatcombineDUBandE3ligaseactivityhavebeendescribed24,48andmanyDUBsinteractwithE3ligases.49

Sequence Specific Deubiquitination

Theremay beDUBs that act onmonoubiquitinated targets, e.g., those left bypriorchaindeubiquitination(seeabove).TheseDUBsmayspecificallyrecognizeaubiquitinatedsequencecontextintargetproteinsandhencehydrolyzemonoubiquitin,orevenentireubiquitinchainsenbloc.Thiswouldallowforagreatlevelofspecificity,yet such sequence specificDUBshavenot been formally describedyet.However,nonspecific DUBs such as USP family members, may be able to accommodate awiderrangeofsequences in theirproximalbindingsiteandhencemaycompletelydeubiquitinatesubstrates.

Substrate Recognition and Specificity

In order to function within a particular pathway, DUBs need to select theirsubstrate proteins.ManyDUBs contain additional protein interaction domains tofacilitate direct substrate interaction, yet also indirect means, e.g., by localizingDUBstospecificplacesinthecellmayaidsuchselectivity.7LocalisationofaDUBviaproteininteractiondomainsmayaffectotherlayersofspecificity,suchaslinkagepreference.FormationofaDUB‑substratecomplexwouldsignificantlyincreasethelocalconcentrationofparticularubiquitinlinkages,potentiallyoverridingtheintrinsiclinkagepreferenceoftheDUB.

THE FIVE HUMAN DUB FAMILIES

AsurgeofdatainthelastyearshasrevealedmanyaspectsofDUBbiologyandinparticularstructuralstudiesbyX‑raycrystallographyandNMRhaveyieldedimportantinsightsinDUBactivity,specificityandregulation.Inthefollowingsection,thefivehumanDUBfamiliesarediscussedindividuallyandrecurrentmechanismsofregulationandspecificityareoutlined.



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USP Domain DUBs

USPfamilyDUBscomprisethelargestandmostdiversefamilyofdeubiquitinasesin mammalian cells with 56 distinct members. Another 12 USP17 (also known asDUB3)‑likeUSPgenesexist.USPdomainDUBsareusuallylargeproteins(between350and3400aminoacids(Aa),averagesize∼1000Aa)withacorecatalyticdomainof ∼350Aa.Outsideof their catalytic core,USPenzymescomprisenumerousotherdomains, including protein interaction domains that facilitate substrate binding, ordomainsdeterminingsubcellularlocalization.OnlyUSP19,USP30andUSP48containpredictedtransmembraneregions.USP19isanchoredattheendoplasmicreticulum,50 whileUSP30islocalizedintheoutermembraneofmitochondria.51Inaddition,ubiquitinbindingdomains (UBDs) suchas zinc‑fingerubiquitin specificprotease (ZnFUBP),ubiquitininteractingmotifs(UIM)andubiquitinassociated(UBA)domainsarefoundinseveralenzymes.6,7Finally,ubiquitin‑like(UBL)domainsarefoundinatleast18USPdomainDUBs.52ThepresenceofUBLdomainsmightsuggestacommonautoregulatorymechanismthatremainsunstudiedtodate.

TheUSPdomainitselfconsistof threesub‑domains,Palm,ThumbandFingers,resemblingarighthand35(Fig.1A).ThecatalyticcentreliesattheinterfacebetweenPalmandThumb,whiletheFingersdomaingripthedistalubiquitin.DramaticconformationalchangesarepresentinUSPdomainsuponubiquitinbinding.35,47InUSP7,thecatalyticCysshiftsuponubiquitinbindingfromacatalyticallyunproductivepositiontoanactivepositionwhereitinteractswiththecatalyticHisresidue35(Fig.1B).Incontrast,thecatalyticmachineriesofUSP14andUSP8areproperlyalignedforcatalysisinabsenceofubiquitin,howeverubiquitin‑bindingsurfaceloopsblocktheubiquitinbindingsite47,53andtheseloopsundergoconformationalchangesuponubiquitinbindinginUSP14.47Furthermore,inUSP8,whichhassofaronlybeencrystallizedwithoutubiquitin,theFingersdomainistightenedinward,additionallyblockingtheubiquitinbindingsite(Fig.1C).53InactiveconformationsarenotaglobalfeatureofUSPs,astheCYLDUSPdomainwaspoisedforcatalysisanddidnotshowablockedactivesitecleft(Fig.1D).54

Most of the analyzed USP family enzymes are nonspecific and will cleave anychaintype,20yetsomemembersshowdistinctspecificities.USP14preferentiallycleavesLys48‑linkedubiquitinchains,47whileCYLDspecificallyhydrolyzesLys63‑linkedandlinearchains.20ThestructuresofUSP14andCYLDhavegiveninsightsintotheirmechanismofactionandspecificity.ThestructureoftheLys63‑specificenzymeCYLDhasrevealedthattheproximalubiquitinbindingsiteandinparticularanextendedloopinthisregion,contributetotheobservedlinkagespecificity(Fig.1D).19USPdomainscanhaveendo‑andexo‑activityagainstpolyubiquitinchains.TheFingers‑subdomainofUSP7andUSP14wrapsaroundthedistalubiquitin,restrictingaccesstoLys48andLys63(Fig.1A).ThisallowstheseUSPstobindtothedistalendofachainonlyandconsistently,USP14actsprimarilyasanexo‑DUB.47Incontrast,CYLDlackstheFingerssubdomain,allowingLys63(andlinear)chainstocontinuefromthedistalubiquitin(Fig.1D).HenceCYLDcaninteractwithaubiquitinchainatanypointincludingatinternalpositionsandhasendo‑activity.19 SeveralUSPdomainsarecross‑reactivewithotherUBLmodifiers.55,56TheseenzymesincludeUSP18andUSP13thatinteractwithISG15suicideprobes(ISG15‑vinylsulfone,similartoUbVS,seeabove)betterthanwithubiquitinprobesandseveralotherUSPdomainsthatbindtobothubiquitinandISG15probes.55EquivalentstudiesareimportantforotherUBLmodifierswithmoreelusiveroles.



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AnintriguingstructuralfeatureofUSPdomainsistheirdisruptedcatalyticdomain.ThecatalyticcoreofUSPdomainscomprises∼350residues,yetmorethanhalfofthehumanUSPshavecatalyticdomainsofmuch larger sizes (400‑850Aa)annotated.57 Thisisanartefactfromthebioinformaticannotation,whichdefinesUSPdomainsasthe regionbetween theN‑terminalCys‑boxandC‑terminalHis‑andAsp‑boxes thatcontaintheresiduesofthecatalytictriad.MoredetailedanalysisshowsthattheUSPdomaincorecanbesubdividedintosixconservedsequenceboxes,spanning∼350‑400residues,inallhumanUSPdomains.57Thefiveboundariesbetweenboxesarepointswherelargeinsertionsoccur.Theseinsertedsequencescontainadditionalindependently

Figure 1. Structures of USP domain deubiquitinases. A) Structure of USP7 (also known as HAUSP)bound to ubiquitin (pdb‑id 1nbf).35 The USP domain (white) is shown in cartoon representation andthecatalyticcentreresiduesareshownasstickmodels ingreycolors.Ubiquitin isshownunderagreysemitransparent surface.Hydrogenbonds are indicatedbydotted lines.TheFingers,PalmandThumbdomains are indicated.B)Close‑up view of the active site ofUSP7 bound to ubiquitin. The catalytictriadresiduesandtheir interactionsareshown.C)StructureofUSP8(pdb‑id2gfo).53In theabsenceofubiquitin,theFingerssubdomainisclosertotheThumb/Palmpreventingubiquitinbinding.TheFingerssubdomains of 45 out of 56 USP domain DUBs including USP8 comprise a functional zinc‑bindingsite (zinc indicatedasagreysphere).57D)StructureofCYLD(pdb‑id2vhf).19CYLDdoesnotcontaina Fingers subdomain, allowing it to act as an endo‑deubiquitinase against Lys63‑linked and linearchains. A specificity determining loop near the active site disfavors Lys48‑chain binding. TheCYLDUSPdomains contains a zinc‑bindingB‑box domain inserted in its sequence.



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foldeddomains,includingproteininteractiondomains(e.g.,B‑boxinCYLD(Fig.1D)19 andMYNDdomaininUSP19)50andubiquitinbindingdomains(e.g.,UBAdomainsinUSP5,58orUIMmotifsinUSP37).57SevenUSPscontainubiquitin‑likefoldsasaninsertion.52,57Althoughnotyetbackedupbystructuralwork, theUBLinsertionsarelikelypositionednearthedistalubiquitinbindingsite,wheretheymaydirectlyalterUSPfunction.52StructuresofUSPdomainscontaininganinsertionwilllikelyyieldinterestinginsightsregardingregulationoftheseenzymes.

FurtherregulationofUSPdomainDUBsisprovidedbyinteractingproteins,andmorethan770DUBinteractingproteinshaverecentlybeenrevealed.49ManyUSPfamilymembers interactwithWD40 repeat containing proteins. TheWD40 proteinUAF1(USP1associatedfactor,alsoknownasWDR48)wasshownpreviouslytointeractwithUSP1,USP12andUSP46andmore importantly toallostericallyactivate theseUSPenzymes.59,60AnothercommonlyobservedinteractionexistsbetweenDUBs(notonlyUSPdomains,butalsootherclasses)andE3ubiquitinligases.7,49DUBactivitymaypreventautoubiquitination,acommonfeatureofE3ligases,oralternatively,E3ligasesmightdown‑regulateDUBs.Thisyetagainillustratesintricateinterplaybetweenubiquitinationanddeubiquitination.

Todate,mostUSPdomaincontainingenzymesremainpoorlycharacterizedandvirtuallynoliteratureexistsformorethan25%oftheUSPproteins.Thisislikelytochangewithnewgenomewidescreens,whichhaveprovenhighlysuccessfulinidentifyingnewDUBfunctions(seeref.61foranexample).Still,biochemicalcharacterisationisimportanttounderstandmoreaboutthisenzymefamily.

OTU Domain DUBs

Humancellscontain15OTUdomainDUBs,onlyhalfofwhichhavebeenstudiedtodate.SeveralOTUenzymesareinvolvedincellsignallingprocesses,regulatingNF‑κBsignalling (A20, Cezanne1/2),24,62 Wnt signalling (TRABID)25 and IRF3 signalling(OTUD5,alsoknownasDUBA).26OtherOTUmembershavemoreelusiveroles.OTUfamilyproteins range in size from230Aa to1222Aaand likeUSPdomains,oftencontainadditionaldomainswithlinkstotheubiquitinsystem,includingUIMandUBAdomainsandUBLfolds.7

ThestructureoftheOTUdomaindoesnotresemblethatofUSPdomains,yetthecatalyticresiduesoftheactiveenzymessuperposewell(Fig.2A,B).54TheOTUdomaincorecomprises∼150‑200residues,63however,asubclassofenzymes,(A20,Cezanne1/2,TRABID,VCIP135)containanextendedcatalyticcoreof∼360residues(Fig.2D54 and D.K.,unpublished).LikesomeUSPdomains,thedistalbindingsiteofOTUdomainsundergoesadisorder‑to‑ordertransitionuponubiquitinbinding.37Atleastinonecase(OTUB1),38theactivesiteisinanunproductiveconfigurationandrequiresconformationalchangespriortoactivation(Fig.2C).ThecatalyticallyinactiverestingstatefoundinmanyDUBs,notonlyOTUmembers,mayprotectthecatalyticCysresiduefromoxidativestress.AlowpKaCysresidueintheactivesitewouldbeattackedbyreactiveoxygenspecies(ROS)andithasbeensuggestedthathighlevelsofROSaffectthefunctionoftheOTUDUBCezanne.64ROSmayalsoregulateotherdeubiquitinaseclasses.

OTU family enzymes displaymarked chain linkage specificity. TRABID andDUBA are Lys63‑specific,20,26 while OTUB1 is Lys48‑specific.38 The A20 OTUdomain is Lys48‑specific in vitro,54,65 yet the substrates of A20 aremodifiedwithLys63‑linkedchains.A20was shown to actonLys63‑polyubiquitinated substrates



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suchasTRAF6,releasingwholechainsfromtheproteins,potentiallybycleavingtheproximalubiquitin.65MostOTUdomainsdonotcleavelinearchainsefficientlyandhencemaybestrictisopeptidases,20however,OTUB1wassuggestedtocleavebothubiquitinandNedd8conjugates.38

UCH Domain DUBs

TheUCHfamilyofdeubiquitinasescontainsfourmembers,twoofwhichconsistofonlyacatalyticdomain(UCHL1andUCHL3,∼200Aa).6,7UCHL1andUCHL3haverolesinbrainfunction66‑68andtheIle93MetpointmutantofUCHL1isassociatedwithfamilialParkinson’sdisease.69Athirdmember,UCHL5(alsoknownasUCH37)contains

Figure 2. Structures ofOTUdomain deubiquitinases.A)Structure ofOTU1bound to ubiquitin (pdb‑id3by4).37TheOTUdomain(white)isshownincartoonrepresentationandthecatalyticcentreresiduesareshownasstickmodelsingreycolors.Ubiquitinisshownunderagreysemitransparentsurface.Hydrogenbonds are indicated by dotted lines. B) Close‑up view of the active site of OTU1 bound to ubiquitin.Thecatalytictriadresiduesandtheirinteractionsareshown.C)StructureofOTUB1(pdb‑id2zfy).38 The Otubains (OTUB1 andOTUB2) contain several additional helices. D) Structure of A20 (pdb‑id 2vfj).54 TheA20 catalytic domain is∼150 residues longer and contains additional structural elements.



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a 100Aa extensionwhich is essential to bind to the proteasome subunitRpn13.70‑72 Proteasome‑boundUCHL5 is oneof threeDUBs that recycle ubiquitin chains fromproteasomesubstrates.10ThefourthhumanUCHenzyme,BAP1(BRCA1associatedprotein‑1),containsaC‑terminalextensionof>500Aa.BAP1isatumorsuppressorandinteractswiththeBRCA1/BARD1E3ubiquitinligaseinvolvedinDNArepair,yetitsrolesintheDNAdamageresponsearedebated.73‑75RecentdatashowsthatBAP1alsointeractswiththecellcycleregulatorhostcellfactor‑1(HCF1).76,77HumanNCI‑H226squamouslungcarcinomacellsharboradeletionofBAP1andoverexpressionofBAP1inthiscelllineblockstheirproliferationandtumorgrowthinmice.77

Structures ofUCHdomain reiterate commonprinciples ofDUB regulation andspecificity. The catalytic residues in ubiquitin‑free UCHL1 are in a nonproductiveconformation78andneedtoundergoaconformationalchangeuponbindingtoubiquitin.IntheactiveubiquitinboundconformationofUCHL3(Fig.3A)orYuh1(thesingleyeastUCHenzyme),thecatalytictriadresiduessuperposewellwithotherDUBclassesandseveralloopsareremodelleduponubiquitinbinding(Fig.3B).36,79Themoststriking

Figure 3. Structure of a UCH domain deubiquitinase. A) Structure of UCHL3 bound to ubiquitin(pdb‑id 1xd3).79 TheUCH domain (white) is shown in cartoon representation and the catalytic centreresidues are shown as stick models in grey colors. Ubiquitin is shown under a grey semitransparentsurface. Hydrogen bonds are indicated by dotted lines. The active site crossover loop forming acrosstheubiquitinC‑terminus at the active site is indicated.B)Close‑upviewof the active site ofUCHL3boundtoubiquitin.Thecatalytictriadresiduesandtheirinteractionsareshown.C)StructureofUCHL3in the apo formwithout ubiquitin (pdb‑id 1uch).30 The active site crossover loop is disordered.



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featureofUCHenzymesisalargesurfaceloop,theactivesitecrossoverloop,whichformsuponubiquitinbinding(Fig.3A,C).36,79TheubiquitinC‑terminushastothreadthroughthisloopinordertoreachtheactivesite.Thisposesasignificantstericconstraintanddoesnotallowbindingoffoldedubiquitinatedproteinsofmorethanapproximately10Åindiameter.Thisstructuralfeatureexcludesubiquitinchains,whichwouldbetoobigtoenterthroughthecrossoverloop.Indeed,UCHenzymeshavenegligibleactivityagainstubiquitinpolymersofanylinkagetypeinvitro.20,80Onlysignificantextensionofthecrossoverloopallowspolyubiquitincleavage.80Hence,UCHenzymeswiththeirrestrictedaccessibilitytotheactivesite,canactonubiquitinationsitesinunfoldedregionsofproteins(andmaybeperformchainamputation)andonubiquitin‑peptideconjugateswhichmaybeaby‑productofproteasomaldegradation.Interestingly,proteasome‑boundUCHL5canactagainstpolyubiquitinchains,despiteapredictedanalogousactive‑sitecrossoverloop.81Hence,eitherproteasomeinteractioninducesaconformationalchangeinUCHL5toremodeltheobstructingloop,ortheproteasomeunfoldsubiquitinpolymerssignificantlysotheycanenterthroughthecross‑overloop.UCHL3butnotUCHL1isinhibitedbydiubiquitin82andUCHL5alsodoesnothydrolyzediubiquitinefficiently.70 Themolecularbasisforthisinhibitionisnotclearatthemoment.

Josephin Domain DUBs

Four human DUBs contain a catalytic Josephin domain, which was identifiedbybioinformatics83 and subsequentlyvalidated tobe catalytically active.84ThemostprominentmemberofJosephinDUBsisAtaxin‑3.Ataxin‑3istheproteinmutatedinMachado‑Joseph disease (MJD, SCA3), the most common form of spinocerebellarataxias.85Ataxin‑3 contains a stretchofGln residues (polyQ),which is significantlyextendedinthediseasestateastheconsequenceofamplificationofanunstableCAGtripletrepeat.TheresultingpolyQstretchleadstoproteinaggregationintheformofintracellularinclusionbodies.86

Josephindomainshavebeenstudiedbynuclearmagneticresonance(NMR)techniquesand currently several inactive structures are available,where the catalytic triad is innonproductiveconformations(Fig.4).87‑90ThekeyfeatureofJosephindomainsisalargehelicalleverthatrestrictsaccesstotheactivesiteinabsenceofubiquitin(Fig.4A,C,D).87,88NMR‑baseddockinganalyzesofdiubiquitinontoAtaxin‑3suggestthatubiquitinbindingstabilizesanactiveconformationofAtaxin‑3.90Interestingly,Ataxin‑3catalyticactivityisactivatedbyubiquitinationoftheJosephindomainitselfbyanunknownE3ligase.91Itistemptingtospeculatethatubiquitinationstabilizesthehelicalleverinanopenconformation.

Ataxin‑3containsthreeUIMmotifsinitsC‑terminalpart.92ThetwoJosephin‑proximalUIMswererecentlyshowntopreferentiallyinteractwithLys48‑linkedubiquitinchains,93 however,Ataxin‑3wasalsosuggestedtoeditLys63‑linkagesinmixedlinkagechains.94 ThesubstratesofAtaxin‑3andtherolesoftheremainingJosephindomainproteinsarecurrentlyunclear.

JAMM/MPN+ Domain DUBs

Eight humanDUBs contain a JAMM/MPN+metalloprotease domain and theseproteinsoftenoperateaspartofmulti‑subunitproteincomplexes.AJAMM/MPN+DUBintheproteasome,POH1,contributestorecyclingubiquitinchains,10whileAMSHand



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AMSH‑LPareassociatedwiththeESCRTmachineryandareinvolvedinmembranereceptortrafficking.95BRCC3hasbeenfoundintwoDNArepaircomplexes,theBRISCcomplexandtheBRCA1Acomplex.96‑99CSN5isacomponentoftheCOP9signalosomeandactsasdeneddylatingenzymetoremovetheactivatingNedd8modificationfromCullinE3ligases.100MYSM1ispartofahistonedeubiquitinasecomplex.101PRPF8,asplicingfactor,containsanimpairedmetalbindingsiteandhencemayhavelostDUBactivity.102Theremainingenzyme,MPNDhasnotbeenstudiedtodate.

Most JAMM/MPN+ DUBs cleave Lys63 ubiquitin chains and some (AMSH,AMSH‑LP,BRCC3)withexquisitespecificity.96,103Themolecularbasisforthislinkage

Figure 4. Structure of a Josephin domain deubiquitinase.A)Structure ofAtaxin‑3 bound to ubiquitin(pdb‑id1jri).90TheJosephindomain(white) isshownincartoonrepresentationandthecatalyticcentreresidues are shown as stick models in grey colors. Ubiquitin is shown under a grey semitransparentsurface.Thehelicalleverregulatingaccesstotheactivesiteislabelled.B)Close‑upviewoftheactivesite of Ataxin‑3. The invariant catalytic residues have been verified by mutagenesis, but are in anunproductiveconformationinallstructures.C,D)StructureofAtaxin‑3inabsenceofubiquitin(pdb‑id2aga,)87 and in presence of diubiquitin (2jri).90 SeveralNMRmodels have indicated highflexibility ofthe helical lever that moves between closed (C) and open (D) conformations. Diubiquitin has beenomitted fromD for clarity.



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specificitywasrevealedinthecrystalstructureofAMSH‑LPboundtoLys63‑linkeddiubiquitin(Fig.5).41ApartfromrepresentingthefirstDUBstructurewithasubstratechainboundacross the active site, this structure alsogave important insights intoLys63 specificity of DUBs. Lys63‑linked polyubiquitin chains show an extendedconformation20andAMSH‑LPexploitsthis,bystretchingtheLys63‑linkagemaximally(Fig.5A,C).41Thelinkerresiduesarecontactedbytheproteinandfurthermore,thesequencecontextoftheLys63residue,Gln62andGlu64,arespecificallycontacted

Figure 5. Structure of a JAMM/MPN+ domain deubiquitinase. A) Structure of AMSH‑LP boundto Lys63‑linked diubiquitin (pdb‑id 2znv).41 The JAMM/MPN+ domain (white) is shown in cartoonrepresentation and the catalytic centre residues are shown as stickmodels in grey and zinc ions asgreyspheres.TheLys63‑linkeddiubiquitinisshownunderasemitransparentsurfaceandbindsacrossthe active site. The complexwas obtained by disrupting the primary zinc binding site andmutationof the catalyticGlu residue.B) Structure of theAMSH‑LP JAMM/MPN+ domainwithout ubiquitin(pdb‑id 2znv).41 The enzyme is in an active configuration with two zinc ions. C) Catalytic centreof the AMSH‑LP enzyme. The active zinc‑bound form is superposed onto the ubiquitin complex.The catalytic residues and their interactions are shown. Also the Lys63‑adjacent residues Gln62andGlu64 are shown in the proximal ubiquitin,whichmake specificity‑determining contacts to theAMSH‑LP protein.



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by theAMSH‑LPJAMM/MPN+ core (Fig.5C).41Hencesimilarly toCYLD,19 the proximalubiquitincontainingtheLysresidueofthelinkageplaysanimportantpartindeterminingthelinkagespecificityoftheDUB.ThemoleculardetailsforNedd8cleavage by the CSN5 JAMM/MPN+ domain, or for the activity of POH1 in theproteasomearelessclear.

CONCLUSION AND FUTURE PERSPECTIVES

Proteindeubiquitinationisbeingrecognizedasakeyinstrumenttounderstandthecomplexubiquitinsystem.ThesystematicanalysisofDUBinvolvementinbiologicalprocesses, facilitated by powerful siRNA screeningmethods16,26,61 and by the recentcomprehensiveanalysisofDUBinteractingproteins,49alloweddeepinsightsintoubiquitinmediated regulatory cascades. The prevalent idea that ubiquitination is primarily adegradationsignalhasbeenchallengedbyidentificationofDUBssuchasTRABID25 and DUBA,26whicharespecificfornondegradativeLys63‑chains.ThischaintypewasnotknowntobeinvolvedinthepathwaysregulatedbytheseDUBs(Wnt‑andIRFsignalling,respectively)openingnewavenuesforunderstandingof,butalsoforinterferingwith,thesepathways.

Chainlinkagespecificitywillbeahottopicintheyearstocome,astheabundanceof atypical chain types has just been realized through powerful developments inproteomics.104,105However,inordertogainfurtherinsight,noveltoolshavetobedeveloped.Mostimportantly,chainsynthesisoftheremainingchaintypeshastobeachieved.DUBswillundoubtedlyplayamajorroletounraveltherolesofnovelubiquitinmodificationandfurthermore,thespecificmembershavegreatpotentialtobecomeimportanttoolsinubiquitinresearch.

Despite much progress to understand the deubiquitinases at a structural level,moreworkliesahead.ThekeytounderstandingDUBspecificityis toobtainfurtherstructuresofDUBsboundtoubiquitinpolymersofdifferentlinkages.AlsotherecentidentificationsofallostericDUBactivatorsrequirefurtherstudies.MostDUBsarepoorenzymesandhydrolyzeubiquitinpolymerswithslowkinetics.Thereasonsforthismaybenon‑idealsubstrates,orgeneralallostericmechanismsregulatingDUBactivitythathavenotbeenuncovered.WithmoreDUBstructuresavailable,thesubtledifferenceswillbecomeapparent.

NumerousDUBshavetightlinkswithhumandisease.Asproteaseswereinthefocusofpharmaceuticalinterventionforalongtime,itissurprisingthattherehasbeenrelativelylittleprogressonthedevelopmentofDUBinhibitors(forarecentreviewseeref.32).ThepotentialofDUBsasdrugtargetsisbeingrealized,butrequirescarefulbiochemicalandgeneticanalysis,aswellasbetterassaytechnologies.106Thisareaofresearchpromisestoyieldexcitingandinterestinginsightsintheyearstocome.

ACKNOWLEDGEMENTS

IwouldliketothankmembersofmygroupfortheircontributionsandSonjaFlottandYogeshKulathuforcriticalcommentsonthemanuscript.WorkinmylabisfundedbytheMedicalResearchCouncil.



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