The immunoproteasome in antigen processing and other immunological functions

The immunoproteasome in antigen processing and other immunological functions

Michael Basler

, Christopher J Kirk

and Marcus Groettrup

Treatmentofcellswithinterferon-gleadstothereplacementof theconstitutivecatalyticproteasomesubunitsb1,b2,andb5 bytheinduciblesubunitsLMP2(b1i),MECL-1(b2i),andLMP7 (b5i),respectively,buildingtheso-calledimmunoproteasome.

Theincorporationofthesesubunitsisrequiredforthe productionofnumerousMHCclass-IrestrictedTcellepitopes.

Recently,newevidenceforaninvolvementofthe

immunoproteasomeinotherfacetsoftheimmuneresponse emerged.Investigationsofautoimmunediseasesinanimal modelsandageneticpredispositionofb5iinhuman autoimmunedisorderssuggestacrucialfunctionofthe immunoproteasomeinproinflammatorydiseases.Therecent elucidationofthehigh-resolutionstructureofthe

immunoproteasomewillfacilitatethedesignof

immunoproteasomeselectiveinhibitorsforpharmacological intervention.

Addresses

1DivisionofImmunology,DepartmentofBiology,UniversityofKonstanz, Universita¨tsstrasse10,D-78457Konstanz,Germany

2BiotechnologyInstituteThurgau(BITg)attheUniversityofKonstanz, CH-8280Kreuzlingen,Switzerland

3OnyxPharmaceuticals,SouthSanFrancisco,CA94080,USA

Introduction

The20Sproteasomeisalargeintracellularmulticatalytic proteaseconsistingofaandbsubunitsthatbuildabarrel- shapedcomplex of four ringswith seven subunits each [1,2]. In cells of hematopoietic origin, or during an immuneresponse inthecontextofinterferon-g(IFN-g) ortumornecrosisfactor-a(TNF-a)stimulation,thethree catalyticallyactivebsubunits(b1,b2,andb5)arereplaced bytheinduciblecatalyticsubunitsLMP2(b1i),MECL-1 (b2i),andLMP7 (b5i) duringproteasome neosynthesis.

Theimmunologicalbenefitoftheresulting‘immunopro- teasome’ isattributedto structural changes in substrate bindingpockets[2]andanalteredcleavagepatternofthe multicatalytic complex, thus optimizing quality and

quantity of the generated peptides for presentation on MHC class Imolecules[3–6]. Becauseof a pivotalrole inclass-Iligandgeneration,theimmunoproteasomeshapes thenaı¨veCD8-T-cellrepertoireinthethymusandcyto- toxic T-cell responses in the periphery [7,8,9,10–12].

Recently,novelfunctionsofimmunoproteasomesinauto- immunediseases,virusinducedneuroinflammation,Tcell expansion, T helper cell differentiation, and cytokine productionhavebeenproposed[13,14–16].Inthisreview wediscussthelatestfindingsontheimmunoproteasomein antigenprocessingaswellasthesenovelfunctions.

Theimmunoproteasomeinantigenprocessing

The major histocompatibility complex (MHC) class-I restrictedpathwayofantigenprocessingallowsthepres- entation ofintracellularantigenstocytotoxicTlympho- cytes.The main proteaseinvolvedinthisprocessisthe proteasome [17–19]. It is generally assumed that the immunoproteasome improves quality and quantity of generatedclass-Iligands.Indeed,aproteomicanalysisof MHC-Iassociatedpeptidesderivedfromwildtype(WT) andb2i^//b5i^/-doubledeficientmousedendriticcells (DC)demonstratedthatimmunoproteasomesdramatically increasetheabundanceanddiversityofclass-Iligands[20].

The recentlysolvedcrystalstructuresoftheconstitutive proteasomeandimmunoproteasomeofthemouseat2.9A˚ providesanexplanationforenhancedantigenprocessing byimmunoproteasomes[2](Table1).Theb1isubstrate- binding channel islinedwith hydrophobicamino acids, whichenhancestheproductionofMHC-Iepitopesending withsmall,nonpolarresidues.Theb5i-mediatedpeptide bond hydrolysis might be kinetically favored by an increasedhydrophilicityoftheactivesiteandadditional hydrogenbondsshapingtheoxyanionhole.Fromastruc- turalpointofview,theexchangeofb2/b2iisnotobvious, anditisquiteanenigmawhy,nevertheless,b2ideficient miceareprotectedfromexperimentalcolitis[14]andwhy b2iinfluenceshomeostaticproliferation[21].

AnalysisoftheTcellresponseinmurinecytomegalovirus (MCMV)infectedb5i-deficient micerevealed a critical role for immunoproteasomes [22]. Interestingly, all MCMV-derived CD8⁺ T cell epitopes tested were affected by the loss of b5i, suggesting that the virus has evolved a primary sequence poorly processed by constitutiveproteasomes.Theauthorshypothesizedthat DCscontainingbothimmunoproteasomesandconstitu- tive proteasomeselicittheacuteMCMV-specificT cell response,whereasthechronicMCMVinfectionismain- tained in cells expressing constitutive proteasomes.

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-219861

Hence, with the help of expression of immunoprotea- some-dependentepitopes, MCMV mayevade immune recognitionleadingtoviralpersistence.

The structural properties rather than the proteolytic activity of immunoproteasome subunits are needed for thegenerationofsome epitopes[23,24],buttheunder- lying mechanisms have remained elusive. In a recent study,thepresentationofthemaleHYAg-derivedepi- topeUTY_246–254andtheinfluenzavirusmatrixM158–66 epitopewereanalyzed, whichboth weredependent on thestructureofb1iorb5i,respectively,butnotontheir catalyticactivity[25].Withdifferentproteasomeinhibi- torsitwasshownthatb5iprotectsmatrixM158–66from cleavagebyb5andb1iprotectsUTY_246–254fromcleavage byb1,proposinganovelmechanisticbasisforthefunc- tionofimmunoproteasomesubunits (Figure 1).

Using newly developed immunoproteasome subunit- specificantibodies,Guillaume etal.isolatedandcharac- terized human 20S proteasomes that are intermediate between the standard and the immunoproteasome [26]. Rather than jointly incorporating b1i, b2i, and b5iinto immunoproteasomes,intermediateproteasomes incorporateonlyone(b5i)ortwo(b2iandb5i)immuno- proteasomesubunits.Theexistenceofintermediatepro- teasomes is consistent with the rules of cooperative assembly of immunoproteasome subunits [27–29].

Dependingon the investigated organ, the intermediate

proteasomesrepresentbetween30%and50%ofthetotal proteasomecontent.Notunexpectedly,theintermediate proteasomes have different cleavage properties in the generationofclassIpeptides[26,30].Theexistenceof 4differenttypesofproteasomeswithincellsbroadensthe MHC-I-presented peptidome. It is conceivable that an asymmetrichybridproteasome,consistingofimmunopro- teasome and constitutive proteasome, exists. Neverthe- less,Guilllaume et al. didnot find asymmetrical b5/b5i proteasomesinmelanomacellsandkidneysamples[26].

Micedeficientforoneortwoimmunoproteasomecatalytic subunitshaverelativelymodestchangesinantigenpres- entation(summarizedin[10]).Toinvestigatetheantiviral immuneresponseinmicedevoid ofimmunoproteasome activity, we analyzed the lymphocytic choriomeningitis virus specific T cell response in b1i^//b2i^/ double- deficient mice treated with the b5i-selective inhibitor ONX0914togeneratemicedevoidofimmunoproteasome activity[11].Micedevoidofimmunoproteasomeactivity couldmountastrongCTL-response,althoughtheTcell responsetosomeepitopeswasslightlyalteredcomparedto WTmice. Interestingly,b1i andb2i areneededforthe generation of the lymphocytic choriomeningitis virus (LCMV)-derivedepitopeNP_205–212,whereasb5idestroys NP_205–212 inb1i/b2i deficientcells.Amorepronounced phenotype with respect to antigen presentation was observed in genetically engineered mice completely lacking immunoproteasome subunits [31]. Similar to

Table1

Immunoproteasomesubunit-inducedalterationsinthe20Sproteasome(informationderivedfromRef.[2]) Immunoproteasome Alteration–immunovs.

constitutivesubunit

Alterationinsubstratebindingchannel Consequences

MECL-1(b2i) D53E Identicalsubstratebindingchannel. Therationalefortheincorporationof subunitb2iintothe

immunoproteasomeremainselusive.

LMP2(b1i) T20V,T31F,R45L,andT52A IncreaseinhydrophobicityofS1pocket.

DiminishesS1pocketinsize.

CD8⁺Tcellepitopeswithnon-polarC- terminisuchasIle,Leu,orValare produced.Theseepitopesarebetter suitedforpresentationonMHC-I molecules.Peptidebondhydrolysis preferentiallyoccursaftersmall, hydrophobic,andbranchedresidues.

T22A,andA27VinLMP2;

Y114Hinb2i

DecreasesizeandincreasepolarityofS3 pocket.

AlteredaminoacidpreferenceatP3.

LMP7(b5i) Ala20,Met45,Ala49,and Cys52inS1pocketare unchanged.

HydrophobiccharacterofS1pocketis maintained.

Bothb5andb5iareresponsibleforthe chymotrypsin-likeactivityofthe proteasome.

Gly48SerorCys ShallowS2pocket. LimitssizeofP2aminoacids.

Ala27Ser RestrictssizeofS3pocketandendowsit withamorehydrophiliccharacter.

LimitsP3aminoacidstosmall hydrophilicaminoacids.

Distinctconformation ofMet45

ResultsinspaciousS1pocketinb5i. b5icanaccommodatelargeramino acidsinS1comparedtob5.

A46S,V127T Increasethehydrophilicitysurrounding theactivesitenucleophilicThr1Ogofb5i.

Elevatedpolaritymightfavorpeptide bondhydrolysis.

SerOg,Thr127Og,and Gly47NH

Builduniquehydrogennetwork. Stabilizationofthetetrahedral transitionstateduringcatalysis.

b5i-deficientmice[32],MHC-Isurfaceexpressionintriply deficientmicewasreducedbyapprox.50%.Presentation ofnumerousCD8⁺Tcellepitopesderivedfromdifferent antigenswas markedlychanged intriple-deficientmice.

Interestingly,mostinvestigatedepitopeswerepoorlypre- sented in cells completely lacking immunoproteasome subunits,except forthe LCMV-derived epitope GP_276–

286,whichelicitedasignificantlyincreasedCTL-response in LCMV-infected triple-deficient mice. An increased presentationofthisTcellepitopewasalreadypreviously observed in b1i and b5i single-deficient mice [7,33], whereas b2i-deficient mice demonstrated an increased GP_276–286-CTL-responseowingtoalterationsintheTcell repertoire [8]. Mass spectrometric analysis of MHC-I boundpeptidesonsplenocytesderivedfromb1i/b2i/b5i triple-deficientorWTmicerevealedmarkedchangesin theMHC-Ipeptiderepertoire[31].Approx.1/3 ofthe detectedpeptideswereuniquelypresentedonWTcells,

1/3uniquelyontriple-deficientcells,and1/3waspresented on both cell types. Interestingly, triple-deficient mice rejectedadoptivelytransferredWTsplenocytes,whereas adoptivelytransferredimmunoproteasome-deficientcells weretoleratedinWTmice.Asimilarobservationwasmade withWTskingraftedontob5i^/mice,butnotviceversa [4].Whytheimmunoproteasome-deficienttransplantsare notrejectedfromWTmice,althoughtheypresentapprox.

1/3 unique MHC-I peptides, has remained elusive and needsfurtherinvestigation.

Otherimmunologicalfunctionsofthe immunoproteasome

In recentyearsit became apparentthatimmunoprotea- somesdo notonly function tochange theprocessing of MHC-Iligands,butalsopossessadditionalimmunological functions. Aninvolvementoftheimmunoproteasome in NF-kB activation has remained controversial [34–37].

Figure1

constitutive proteasome

proteasome cleavage

no presentation due to epitope destruction

presentation on MHC-I

TCD8+

proteasome cleavage

protein containing CD8⁺ T cell epitope CD8⁺ T cell epitope

MHC-I T cell receptor

proteasome cleavage immunoproteasome

Current Opinion in Immunology

TheimmunoproteasomeprotectsaCD8⁺Tcellepitope.AproteincontainingaCD8⁺Tcellepitope(inred)isdestroyedbytheconstitutive proteasome.TheinductionoftheimmunoproteasomesubunitsandthereplacementoftheircorrespondingconstitutivesubunitsprotectsthisTcell epitopefromthedestructionbytheconstitutiveproteasomeandthepeptidecanbepresentedtocytotoxicTcells(T_CD8+)[25].

Usingb1iandb5ispecificinhibitors,Jangetal.recently demonstratedthatimmunoproteasomesarenotessential forcanonicalNF-kBactivation[38].In2001,Chenetal.

reportedthatimmunoproteasomesaremajordeterminants ofthehierarchyofTcellepitopesduringantiviralCTL responses. Alreadyinthis study itwas notedthat adop- tively transferred b1i-deficient T cells werenot ableto expandininfluenzavirusinfectedWThosts[9],butthis phenomenonwassuspectedtorelyontherejectionofthe adoptivelytransferredcells[39]. Theinabilityofimmu- noproteasomesubunit-deficientTcellstoexpandinavirus infectedWThostwasalsoobservedbyMoebiusetal.,who providedstrongevidencethatthelossofb5i-deficientT cellsaftertransferwasnotaconsequenceofgraftrejection bythehost[15].Hence,theimmunoproteasomepossesses a so far uncharacterized function in controlling T cell expansioninaninfectedhostandthereforemightqualify asapotentialnewtargetforthesuppressionofundesired pro-inflammatoryTcellresponses.Indeed,withthehelp of a b5i-selective inhibitor (named PR-957 and later renamed to ONX 0914), the autoreactive immune responsesintwomousemodelsofarthritisanda model ofdiabetescouldbesuppressed[13].Additionally,anew function of immunoproteasomesin cytokine production and T helper cell differentiation was proposed [13].

Furthermore,b5iinhibitionpreventedexperimentalcoli- tis[14],murinelupuslikedisease[40],andHashimoto’s thyroiditis [41]. Not merely inhibition, but also genetic deficiency of immunoproteasome subunits attenuates inflammatoryboweldiseaseinmousemodels,suggesting a special contribution of the immunoproteasome in the etiologyofinflammatory boweldiseases[14,42,43]. Dis- parate results have been obtained in mouse models of murine autoimmune encephalomyelitis (EAE). Frausto et al. demonstrated that the immunoproteasome is not requiredfortheestablishmentofmyelinoligodendrocyte glycoprotein-induced EAE in b1i-deficient mice [44], whereas Seifert et al. reported anexacerbation of EAE symptoms in b5i^/ mice [45]. Additionally, it was demonstratedthat immunoproteasomes arerequiredfor the efficient degradation of poly-ubiquitylated proteins and the preservation of cell viability under cytokine- inducedoxidativestress[45,46].However,howanimmu- noproteasomesubunit shouldcontrolsubstrateaccessto the 26S proteasomes has remained elusive especially becausethehigh-resolutioncrystal structuresof the20S constitutive–andimmunoproteasomeofthemousedid notrevealanydifferenceinthea-ringswhereproteasome regulatorsbind[2].

Severalrecenthumangeneticsstudiessupporttheinvol- vementofimmunoproteasomesininflammatorydisorders [47,48,49]. Geneticmapping ofpatients with anauto- somal-recessive auto-inflammatory syndrome character- ized by joint contractures, muscle atrophy, microcytic anemia,andpanniculitis-inducedlipodystrophysyndrome (JMP syndrome) revealed a point mutation (T75M) in

PSMB8,thegeneencodingforb5i,leadingtoadisruption of the tertiary structure of b5i [47]. Patients bearing a G176V mutation in the PSMB8 gene, suffered from a newlyrecognizedtypeofJapaneseautoinflammatorysyn- drome with lipodystrophy (JASL). Themutation mani- fested in low b5i expression causing increased p38 phosphorylation, which resulted in increased IL-6 pro- duction[48].Similarly,Arimaetal.foundthataG201V mutationinthePSMB8genecausestheautoinflammatory disorderNakajo-Nishimurasyndrome[49].Themutation disruptstheb-sheetstructureofb5i,resultinginaccumu- lationofpoly-ubiquitylatedandoxidizedproteinswithin cells expressing immunoproteasomes. Furthermore, a strong associationbetween human type 1 diabetes and twosinglenucleotidepolymorphismsinthePSMB8gene demonstrateda correlationofautoimmunediseaseswith geneticalterationofb5i[50]. Theauthorsalso showed thatb2i/b5idouble-deficientmicedevelopCD8⁺Tcell- mediatedearly-stagemultiorganautoimmunityfollowing irradiationandbonemarrowreconstitution,suggestingthat immunosubunitsalso playanimportant function in the prevention ofCD8⁺ Tcell-mediated autoimmune reac- tions[50]ashasbeenhypothesizedpreviously[51].

Figure2

Th1

β5i inhibition

IFN- γ, IL-12

IL-4

TGF-β, IL-6

TGF- β Th0

Th17 Th2

Treg

Current Opinion in Immunology

InfluenceofLMP7onThelpercelldifferentiation.Dependingonthe cytokineenvironmentnaı¨veThelpercells(Th0)differentiateintoTh1, Th2,Th17,orregulatoryTcells(Treg).":enhanceddifferentiation;!:no influence;#:reduceddifferentiation[52].

How does the immunoproteasome exert its effect in autoimmune diseases? A likely explanation could be through the regulation of inflammatory cytokines or T helpercelldifferentiation.Indeed,selectiveinhibitionor geneticablation ofb5i resultedindiminished Th1and Th17differentiation,enhanceddevelopmentofregulat- ory T cells, but no effect on Th2 differentiation [13,43,52](Figure 2). Muchamuel et al. first demon- stratedthatselective inhibition ofb5i blockedthe pro- duction of IL-23 by activated monocytes and the productionof IFN-g and IL-2 by T cells, whereas the inhibition of b5 did not substantially affect cytokine release [13]. Mixed proteasomes expressed in b1i^/ micedecreasecytokineproduction byDCs,whichsup- portsthenotionofimmunoproteasomesplayingarolein cytokineproduction[53].PMA/ionomycinstimulationof b2i^//b5i^/ derived splenocytes demonstrated that immunoproteasomes regulate the expression of IFN-g, IL-4,IL-10, IL-2Rb,GATA3,andT-bet[54].Further- more, LPS-stimulated thioglycollate-elicited macro- phages from immunoproteasome-deficient mice were found to produce markedly reduced NO levels owing todefectsintheTRIF/TRAMandIRF-3pathway[55].

Conclusions

Thesevere phenotype in MHC-I ligand generation of triplyimmunoproteasome-deficientmice [31]and the existenceofintermediateimmunoproteasomesdiversify- ingtheMHC-Irepertoire[26]emphasizetheimportant role of the immunoproteasome in antigen processing.

The development of a specific inhibitor of b5i has revealed a new function of immunoproteasomes in inflammatoryautoimmunedisorders. However,howthe immunoproteasome is mechanistically involved in the newlydescribedprocesses has remained unclearso far.

We propose that the immunoproteasome might selec- tively processes a factor that is required for regulating cytokineproductionandThelpercelldifferentiation,but suchafactorremainstobeidentified.Thoughselective inhibitors have been described, the recently solved immunoproteasome crystal structures will promote the structure-guideddesignofnewinhibitoryleadstructures [2]. Finally, clinical investigations, with either ONX 0914 or other immunoproteasome inhibitors will show whetherthepromisingpre-clinicalfindingscanbetrans- latedtohumanmedicine.

Conflictofinterest

C.J.K.isanemployeeofandshareholderinOnyxPhar- maceuticals.M.B.andM.G.havenofinancialconflictsof interest.

Acknowledgements

ThisworkwasfundedbytheGermanResearchFoundationgrantGR1517/

12-1,theKonstanzResearchSchoolChemicalBiology,theFritzThyssen FoundationgrantAZ10.10.2.122,andtheSwissNationalScience Foundationgrant31003A_138451.

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Immunoproteasomespreserveproteinhomeostasisupon interferon-inducedoxidativestress.Cell2010,142:613-624.

Thisstudy proposes thatimmunoproteasomes are more capable to degradepoly-ubiquitylatedoroxidizedproteinsthanconstitutiveprotea- somes.

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This publicationshows thata mutation in the LMP7 gene leads to autoinflammationinhumans.

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ThisstudydemonstratesthatimmunoproteasomesplayacrucialroleinT helpercelldifferentiation.

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