Immunoproteasome subunit deficiency has no influence on the canonical pathway of NF-κB activation

Immunoproteasome subunit deficiency has no influence on the canonical pathway of NF- ␬ B activation

Annegret Bitzer

, Michael Basler

, Daniel Krappmann

, Marcus Groettrup

aDivisionofImmunology,DepartmentofBiology,UniversityofKonstanz,D-78457Konstanz,Germany

bBiotechnologyInstituteThurgau(BITg)attheUniversityofKonstanz,CH-8280Kreuzlingen,Switzerland

cResearchUnitCellularSignalIntegration,InstituteofMolecularToxicologyandPharmacology,HelmholtzZentrumMünchen–GermanResearchCenter forEnvironmentalHealth,IngolstädterLandstr.1,D-85764Neuherberg,Germany

a r t i c l e i n f o

Keywords:

NF-␬B

Immunoproteasome LMP2

LMP7 Inflammation

a b s t r a c t

Activationofthepro-inflammatorytranscriptionfactorNF-␬Brequiressignal-induced proteasomal degradationoftheinhibitorofNF-␬B(I␬B)inordertoallow nucleartranslocation.Mostcelltypes arecapableofexpressingtwotypesof20Sproteasomecoreparticles,theconstitutiveproteasomeand immunoproteasome.Inducibleunderinflammatoryconditions,theimmunoproteasomeismainlychar- acterizedthroughanalteredcleavagespecificitycomparedtotheconstitutiveproteasome.However,the questionwhetherimmunoproteasomesubunitsaffectNF-␬Bsignaltransductiondifferentlyfromconsti- tutivesubunitsisstillupfordebate.TostudytheeffectofimmunoproteasomesonLPS-orTNF-␣-induced NF-␬Bactivation,weusedIFN-␥stimulatedperitonealmacrophagesandmouseembryonicfibroblasts derivedfrommicedeficientfortheimmunoproteasomesubunitslowmolecularmasspolypeptide(LMP) 2,orLMP7andmulticatalyticendopeptidasecomplex-like1(MECL-1).Alongthecanonicalsignaling pathwayofNF-␬BactivationnodifferencesintheextentandkineticofI␬Bdegradationwereobserved.

NeitherthenucleartranslocationandDNAbindingofNF-␬BnortheproductionoftheNF-␬Bdependent cytokinesTNF-␣,IL-6,andIL-10differedbetweenimmunoproteasomedeficientandproficientcells.

Hence,weconcludethatimmunoproteasomesubunitshavenospecializedfunctionforcanonicalNF-␬B activation.

1. Introduction

NuclearfactorkappaB(NF-␬B)isacentralpro-inflammatory transcriptionfactor,whichisubiquitouslyexpressedandamas- terswitchbetweeninitiationandmaintenanceofinflammation.As such,NF-␬Bcontrolsexpressionofalargenumberofimmunoreg- ulatoryproteinslikecytokines,chemokinesandtheirreceptors,as wellasregulatorsofapoptosisandproliferation(Pahl,1999).In unstimulatedcells,NF-␬Bissequesteredinthecytoplasmasitis boundtoinhibitorsof␬B(I␬Bs)(Hinzetal.,2012).Extensivesignal transductioncascadesintegrateextracellularand/orintracellular signalsultimatelyleadingtophosphorylation,polyubiquitylation, andproteasomaldegradationofI␬Bs(HaydenandGhosh,2008).

FreeNF-␬Bthenmigratesintothenucleuswhereitbindstocon- sensussitesinpromotorsofnumerousgenes(Brasier,2006).

∗Correspondingauthorat:DivisionofImmunology,DepartmentofBiology,Uni- versityofKonstanz,Universitaetsstrasse10,D-78457,Konstanz,Germany.

E-mailaddress:Marcus.Groettrup@uni-konstanz.de(M.Groettrup).

NF-␬B signaling can be subdivided into two distinct path- ways,i.e.thecanonicalandnoncanoncial/alternativepathway.The canonicalpathwaycanbeactivatedbyavarietyofsignalsinclud- ingcytokines,pathogens,stresssignals,andradiation.Generally, receptorproximalsignalingadaptermoleculesinitiateasignaling cascadeultimatelyleadingtoakeystepinthecanonicalNF-␬B signalingpathway,theactivationoftheinhibitor␬Bkinase(IKK) complex(HaydenandGhosh,2008).Onceactivated,theIKKcom- plexphosphorylatesI␬B␣,whichissubsequentlymodified with ubiquitinbytheSCF-␤-TrCPcomplexanddegradedbytheprotea- some(KanarekandBen-Neriah,2012).

SincedegradationofI␬B␣isanessentialstepinNF-␬Bactiva- tion,theproteasomecomplexisakeyplayerinNF-␬Bsignaling.

Structurally, the 20S proteasome core particle consists of four stackedrings,eachconsistingofsevensubunits.Whiletheouter ringscontaincatalyticallyinactivealphasubunits,theinnerrings containsevendifferentbetasubunits,threeofwhichdisplayN- terminalthreonineproteaseactivity(␤1,␤2,␤5)(Tanaka,1998).

CellsofhematopoieticoriginandcellsstimulatedwithIFN-␥or, toa lesserextent,withtype Iinterferonsexpressanadditional

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-g7s5l3w4p0dj7 Erschienen in: Molecular Immunology ; 83 (2017). - S. 147-153

https://dx.doi.org/10.1016/j.molimm.2017.01.019

set of catalytically active proteasome subunits: low molecular masspolypeptide2(LMP2or␤1i),multicatalyticendopeptidase complex-like1(MECL-1, or␤2i),and LMP7(␤5i)(Barton etal., 2002;Khanetal.,2001;Shinetal.,2006).Duringdenovosynthe- sisofproteasomes,thesesubunitsareincorporatedandformthe immunoproteasome,whereasincorporation ofconstitutive sub- unitsis proportionally reduced (Griffinet al., 1998).Compared totheconstitutiveproteasome,theimmunoproteasomeismainly characterizedby an altered cleavage specificity (Driscoll et al., 1993; Gaczynska et al., 1994; Van Kaer et al., 1994). Overall, immunoproteasomesproducemorepeptideswithahydrophobic C-terminus,whicharewellsuitableforthepresentationonMHC classImolecules(Strehletal.,2005;Toesetal.,2001).

BesidesshapingtheimmunopeptidomepresentedonMHCclass Imolecules,severalstudiessupportabroaderimmunologicalrole oftheimmunoproteasome(Groettrupetal.,2010).Micedeficient foranyoftheimmunoproteasomesubunitsareprotectedfromdex- transulphatesodium-inducedcolitis(Basleretal.,2010;Fitzpatrick etal.,2006;Schmidtetal.,2010).Moreover,inhibitionofLMP7 hasbeenproveneffectiveforthetreatmentofautoimmunecondi- tionsindifferentmousemodels(Basleretal.,2015)andattenuates LCMV-inducedmeningitis(Mundtetal.,2016).However,inhibi- tionofthechymotrypsin-likeactivityinhematopoieticcellsrather thanthecatalyticspecificityofLMP7seemstobetheunderlying mechanisminthesemodels(Basleretal.,2014).Nevertheless,the mechanismbywhichLMP7deficiencyinfluencesimmuneregula- torymechanismsisstillnotfullyunderstood.Ultimatelydepending onthe proteasome for activation, NF-␬B is an important tran- scriptionfactorininflammatoryconditions.Still,previousstudies concernedwiththeeffectofimmunoproteasomesubunitsonNF-

␬Bactivationyieldedcontradictoryresults(HayashiandFaustman, 2000,1999;Hensleyetal.,2010;Kessleretal.,2000;Maldonado etal.,2013;Visekrunaetal.,2006).

Inthisstudy,wehaverevisitedtheinfluenceofLMP2knock- outandLMP7/MECL-1doubleknockout(L7M)onNF-␬Bactivation along thecanonical signalingpathway. Peritoneal macrophages andmouseembryonicfibroblasts(MEFs)derivedfromknockout mice were stimulated with LPS or TNF-␣ and I␬B␣ degrada- tion,cytokinesecretion,andnucleartranslocationofNF-␬Bwere assessed.Wefoundnoinfluenceofimmunoproteasomesubunit deficiencyonanyoftheseparametersandthereforeproposeNF-␬B signalingtobeindependentofproteasomesubunitcomposition.

2. Materialandmethods 2.1. Mice,celllinesandcytokines

C57BL/6mice(H-2b)wereoriginallypurchasedfromCharles River.LMP2(VanKaeretal.,1994),LMP7(Fehlingetal.,1994), andMECL-1(Basleretal.,2006)gene-targetedmicewereprovided byJ.Monaco(Cincinnati,OH,USA).LMP7^−/−/MECL-1^−/− double deficientmice(L7M^−/−)weregeneratedbycrossingtheF1gen- erationofLMP7^−/−xMECL-1^−/−mice.Micewerekeptinaspecific pathogen-freefacilityandusedat8–12weeksofage.Animalexper- imentswereapprovedbythereviewboardofRegierungspr ¨asidium Freiburg.Peritonealmacrophagesandprimarymouseembryonic fibroblasts(MEFs)wereculturedinDMEMwithGlutaMAXsupple- mentedwith10%FCSand100U/mlpenicillin/streptomycin.Media andsupplementswerepurchasedfromInvitrogen-LifeTechnolo- gies.RecombinantmurineIFN-␥andTNF-␣waspurchasedfrom Peprotechandusedat200U/mland100U/ml,respectively.

2.2. Generationofperitonealmacrophagesandmouseembryonic fibroblasts(MEFs)

Peritoneal macrophages were generated by i.p. injection of 0.5ml3%thioglycolatebroth.After4days,cellswerewashedout oftheabdominalcavitybyperitoneallavageusingPBS.Cellswere platedovernightandadherentcellswerefurtherculturedinthe presenceofIFN-␥for2daysorleftuntreated.

MEFs were prepared from embryos onday 14 of gestation.

After removal of head and liver, embryos were finely minced anddigestedintrypsin/EDTAsolution(Invitrogen-LifeTechnolo- gies) for 15min at 37^◦C. Trypsin was inactivated withculture mediumandremovedbycentrifugation.Cellswerepassedthrough a100␮mfilterandplatedfortwodaysat37^◦Cand5%CO₂.Aliquots ofcellswerestoredat−150^◦Candadifferentbatchofcellswas usedforeachreplicationofanexperiment.

2.3. Stimulationofcells

Peritoneal macrophages or MEFs were seeded into 12-well platesandstimulatedwith200U/mlIFN-␥fortwodaystoinduce immunoproteasomeexpression.Controlcellswereleftuntreated.

Cells were then stimulated with 100U/ml TNF-␣ or 200ng/ml LPSandharvestedafterdifferenttimepoints.Cellswerewashed withcoldPBS andusedfor SDS-PAGEand westernblot, EMSA, or TransAM NF-␬BELISA. Cells used for expression analysis of immunoproteasomesubunitsweredirectlyharvestedafterIFN-␥

treatment.Theproteinconcentrationofsampleswasdetermined usingDCproteinassay(Bio-Rad)accordingtothemanufacturer’s instruction.

Toanalyzecytokinesecretionperitonealmacrophagesstimu- latedwith200U/mlIFN-␥ for 2days wereseededinto 96-well plates. Two hours after seeding, cells were stimulated with 200ng/mlLPSorleftuntreated.Supernatantsoftriplicateswere collected24hlaterandanalyzedusingIL-6,TNF-␣,orIL-10ELISA accordingtothemanufacturer’sinstruction(ebiosciences).

2.4. SDSPAGEandwesternblot

CellswerelysedinRIPAbuffer(150mMNaCl,50mMTrispH8, 1%(v/v)TritonX-100,0.5%(v/v)sodiumdesoxycholate,0.1%(w/v) SDS)includingproteaseinhibitors(cOmpleteEDTA-free,Roche)for 30minonice.Lysateswerecentrifugedat14,000rpmfor15min andsupernatantswereboiledwithSDSsamplebufferfor5min at95^◦C.ProteinswereseparatedbySDS-PAGEandblottedonto nitrocellulosemembranes(Whatman).AfterblockinginRoti-Block solution(Roth)for1hatroomtemperature,membraneswereincu- batedwithprimaryantibodiesat4^◦Covernight.Membraneswere washed and incubated withappropriate peroxidase-conjugated secondaryantibodies(Dako)for2hatroomtemperature.Mem- branes were washed again and proteins were visualized with enhancedchemiluminescence.Primaryantibodiesusedwere:anti- LMP7andanti-LMP2(Kremeretal.,2010),anti-␤5(D1H6B,Cell SignalingTechnology),anti-␤1(cloneE1K90,CellSignalingTech- nology),anti-␣1(cloneIB5,K.Scherrer,Paris,France),anti-I␬B␣ (clone L35A5, Cell Signaling Technology), anti-␣-tubulin (clone AA13,Sigma).

2.5. Electrophoreticmobilityshiftassay(EMSA)

Nuclear extracts prepared from MEFs were used for EMSA.

MEFswerelysedinhypotoniclysisbuffer(10mMHEPESpH7.9, 10mMKCl,0.1mMEDTA)includingproteaseinhibitors(cOmplete EDTA-free,Roche)andphosphataseinhibitors(PhosSTOP,Roche) for10minonice.NP-40wasaddedtoreachafinalconcentration of0.2%.Lysateswerevortexedandcentrifugedat13,000rpmand

4^◦Cfor20s.Pelletscontainingthenuclearfractionwerelysedin nuclearextractionbuffer(20mMHEPESpH7.9,10%(v/v)glycerol, 0.4MNaCl,1mMEGTA,0.1mMEDTA)includingphosphataseand proteaseinhibitorsfor 30minonice.Nuclear lysateswerecen- trifugedat14,000rpmand4^◦Cfor14min.Theproteincontentof supernatantswasdeterminedwithaDCproteinassay(Bio-Rad) andfurtherusedforshiftassays.ToensureequalloadingofEMSA reactions,aliquotsofnuclearlysateswereboiledwithSDSsample bufferfor5minat95^◦CandsubjectedtoSDS-PAGEandwestern blotusinganti-LaminA/Cantibody.

EMSAwasperformedusinga³²P-labeleddouble-strandedDNA probe containingtheNF-␬Bbindingsite fromthemouseH–2K promotor (5-CAGGGCTGGGGATTCCCCATCTCCACAGG-3). Com- plementaryoligonucleotidescontaining5GATCoverhangswere mixedinannealingbuffer(50mMTrispH8,70mMNaCl)atacon- centrationof200ng/ml,incubatedat90^◦Cfor10minandslowly cooleddowntoallowoligonucleotideannealing.Oligonucleotides were then radioactively labeled with dATP[␣-³²P] using DNA polymeraseIKlenowfragment(NEB)accordingtothemanufac- turer´ısinstructions.LabeledprobeswerepurifiedusingQIAquick nucleotideremovalkit(Qiagen).Shiftreactions(20␮l)contained 5␮gnuclearextracts,0.5␮gBSA,5mM DTT,0.1␮g/␮lpoly(dI- dC) (Affymetrix)and0.5ng probein shiftbuffer(20mMHEPES pH7.9,60mMKCL,4%Ficoll)andwereincubatedfor30minat roomtemperature.Thecomplexeswereseparatedona5%native polyacrylamidegelinTBEbuffer(100mMTris,90mMboricacid, 1mMEDTA,pH8.2).Gelswerevacuum-driedontowhatmanpaper, exposedtophosphorscreens,andthebandswerevisualizedusing aphosphorimager.

2.6. NF-BELISA

TheDNA-bindingactivityofp65innuclearextractsprepared from stimulated MEFs was measured using a TransAM^® NF-

␬BfamilyELISA kit(Active Motif)followingthemanufacturer’s instruction.Allsampleswereanalyzedinduplicatesusing10␮g proteinperwell.

2.7. Statisticalanalysis

Theunpairedtwo-tailedStudent‘sttestandtwo-wayANOVA with Tukey correction were used for statistical analysis using GraphPadPrismsoftware.

3. Results

3.1. Proteasomesubunitcompositioninperitonealmacrophages andMEFs

Asanapproachforassessingtheinfluenceofimmunoprotea- somesubunitsonNF-␬Bactivationtwodifferenttypesofprimary cells were isolatedfrom wildtype, LMP7^−/−/MECL-1^−/− double knockout mice (L7M^−/−), and LMP2^−/− mice. Thioglycollate- elicited peritonealmacrophages (pMs) and mouse embryonic fibroblasts(MEFs)werechoseninordertoincludecelltypesofthe hematopoieticandnon-hematopoieticlineage,respectively. Iso- latedpMswereculturedinthepresenceofIFN-␥fortwodays tofurtherupregulateimmunoproteasomeexpression.Thesubunit compositionwasthenanalyzedwithSDS-PAGEandwesternblot (Fig.1A).UnstimulatedwildtypepMsstronglyexpressLMP7, whichappearsnottobefurtherupregulatedafterstimulationwith IFN-␥.Incontrast,expressionofLMP2isratherlowinunstimu- latedcellsandisstronglyinducedafterstimulation.Theseresults indicatethataproportionofproteasomesinunstimulatedpMs hasamixedsubunitcompositionconsistingofLMP7togetherwith theconstitutivesubunits␤1and␤2.Thisisinaccordance with

Fig.1.Proteasomesubunitcompositioninperitonealmacrophages(pMs)and mouseembryonicfibroblasts(MEFs).pMs(A)orMEFs(B)isolatedfromC57BL/6, LMP7^−/−MECL-1^−/−(L7M^−/−),andLMP2^−/−micewereculturedinthepresenceof 200U/mlIFN-␥for2daysorleftuntreated.Wholecelllysateswerepreparedand analyzedbywesternblotting.Thecatalyticallyinactiveproteasomesubunit␣1(iota) wasusedasloadingcontrol.Oneoutofthreeindependentexperimentswithsimilar outcomeisshown.

datapublishedbyGuillaumeetal.,whofoundabout40%ofprotea- somespresentindendriticcellstohaveincorporatedonlyLMP7 butnotLMP2orMECL-1(Guillaumeetal.,2010).Afterstimulation, thesubunit composition shiftsfurther towardsimmunoprotea- somes.However,expressionofMECL-1and␤2wasnottestedand itisthereforenotpossibletodiscriminatetheproportionoffull immunoproteasomesandcomplexescontainingLMP7andLMP2 togetherwith ␤2. In agreementwithpreviousreports, L7M^−/−

pMsshowastronglyreducedincorporationofLMP2andanaccu- mulationoftheunprocessedLMP2precursor(Griffinetal.,1998;

Deetal.,2003).Hence,L7M^−/−pMalmostexclusivelyexpress constitutiveproteasomes.Incontrast,LMP2^−/−cells showLMP7 expressionlevelssimilartowildtypecells.

UnlikepMs,MEFsdonotexpressimmunoproteasomesub- units inthe absenceof IFN-␥ stimulation(Fig.1B). AfterIFN-␥ stimulation,expressionofLMP7andLMP2isinduced,whilethe respectiveconstitutive subunits ␤5 and ␤1 are downregulated.

SimilartopMs,L7M^−/−MEFsdisplaystronglyreducedincorpo- rationofLMP2,whereasLMP2^−/−MEFsshowreducedexpression ofLMP7.

3.2. DegradationofIB˛isnotalteredin immunoproteasome-deficientcells

AfterhavinganalyzedtheproteasomecompositioninpMs andMEFs,stimulus-induceddegradationofI␬B␣wasinvestigated next.Atthispoint,theproteasomeisdirectlyinvolvedinthesig- nalingcascade.IncompleteordelayeddegradationofI␬B␣leadsto

Fig.2. Stimulus-induceddegradationofI␬B␣inpMsandMEFs.pMs(AandB)orMEFs(C)isolatedfromC57BL/6,L7M^−/−,andLMP2^−/−micewereculturedinthe presenceof200U/mlIFN-␥for2days.Cellswerethenstimulatedwith200ng/mlLPS(A)or100U/mlTNF-␣(BandC).Wholecelllysateswerepreparedattheindicatedtime pointsandsubjectedtowesternblotanalysis.Tubulinwasusedasloadingcontrol.Oneoutofatleastthreeindependentexperimentswithsimilaroutcomeisshown.The meansofrelativeI␬B␣signalintensitiesfromatleastthreeindependentexperimentswerecalculatedandstatisticallyanalyzedbytwo-wayANOVAwithTukeycorrection;

ns(notsignificant)P>0.05.

retentionofNF-␬Binthecytoplasm.Thus,anyalterationaffecting theextentorkineticofI␬B␣degradationwouldimminentlyaffect alldownstreamsignalingevents.pMsfromC57BL/6,L7M^−/−,and LMP2^−/−micewereincubatedinthepresenceofIFN-␥fortwodays tofurtherupregulateimmunoproteasomeexpression.Degradation ofI␬B␣wasinducedbystimulatingthecellswiththeTLR4ligand LPSandmonitoredbySDS-PAGEandwesternblotting.Nodiffer- enceinI␬B␣degradationcouldbedetectedbetweencellsderived fromwildtypeC57BL/6miceandeitherL7M^−/−orLMP2^−/−mice (Fig.2A).ThesameresultwasobtainedforpMsthatwerenotpre- treatedwithIFN-␥invitro(datanotshown).Inordernottorely onlyonasinglestimulus,pMswerealsostimulatedwithtumor necrosisfactor(TNF)-␣.Duetoadifferentupstreamsignalingcas- cade,receptorbindingofTNF-␣triggersafasterresponsecompared toLPS.Here,maximumI␬B␣degradationisalreadyreachedafter 15min(Fig.2B).Nevertheless,immunoproteasomedeficiencyhad noapparentinfluenceonI␬B␣degradation.

SimilartopMs,MEFsstimulatedwithTNF-␣displayedafast response,which is evidenced bytherapid degradationof I␬B␣

(Fig.2C).AlthoughMEFswerepre-stimulatedwithIFN-␥toupreg- ulateimmunoproteasomeexpression,I␬B␣degradationwasnot influencedbyeitherLMP7andMECL-1orLMP2deficiency.Even thoughincorporationofimmunoproteasomesubunitsaltersthe cleavagespecificityandsubstratebindingpocketsofthe20Score

particle(Huberetal.,2012;Toesetal.,2001)thereseemstobeno influenceofthesesubunitsonthedegradationofI␬B␣.

3.3. NucleartranslocationoffreeNF-Bandtransactivationof targetgenesisnormalinimmunoproteasomeknockoutcells

The final steps in NF-␬Bsignaling are nuclear translocation offreeNF-␬Bdimersandtransactivationoftargetgenes.Totest whethertheimmunoproteasomehasanyinfluenceonNF-␬Bsig- nalingdownstreamofI␬B␣,activenuclearNF-␬Bwasquantified usinganelectrophoreticmobilityshiftassay(EMSA).MEFsderived fromC57BL/6,L7M^−/−,andLMP2^−/−micewereincubatedinthe presenceofIFN-␥fortwodaysfollowedbystimulationwithTNF-

␣.Nuclearextractswerepreparedafterdifferenttimepointsand subjectedtoEMSAusinganNF-␬Bbindingsiteprobe(Fig.3A).As expectedfromtheresultsobtainedforI␬B␣degradation,nodif- ferenceintheamountofactiveNF-␬Bcouldbedetectedbetween wildtypeandimmunoproteasomeknockoutcelllines.Likewise, theamountofp65boundtooligonucleotidecontaininganNF-␬B consensussequenceasdeterminedbyELISAdidnotdifferinthese nuclearextractsatanytimepointafterstimulation(Fig.3B).

Cytokinesmediateimportanteffectorfunctionsoftheimmune system. Hence, many genes encoding cytokines contain NF-␬B responsiveelementsintheirpromotorregion.Asa measurefor

Fig.3. NucleartranslocationoffreeNF-␬B.MEFsisolatedfromC57BL/6,L7M^−/−, andLMP2^−/−micewereculturedinthepresenceof200U/mlIFN-␥for2days.Cells werethenstimulatedwith100U/mlTNF-␣fortheindicatedtimeperiods.Nuclear extractswerepreparedandanalyzedbyelectrophoreticmobilityshiftassay(EMSA) usinganNF-␬Bbindingsiteprobe(A)orTransAM^® NF-␬BELISA(ActiveMotif) employinganti-p65antibody(B).(A)Asaloadingcontrol,nuclearextractswerealso subjectedtowesternblot(WB)analysisusingananti-LaminA/Cantibody.Oneout ofthreeindependentexperimentswithsimilaroutcomeisshown.(B)Thegraph showspooleddatafromthreeindependentexperimentsincludingmean;ns(not significant)P>0.05(two-wayANOVAwithTukeycorrection).

transactivation,secretionofthepro-inflammatorycytokinesTNF-

␣andIL-6aswellastheanti-inflammatorycytokineIL-10byIFN-␥

–pre-conditionedandLPS-stimulatedpMswasquantifiedusing ELISA(Fig.4).Concentrationsof noneof thecytokinesdiffered inastatisticallysignificantwaybetweensupernatantsofpMs derived fromC57BL/6,L7M^−/−,and LMP2^−/− mice.Thus,NF-␬B mediatedtransactivationisnotinfluencedbyimmunoproteasome deficiency.

Overall,I␬Bdegradation andsignaling eventsfurtherdown- streamofthissteparenotinfluencedbytheimmunoproteasome subunitsLMP7,MECL-1,andLMP2.Collectively,thesedatastrongly supportamodelofNF-␬Bactivationbeingindependentofthe20S proteasomesubunitcomposition.

4. Discussion

Two decades of immunoproteasome research solidified the hypothesis of immunoproteasome particles shaping the immunopeptideomepresentedonMHCclassImolecules(Basler etal.,2011;Basleretal.,2006;Basleretal.,2004;Kincaidetal., 2012;Mishtoetal.,2014;Moreletal.,2000;Osterlohetal.,2006;

Sijtsetal.,2000;Toesetal.,2001).Moreover,itbecameevident thattheimmunoproteasomehas,apartfromantigenprocessing,

Fig.4.TransactivationofNF-␬Btargetgenes.pMsisolatedfromC57BL/6,L7M^−/−, andLMP2^−/−micewereculturedinthepresenceof200U/mlIFN-␥for2days.Cells werethenstimulatedwith200ng/mlLPSfor20horleftuntreated(control).Super- natantswerecollectedandconcentrationsofcytokinesTNF-␣,IL-6,andIL-10were determinedbyELISAintriplicates.Graphsshowpooleddatafromatleastfive independentexperimentsincludingmean;ns(notsignificant)P>0.05(unpaired two-tailedStudent´ısttest).

additional immunological functions. Immunoproteasome defi- ciency or inhibition affects T cell survival, expansion, and differentiation (Basler et al., 2004; Chen et al., 2001; Kalim etal.,2012;Moebiusetal.,2010;Muchamueletal.,2009;Zaiss etal.,2008),cytokineproduction(Basleretal.,2011;Basleretal., 2010;Basleretal.,2014;Muchamueletal.,2009),andprogression ofautoimmuneconditions(Basleretal.,2015).Moreover,muta- tionsinLMP7andLMP2inhumanscausecomplexautoimmune andinflammatoryphenotypes(BrehmandKruger,2015;Brehm et al.,2015).The impactof theimmunoproteasomeonvarious immunologicalaspectsrenderedthiscomplexanemergingphar- macolocigaltargetforvariousdiseasesandraisedanevenhigher interest in understandingits exact cellular functionin autoim- munityandinflammation.SinceNF-␬Bisamasterswitchinthe initiation and maintenance of inflammation, it is of particular interesttoclarifywhetheralteredNF-␬Bsignaltransductionisthe underlyingmechanismoftheobservedphenomena.

Inourstudy,theexpressionofdifferentproteasomesubunits wasfirst determined by SDS-PAGE and westernblotting. After stimulationwithIFN-␥,bothpMsandMEFsexpressedimmuno- proteasomes. Still, expression of constitutive subunits was not completelydownregulated indicating a mix of constitutive and immunoproteasomes present in the cells. Due tothe assembly defectinL7M^−/−cellstheparticlespresentarealmostexclusively composedofconstitutivesubunits.Incontrast,LMP2^−/−cellsseem toexpressLMP7butduetothedefectinMECL-1incorporationmost likelycontainconstitutiveproteasomesaswellasmixedparticles containing␤1,␤2,andLMP7.

Oneofthebest-studiedsignaltransductionpathwaysultimately dependingontheproteasomeistheNF-␬Bpathway.Here,protea- somesarerequiredfortheprocessingofNF-␬Bprecursorsaswell asforthesignal-induceddegradationofI␬B␣.Theliteratureabout theroleoftheimmunoproteasomeinNF-␬Bactivationisquitecon- troversial.AnearlystudyperformedwithsplenocytesfromBALB/c, NOD,andLMP2^−/−micebyHayashiandFaustmansuggestedthat spleencellsfromNODmicelackLMP2andthatLMP2isrequired fortheprocessingoftheNF-␬Bp50subunitfromthep105precur- sorandforthedegradationofI␬B(HayashiandFaustman,1999).

Thelatternotionwassubsequentlyconfirmedbythesameauthors in theLMP2/LMP7 double deficient human lymphoblastoidcell lineT2whenthismutantwascomparedtotheLMP2/LMP7profi- cientparentalcelllineT1(HayashiandFaustman,2000).However, twoindependentgroupscouldnotconfirmalowerexpressionof LMP2mRNAorproteininNODascomparedtoBALB/csplenocytes.

(Kessleretal.,2000;Runnelsetal.,2000).Moreover,areconstitu- tionofT2cellswithLMP2and/orLMP7didnotchangethequantity ofthematurep50subunitof NF-␬B(Runnelsetal., 2000).The follow-upstudybyHayashiandFaustmanalsofoundreducedI␬B␣

degradationinLMP2^−/−lymphocytesuponstimulationwithTNF-

␣(HayashiandFaustman,2000).Thisfindingcouldbeconfirmed withLPS-stimulatedBcellsderivedfromLMP2^−/−micebyanother group,althoughthe observedeffectwasrather minor(Hensley etal.,2010).Inastudyinvestigatinghumancolitis,Visekrunaetal.

showedenhancedprocessingofp105top50incytosolicextracts fromthecolonicmucosa ofpatients withCrohn’sdisease(CD), whichexpressedhigherlevelsofimmunoproteasome,ascompared tomucosalextractsfrompatientswithulcerativecolitis(UC)con- taininglessimmunoproteasome(Visekrunaetal.,2006).However, thisfindingwasmerelycorrelativeanditcouldnotberuledoutthat otherfactorsthatdifferintheCDandUCextractsaccountedfordif- ferentp105processingrates.Thesamestudyalsofoundenhanced invitrodegradationofI␬B␣inthepresenceofpurified20Simmuno- proteasomes compared toconstitutive proteasomes. While free I␬B␣canindeedbedegradedinaubiquitin-independentfashion (Mathesetal.,2008)thisdegradationpathwaymightnotberep- resentativeforthecanonicalubiquitin-dependentdegradationof NF-␬B-boundI␬B␣viathe26Sproteasome.Incontrast,arecent reportbyMaldonadoandcolleaguesfoundthatactivationofthe canonicalpathwaywasnotaffectedinLMP2^−/−andL7M^−/−cells whilethealternativepathwayseemedtobe“aberrant”inLMP2^−/−

cells(Maldonadoetal.,2013).Furthermore,ithasbeenproposed thattheimmunoproteasomehasahighercapacitytoclearubiqui- tylatedproteinsaccumulatingafterstimulationwithIFN-␥(Seifert etal.,2010).However,thisfindingtoocouldnotbereproduced by others (Nathanet al.,2013; Kincaid et al., 2012).Unlike in thepreviousstudies,weusedtwodifferenttypesofprimarycells directlyderivedfromknockoutmice.BychoosingpMsandMEFs weincludedcellsfromhematopoieticandnon-hematopoieticori- gin,respectively,andourexperimentscouldnotbecomplicated bypotentialdifferencesinthedistributionoflymphocytesubpop- ulationsthatoccurinimmunoproteasomeknockoutmice(Basler etal.,2006;VanKaeretal.,1994).Thus,theavoidanceofhighly

specializedcelltypes,totallymphocytes,andgeneticallymodified celllinesfurtherstrengthensourdataset.

Inconclusion,theresultspresentedinourstudyclearlyargue againstaninfluenceofimmunoproteasomesonthecanonicalpath- wayofNF-␬BactivationatleastinMEFsandpMs.Twodifferent typesofprimarycellspreparedfromLMP2^−/−,L7M^−/−,andwild type micedisplayednodifferencesin theextentandkinetic of I␬B␣ degradation when stimulatedwithLPS or TNF-␣. Consis- tentwiththisfinding,theamountofactiveNF-␬Binthenucleus ofknockoutcellsaswellasthetransactivationactivitywasnor- mal.Althoughgenerationofp50fromthep105precursorwasnot analyzedhere,ourresultsdonotindicateadeficitinmaturedNF-

␬Bsubunitsintheknockoutcells.Astudyconductedwithsmall moleculeinhibitorstargetingLMP2orLMP7furthersupportsthe conceptionofNF-␬Bactivationbeingindependentofproteasome composition.InhibitionofLMP2,LMP7,orevenbothhadnoinflu- enceonI␬B␣degradation incells stimulatedwithTNF-␣(Jang et al., 2012). Given that neither geneticdeletion nor chemical inhibition ofimmunoproteasomesubunitsaffects canonicalNF-

␬Bactivation,theelucidationofalternativemechanismshowthe immunoproteasomeinfluencescytokineproduction,Thelpercell differentiation,and autoimmunity requires substantialresearch effortsinthefuture.

Conflictofinterest

Theauthorsdeclarenoconflictsofinterest.

Funding

ThisworkwassupportedbygrantsBA4199/2-1andGR1517/14- 1fromtheGermanResearchFoundation(DFG).A.B.wasamember oftheDFGResearchTrainingGroup1331.Thefundershadnorolein thestudydesign,norinthecollection,analysis,andinterpretation ofthedata,writingofthereport,anddecisiontosubmitthearticle forpublication.

Acknowledgements

WethankM.VincendeauforhelpwithEMSAsandG.Schmidtke forhelpwithradioactivework.

References

Barton,L.F.,Cruz,M.,Rangwala,R.,DeepeJr.,G.S.,Monaco,J.J.,2002.Regulationof immunoproteasomesubunitexpressioninvivofollowingpathogenicfungal infection.J.Immunol.169,3046–3052.

Basler,M.,Youhnovski,N.,VanDenBroek,M.,Przybylski,M.,Groettrup,M.,2004.

Immunoproteasomesdown-regulatepresentationofasubdominantTcell epitopefromlymphocyticchoriomeningitisvirus.J.Immunol.173,3925–3934.

Basler,M.,Moebius,J.,Elenich,L.,Groettrup,M.,Monaco,J.J.,2006.AnalteredTcell repertoireinMECL-1-deficientmice.J.Immunol.176,6665–6672.

Basler,M.,Dajee,M.,Moll,C.,Groettrup,M.,Kirk,C.J.,2010.Preventionof experimentalcolitisbyaselectiveinhibitoroftheimmunoproteasome.J.

Immunol.185,634–641.

Basler,M.,Beck,U.,Kirk,C.J.,Groettrup,M.,2011.Theantiviralimmuneresponse inmicedevoidofimmunoproteasomeactivity.J.Immunol.187,5548–5557.

Basler,M.,Mundt,S.,Muchamuel,T.,Moll,C.,Jiang,J.,Groettrup,M.,Kirk,C.J., 2014.Inhibitionoftheimmunoproteasomeamelioratesexperimental autoimmuneencephalomyelitis.EMBOMol.Med.6,226–238.

Basler,M.,Mundt,S.,Bitzer,A.,Schmidt,C.,Groettrup,M.,2015.The

immunoproteasome:anoveldrugtargetforautoimmunediseases.Clin.Exp.

Rheumatol.33,S74–S79.

Brasier,A.R.,2006.TheNF-kappaBregulatorynetwork.Cardiovasc.Toxicol.6, 111–130.

Brehm,A.,Kruger,E.,2015.Dysfunctioninproteinclearancebytheproteasome:

impactonautoinflammatorydiseases.Semin.Immunopathol.37,323–333.

Brehm,A.,Liu,Y.,Sheikh,A.,Marrero,B.,Omoyinmi,E.,Zhou,Q.,Montealegre,G., Biancotto,A.,Reinhardt,A.,AlmeidadeJesus,A.,Pelletier,M.,Tsai,W.L., Remmers,E.F.,Kardava,L.,Hill,S.,Kim,H.,Lachmann,H.J.,Megarbane,A.,Chae, J.J.,Brady,J.,Castillo,R.D.,Brown,D.,Casano,A.V.,Gao,L.,Chapelle,D.,Huang, Y.,Stone,D.,Chen,Y.,Sotzny,F.,Lee,C.C.,Kastner,D.L.,Torrelo,A.,Zlotogorski,

A.,Moir,S.,Gadina,M.,McCoy,P.,Wesley,R.,Rother,K.I.,Hildebrand,P.W., Brogan,P.,Kruger,E.,Aksentijevich,I.,Goldbach-Mansky,R.,2015.Additive loss-of-functionproteasomesubunitmutationsinCANDLE/PRAASpatients promotetypeIIFNproduction.J.Clin.Invest.125,4196–4211.

Chen,W.,Norbury,C.C.,Cho,Y.,Yewdell,J.W.,Bennink,J.R.,2001.

ImmunoproteasomesshapeimmunodominancehierarchiesofantiviralCD8(+) TcellsatthelevelsofTcellrepertoireandpresentationofviralantigens.J.Exp.

Med.193,1319–1326.

De,M.,Jayarapu,K.,Elenich,L.,Monaco,J.J.,Colbert,R.A.,Griffin,T.A.,2003.Beta2 subunitpropeptidesinfluencecooperativeproteasomeassembly.J.Biol.Chem.

278,6153–6159.

Driscoll,J.,Brown,M.G.,Finley,D.,Monaco,J.J.,1993.MHC-linkedLMPgene productsspecificallyalterpeptidaseactivitiesoftheproteasome.Nature365, 262–264.

Fehling,H.J.,Swat,W.,Laplace,C.,Kuhn,R.,Rajewsky,K.,Muller,U.,vonBoehmer, H.,1994.MHCclassIexpressioninmicelackingtheproteasomesubunit LMP-7.Science265,1234–1237.

Fitzpatrick,L.R.,Khare,V.,Small,J.S.,Koltun,W.A.,2006.Dextransulfate sodium-inducedcolitisisassociatedwithenhancedlowmolecularmass polypeptide2(LMP2)expressionandisattenuatedinLMP2knockoutmice.

Dig.Dis.Sci.51,1269–1276.

Gaczynska,M.,Rock,K.L.,Spies,T.,Goldberg,A.L.,1994.Peptidaseactivitiesof proteasomesaredifferentiallyregulatedbythemajorhistocompatibility complex-encodedgenesforLMP2andLMP7.Proc.Natl.Acad.Sci.USA.91, 9213–9217.

Griffin,T.A.,Nandi,D.,Cruz,M.,Fehling,H.J.,Kaer,L.V.,Monaco,J.J.,Colbert,R.A., 1998.Immunoproteasomeassembly:cooperativeincorporationofinterferon gamma(IFN-gamma)-induciblesubunits.J.Exp.Med.187,97–104.

Groettrup,M.,Kirk,C.J.,Basler,M.,2010.Proteasomesinimmunecells:morethan peptideproducers?Nat.Rev.Immunol.10,73–78.

Guillaume,B.,Chapiro,J.,Stroobant,V.,Colau,D.,VanHolle,B.,Parvizi,G., Bousquet-Dubouch,M.P.,Theate,I.,Parmentier,N.,VandenEynde,B.J.,2010.

Twoabundantproteasomesubtypesthatuniquelyprocesssomeantigens presentedbyHLAclassImolecules.Proc.Natl.Acad.Sci.U.S.A.107, 18599–18604.

Hayashi,T.,Faustman,D.,1999.NODmicearedefectiveinproteasomeproduction andactivationofNF-kappaB.Mol.CellBiol.19,8646–8659.

Hayashi,T.,Faustman,D.,2000.Essentialroleofhumanleukocyteantigen-encoded proteasomesubunitsinNF-kappaBactivationandpreventionoftumor necrosisfactor-alpha-inducedapoptosis.J.Biol.Chem.275,5238–5247.

Hayden,M.S.,Ghosh,S.,2008.SharedprinciplesinNF-kappaBsignaling.Cell132, 344–362.

Hensley,S.E.,Zanker,D.,Dolan,B.P.,David,A.,Hickman,H.D.,Embry,A.C.,Skon, C.N.,Grebe,K.M.,Griffin,T.A.,Chen,W.,Bennink,J.R.,Yewdell,J.W.,2010.

UnexpectedrolefortheimmunoproteasomesubunitLMP2inantiviral humoralandinnateimmuneresponses.J.Immunol.184,4115–4122.

Hinz,M.,Arslan,S.C.,Scheidereit,C.,2012.IttakestwototangoIkappaBs,the multifunctionalpartnersofNF-kappaB.Immunol.Rev.246,59–76.

Huber,E.M.,Basler,M.,Schwab,R.,Heinemeyer,W.,Kirk,C.J.,Groettrup,M.,Groll, M.,2012.Immuno-andconstitutiveproteasomecrystalstructuresreveal differencesinsubstrateandinhibitorspecificity.Cell148,727–738.

Jang,E.R.,Lee,N.R.,Han,S.,Wu,Y.,Sharma,L.K.,Carmony,K.C.,Marks,J.,Lee,D.M., Ban,J.O.,Wehenkel,M.,Hong,J.T.,Kim,K.B.,Lee,W.,2012.Revisitingtherole oftheimmunoproteasomeintheactivationofthecanonicalNF-kappaB pathway.Mol.Biosyst.8,2295–2302.

Kalim,K.W.,Basler,M.,Kirk,C.J.,Groettrup,M.,2012.Immunoproteasomesubunit LMP7deficiencyandinhibitionsuppressesTh1andTh17butenhances regulatoryTcelldifferentiation.J.Immunol.189,4182–4193.

Kanarek,N.,Ben-Neriah,Y.,2012.RegulationofNF-kappaBbyubiquitinationand degradationoftheIkappaBs.Immunol.Rev.246,77–94.

Kessler,B.M.,Lennon-Dumenil,A.M.,Shinohara,M.L.,Lipes,M.A.,Ploegh,H.L., 2000.LMP2expressionandproteasomeactivityinNODmice.Nat.Med.6, 1064.

Khan,S.,vandenBroek,M.,Schwarz,K.,deGiuli,R.,Diener,P.A.,Groettrup,M., 2001.Immunoproteasomeslargelyreplaceconstitutiveproteasomesduringan antiviralandantibacterialimmuneresponseintheliver.J.Immunol.167, 6859–6868.

Kincaid,E.Z.,Che,J.W.,York,I.,Escobar,H.,Reyes-Vargas,E.,Delgado,J.C.,Welsh, R.M.,Karow,M.L.,Murphy,A.J.,Valenzuela,D.M.,Yancopoulos,G.D.,Rock,K.L., 2012.Micecompletelylackingimmunoproteasomesshowmajorchangesin antigenpresentation.Nat.Immunol.13,129–135.

Kremer,M.,Henn,A.,Kolb,C.,Basler,M.,Moebius,J.,Guillaume,B.,Leist,M.,Van denEynde,B.J.,Groettrup,M.,2010.Reducedimmunoproteasomeformation andaccumulationofimmunoproteasomalprecursorsinthebrainsof

lymphocyticchoriomeningitisvirus-infectedmice.J.Immunol.185, 5549–5560.

Maldonado,M.,Kapphahn,R.J.,Terluk,M.R.,Heuss,N.D.,Yuan,C.,Gregerson,D.S., Ferrington,D.A.,2013.Immunoproteasomedeficiencymodifiesthealternative pathwayofNFkappaBsignaling.PLoSOne8,e56187.

Mathes,E.,O’Dea,E.L.,Hoffmann,A.,Ghosh,G.,2008.NF-kappaBdictatesthe degradationpathwayofIkappaBalpha.EMBOJ.27,1357–1367.

Mishto,M.,Liepe,J.,Textoris-Taube,K.,Keller,C.,Henklein,P.,Weberruss,M., Dahlmann,B.,Enenkel,C.,Voigt,A.,Kuckelkorn,U.,Stumpf,M.P.H.,Kloetzel, P.M.,2014.Proteasomeisoformsexhibitonlyquantitativedifferencesin cleavageandepitopegeneration.Eur.J.Immunol.44,3508–3521.

Moebius,J.,vandenBroek,M.,Groettrup,M.,Basler,M.,2010.

ImmunoproteasomesareessentialforsurvivalandexpansionofTcellsin virus-infectedmice.Eur.J.Immunol.40,3439–3449.

Morel,S.,Levy,F.,Burlet-Schiltz,O.,Brasseur,F.,Probst-Kepper,M.,Peitrequin,A.L., Monsarrat,B.,VanVelthoven,R.,Cerottini,J.C.,Boon,T.,Gairin,J.E.,Vanden Eynde,B.J.,2000.Processingofsomeantigensbythestandardproteasomebut notbytheimmunoproteasomeresultsinpoorpresentationbydendriticcells.

Immunity12,107–117.

Muchamuel,T.,Basler,M.,Aujay,M.A.,Suzuki,E.,Kalim,K.W.,Lauer,C.,Sylvain,C., Ring,E.R.,Shields,J.,Jiang,J.,Shwonek,P.,Parlati,F.,Demo,S.D.,Bennett,M.K., Kirk,C.J.,Groettrup,M.,2009.Aselectiveinhibitoroftheimmunoproteasome subunitLMP7blockscytokineproductionandattenuatesprogressionof experimentalarthritis.Nat.Med.15,781–787.

Mundt,S.,Engelhardt,B.,Kirk,C.J.,Groettrup,M.,Basler,M.,2016.Inhibitionand deficiencyoftheimmunoproteasomesubunitLMP7attenuatesLCMV-induced meningitis.Eur.J.Immunol.46,104–113.

Nathan,J.A.,Spinnenhirn,V.,Schmidtke,G.,Basler,M.,Groettrup,M.,Goldberg, A.L.,2013.Immuno-andconstitutiveproteasomesdonotdifferintheir abilitiestodegradeubiquitinatedproteins.Cell152,1184–1194.

Osterloh,P.,Linkemann,K.,Tenzer,S.,Rammensee,H.G.,Radsak,M.P.,Busch,D.H., Schild,H.,2006.ProteasomesshapetherepertoireofTcellsparticipatingin antigen-specificimmuneresponses.Proc.Natl.Acad.Sci.U.S.A.103, 5042–5047.

Pahl,H.L.,1999.ActivatorsandtargetgenesofRel/NF-kappaBtranscriptionfactors.

Oncogene18,6853–6866.

Runnels,H.A.,Watkins,W.A.,Monaco,J.J.,2000.LMP2expressionandproteasome activityinNODmice.Nat.Med.6,1064–1065.

Schmidt,N.,Gonzalez,E.,Visekruna,A.,Kuhl,A.A.,Loddenkemper,C.,Mollenkopf, H.,Kaufmann,S.H.,Steinhoff,U.,Joeris,T.,2010.Targetingtheproteasome:

partialinhibitionoftheproteasomebybortezomibordeletionofthe immunosubunitLMP7attenuatesexperimentalcolitis.Gut59,896–906.

Seifert,U.,Bialy,L.P.,Ebstein,F.,Bech-Otschir,D.,Voigt,A.,Schroter,F., Prozorovski,T.,Lange,N.,Steffen,J.,Rieger,M.,Kuckelkorn,U.,Aktas,O., Kloetzel,P.M.,Kruger,E.,2010.Immunoproteasomespreserveprotein homeostasisuponinterferon-inducedoxidativestress.Cell142,613–624.

Shin,E.C.,Seifert,U.,Kato,T.,Rice,C.M.,Feinstone,S.M.,Kloetzel,P.M.,Rehermann, B.,2006.Virus-inducedtypeIIFNstimulatesgenerationof

immunoproteasomesatthesiteofinfection.J.Clin.Invest.116,3006–3014.

Sijts,A.J.,Standera,S.,Toes,R.E.,Ruppert,T.,Beekman,N.J.,vanVeelen,P.A., Ossendorp,F.A.,Melief,C.J.,Kloetzel,P.M.,2000.MHCclassIantigen processingofanadenovirusCTLepitopeislinkedtothelevelsof immunoproteasomesininfectedcells.J.Immunol.164,4500–4506.

Strehl,B.,Seifert,U.,Kruger,E.,Heink,S.,Kuckelkorn,U.,Kloetzel,P.M.,2005.

Interferon-gamma,thefunctionalplasticityoftheubiquitin-proteasome system,andMHCclassIantigenprocessing.Immunol.Rev.207,19–30.

Tanaka,K.,1998.Molecularbiologyoftheproteasome.Biochem.Biophys.Res.

Commun.247,537–541.

Toes,R.E.,Nussbaum,A.K.,Degermann,S.,Schirle,M.,Emmerich,N.P.,Kraft,M., Laplace,C.,Zwinderman,A.,Dick,T.P.,Muller,J.,Schonfisch,B.,Schmid,C., Fehling,H.J.,Stevanovic,S.,Rammensee,H.G.,Schild,H.,2001.Discrete cleavagemotifsofconstitutiveandimmunoproteasomesrevealedby quantitativeanalysisofcleavageproducts.J.Exp.Med.194,1–12.

VanKaer,L.,Ashton-Rickardt,P.G.,Eichelberger,M.,Gaczynska,M.,Nagashima,K., Rock,K.L.,Goldberg,A.L.,Doherty,P.C.,Tonegawa,S.,1994.Alteredpeptidase andviral-specificTcellresponseinLMP2mutantmice.Immunity1,533–541.

Visekruna,A.,Joeris,T.,Seidel,D.,Kroesen,A.,Loddenkemper,C.,Zeitz,M., Kaufmann,S.H.,Schmidt-Ullrich,R.,Steinhoff,U.,2006.Proteasome-mediated degradationofIkappaBalphaandprocessingofp105inCrohndiseaseand ulcerativecolitis.J.Clin.Invest.116,3195–3203.

Zaiss,D.M.,deGraaf,N.,Sijts,A.J.,2008.Theproteasomeimmunosubunit multicatalyticendopeptidasecomplex-like1isaT-cell-intrinsicfactor influencinghomeostaticexpansion.Infect.Immun.76,1207–1213.