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Chaperone BAG6 is dispensable for MHC class I antigen processing and presentation

Annegret Bitzer

a

, Michael Basler

a,b

, Marcus Groettrup

a,b,∗

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

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

Keywords:

BAG6

Antigenprocessing Antigenpresentation MHCclassI

a b s t r a c t

AntigenprocessingfordirectpresentationonMHCclassImoleculesisamultistepprocessrequiring theconcertedactivityofseveralcellularcomplexes.Theessentialstepsatthebeginningofthispathway, namelyproteinsynthesisattheribosomeanddegradationviatheproteasome,havebeenknownforyears.

Nevertheless,thereisaconsiderablelackoffactorsidentifiedtofunctionbetweenproteinsynthesisand degradationduringantigenprocessing.Here,weanalyzedtheimpactofthechaperoneBAG6onMHCclass Icellsurfaceexpressionandpresentationofvirus-derivedpeptides.AlthoughanessentialroleofBAG6 inantigenprocessinghasbeenproposedpreviously,wefoundBAG6tobedispensableinthispathway.

Still,interactionofBAG6andthemodelantigentyrosinasewasenhancedduringproteasomeinhibition pointingtowardsaroleofBAG6inantigendegradation.Redundantchaperonepathwayspotentiallymask thecontributionofBAG6toantigenprocessingandpresentation.

1. Introduction

Antigen processing and presentation in the MHC class I restricted pathway is a process comprising severalsubsequent stepsofpolypeptideprocessingandtransportation.Generally,pro- teinsare degradedtopeptidesby theproteasome,loadedonto MHCclassImolecules,andpresentedasacomplextocytotoxic Tlymphocytes(CTLs)atthecellsurface(RockandGoldberg,1999).

Basedonthediscoveryofanintimateconnectionbetweenantigen presentationandproteinsynthesisYewdellandcolleaguespostu- latedthedefectiveribosomalproduct(DRiP)hypothesis(Yewdell etal.,1996).DRiPsarethoughttobethemainsourceofantigenand aredefinedasrapidlydegradednascentpolypeptidesduetotheir inabilitytoachieveafunctionalstate.AlthoughtheDRiPhypothesis waspostulatedalmosttwodecadesagotherearestillconsider- ablegapsintheknowledgeaboutantigenprocessingupstreamof proteasomaldegradation.Interestingly,areportbyMinamietal.

(2010)foundthechaperoneBCL2-associatedathanogene6(BAG6;

also named Scythe or BAT3) tobe essential for DRiP degrada- tionandMHCclassIcellsurfaceexpression(Minamietal.,2010).

BAG6isconservedinhighereukaryotes,ubiquitouslyexpressed, andencodedintheMHCclassIIIlocus(Banerjietal.,1990;Ozaki etal., 1999;Wangand Liew, 1994).Eventhough BAG6isclas-

Correspondingauthorat:DepartmentofBiology,UniversityofKonstanz,Uni- versitaetsstrasse10,D-78457Konstanz,Germany,Fax:+497531883102.

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

sifiedasachaperone,it hasnoapparentfoldingactivity(Wang etal.,2011).TogetherwithitsbindingpartnersTRC35andUBL4A itassociateswithlonghydrophobicpatchesinclientproteinsand preventsaggregation(Leznickietal.,2013;Mariappanetal.,2010;

Minamietal.,2010;Wangetal.,2011).Therefore,BAG6hasalso beendescribedasa“holdase”whichkeepsitssubstrateinasoluble state(Wangetal.,2011).Becauseofitsbindingproperties,BAG6is especiallyinvolvedinthebiogenesisanddegradationofpolypep- tidescarryinghydrophobicdomains(Kawaharaetal.,2013;Leeand Ye,2013).

Inthisstudy,werevisitedtheroleofBAG6inMHCclassIanti- genprocessingproposedbyMinamietal.(2010).However,we foundnoinfluenceofBAG6knockdownonMHCclassIcellsurface expression.Moreover,BAG6wasnot transcriptionallyregulated afterstimulationwithIFN-␥andpresentationofvirus-derivedpep- tideswasnot alteredinBAG6knockdowncells. Together,these resultssuggestthatBAG6isdispensableorredundantforantigen processingandpresentationonMHCclassI.

2. Materialsandmethods

2.1. Mice,viruses,celllines,andcytokines

C57BL/6mice(H-2b),BALB/cmice(H-2d)andAADmice(H-2b, transgenicforanHLA-A*0201/H-2Ddchimericprotein(Newberg etal.,1996))wereoriginallypurchasedfromCharlesRiver.Mice werekeptin a specificpathogen-free facilityand usedat 8–12

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-322750

Erschienen in: Molecular Immunology ; 69 (2016). - S. 99-105 https://dx.doi.org/10.1016/j.molimm.2015.11.004

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weeksofage.Animalexperimentswereapprovedbythereview boardofRegierungspräsidiumFreiburg.

LCMV-WE was originally obtained from F. Lehmann-Grube (Hamburg, Germany) and propagated on the fibroblast line L929.VacciniavirusWesternReservestrain(VV-WR)wasorig- inallyobtainedfromH. Hengartner (UniversityHospitalZürich, Switzerland).Recombinantvacciniavirus(rVV)expressingtyrosi- nase(rVV-Tyr)wasakindgiftfromV.H.Engelhard(Universityof Virginia,VA,USA).Bothvacciniavirusstrainswerepropagatedon BSC-40cells.

B8-Db(H-2d)isamurinefibroblastcelllinestablytransfected withH-2Db (Basler et al., 2004). B8-Db cells were cultured in IMDM. Primary murine embryonic fibroblasts from AAD mice, HeLacells, HEK293cells, andMelJuSocells weremaintainedin DMEM.ThehumanlungfibroblastcelllineIMRSwasmaintained inMEMincluding15%FCS.MelJuSocellsandIMRScellswerea kindgiftfromN.Koch(UniversityofBonn,Germany).Allmedia werepurchasedfromInvitrogen-LifeTechnologiesandcontained GlutaMAX,10%FCSand100U/mlpenicillin/streptomycin.

Recombinanthumanandmurineinterferon-␥(IFN-␥)waspur- chasedfromPeprotech(Hamburg,Germany)andusedat200U/ml fortwodays.

2.2. Transfections

Knockdown of BAG6 was performed through simultaneous transfectionoffourdifferentsiRNAstargetingBAG6.At80%con- fluence,cellsweretransfectedwithmouseorhumanBAG6siRNA (ON-TARGETplus SMART pool siRNA, Thermo Scientific) using DharmaFECT1transfectionreagent(ThermoScientific)according to the manufacturer’s instruction. After 48h, cells were trans- fectedasecondtimetoachievemaximumefficiency.Controlcells weretransfectedwitha mixof fournon-targetingsiRNAs(ON- TARGETplusNon-TargetingpoolsiRNA,ThermoScientific).

For transient overexpression of BAG6, HeLa cells were transfected with a pCMV6-Entry expression plasmid encoding BAG6-Myc-DDK(OriGeneTechnologies).Controlcellsweretrans- fected with empty vector. Transfection was performed at 80%

confluence using FuGENE HD transfection reagent (Promega) according to the manufacturer’s instructions. Cells were fur- theranalyzed24hposttransfection.Transientoverexpressionof tyrosinase(Tyr) inHEK293 cells wasachievedlikewise using a pCMV6-Entryexpressionconstructencodingtyrosinase-Myc-Flag andTrans-IT-LT1transfectionreagent(MirusBioLLC.)accordingto themanufacturer’sinstructions.

2.3. Flowcytometry

MHCclassIsurfaceexpressionwasanalyzedbyflowcytom- etryusingfluorescently-labeledantibodies.Cellswereincubated togetherwithantibodiesdiluted1:150inPBS,2%(v/v)FCS,2mM EDTAfor20minat4C.Cellswerewashedandacquiredwiththe useoftheAccuriC6flowcytometersystem(BDBiosciences).Data wereanalyzedwithFlowJosoftware(TreeStar).Anti-H-2DbFITC (cloneKH95),anti-H-2DdPE(clone43-2-12)andanti-HLA-ABCAPC (cloneG46-2.6)antibodieswereobtainedfromBDBiosciences.

2.4. Real-timeRT-PCR

Total RNA was extracted using NucleoSpin RNA II kit (Macherey-Nagel) according tothe manufacturer’s instructions.

ExtractedRNAwasreverse-transcribed usingtheReverseTran- scription System (Promega) according to the manufacturer’s instruction.Quantitative real-time RT-PCRwas performedwith the LightCycler Instrument II (Roche) and LightCycler Fast- Start DNA Master SYBR Green I kit (Roche). Gene expression

was calculated relative to control cells and normalized to HPRT(hypoxanthineguaninephosphoribosyltransferase)expres- sionusingREST-384Osoftwareversion2 (GeneQuantification).

Sense and antisense primers used for amplification were as follows:BAG6(human)TACAATAACAATCACGAGGGCC,GGTGGT- GTAGTGAGACATAGG;TRC53(human)CGTGACCTTTGTGTCCAGAG, TTAAACAGAGAAACTGTAGCACGG;HPRT(human)TGGACAGGACT- GAACGTCTTG, CCAGCAGGTCAGCAAAGAATTTA; LMP7 (human) AATGCAGGCTGTACTATCTGCG,TGCAGCAGGTCACTGACATCTG.

2.5. SDSPAGEandwesternblot

CellswerelysedinRIPAbuffer(150mMNaCl,50mMTrispH8, 1%(v/v)TritonX-100,0.5%(v/v)sodiumdesoxycholate,0.1%(w/v) SDS)includingproteaseinhibitors(cOmpleteEDTA-free, Roche) for30minonice.Lysateswerecentrifugedat20,000×gand4C for15min.SupernatantsweremixedwithSDSsamplebufferand boiledfor5minat95C.ProteinswereseparatedbySDS-PAGEand blotted onto nitrocellulose membrane (Whatman). Membranes were blockedfor 1hin Roti-Block solution (Roth) followed by overnightincubation in primary antibodiesat 4C. Membranes werewashedandincubatedfor2hwithappropriateperoxidase- conjugatedsecondaryantibodies(Dako).Membraneswerewashed andproteinswerevisualizedwithenhancedchemiluminescence.

Primaryantibodiesused:Rabbit anti-BAG6(kindgift fromR.S.

Hegde,Cambridge,UK),mouseanti-␣-tubulin(cloneAA13,Sigma) andmouseanti-FLAG(cloneM2,Sigma).

2.6. Generationofepitope-specificcytotoxicTlymphocyte(CTL) lines

LCMV-specificCTLlinesweregeneratedfromfemaleC57BL/6 orBALB/cmiceinfectedi.v.with200PFULCMV-WE.Fourweeks postinfectionsplenocytes from infected micewerecultured in IMDM 10% FCS, P/S, supplemented with 40U/ml IL-2, 100␮M 2-mercaptoethanoland10−6Mpeptide.Cytokine-supplemented mediumwasaddedeveryotherdayfor8–14days.BeforeCTLs wereusedinpresentationassays,deadcellswereremovedbyFicoll densitycentrifugation.

Tyr-specific CTL-lines were generated from male AAD mice infectedi.p.with2×106PFUrVV-Tyr.Splenocytesfrommemory micewereculturedforthreeweeksasdescribedabove.Toincrease percentageofspecificTcells,CTLswererestimulatedtwicewith peptide-loaded,irradiated(20Gy)AADsplenocytesonday8and 16.

2.7. Syntheticpeptides

ThesyntheticpeptidesGP276-286(SGVENPGGYCL),NP118-126 (RPQASGYVM)andNP396-404(FQPQNGQFI)wereobtainedfromP.

Henklein(Charité,Berlin,Germany).ThesyntheticpeptideTyr369- 377(YMDGTMSQV)wasobtainedfromSigma.

2.8. Antigenpresentationassay

B8-DbcellsorAADmouseembryonicfibroblasts(MEFs)were transfectedwithBAG6siRNAasdescribedabove.Transfectedcells wereharvestedandinfectedwithLCMVorrVVwithamultiplic- ityofinfection(MOI)of10for 2h,4h, or5h. Serialdilutionof infectedcellswasperformedtoachievedifferenteffectortostimu- latorratios(E/S).Epitope-specificCTLlineswereaddedtoinfected cellsandincubatedinpresenceof10␮g/mlbrefeldinAfor3hat 37C. Activationof CTLswas determinedbyintracellular IFN-␥ stainingandflowcytometry.Allsampleswerepreparedasdupli- cates.

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2.9. IntracellularIFN-staining

IntracellularIFN-␥stainingwasperformedasdescribedprevi- ously(Basleretal.,2004)exceptthatanti-CD8aAPC(clone53-6.7, eBioscience)(1:150)wasusedtostaincytotoxicTcells.

2.10. Immunoprecipitation

Cellstransientlytransfected withanexpressionconstructfor tyrosinase-Myc-Flagormocktransfectedcellswereincubatedwith 10␮MMG132(Sigma)orDMSO(1:1000)for5hat37C.Cellswere thenharvestedandlysedinlysisbuffer(20mMTrispH7.8,50mM NaCl,10mMMgCl2,1%(v/v)NP40)includingproteaseinhibitors (cOmpleteEDTA-free,Roche)for30minonice.Lysateswerecen- trifugedat20,000×gand4Cfor15min.Supernatantswereloaded ontoproteinGaffinitygel(Sigma)togetherwithanti-Mycantibody (clone9E10,Sigma)andincubatedovernightat4Conarotator.

Theaffinitygelwaswashedthreetimesinhighsaltbuffer(50mM TrispH8,650mMNaCl,5mMEDTA,0.5%(v/v)TritonX-100)and threetimesinlowsaltbuffer(50mMTrispH8,150mMNaCl,5mM EDTA,0.5%(v/v)TritonX-100).Then,precipitatedproteinswere

heatedinSDSsamplebufferfor5minat95Candsubsequently analyzedbySDS-PAGEandwesternblotasdescribedabove.

2.11. Statisticalanalysis

Theunpairedtwo-tailedStudent’sttestwasappliedforstatis- ticalanalysisusingGraphPadPrismsoftware.

3. Results

3.1. MHCclassIsurfaceexpressionisindependentofBAG6

IfDRiPsresultfromincompletecaptureofnascentpolypeptides bychaperones,deletionofchaperonesshouldincreaseDRiPrates.

AhigherproportionofDRiPsshouldinturnincreaseMHCclassI surfaceexpressionduetoincreasedpeptidesupply.Ontheother hand,deletionofchaperonescouldleadtoinefficientshuttlingand degradationofantigen.ToanalyzetheinfluenceofBAG6onMHC classIsurfaceexpression,themurinefibroblastcelllineB8-Db(H- 2d+H-2Db)andHeLacells weresubjectedtoBAG6knockdown.

Subsequently, surfaceexpressionof MHC classImolecules was

Fig.1. KnockdownoroverexpressionofBAG6hasnoinfluenceonMHCclassIsurfaceexpression.(AandC)B8-Dbcells(A)andHeLacells(C)weretransfectedwithBAG6 siRNAorcontrolsiRNAandsurfaceexpressionofMHCclassImoleculesH-2DbandH-2Dd(A)orHLA-A,B,C(C)wasanalyzedbyflowcytometry.(BandD)Westernblotanalysis ofwholecelllysatespreparedfromB8-Dbcells(B)orHeLacells(D)transfectedwithBAG6siRNAorcontrolsiRNA.(E)HeLacellsweretransfectedwithaBAG6expression constructoranemptyplasmid(control)andsurfaceexpressionofMHCclassImoleculesHLA-A,B,Cwasanalyzedbyflowcytometry.(F)Westernblotanalysisofwholecell lysatespreparedfromHeLacellstransfectedwithaBAG6expressionconstructoranemptycontrolplasmid.Tubulinwasusedasloadingcontrol.Forallexperimentsone outoftwoindependentexperimentsisshown.

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Fig.2.BAG6isnottranscriptionallyregulatedafterIFN-␥stimulation.HeLacells(A),IMRScells(B)orMelJuSocells(C)werestimulatedwithIFN-␥for24hor48horwere leftunstimulatedandmRNAlevelsofBAG6andLMP7weredeterminedbyreal-timeRT-PCR.Expressionlevelswerecalculatedrelativetounstimulatedcellsandnormalized tohypoxanthineguaninephosphoribosyltransferase(HPRT)expression.Bargraphsshowmeans±SDfromthreeindependentexperiments.ns(notsignificant)P>0.05,

**P<0.01,***P<0.001(unpairedtwo-tailedStudent’sttest).

determinedbyflowcytometry.Nodifferenceinsurfaceexpression ofthetransfectedMHCclassImoleculeH-2Dbandtheendogenous moleculeH-2Dd could beobserved between B8-Db cells trans- fectedwithBAG6siRNAandcellstransfectedwithcontrolsiRNA (Fig.1A).SuccessfulknockdownleadingtoablationoftheBAG6 proteinbelow thedetectionlimit ofwesternanalysiswascon- firmedbySDS-PAGEandimmunoblotting(Fig.1B).Thesameresult wasobtainedwithHeLacellsinwhichtotalMHCclassIsurface expression(HLA-A,B,C)didnotdifferbetweenBAG6knockdown andcontrolcells(Fig.1C).VerysuccessfulknockdowninHeLacells wasagainconfirmedbySDS-PAGEandimmunoblotting(Fig.1D).

Torule out limiting effects of BAG6onMHC class I surface expression,overexpressionwasperformedinHeLacells.Cellswere transientlytransfectedwithanexpressionconstructencodingfor BAG6andsubjectedtoflowcytometricanalysis24hposttransfec- tion(Fig.1E).NoinfluenceonHLA-A,B,Csurfaceexpressioncould bedetectedalthoughlysatesoftransfectedcellssubjectedtoSDS- PAGEandimmunoblottingrevealedveryhighexpressionlevelsof BAG6comparedtocontrolcells(Fig.1F).

Takentogether,wecouldnotobserveaninfluenceofBAG6on MHCclassIsurfaceexpression.LossofBAG6mightbeeffectively compensatedbyothercytosolicchaperonesinthesettingapplied here.Ontheotherhand,BAG6isnotalimitingfactorinantigen

processingsinceitsoverexpressionhadnoeffectonMHCclassI surfaceexpression.

3.2. BAG6isnottranscriptionallyregulatedafterinterferon gamma(IFN-)stimulation

StimulationofcellswiththecytokineIFN-␥inducesorenhances transcriptionofseveralgeneswhoseproductsdelayviralreplica- tionandenhancepeptidepresentationonMHCclassI(Platanias, 2005;Senand Lengyel,1992).Like many ofthesegenes, BAG6 isencodedin theMHC locusand cytokineregulatedexpression controlcouldbepossible(Banerjietal.,1990).Yet,stimulationof HeLacellswithIFN-␥for24hor48hdidnotinduceupregulation ofBAG6mRNA(Fig.2A,leftside).Incontrast,expressionofthe immunoproteasomesubunitLMP7,a well-known IFN-regulated gene, wasstrongly upregulated afterstimulation (Fig. 2A,right side).AlthoughKamperetal.foundexpressionofthebag6geneto beupregulatedafterstimulationofthemelanomacelllineMelJuSo andthelungfibroblastcelllineIMRSwithIFN-␥wecouldneither confirmthisfindingforHeLacells,norfortheothertwocelllines (Fig.2BandC)(Kamperetal.,2012).Overall,ourresultsdonot pointtowardsacentralroleofBAG6inatypeIIinterferonregulated antiviralimmuneresponse.

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Fig.3.BAG6isdispensableforpresentationofvirus-derivedepitopes.(AandB)CellsweretransfectedwithBAG6siRNAorcontrolsiRNAandin(A)infectedwithlymphocytic choriomeningitisvirus(LCMV),(MOI=10).Epitope-specificCTLswereaddedtoinfectedcellsindifferenteffectortostimulatorratios(E/S)for3handthepercentageof IFN-␥+ofCD8+cellswasdeterminedbyflowcytometryasameasureforepitopepresentation.Valuesrepresentmean±SDofduplicatedeterminations.(A)B8-Dbcells infectedwithLCMVfor2,4or5hwereanalyzedforpresentationofNP396-404,GP276-286orNP118-126.(B)TransfectedMEFsfromAADmicewerestimulatedwithIFN-␥

for2days,infectedfor5hwithrVV-TyrorVV-WRandthenanalyzedforthepresentationofTyr369-377(D).(C)WesternblotanalysisofwholecelllysatespreparedfromAAD MEFstransfectedwithBAG6siRNAorcontrolsiRNAandstimulatedwithIFN-␥for2days.Tubulinwasusedasloadingcontrol.(D)HEK293cellsweretransientlytransfected

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3.3. PresentationofLCMV-derivedepitopesisindependentof BAG6

Toaddresswhethergenerationofvirus-derivedpeptideepi- topesisdependentonBAG6,weinvestigatedantigenpresentation inlymphocyticchoriomeningitisvirus(LCMV)-infectedcellsafter BAG6knockdown.B8-DbcellsweretransfectedwithBAG6siRNA andsubsequentlyinfectedwithLCMV-WEfor2,4or5h.Peptides presentedonMHCclassIweredetectedwithpeptide-specificCTL linesspecificfortheepitopesnucleoprotein(NP)118–126(H-2Ld), NP396-404 (H-2Db)and glycoprotein(GP) 276–286 (H-2Db)by stainingfortheCTLsurfacemarkerCD8andintracellularIFN-␥.

CTLsproduceIFN-␥uponstimulationoftheirreceptorwiththe appropriatepeptide-MHCcomplex.Thus,theproportionof IFN-

␥producingCTLsofallCD8bearingCTLsreflectstheamountof peptidespresentedbyinfectedcells.GP276-286representsanepi- topederivedfromthesecretorypathway,whereasNP118-126and NP396-404arederivedfromacytosolicprotein.Comparedtocon- trolcells,BAG6knockdowncellsarenotconfinedintheirabilityto presentLCMV-derivedepitopes(Fig.3A).

3.4. Presentationofretrotranslocatedtyrosinase-derivedepitope isnotaffectedbyBAG6knockdown

BAG6maintainsretrotranslocatedER-associatedproteindegra- dation(ERAD) substratesin a soluble state and thereby allows proteasomaldegradationofthesesubstrates(ClaessenandPloegh, 2011;Wangetal.,2011).Tostudythepresentationofanepitope derivedfromaretrotranslocatedproteinwechosethetyrosinase epitopeTyr369-377(D).Tyrosinaseisatransmembraneglycopro- teinproducedinmelanocytesandmelanomacellsthatistargeted totheER(Jimbowetal.,2000;Petrescuetal.,2000).Misfolded tyrosinaseisexportedbacktothecytosolwhereitisdeglycosylated leadingtotheconversionofN371toD371throughdeamination (Skipperetal.,1996).TheHLA-A*0201-restrictedepitopeTyr369- 377(D)spansacrossthismodifiedaminoacidandallowsanalysis ofanepitopederived exclusivelyfromaretrotranslocatedanti- gen.TostudypresentationofTyr369-377(D),wemadeuseofthe transgenicmousestrainAAD(H-2b,HLA-A2.1/H2-Dd)expressinga hybridMHCclassImoleculeconsistingofthealpha-1andalpha- 2domainofhumanHLA-A*0201andthealpha-3transmembrane andcytoplasmicdomainofmouseH-2Dd(Newbergetal.,1996).

AADmouseembryonic fibroblasts(MEFs)weretransfectedwith BAG6siRNA and stimulatedwith IFN-␥ to induce MHC class I expression.Cellswerethen infected withrecombinantvaccinia virusexpressingtyrosinase(rVV-Tyr)for5h.Tyr369-377(D)-MHC complexesweredetectedwithspecificCTLlinesgeneratedfrom AAD mice and analyzed by staining for CD8 and intracellular IFN-␥. BAG6knockdowndidnot alter Tyr369-377(D) presenta- tion (Fig. 3B) although no detectable BAG6 protein remained afterknockdown (Fig.3C). While BAG6seemed to bedispens- ableforTyr369-377(D)presentation,endogenousBAG6couldbe pulleddownwithtransientlyexpressedtyrosinaseinHEK293cells (Fig.3D).Thisinteractionwasevenenhancedwhenproteasomal degradationwasblockedbyMG132treatmentindicatingaroleof BAG6intyrosinasedegradation.

Overall,wefoundnoevidenceforanessentialroleofBAG6in generatingLCMV-ortyrosinase-derived ligandsfor thepresen- tationonMHCclassI.However,wecannotformallyexcludethe possibilitythatotherepitopesmightdependonBAG6foreffective processingandpresentation.

4. Discussion

Antigenprocessingandpresentationhavecrucial impactson theefficiencyandoutcomeofanimmuneresponse.Apossibilityto rapidlydetecttranslationofforeignproteinswithinacellistheuse ofDRiPsassubstratesforantigenprocessing.Asaconsequence,the

“immunopeptidome”surveyedbyCTLsmirrorstranslationrates ratherthanproteinconcentrations(Caronetal.,2011;Fortieretal., 2008;Khanetal.,2001).Bourdetskyandcolleaguescouldindeed showthatpeptidespresentedonMHCclassIshiftedfasterfrom lighttoheavyisotopeformsthantheirsourceproteinswhencells wereswitchedtomediacontainingaminoacidswithheavyiso- topes(Bourdetskyetal.,2014).Thisraisesthequestionwhether DRiPsandDRiP-derivedpeptidesaresimpleby-productsofpro- teinsynthesisorifcellshaveevolvedmechanismsregulatingDRiP formationand/orprocessing.Yewdell andNicchitta(2006)sug- gested the existenceof “immunoribosomes” specialized onthe production of substratesfor the MHC class Irestricted antigen processingpathway(YewdellandNicchitta,2006).Suchimmunori- bosomesmayhaveadifferentsubunitcompositionorlocalizeina differentintracellularcompartmentthanconventionalribosomes.

Althoughintellectuallyattractive,thishypothesishasnotyetbeen confirmedexperimentally.Instead,ribosomesaremorelikelyto functionasaplatformforassociatedfactorsactinguponthenascent chainandinfluencing itsfate(Krameret al.,2009).Whilemore andmorefactorsarebeingidentifiedtomediateco-translational andpost-translationalproteinqualitycontrollittleisknownabout thecontributionof thesefactors toantigen processing.Specifi- callydesigned torecognize non-nativepolypeptides chaperones are amongst the candidates ideally suited for DRiP processing.

Chaperonesmediateanimportantlayerofqualitycontrolandcan facilitatedegradationof non-nativepolypeptidesthrough inter- actionwithfactorsoftheubiquitin-proteasomesystem(Hohfeld etal.,2001;Rodrigo-BrenniandHegde,2012).

The chaperone BAG6is involved in post-translational inser- tion of tail-anchored proteins into the ERmembrane (Leznicki etal., 2010;Mariappan et al.,2010).While assistingin protein biosynthesis ontheone hand,BAG6alsomediates degradation ofmislocalizednascentchains(Hessaetal.,2011)andERADsub- strates(ClaessenandPloegh,2011;Claessenetal.,2014;Payapilly andHigh,2014;Wangetal.,2011).Moreover,Rodrigo-Brennietal.

(2014)recentlydescribedRNF126asanE3ligaseinteractingwith BAG6andspecificallyubiquitylatingchaperoneboundclientpro- teins(Rodrigo-Brennietal.,2014).ThefactthatBAG6hasbeen describedasa“holdase”conferringnoevidentfoldingactivityonto itssubstratesmakesthis chaperoneevenmoreinteresting with regard toproteinquality controland degradation (Wang etal., 2011).

Here,weaddressedwhetherBAG6caninfluenceantigenpro- cessingand presentationintheMHCclassIrestrictedpathway.

Knockdownof BAG6had noinfluence onMHC classI cellsur- face expression indicating that loss of BAG6 can be effectively counterbalancedbyothercytosolicchaperonesunderphysiological conditions.ThisresultisincontrasttoapreviousreportbyMinami etal.(2010)whofoundreducedMHCclassIcellsurfaceexpression inBAG6knockdowncells(Minamietal.,2010)butinagreement withamorerecentstudybyKochandcolleagueswhodidnotfind suchaneffect(Kamperetal.,2012).Wehavecurrentlynoexplana- tionforthisdiscrepancy,especially,sincewecouldnotdetectany residualBAG6expressionaftertransfectionwithBAG6siRNA.Pre- sentationofthreeLCMV-derivedepitopeswasentirelyunaffected

withanexpressionconstructfortyrosinase(Tyr)-Myc-Flagoremptyplasmid(control)andtreatedwith10␮MMG132orsolventDMSOfor5h.Celllysatesweresubjected toimmunoprecipitation(IP)withanti-Mycantibodyfollowedbywesternblot(WB)analysiswithBAG6orFlagspecificantibodies.Loadindicatesproteinexpressionlevels intotalproteinlysates.Forallexperimentsoneoutoftwoindependentexperimentsisshown.

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althoughoneoftheepitopesanalyzed(GP276-286)derivesfrom anER-targetedglycoprotein.Hence,degradationoftheGP276-286 precursorcouldbehandledbytheERADpathwayorthemislocal- izedproteindisposalpathwayinbothofwhichBAG6isinvolved.

Strikingly,thetyrosinase-derived epitopeTyr369-377(D), which strictly requires retrotranslocation from the ER in order to be presented,wasalsonotaffectedbyBAG6knockdown.Together, theseresultsindicate thatBAG6isdispensableforantigenpro- cessingandpresentationonMHCclassI.Enhancedinteractionof BAG6and tyrosinaseunderproteasome inhibitionontheother handstillindicatesthatBAG6couldbeapotentialplayerinanti- genprocessing.Theredundancyincellularchaperonenetworks could,however,maskthecontributionofBAG6tothispathway.

BAG6togetherwithitsE3 ligaseRNF126indeed resemblesthe HSP70/HSP90chaperonesystem,whichcooperateswiththeE3lig- aseCHIPtopromotesubstratedegradation(Connelletal.,2001;

Rodrigo-Brennietal.,2014).ThefindingthatBAG6substratescould stillbedegradedin theabsenceofBAG6and/orRNF126,albeit moreslowlyandlessefficiently,furtherindicatestheexistenceof oneorseveralredundantpathways(Rodrigo-Brennietal.,2014).

Moreover,BAG6andHSP70seemtocompeteforsubstratebind- ingasBAG6couldpreventHSP70-mediatedrefoldingofdenatured luciferaseinaninvitroassay(Wangetal.,2011).AlthoughBAG6 preferablybindstolongerhydrophobicpatches(Mariappanetal., 2010)andHSP70/HSP90toshorterhydrophobicpatches(Jackson, 2013;Rudigeretal.,1997)considerableoverlapinbothsystems islikely.Definitionofthepotentialproportionofantigenthatis handledbyBAG6wouldthereforebepossibleifBAG6-mediated degradationcouldbeinhibitedaftersubstratebindingtothechap- erone.Thereby,antigencouldbetrappedonBAG6anddegradation viaadifferentroutewouldbeblocked.

Compartmentalizedantigenprocessingwasfirstsuggestedby Levetal.(2010)(Levetal.,2010)andcouldbeanotherexplanation forourfindings.Inthiscase,BAG6mightbeinvolvedindegradation ofbulkproteinswhilebeingdispensableforepitopegeneration.

Takentogether,weproposethatBAG6isdispensableforantigen processingandpresentationonMHCclassIbutitmightbecome essentialunderconditionsoflimitedchaperoneavailability.

Conflictofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WethankRamanujanS.Hegde,VictorH.EngelhardandNorbert Kochforsharingantibodies,virusstrainsandcelllineswithus.

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