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The unique functions of tissue-specific proteasomes

Andrea Kniepert

1

and Marcus Groettrup

1,2

1DivisionofImmunology,DepartmentofBiology,UniversityofKonstanz,D-78457Konstanz,Germany

2BiotechnologyInstituteThurgauattheUniversityofKonstanz,CH-8280Kreuzlingen,Switzerland

The26Sproteasomeisthemainproteaseineukaryotes.

Proteolysisoccurswithinthecylindrical20Sproteasome thatisconstitutivelyexpressedinmosttissues.Howev- er,threetissue-specificversionsofthe20Sproteasome havebeendiscoveredtodate.Theimmunoproteasome isoptimizedtoprocessantigensanditdirects thedif- ferentiationofThelper(Th)cells.Thethymoproteasome isselectivelyexpressedincorticalepithelialcellsofthe thymuswhereitplays anessentialrole inthe positive selectionofTlymphocytes.Finally,thespermatoprotea- someisfound inthetesteswhereitisrequiredduring spermatogenesis. Here, weoutline howtissue-specific proteasomesadapt tofunctionalneedsintheirrespec- tive tissues andhow their selective inhibition may be usedtointerferewithautoimmunediseasesandcancer.

Theproteasome:evolutiontowardscomplexityand specialization

The proteasome is an ancient enzyme that has steadily evolvedtowardsahighercomplexityofsubunitsandreg- ulatorswhile preserving itscylindrical architecture con- stituted from fourstacked rings. In the archebacterium Thermoplasmaacidophilum,the20Sproteasomeconsists of two outer rings with seven copies of a single a-type subunitandtwoinnerringswithsevencopiesofthesame b-typesubunit[1](Figure1).Intheeubacterialactinomy- ceteRhodococcus sp.,the20Sproteasomeisconstructed fromtwodifferenta-typeandtwodifferentb-typesubunits [2],whereasintheeukaryoticyeast Saccharomycescere- visiae, it consists of seven different a-type and seven different b-type subunits [3]. Proteolysis takes place in theinnerchamberoftheproteasomeformedbythetwob rings.Althougheachofthebsubunitsofthearchebacterial andeubacterialproteasomescarrypeptidolyticallyactive centers,onlythreeoftheseveneukaryoticb-typesubunits are catalytically active. It appears that the increase in complexity is accompanied with a reduction in the number of active b subunits;thedrivingforcefor which has remained elusive. This tendency goes along with a

specializationofsubstratespecificityofthesethreebsub- units,whichismeasuredviathehydrolysisofsmallfluoro- genicpeptideswithcertainaminoacidsintheP1position.

Thesedefinethechymotrypsin-likeactivity(cleavageafter hydrophobicresidues), thetrypsin-likeactivity(cleavage after basicresidues),andthecaspase-likeactivityofthe proteasome(cleavageafteracidicresidues).Althoughthe archaealandbacterialproteasomesonlyshowchymotryp- sin-likeactivity,theeukaryoticproteasomesshowallthree activities that are associated with the b1 (caspase-like activity), b2 (trypsin-like activity), and b5 (chymotryp- sin-likeactivity)subunits.Theseb-typesubunitsofeukar- yoteshavebeennamedaccordingtotheirpositionintheb ring[3](Figure1).

Interestingly,for thenormalhousekeeping functionof theproteasome inproteolysis of regulatory or misfolded proteins, this diversification is not necessary. Yeast mutants thatonly harborthechymotrypsin-like activity remainedviable[4].Whetherspecializedproteasomesub- unitsareneededtocopewithanenhancedproteindegra- dation load is currently debated (see below). However, whenlooking atthepeptideproductsof theproteasome, the cleavage preferences of b1, b2, and b5 make a big difference.Proteasomeproductsareusedbytheimmune systemtoenableantigenrecognitionbyTlymphocytesand proteasomal peptideproducts mayeven have as yetun- known signaling functions in spermatogenesis or other aspects of cell biology. The functional specialization of b1, b2, andb5 enablesthe modification of the cleavage preferences of the proteasome by replacing them with inducibleortissue-specifichomologs.Nexttotheb1–b2–

b5containing‘constitutive’proteasome,threefurtherpro- teasomesubtypeswithdifferingsubunitcompositionhave been found: the immunoproteasome, the thymoprotea- some, and the spermatoproteasome (Figure 2). Recent insights, such as how their special subunit composition enablesthemtoservefunctionsthattheconstitutivepro- teasomecanonlyexertsuboptimally,arediscussedinthis review.

Theimmunoproteasomeinantigenprocessing

The immunoproteasome is a tissue-specific complex be- causeitiscontinuouslyexpressedincellsoftheimmune systemsuchasTcells,Bcells,monocytes,macrophages, dendritic cells, or medullary thymic epithelial cells [5].

However,itisalsostronglyinduciblebytheproinflamma- torycytokinesinterferon(IFN)-gandtumornecrosisfactor (TNF)-ainvirtuallyalltissuesexceptforthebrain,where

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immunoproteasome expression is largely confined to microgliaandinvadingleukocytes[6].Remarkably,immu- noproteasomesreplace up to 90%of constitutiveprotea- somesintissuesinthecourseofviral,bacterial,orfungal infections[5–7],whichraisesthequestionwhytheimmune system induces suchan extensivechangeinproteasome composition. Interestingly, this arguesthat immunopro- teasomes can take over the housekeeping functions of constitutiveproteasomesforseveraldayswithoutharming theinflamedtissues.

Inthe immunoproteasomeallthreeactivesitebearing subunitsoftheconstitutive20Sproteasome(b1,b2,andb5) are replaced by homologous, cytokine-inducible subunits namedb1i[lowmolecularmasspolypeptide(LMP)2],b2i [(multicatalytic endopeptidase complex-like-1 (MECL-1)], andb5i(LMP7),respectively.Twosubunits oftheimmu- noproteasome,b1iandb5i,werediscoveredduringgenomic sequencingofthemajorhistocompatibilitycomplex(MHC) genelocusovertwodecadesago,whereastheb2isubunit wasdiscoveredlaterbecauseitisnotencodedintheMHC locus[8].ManymembersoftheMHCclassIandIIantigen presentationprocessing pathwaysareencodedwithinthe MHClocus,whichhadfocusedimmunoproteasomeresearch ontheirroleinantigenprocessing[9].

ProteasomeactivityisrequiredforMHCclassIbutnot classII restrictedantigen presentation.This wasshown withbroadspectrumproteasomeinhibitorsthatinterfered withMHCclassIreleasefromtheendoplasmicreticulum (ER)andclassIcellsurfaceexpression[10–12],whereas theclassicalMHCclassIIpathwaywasbarelyaffectedby them. Theproteasomegeneratespeptidefragments from proteinantigensthatbindintothepeptide-bindinggroove of classImolecules.Theycanbegeneratedintheirfinal lengthby proteasomes eitherinthecytoplasm orin the nucleus. Theycanalso begenerated asN-terminally ex- tendedprecursorsthataresubsequentlytrimmedeitherin thecytoplasmoraftertransportintothelumenoftheER where theymeet partiallyfolded MHC classI/b2-micro- globulincomplexes[8].Onlyifagivenpeptidebindstightly intothepeptideligandcleftofclassImoleculesdoesthe trimericcomplexfinallyfoldandgetreleasedfromtheER

tothecellsurface.There,theantigenreceptorofcytotoxic TlymphocytescanrecognizepeptideplusMHCandkillthe cellthatpresentstheircognate antigens.The criteriafor tightpeptide–classIbindingare(i)alengthof8–9amino acids and (ii) the presence of anchor residues at the C terminus of the peptide and somewhere else within the peptide sequence. The C-terminal anchor residues in humans can beof basic orhydrophobic nature, whereas mouseclassImoleculesonlyacceptpeptideswithhydro- phobic C termini. Theserather tightacceptance criteria render it difficult to cleave out a sufficient number of peptideligandstomountanimmuneresponseespecially againstsmallvirusesthatencodeonlyafewproteins.Itis certainly one of the contributions of the immunoprotea- some to a successful cellular immune response against virusesorintracellularbacteria thatitsalteredcleavage prioritiesenhancetheversatilityofantigenprocessingand thechancetogenerateapivotalantigenicpeptide[8].

The recently reported high-resolution X-ray crystallo- graphicstructures of themouseconstitutiveproteasome andimmunoproteasomeallowasidebysidecomparisonof thethree differentsubstrate-binding pockets thatdefine thecleavageprioritiesofthetwocomplexes[13].Themost striking differenceisthatbetweenb1andb1i.Although theformeraccommodatespeptideswithanacidicP1resi- due(caspase-likeactivity),thelatterbindspeptideswitha hydrophobicP1 residue.For unknownreasons, thehun- dredsofallelicvariantsofmouseandhumanMHCclass I molecules do not accept peptide ligands with acidic C-terminal anchorresidues.The proteasomeisthemain determineroftheCterminusofantigenicpeptides,there- fore, b1 products are lost for antigen presentation. The replacement of b1 with b1i avoids this loss, generating usefulclassIligandswithhydrophobicCtermini,there- fore enough ligands becomeavailable to sustaintheap- proximately tenfold upregulation of class I cell surface expression afterstimulationwithIFN-g.Thisadvantage most likely explains, at least in part, a phenotype of immunoproteasome-deficient mice: they show a 50% re- ducedMHCclassIsurfaceexpressionoflymphocytesand dendriticcells[14–16].Alsothereplacementofb5byb5i (A)

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Figure 1.Theproteasomein threekingdoms oflife. 20Sproteasome modelsinthebarrel-shapeda1–71–7b1–7a1–7pattern.(A)Proteasomefromarchaebacterium Thermoplasmaacidophilum.Thearchaebacterialproteasomeiscomposedoftwoouterandtwoinnerrings;eachofwhichcontainssevenidenticalasubunits(inred)orb subunit(gray),respectively.(B)Amongbacteria,proteasomeshavebeenfoundineubacterialactinomycetales.TheproteasomefromRhodococcuserythropolisisbuiltof twodifferenta(redandorange)andtwodifferentbsubunits(grayandgreen),whicharemostlikelyrandomlyarrangedintherespectiveaandbrings.(C)Proteasome fromtheeukaryoteSaccharomycescerevisiae.Sevendifferentaandsevendifferentbsubunitshavetheirdefinedpositionsintheeukaryotic20Sproteasome.

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likely contributes to this phenomenon because the S1 pocket of b5i better accommodates larger hydrophobic aminoacidsidechains,whicharethepreferredC-terminal anchorresiduesformanyclassImolecules.Moreover,b5i has a more hydrophilic surrounding for the active site threonineandformsaspecifichydrogenbondthatstabi- lizes the tetrahedral transition state during catalysis, which could together kinetically favour the peptidolytic activityofb5i[13].Bycontrast,thereplacementofb2with b2iisenigmatic,becausebothsubstratepocketsarevirtu- allyidenticalandtheyshowthesametrypsin-likeactivity [17,18].Nevertheless, b2iknockoutmice haveclearphe- notypes(seebelow),whichmay haveastructuralrather thanacatalyticbasis[19,20].

Comparedtotherelativelyminorconsequenceofimmu- noproteasomedeficiencyonbulkMHCclassIpeptideload- ingandsurfaceexpression,thelossofb1i,b2i,orb5ican haveenormouspositiveandnegativeconsequencesforsin- gleepitopes.Someepitopesareimmunoproteasomedepen- dentintheirgeneration[21–24],whereasothers–including importantTcellepitopesoftumorantigens–aredestroyed byimmunoproteasomes[25,26].Aspecialcase,wherethe expression of a catalytically inactive b1i subunit could rescue an immunoproteasome-dependent T cell epitope, wasrecentlyexplained:theinactiveb1itakestheplaceof b1,whichwouldhavedestroyedtheepitopewithitscaspase- likeactivity[27].Previously,theimpactofimmunoprotea- somesonthepeptiderepertoirepresentedbyMHCclassI molecules has generally been underestimated [28]. The proteomicanalysisofpeptideselutedfromclassImolecules

ofsplenocytesfrommiceeitherproficientordeficientforall three immunoproteasome subunits revealed that about one-thirdofpeptideswereonlypresentedbywildtypecells, one-thirdwerepresentedonlybyb1i / b2i / b5i / cells, andanotherthirdwerepresentedbysplenocytesfromboth mouse strains[16]. This result implies that more T cell epitopesmustbeaffectedbyimmunoproteasomeinduction ininflamedtissuesthanpreviouslyexpected.

Initialreportsofthebiologicaleffectsofimmunoprotea- somedeficiencyweredisappointing.Lymphocyticchoriome- ningitisviruswasclearednormallyinimmunoproteasome- deficientmice[15],whereastherewasadefectreportedto clear Listeria monocytogenes from the liver but not the spleenofb5i / mice[29].However,arecentstudyrevealed that b5i-deficient mice succumb to an infection with Toxoplasmagondii that wildtype mice can survive[30].

Therefore, it is warranted to test further pathogens in immunoproteasome-deficient mice including the recently generatedtripleknockoutmice.Ifmorepathogensthatrely onimmunoproteasomesfortheirclearancecanbeidentified, andifimmunoproteasomesfulfilltheproteolytichousekeep- ingfunctionsasefficientlyasconstitutiveproteasomes,one maywonderwhyimmunoproteasomesarenotconstitutive- lyexpressedinalltissues.

Severalyearsagoweproposedthe hypothesisthatthe induction of immunoproteasome expression in inflamed tissues would focus the effector phase of the cytotoxic Tcellresponseonimmunoproteasome-dependentepitopes that would not bepresented inuninflamedtissues. This couldpreventaccidentallyactivatedself-reactivebystander (A)

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Figure2.Eukaryotic20Sproteasomesubtypes.(A)Constitutiveproteasome;subunitsthatdifferintissuespecific20Sproteasomesarehighlightedindifferentcolors.(B) Vertebratespossessinganadaptiveimmunesystemcanadditionallyexpresstheinducibleproteolyticsubunits b1i[lowmolecularmasspolypeptide(LMP)2],b2i (multicatalyticendopeptidasecomplex-like-1;MECL-1),andb5i(LMP7)thatareincorporatedintoimmunoproteasomes.(C)Thethymoproteasomeexpressedincortical thymicepithelialcells(cTECs)containstheproteolyticsubunitsb1i,b2i,andthethymus-specificsubunitb5t.(D)SpermatoproteasomesfromDrosophilamelanogasterare composed of several alternative subunits. Testis-specific subunits are: a3T, a4T1/2, a6T, b2R1/2, b4R1/2, and b5R1/2. (E) Mammalian spermatoproteasomes characteristicallyincorporatethetestis-specifica4ssubunit.

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T cells in inflamed tissues from finding the same self- epitopes elsewhere in the body [31]. We predicted that immunoproteasomes wouldthus contribute to protection fromautoimmunediseases.Recently,Zaissetal.reported evidence supporting this hypothesis. They showed that b2i / b5i / mice,whenirradiatedandreconstitutedwith bone marrow from the same strain, developed insulin- dependentdiabetesmellitusthatwascausedbycytotoxic Tlymphocytesrecognizingb-isletantigens[32].Moreover,it wasshownthatcertainallelicvariantsofb5iorb1iwere associatedwithhumantype1diabetes,andotherhuman autoimmuneorautoinflammatorydiseases[33–36].Taken together,itappearsthatontheonehandtheimmunopro- teasomeenhancesanddiversifiesthesetofclassIligands andontheotherhanditmaycontributetotheavoidanceof autoimmunity.

TheimmunoproteasomeinThcelldifferentiationand thepathogenesisofautoimmunity

A new unexpected function of the immunoproteasome, which isunrelatedto itsrole inantigen processing, has recentlybeendiscovered.WhenTcellslackingb2i,b5ior, toalesserextent,b1iweretransferredintovirus-infected recipientmice,theyfailedtosurviveincontrasttoimmu- noproteasome-proficientTcells[19,23].Thiswastruefor bothThcellsandcytotoxicTcells,therefore,thisphenom- enoncould notberelatedtoMHCclassIantigenproces- sing.ThenotionthatTcellsneedtheimmunoproteasome to survive inan inflammatory environmentsparked the idea thataselective inhibition of b5i may interrupt the pathogenetic function of T cellsinchronic inflammatory diseaseslikeautoimmunediseases.Remarkably,thetreat- ment of mice withtheb5i selective inhibitor ONX 0914 (formerlycalledPR-957)wasabletopreventtheprogres- sionofrheumatoidarthritisintwopreclinicalmousemod- elsand theinduction of Tcell-mediated type 1diabetes [37].Moreover,theinhibitionorthedeletionofb5iorb2i abrogatedthedextran-sulfate-sodium-mediatedinduction of colitis [20,38,39],andb5iinhibition preventedthede- velopment of lupus-erythematosus-like disease in lupus- pronemice[40].Thesefindingsmayopenanewapproach for the treatment of proinflammatory autoimmune dis- eases using immunoproteasome selective inhibitors. In addition,theyraisetheinteresting questionwhether the immunoproteasome is mechanistically involved inthese diseases. A firststep in supportof this ideamay bethe observationthattheinhibitionordeletionofb5ipartially suppressed the differentiation of the proinflammatory Th1-andTh17-typecells,while thedevelopmentofanti- inflammatory regulatory T (Treg) cells was suppressed [41].BothTh1andTh17cellsareinvolvedinthedevelop- mentofautoimmunediseases,whereasTregcellssuppress them.Mechanistically,silencingb5isuppressesphosphor- ylationofthetranscriptionfactorssignaltransducerand activatoroftranscription(STAT)1andSTAT3,whichdrive thedifferentiationofnaı¨veThelpercellstoTh1andTh17 cells,respectively[41,42].Howexactlyb5ienablesSTAT1 andSTAT3phosphorylationremainstobedetermined.An intriguing hypothesis isthat b5i processesa factor that promotesTh1andTh17differentiationdifferentlythanb5.

Ingeneral,theproteasomeisabletogeneratefactorsfrom

precursor proteins by limited proteolysis as has been shown for the nuclear factor (NF)-kB precursor protein p105[43]andtheplasmamembraneresidenttranscription factor precursor Spt23/MGA2 [44]. In fact, it has been reported that processing of p105 to p50 is specifically performedbytheimmunoproteasome[45],butthisconcept could notbe confirmedby others [46,47] and remains a matterofexperimentationanddebate.

Aspecialroleoftheimmunoproteasomeinthe accelerateddegradationofpolyubiquitinconjugates?

Stillanotherspecialfunctionoftheimmunoproteasomeis the enhanced degradation of polyubiquitin conjugates, whichwasrecentlyproposedbySeifertetal.[48].Itwas reportedthatafterstimulationoffibroblastsorHeLacells withIFN-gpolyubiquitinconjugatesaccumulatedfor8h and declined thereafter. The decline of ubiquitin conju- gatescorrelatedwiththeinductionofimmunoproteasomes inthesecells,whichledto thehypothesisthatimmuno- proteasomes might be needed to handle the excess of ubiquitin conjugates in IFN-g-stimulated cells. Such a transient rise and decline in polyubiquitin conjugates was not observed by Seifert et al. in IFN-g-stimulated mouse embryonal fibroblasts (MEFs) from b5i-deficient mice, supporting this concept. Instead, an accumulation ofubiquitin-containingproteinaggregateswasobservedin b5i-deficientMEFsaswellasinthebrainofb5i / mice sufferingfromexperimentalautoimmuneencephalomyeli- tis (EAE); a model for multiple sclerosis. In apparent agreement with the latter result, higher EAE disease scores wererecordedfor b5i / ascomparedtowildtype mice.The notionthatthe26Simmunoproteasomewould degradepolyubiquitylatedproteinsfasterthantheconsti- tutive26Sproteasomewasthenconfirmedbymonitoring the degradation of polyubiquitylated mucin by the two proteasesinvitro[48].

That IFN-g stimulationmight induceaprotease that wouldmoreefficientlydegradethemisfoldedproteinsthat italsoinducesisanappealingnotion.However,noneofthe aforementionedresultsbySeifertetal.[48]thatindicated a higher capacity of the immunoproteasome to degrade polyubiquitylatedproteinscouldbeconfirmedinthesame or very similarexperiments performed byNathan etal.

[49]. Instead,it was found that the immunoproteasome and the constitutive proteasome do not differ in their abilitytobindandtodegradepolyubiquitylatedproteins.

To identifydetails inexperimentalprocedures thatmay accountforthestrikinglydifferentoutcomesoftheexperi- ments in these two studies is beyond the scope of this review.However,itshouldbementionedthatnodifference in the amounts of polyubiquitylated proteins in spleno- cytesfromwildtypemiceandmicetriplydeficientforb1i, b2i,andb5icouldbedetectedbyRockandcolleagues[16].

Furthermore, there is no evidence that immunoprotea- somes associate preferentially with any of the known proteasomeregulatorsincludingthe19Sregulator,which polyubiquitylatedproteinsneedtodockontoinordertobe unfolded under ATP hydrolysis toenter the20Sprotea- some or20S immunoproteasome [50]. This isconsistent withtherecentlyelucidatedhighresolutioncrystalstruc- tures of mouse constitutive- and immunoproteasomes,

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whichstructurallydonotdifferintheira-endplateswhere allknownregulatorsbind[13].Therefore,itisdifficultto envisionhow immunoproteasomeswould degradepolyu- biquitylatedproteinsfasterthanconstitutiveproteasomes do.Takentogether,structuralconsiderationsandcompel- lingexperimentaldata[16,49]arguethatimmunoprotea- somes do not degrade ubiquitin conjugates faster than constitutiveproteasomes.

Theroleofthethymoproteasomeinselecting developingTlymphocytes

Sometimesdataminingcanbemorerewardingthangold mining.When Shigeo Murata,Keiji Tanaka, andcollea- guesweresearchingagenomedatabasefor proteasome- related genes they found an open reading frame with homology to b5 and b5i. This gene was exclusively expressed in the thymus, which led to its designation b5t (tfor thymus)[51]. Amoredetailed analysisof sub- populationsofcellsinthethymusrevealedthatb5twas exclusively expressed in epithelial cells of the thymic cortex. The thymusis aprimary lymphoid organ where immatureTcellsgaintheirantigenreceptorspecificityand where those T cells that bind to the MHC molecules encodedbytheindividual(self-MHC)arepositivelyselect- ed to survive and mature [52]. This positive selection occurs in the cortex of the thymus and is mediated by the highly specialized cortical thymic epithelial cells (cTECs). By contrast, T cells that recognize self-MHC andaself-peptidehavethepotentialtocauseautoimmu- nity,thustheyneedtobeselectedagainstandinducedto die by apoptosis. This process of negative selection is mediated by medullary thymic epithelial cells (mTECs) anddendritic cells,whichboth expressaboutequivalent levelsofconstitutiveandimmunoproteasomes[53,54].To determinethefunctionofthenewproteasomesubunitb5t, Murataetal.generatedab5t-deficientmousethatturned outtohaveadramaticphenotype.Althoughtheselectionof mature CD4+T helpercellswasnormal,theselection of matureCD8+cytotoxicTcellswasreducedby80%,leading toasimilarlystrongreductionofCD8+Tcellsinthespleen [51].Consequently,b5t / micesuccumbedtoaninfluenza virusinfectionthatwildtypemicesurvived[55].

Thestrongphenotypeofb5t / miceraisedthequestion ofwhytheb5orb5isubunitscannotcompensatefortheloss ofb5t.Aproblemwithproteasomeassemblycouldbeex- cludedbecausewithb5tdeficiencytheso-calledthymopro- teasomeofmousecTECsusestheb5iactivesitesubunitin its place[55]. Insight intothe unique function of b5t in thymicpositiveselectionwasderivedwhencomparingthe peptidolytic activities of b5t- and b5-containing protea- somes.Theb5t+proteasomeshadamuchlowerchymotryp- sin-like activity than b5+ proteasomes, whereas their caspase-like and trypsin-like activities were not affected byb5tincorporationintotheproteasome[51].Thischange in activity profile can be explained when looking at the aminoacidsthatlinethesubstratebindingpocketofb5t.

TheS1pocketsofb5andb5iaccommodatelargerhydro- phobicaminoacids, whereas fourreplacementsofhydro- phobicaminoacidsinthe S1pocketofb5andb5ibythe hydrophilicaminoacidsserineorthreonineinb5tpredict a cleavage preference for b5t after hydrophilic or less

hydrophobicamino acids. The resulting peptideproducts of b5t+ proteasomes are expected to beweak ligands of mouseMHCclassImolecules.Murataetal.proposedthat suchweakb5t-dependentclassIligands,whichhaveahigh offrate, might beaprerequisitefor asuccessfulpositive selectionofTcellsinaccordancewiththeaffinitymodelof thymicselection.Theaffinitymodelstatesthatlow-affinity bindingoftheTcellreceptor(TCR)toMHC/peptidecom- plexeswillleadtopositiveselection,whereashigh-affinity TCRbindingwillleadtonegativeselection[56].

Thestructural,enzymaticandfunctionaldataonb5tall areremarkablyconsistent.Thereisonlyoneexperimental aspectthatisdifficulttoreconcilewithalargebodyofdata.

IthasbeenshownthatMHCclassIligandswithweakC- terminalanchorresiduesarepoorstabilizersofclassIcell surfaceexpression[57].Nevertheless,adetailedanalysis ofsurfaceexpressionofthreedifferentclassImoleculeson cTECs fromwildtype andb5t / mice revealedthatthe lack ofb5tdid notaffectclassIsurfaceexpression [55].

PerhapscTECshavedevelopedmechanismsthatallowthe stabilizationevenofweakpeptide/classI/b2-microglobulin complexes on the cell surface. However, this and other hypotheseswillbedifficulttoaddressexperimentallybe- causefewcTECscanbeisolatedfromamurinethymus.

Thespermatoproteasomeinthegenerationof spermatids

Themostextensivetissue-specificalterationofproteasome subunitcompositionhasbeenreportedforthetestesandin particular for spermatids [58]. The differentiation from spermatogonial stemcells to mature spermsis ahighly regulatedcellularprocess thatincludesmitosis, meiosis, andmorphologicalchanges,whicharestepsthatmightall be dependent on proteasomal function. The existence of testis-specificproteasomesubunitswasfirstdescribedfor Drosophila melanogaster in the mid-1990s. Belote and coworkers reported two alternative a4-type subunits (a4T1 and a4T2) encoded by paralogous genes thatare exclusively expressed in the male germline [59]. Subse- quent studies revealed that proteasome subunit gene duplicationsarewidespreadinD. melanogaster:todate, 12ofthe3326Sproteasomesubunitshavebeenidentified toexistintwooreventhreedifferentisoforms.Although theconventionalformofeachsubunitcanbedetectedinall tissuesandduringalldevelopmentalstagesexamined,all additionalisoformsarespecificallyexpressedinthetestes (reviewedin[58]).Somealternativesubunitswerestudied inmoredetailusingreportertransgenes.Theseinvestiga- tionsshowedanexpressionpatternlimitedtomalegerm linecellsduringthemidtolatestagesofspermatogenesis, whereas only the conventionalproteasome subunits are expressedintheearlygonialstages[59–61].Inelongated spermatidsproteasomal ‘speckles’ containingalternative subunitscouldbeobservedincloseproximitytotheindi- vidualizationcomplex(IC)[60](Figure3).Thecytoskeletal membranousICharborsaclusterofactinconesandmed- iatesspermindividualizationbymovingdownthesperma- tidbundle, thus expellingcytoplasmand organellesand simultaneouslypackingeachspermatidinitsownplasma membrane.Thefunctionalconsequenceoftheproteasomal subcellularlocalizationattheIChasnotyetbeenclarified

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but,interestingly,knockoutofthealternativesubunita6T inD. melanogaster resultedin malesterility based on a defectivespermindividualization,whichwasaccompanied bydisruptedactinconecoordinationinICs[60].

Recent work detected the alternative a4-type protea- some subunit,a4s/PSMA8,inthemammaliantestes.Its expressionisrestrictedtospermatidsandmaturesperm, whichcorrespondtothelatestageexpressionof alterna- tivesubunitsinD.melanogaster[62,63].Astheproteaso- mal outer a ring lacks catalytic activity, thefunction of alternativeasubunitsislessclear.Onepossibilitycouldbe apreferred interactionwithcertainregulatory particles.

Besidesa4s,mammaliantestisproteasomeswerereported tocontainimmunoproteasomesubunits[63,64].However, b1i,b2i,and b5i showedpartly dissimilarcelltype and differentiation stage expression patterns. Clearly, lower amountsofimmunoproteasomescouldbedetectedintes- tescomparedtospleen[63].

Inadditiontothespecialsubunitcompositionofthe20S complex,theassociatedregulatoryparticlesarealsotestis specific.The proteasomeregulatorPA200isexpressedin differentmammaliantissuesbutitisparticularlyabundant intestis[65],wherePA200-cappedproteasomesarepresent assingleordouble-cappedcomplexesorintheformofhybrid proteasomesthatcarrybothPA200andthe19Sregulator, eachbindingonopposinga-endplatesofthe20Sbarrel[63].

The 200-kDa PA200isamonomeric non-ATPase protea- someactivatorthatattachestotheouteraringsofthe20S complexandenhancestheproteasomaldegradationofsmall peptidesbutnotubiquitylatedproteinsubstrates invitro [65].PA200knockoutmiceareviableandshownoobvious developmental abnormalities. However, PA200-deficient

malesexhibitamarkedreductionoffertilityduetodefective spermatogenesis [66]. A mechanistic explanation was recently suggested when Qianet al. showedthat PA200 promotes the acetylation-dependent degradation of core histonesduringsomaticDNAdamageresponsesandsper- matogenesis[63].Histoneacetylationweakensthehistone–

DNAinteractionandleadstoanopenandtranscriptionally active chromatin structure.Duringspermatogenesis,his- tone acetylation is followedby removal of histones from chromatin and their replacement by transition proteins and subsequently protamines, which allow denser DNA packing[67].Aninterestingquestion,whichhasnotbeen addressed yet, is whether the testis-specific a4s/PSMA8 subunit supports the docking of PA200 more than the conventionala4subunitdoes. Takentogether,PA200-de- pendent(andperhapsa4s/PSMA8dependent)proteasomal degradationofacetylatedhistonesmightrepresentanim- portant mechanismduringspermatogenesis that enables transcriptionarrest,spermatiddifferentiation,andchroma- tincondensation.

Concludingremarksandfutureperspectives

The existence of tissue-specific proteasomes or protea- somes that are only expressed during stimulation with cytokines pose many unanswered questions. Are there selectivesubstratesoftissue-specificproteasomes?Dotis- sue-selectiveproteasomesprocessprecursorproteinsina different manner as compared to constitutive protea- somes? Do tissue-selective proteasomes associate differ- ently with the increasing number of proteasome regulators?Apartfromthesefundamentalbiologicalques- tions tissue-specific proteasomes offer interesting new

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Figure 3.Imagesoftestis-specific proteasomesubunitsa3Tanda6Tintheindividualizationcomplex(IC)ofspermatidsinDrosophilamelanogaster.Duringthe individualizationofspermatidsthesyncytialspermatidbundleisresolvedintoseparatespermcells.OntheleftacartoonoftheICisdepicted;itiscomposedofactincones (orange)withthecysticbulge,whichisformedastheICmovesdownthespermatidbundle.Spermnucleiareatthetop(blue).(A)Specklesofa3T-GFP(green)trailingthe actincones(red).(B)Specklesofa6T-GFP(green)trailingtheactincones(red).(C)Immunostainingofwildtypetestiswithanti-proteasomeantibodies(green).(D) Immunostainingofa6T-GFPinthetestisofana6T-GFPtransgenicflywithanti-proteasomeantibodyshowingthecolocalization(yellow)ofa6T-GFPandproteasome signals.Reproduced,withpermission,from[58].

22

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perspectivesfortheirselectivepharmacologicalinhibition while leaving the activity of constitutive proteasomes intact.Broad-spectrum proteasomeinhibitors likeborte- zomib (Velcade) or carfilzomib (Kyprolis) are currently usedtotreatmultiplemyelomabuttheyneedtobeused closeto themaximallytolerateddosetobeeffective.The dose-limiting sideeffects stemfrom the inhibition of the housekeeping functions of the constitutive proteasome neededinvirtuallyalltissues[68].Subunit-specificprotea- someinhibitors,incontrast,maytargetdiseasesinatissue specificmanner.Theb5iinhibitorONX0914isaprominent examplebecauseitcansuppressautoimmunediseasesat doses far belowthe maximal tolerated dose inmice [37]

(Figure 4). Other immunoproteasome-specific inhibitors suchasUK-101andIPSI,whichbothinhibittheb1isub- unit,havebeendevelopedwiththeaimofimprovingcancer therapy[69,70].Althoughcell-permeableinhibitorsof the trypsin-likeactivityoftheproteasomeandimmunoprotea- somehavebeendevelopedsuccessfully,theydonotdiscrim- inatebetweenb2andb2i,probablybecausetheirsubstrate- bindingpocketissimilar[71].Itwillbeinterestingtofollow thefurtherdevelopmentofsubunit-selectiveinhibitorsand totesttheirtherapeuticpotentialinpreclinicalmodelsand inhumans.Apartfromautoimmunediseasesandcancerthe

in vivo manipulation of antigen presentation and T cell selectioninthethymusmightofferunanticipatedclinical opportunities.Testis-specificproteasomesubunitsorregu- lators,bycontrast,mayserveastargetsforthedevelopment ofmalecontraceptives.

Acknowledgments

WethankJohnM.Beloteforcriticalreadingofthemanuscript.Thiswork wasfundedbytheGermanResearchFoundationgrantGR1517/12-1,the KonstanzResearchSchoolChemicalBiology,theFritzThyssenFounda- tiongrantAZ10.10.2.122,andtheSwissNationalScienceFoundation grant31003A_138451.

References

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resolution.Nature386,463–471

4Heinemeyer,W.etal.(1997)Theactivesitesoftheeukaryotic20S proteasomeandtheirinvolvementinsubunitprecursorprocessing.J.

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5Barton,L.F. etal.(2002)Regulationofimmunoproteasomesubunit expressioninvivofollowingpathogenicfungalinfection.J.Immunol.

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7Khan,S.etal.(2001)Immunoproteasomeslargelyreplaceconstitutive proteasomesduringanantiviralandantibacterialimmuneresponsein theliver.J.Immunol.167,6859–6868

8Groettrup,M.etal.(2010)Proteasomesinimmunecells:morethan peptideproducers?Nat.Rev.Immunol.10,72–77

9Strehl,B.etal.(2005)Interferon-gamma,thefunctionalplasticityof theubiquitin-proteasomesystem,andMHCclassIantigenprocessing.

Immunol.Rev.207,19–30

10 Rock, K.L. et al. (1994) Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presentedonMHCclassImolecules.Cell78,761–771

11 Craiu,A.etal.(1997)Lactacystinandclasto-lactacystinbeta-lactone modifymultipleproteasomebeta-subunits andinhibitintracellular protein degradation and major histocompatibility complex class I antigenpresentation.J.Biol.Chem.272,13437–13445

12 Bai,A.andForman,J.(1997)Theeffectoftheproteasomeinhibitor lactacystinonthepresentationoftransportersassociatedwithantigen processing(TAP)-dependentandTAP-independentpeptideepitopesby classImolecules.J.Immunol.159,2139–2146

13 Huber,E.M.etal.(2012)Immuno-andconstitutiveproteasomecrystal structuresrevealdifferencesinsubstrateandinhibitorspecificity.Cell 148,727–738

14 Fehling,H.J.et al.(1994)MHCclass Iexpressioninmice lacking proteasomesubunitLMP-7.Science265,1234–1237

15 Basler,M.etal.(2011)Theantiviralimmuneresponseinmicedevoidof immunoproteasomeactivity.J.Immunol.187,5548–5557

16 Kincaid, E.Z. et al. (2012) Mice completely lacking immunoproteasomesshow major changes in antigen presentation.

Nat.Immunol.13,129–135

17 Salzmann,U.etal.(1999)Mutationalanalysisofsubunit ibeta2 (MECL-1)demonstratesconservationofcleavagespecificitybetween yeastandmammalianproteasomes.FEBSLett.454,11–15 18 Basler,M.etal.(2006)AnalteredTcellrepertoireinMECL-1-deficient

mice.J.Immunol.176,6665–6672

19 Moebius,J.etal.(2010)Immunoproteasomesareessentialforsurvival andexpansionofTcellsinvirus-infectedmice.Eur.J.Immunol.40, 3439–3449

20 Basler,M.etal.(2010)Preventionofexperimentalcolitisbyaselective inhibitoroftheimmunoproteasome.J.Immunol.185,634–641 21Sijts, A.J.A.M. et al.(2000) MHCclass I antigenprocessing ofan

AdenovirusCTLepitopeislinkedtothelevelsofimmunoproteasomes ininfectedcells.J.Immunol.164,4500–4506

iCP:PR957

β5i

β6

TiBS

Figure4.Structureofthemouseimmunoproteasomewiththeb5i(lowmolecular masspolypeptide7;LMP7)activesiteboundbytheb5iinhibitorONX0914(formerly calledPR-957).SurfacecolorsintheConollysurfacerepresentationofb5iindicate positiveandnegativeelectrostaticpotentialscontouredfrom50kT/e(intenseblue)to –50kT/e(intensered).Inordertoillustrateconformationalchangesinducedbythe inhibitor,theaminoacids46–50wereremoved.Thr1oftheb5isubunitiscoloredin whiteandthecovalentlyboundinhibitorinyellow.Thegreenarrowindicateswhere thephenylsidechainoftheinhibitorwouldclashwiththeconstitutiveb5subunit, whereasintheimmunoproteasomesubunitb5i,areorientationoftheCH3-S-group of Met45 avoidssuch a stericclash, thus explainingthe immunoproteasome selectivityofONX0914.Adapted,withpermission,from[13,72].

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