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OPENREVIEWPost-translationalregulationofinammasomesJieYang1,2,ZhonghuaLiu1andTsanSamXiao1Inammasomesplayessentialrolesinimmuneprotectionagainstmicrobialinfections.
However,excessiveinammationisimplicatedinvarioushumandiseases,includingautoinammatorysyndromes,diabetes,multiplesclerosis,cardiovasculardisordersandneurodegenerativediseases.
Therefore,preciseregulationofinammasomeactivitiesiscriticalforadequateimmuneprotectionwhilelimitingcollateraltissuedamage.
Inthisreview,wefocusontheemergingrolesofpost-translationalmodications(PTMs)thatregulateactivationoftheNLRP3,NLRP1,NLRC4,AIM2andIFI16inammasomes.
WeanticipatethatthesetypesofPTMswillbeidentiedinothertypesofandlesswell-characterizedinammasomes.
BecausethesehighlydiverseandversatilePTMsshapedistinctinammatoryresponsesinresponsetoinfectionsandtissuedamage,targetingtheenzymesinvolvedinthesePTMswillundoubtedlyofferopportunitiesforprecisemodulationofinammasomeactivitiesundervariouspathophysiologicalconditions.
Cellular&MolecularImmunology(2017)14,65–79;doi:10.
1038/cmi.
2016.
29;publishedonline27June2016Keywords:inammasome;phosphorylation;post-translationalmodications;ubiquitinationINTRODUCTIONTheinnateimmunesystemreliesonpatternrecognitionreceptors(PRRs)tosensemicrobialinfectionsandtissuedamageandtoinitiateinammatoryresponses.
1–3Inamma-somesconstituteagroupofPRRsthathaveessentialrolesinimmuneprotectionagainstbacterial,viral,fungalandparasiticinfections.
4–11Dysregulationofinammasomesisimplicatedinvarioushumandiseases,includingautoinammatorydisorders,12metabolicdisorders13,14andcancer.
15,16Eachtypeofinammasomeischaracterizedbyaparticularsensororreceptormoleculefromthenucleotide-bindingdomain(NOD),leucine-richrepeat(LRR)-containingprotein(NLR)family,orpyrinandHINdomain-containingprotein(PYHIN)family.
TheseincludetheNLRP1,4NLRP2,17NLRP3,18NLRC4,19–22NLRP6,23,24NLRP7,25NLRP12,26AIM227–30andIFI1631,32inammasomes.
NLRPproteinscontainanpyrindomain(PYD)attheirN-termini,whereasNLRC4containsacaspaseactivationandrecruitmentdomain(CARD).
33TheAIM2andIFI16proteinsareDNAsensorsthatcontainN-terminalPYDdomainsandC-terminalDNA-bindingHINdomains.
Theadaptermoleculeapoptosis-associatedspeck-likeproteincontainingacaspase-recruitmentdomain(ASC)consistsofN-terminalPYDandaC-terminalCARDdomainsthatfacilitateitsinteractionwithbothupstreamsensormoleculesthroughitsPYDdomainanddownstreamcaspase-1throughitsCARDdomain.
Assemblyofthecanonicalinammasomecomplexescontainingcaspase-1,aswellasthenoncanonicalinammasomescontainingcaspases-4,-5and-11thatareboundtointracellularLPS,34–36leadstoactivationofthesecaspases,whichpromotesmaturationandsecretionofproinammatorycytokines,suchasIL-1β,IL-18andIL-1α.
9,11Activatedcaspase-1andcaspase-11alsocleavegasderminD(GSDMD)andothersubstratesthatinducepyroptoticcelldeaththroughas-yetunknownmechanisms.
37–39Notallcelltypesundergopyroptosisuponinammasomeactivation.
Forexample,activationoftheNLRC4inammasomeinneutrophilsinfectedbySalmonellaleadstotheproductionofIL-1βwithoutapparentpyroptosis,thusfacilitatingclearanceofinvadingpathogensbytheneutrophils.
40–42Furthermore,theAIM2andNLRP3inammasomesarerecruitedtoautophagosomesfordegradation,whichservesasamechanismtoeliminateactivatedinammasomesandterminateinam-matorysignaling.
43Itisunclearwhetherotherinammasomescanalsobedegradedthroughsimilarmechanisms.
Becauseofthehighlyproinammatoryconsequencesofinammasomeactivation,thisprocessistightlyregulatedtolimitcollateraldamage.
Priortotheactivationofinamma-somesbydiversestimuliorligands,aprimingstep,whichcan1DepartmentofPathology,CaseWesternReserveUniversity,Cleveland,OH44106-7288,USAand2GraduatePrograminPhysiologyandBiophysics,DepartmentofPhysiologyandBiophysics,CaseWesternReserveUniversity,Cleveland,OH44106-7288,USACorrespondence:TSXiao,PhD,DepartmentofPathologyCaseWesternReserveUniversity,2103CornellRdWRB,Rm6533,Cleveland,OH44106-7288,USA.
E-mail:tsx@case.
eduReceived:10March2016;Revised:5May2016;Accepted:5May2016Cellular&MolecularImmunology(2017)14,65–79&2017CSIandUSTCAllrightsreserved2042-0226/17www.
nature.
com/cmibeprovidedbyligandsfortheToll-likereceptors(TLRs),isnecessary.
PrimingleadstotheactivationofNF-κBandexpressionofinammasomecomponents,suchassensormoleculesandthepro-formsofinammatorycytokines,suchaspro-IL1βandpro-IL18.
44Primingalsomodulatesthepost-translationalmodications(PTMs)ofNLRP3andASCtofacilitateassemblyofinammasomecomplexes.
45–47Infact,thePTMsofinammasomecomponentshaveemergedasamajorregulatorymechanismforinammatorysignaling.
Virtuallyallcellularprocessesareregulatedbyacombinatorialpost-translationalmodication'code'thatchangesthesurfacefeaturesofproteinstoendowthemwithdiversefunctionalities.
Ofthesemodications,phosphorylationandubiquitinationareamongthebestcharacterized.
48–51PTMscancovalentlymodify15ofthe20aminoacidsorcauseproteolyticalcleavageatspecicresiduestoregulatethesize,conformation,location,turnoverandinteractionoftargetproteins.
ThesePTMsarecatalyzedbyhundredsofenzymesthatarethemselvesmodulatedbybindingpartnersorsmallmoleculeinhibitors.
Asaresult,targetingtheseenzymesmayofferopportunitiesforne-tuninginammatorysignalingprocessesfortherapeuticbenetsininfectiousdiseasesandmetabolic,autoimmuneandautoinammatorydisorders.
52,53Inthisreview,wefocusonrecentdevelopmentsinpost-translationalmodicationofinammasomecomponents,suchassensor/receptormoleculesandtheadapterASC,throughphosphorylation,ubiquitination,proteolyticprocessingandothermechanisms(Table1andFigure1).
Convergenceofthesepost-translationalmodicationscontributestocompre-hensiveandbalancedimmunesurveillancemechanisms.
WeanticipatethatknowledgeregardingPTMswillexpandfromtheseexamplestootherPTMtypesandlesswell-characterizedinammasomes.
BecausehighlydiverseandversatilePTMsshapedistinctinammatoryresponsestowardinfectionsandresultingfromtissuedamage,theroleofpost-translationalmodicationsasaregulatorymechanismforinammasomeactivationwillremainanexcitingareaofresearchfortheforeseeablefuture.
OthermechanismsofinammasomeregulationrelevanttoNLRP3wererecentlyreviewedbyJoetal.
54PHOSPHORYLATIONPhosphorylationisthemostcommonpost-translationalmod-icationandinvolveshundredsofkinasesandphosphatasesthatdynamicallymodulatetheproteome.
ItiswellestablishedthatseveralinammasomesensormoleculesandtheadapterASCareregulatedbydistinctphosphorylationevents,someofwhichhavebeenmappedtospecicresiduesofrelevantinammasomecomponents.
Inmostcases,theexactmechanismsbywhichphosphorylationoftheseproteinsaffectstheirstructureandfunctionremaintobeclaried.
PhosphorylationofASCmodulatestheactivationofmultipleinammasomesASCisanessentialadaptermoleculeformostinammasomesensors.
55PhosphorylationofASCwasreportedsoonafteridenticationoftheinammasome,56buttheroleofitsphosphorylationininammasomeactivationremainedunclear.
Morethanadecadelater,itwasdiscoveredthatinmacrophages,ASCphosphorylationwasrequiredforactivationoftheNLRP3andAIM2inammasomes,butnotforthatofNLRC4.
57,58Specically,SykandJnkkinasephosphorylateTyr144withintheCARDdomainofmurineASC57orTyr146andTyr187inhumanASC.
58ThisphosphorylationeventfacilitatesformationoftheASCspeckintheperinucleararea.
Interestingly,inhibitionofSykandJnkkinaseactivitiesdoesnotdisrupttheinteractionbetweenASCandNLRP3,whichsuggeststhatphosphorylationofASCmayimpactdownstreameventssuchasitsabilitytomigratetotheperinuclearregion,self-associateorrecruitprocaspases-1throughCARD–CARDinteractions.
ThismayalsoexplainthefactthattheNLRC4inammasomeisnotaffectedbytheASC-CARDphosphoryla-tionbecauseNLRC4containsaCARDthatcandirectlyengageprocaspase-1intheabsenceofASC.
20,21,59Incontrasttotheabovestudiesinmacrophages,phosphorylationofASCdoesnotappeartobeimportantforinammasomeactivationinbonemarrow-deriveddendriticcells.
57Themechanismsforsuchcell-typespecicityandinammasomeselectivityremaintobedetermined.
Furthermore,anotherstudyreportedthatSykdeciencyinmacrophagesdidnotreduceactivationoftheNLRP3inammasome.
60Thereasonforthisdiscrepancyisnotclear,butmayberelatedtothedifferentmousestrainsusedinthestudies.
Bruton'styrosinekinase(BTK)wasshowntointeractwithbothASCandNLRP3throughitskinasedomainand,possibly,tophosphorylateASC,whichpromotesactivationoftheNLRP3inammasome,butnottheAIM2inammasome.
61Importantly,treatmentwithanFDA-approvedBTKinhibitorhasaneuroprotectiveeffectinabrainischemiamodelinmice,whichsuggestspotentialrolesforBTKandinammasomesinbrainischemia.
InadditiontotheseexamplesofASCphosphorylationthatenhancesinammasomeactivation,therearealsophosphor-ylationeventsthatsuppressinammasomeactivation.
LossofIκBkinaseα(IKKα)kinaseactivityisassociatedwithsponta-neousinammationandlungcancer.
62,63ThispromptedaninvestigationoftheroleofIKKαininammasomeactivation.
64ItturnedoutthatIKKαfunctionsasanegativeregulatoroftheNLRP3,AIM2andNLRC4inammasomesthroughitsinter-actionwiththeadapterASC,bywhichitreducesthetranslocationofASCfromthenucleustothecytoplasm.
64TheresiduesSer193intheCARDandSer16inthePYDdomainofASCarephosphorylatedbyIKKα,whichisimportantfortheASC–IKKαassociationinthenucleusthatpreventsitstranslocationtothecytoplasmforinammasomeactivation.
MutationsofeitherresidueresultinenhancedactivationofboththeNLRP3andAIM2inammasomes.
InagreementwiththeroleofIKKαasanegativeregulatorofinammasomes,stimulationoftheNLRP3inammasomewithATPornigericinsuppressesthekinaseactivityofIKKαanditsassociationwithASC,perhapsthroughdephosphoryla-tionofIKKαbytheserine/threoninephosphatasePP2A.
Post-translationalmodicationsofinammasomesJYangetal66Cellular&MolecularImmunologyTable1Regulationofinammasomeactivitiesthroughpost-translationalmodicationsPost-translationalmodicationsModicationsites/typesEnzymes/triggersEffectsoninammasomeactivationReferencesPhosphorylationASCTyr144(mouse)Tyr146,Tyr187(human)Syk/Jnk↑NLRP3↑AIM2-NLRC4Haraetal.
,57Linetal.
58BTK↑NLRP3-AIM2Itoetal.
61Ser19andSer193IKKα↓NLRP3↓AIM2↓NLRC4Martinetal.
64Ser58IKKi↑NLRP3↑AIM2Martinetal.
64NLRP3Syk,DAPK,TAK1,ERK1↑NLRP3Grossetal.
,66Said-Sadieretal.
,67Shioetal.
,68Wongetal.
,69Chuangetal.
,70Gongetal.
,71Ghonimeetal.
72PKRNLRP3NLRP1NLRC4AIM2Luetal.
,73Heetal.
,76Hettetal.
,75Yimetal.
,77Boriushkinetal.
74IRAK1,IRAK4↑NLRP3Fernandes-Alnemrietal.
78NEK7↑NLRP3-AIM2-NLRC4Shietal.
,79Schmid-Burgketal.
,80Heetal.
81NLRC4Ser533PKCδ↑NLRC4Quetal.
,100Yangetal.
,93Huetal.
,98Suzukietal.
,94Matusiaketal.
101UbiquitinationASC/K63-linkedpoly-Ub↓NLRP3↓AIM2Shietal.
43/linearpoly-UbLUBAC↑NLRP3↑AIM2-NLRC4Rodgersetal.
47Lys174/K63-linkedpoly-UbTRAF3↑NLRP3-AIM2-NLRC4Guanetal.
109NLRP3Lys689/K48-linkedpoly-UbFBXL2↓NLRP3Hanetal.
112/K48-linkedpoly-UbMARCH7↓NLRP3-AIM2-NLRC4Yanetal.
113XIAP↓NLRP3Yabaletal.
119NLRC4Sug1↑NLRC4Kumaretal.
129IFI16ICP0(HSV-1)↓IFI16-AIM2-NLRP3Orzallietal.
,143Johnsonetal.
,144Kalamvokietal.
,145Cuchet-Lourencoetal.
146Caspase-1↓NLRP3↓AIM2↓NLRC4VanOpdenboschetal.
147DeubiquitinationNLRP3LRRdomain/K63-linkedpoly-UbBRCC3(mouse)/BRCC36(human)↑NLRP3Pyetal.
,46Lopez-Castejonetal.
148(Maytargetpro-IL1β)A20↓NLRP3VandeWalleetal.
,126Duongetal.
127Post-translationalmodicationsofinammasomesJYangetal67Cellular&MolecularImmunologyThisdephosphorylationeventappearstobeuniquetoNLRP3becauseactivationoftheAIM2inammasomeisnotaffectedbythePP2Aknockdown.
64Therefore,activationoftheAIM2inammasomemaybeprecededbyothermechanismsthatinactivateIKKα.
InstrongcontrasttoIKKα,theIKK-relatedkinase(IKKi)phosphorylatesASCatSer58,whichfacilitatesitsnucleus-to-cytoplasmtranslocationandpromotesinamma-someactivation.
64DeciencyofIKKiresultsinreducedactivationofboththeNLRP3andAIM2inammasomes.
Howthesetworelatedkinasesmodulateinammasomeactivationincompletelydifferentmannersremainstobedeterminedbutmayberelatedtotheirparticipationindistinctsignalingpathways:amaindifferencebetweenthefunctionsofIKKαandIKKiisthattheformerisprimarilyinvolvedinactivationoftheNF-κBpathway,whereasthelatterisimplicatedinactivationoftheinterferonresponsefactor(IRF)3and7.
65MultiplekinasesregulatetheNLRP3inammasomeTheNLRP3inammasomerespondstostructurallyandchemicallydiversestimulifrominfectionsandtissuedamage.
6,55SimilartoASC,NLRP3hasbeenreportedtobephosphorylatedbySyk,whichisessentialfortheactivationoftheNLRP3inammasomeuponinfectionbyfungal,malarialandmycobacterialpathogens.
Forexample,activationoftheNLRP3inammasomebythefungalpathogensCandidaalbicansorAspergillusfumigatus,butnotbySalmonellainfec-tionornigericintreatment,wasshowntobedependentonSykkinase,whichinducesROSproductionandpotassiumefux.
66,67Similarly,SykwasphosphorylatedandactivatedbySrckinasesuponexposuretohemozoinduringPlasmodiuminfection,perhapswiththeeffectofinitiatingimmuneresponsesthroughtheNLRP3inammasome.
68Inaddition,activationoftheNLRP3inammasomebytheESX-1secretionsystemanditssubstrateESAT-6fromMycobacteriumtubercu-losiswasreportedtobedependentonSykkinaseactivity.
69ItisnotclearfromthesestudieswhetherSykdirectlyphosphor-ylatesNLRP3,althoughconceivablythephosphorylationofASCbySykcouldbeoneofthemechanismspromotinginammasomeactivation,asdiscussedabove.
57,58SimilartoSyk,DAPKkinasewasshowntoassociatewithNLRP3andcontributetoNLRP3inammasomeassembly.
70However,macrophagesdecientinDAPKstillretainedresi-dualcaspase-1activationandIL-1βmaturationinresponsetostimulationbymonosodiumurate(MSU)andATP.
Becausethekinase-inactiveDAPKmutantwasalsocapableofenhan-cingIL-1βsecretion,itappearsthattheroleofDAPKintheNLRP3inammasomeactivationisindependentofitskinaseactivity.
Inagreementwiththisconcept,theNLRP3LRRwasshowntoco-IPwiththeankyrinrepeats,deathdomainorcytoskeleton-bindingregionofDAPKratherthanwithitskinasedomain.
70AnotherkinaseimplicatedinNLRP3regula-tionisTAK1.
Throughscreeningofnaturalproductsandtestingofkinaseinhibitors,Gongetal.
71identiedTAK1asakinasethatpromotesNLRP3inammasomeactivationinamannerindependentofitsroleintheactivationofgeneexpression.
ItwassuggestedthatTAK1mayregulatetheNLRP3inammasomethroughphosphorylationofNLRP3orothercomponentsoftheinammasomecomplex.
Similarly,theERK1kinase-mediatedphosphorylationeventswerereportedtobeessentialforprimingoftheNLRP3inammasome.
72However,whetherNLRP3isphosphorylatedbythesekinaseswasnotaddressedinthestudiesdescribedabove.
Table1(Continued)Post-translationalmodicationsModicationsites/typesEnzymes/triggersEffectsoninammasomeactivationReferencesProteolyticprocessingNLRP1(b)(murine)Lys44(mouse)/Pro44(rat)LF(anthrax)↑NLRP1(b)Chavarria-Smithetal.
,152Levinsohnetal.
,154Hellmichetal.
,155Boydenetal.
153NLRP3Gly493,Gln2252Aand3C(EV71)↓NLRP3Wangetal.
161Zmp1(mycobacteria)↓NLRP3↓NLRC4Masteretal.
,180Wongetal.
69S-nitrosylationCaspase-1Cys284Nitricoxide↓InammasomeKimetal.
185NLRP3Nitricoxide/SNAP↓NLRP3Mishraetal.
,186Hernandez-Cuellaretal.
,187Guardaetal.
,188Maoetal.
189ADP-ribosylationNLRP3CARDStoxin(mycoplasma)↑NLRP3-NLRP1Boseetal.
194Note:↑,stimulateinammasomeactivation;↓,suppressinammasomeactivation;-,noeffectsoninammasomeactivation;,notdeterminedorconictingreportsoninammasomeregulation.
Post-translationalmodicationsofinammasomesJYangetal68Cellular&MolecularImmunologyPKRisadsRNA-dependentproteinkinase.
ItundergoesautophosphorylationuponinammasomeactivationandisbroadlyimplicatedinpromotingactivationoftheNLRP3,NLRP1,NLRC4andAIM2inammasomes,butnottheNOD1,NLRP12orNLRX1inammasomes.
73–75PKRisphysicallyassociatedwithNLRP3inhumanTHP-1cells,andco-immunoprecipitationexperimentsinwhichPKRandNLRP3wereoverexpressedinHEK293cellsshowedthatthePYD,NODandLRRdomainsofNLRP3wereassociatedwithPKR.
73BycomparingthewildtypewiththemutantPKR,Luetal.
showedthatthekinaseactivityofPKRisrequiredfortheactivationoftheNLRP3inammasome.
Incontrast,Hettetal.
75demonstratedthatknownPKRinhibitorsdidnotreducetheactivationofNLRP3orNLRP1inammasomes.
Inagreementwiththeseresults,Heetal.
76demonstratedthatalthoughPKRenhancedthegenerationofiNOSandbacteria-killingbymacrophages,itwasnotrequiredforactivationoftheNLRP3,NLRC4andAIM2inammasomesintermsofcaspase-1activationandIL-1β/IL-18processing.
Furthermore,Yimetal.
77showedthatthekinaseactivityofPKRsuppressedNLRP3inammasomeactivationbyinhibitingtheexpressionofNLRP3andpro-IL-1β.
Itwasproposedthattheseapparentdiscrepanciesmaybeduetotheuseofdifferentmacrophagecellsorcelllines,aswellasdifferentmousestrains,andtothemultipleeffectsofPKRonproteinexpressionandsignaling.
77Clearly,themechanismsofPKRregulationofinammasomesremaintobefurtherclaried.
TheIL-1R-associatedkinasesIRAK1andIRAK4functiondownstreamoftheTLRsinresponsetoprimingsignalsthatenhancethetranscriptionofinammasomecomponents.
44Fernandes-Alnemrietal.
78showedthatthesetwokinaseswerealsoimplicatedintherapidactivationofNLRP3byListeriamonocytogeneswithin45minofinfection.
TheyfurtherdemonstratedthatthisrapidNLRP3activationwasindepen-dentoftranscriptionbutrequiredgenerationofreactiveoxygenspecies.
Althoughco-immunoprecipitationexperimentshaveshownthatIRAK1associateswithNLRP3,itisnotclearwhetheritskinaseactivityisrequiredforthisrapidNLRP3activationorwhetheranyoftheNLRP3inammasomecomponentsarephosphorylatedbyIRAK1.
78Recently,theserine–threoninekinaseNEK7wasidentiedbythreeindependentgroupsasbeingessentialforactivationoftheNLRP3inammasome,butnottheAIM2orNLRC4inammasomes.
79–81Diversescreeningapproacheswereusedbythesegroups,includingforwardgeneticscreeningforN-ethyl-N-nitrosourea-inducedmutations,79genome-wideCRISPR(clusteredregularlyinterspacedshortpalindromicrepeats)screeninginmacrophages80andproteomicapproachesusingataggedNLRP3protein.
81TheinteractionofNEK7withNLRP3isdependentontheLRRandperhapstheNODdomainofNLRP3andthekinasedomainofNEK7.
However,mutationoftheNEK7catalyticresiduesK63,K64orG43didnotimpairIL-1βsecretionortheNEK7–NLRP3interaction.
79,81TheNEK7–NLRP3associationisnecessaryFigure1Combinatorialpost-translationalmodicationsregulateinammasomes.
Phosphorylation,ubiquitination,de-ubiquitination,proteolyticprocessing,S-nitrosylationandADP-ribosylationareindicatedaccordingtothekeysinthelowerleftcorner.
ThePTMsthatpromoteactivationofinammasomesareshownasblackarrows,whereasthosethatsuppressinammasomeactivationareshownasredblocks.
Post-translationalmodicationsofinammasomesJYangetal69Cellular&MolecularImmunologyfortherecruitmentofASCbyNLRP3andtheformationofASColigomers.
Inaddition,activationoftheNLRP3inam-masomeisblockedduringmitosis,whichsuggeststhatNEK7actsasaswitchbetweeninammasomeactivationandcelldivision.
Itskinaseactivityisdispensablefortheformerbutrequiredforthelatter.
79,82–84TheroleofphosphorylationinNLRC4inammasomeactivationNLRC4isoneofthekeyinammasomesensorsthatisresponsiblefortheimmuneresponsetobacterialinfections.
20–22,85Gain-of-functionmutationsinNLRC4alsoleadtoautoinammatorydisordersinpatients.
86–88MembersofthefamilyofNLRapoptosis-inhibitoryproteins(NAIPs)aredirectreceptorsforbacterialagellinandtypeIIIsecretionsystems.
89–95Ligand-boundNAIPsinduceawheel-likeassem-blyoftheNAIP-NLRC4inammasomesthatmaycatalyzetheactivationofoligomerizedcaspase-1.
96–99AnimportantregulatorymechanismforNLRC4wasrevealedthroughthediscoverythatmouseNLRC4inmacrophagesisphosphory-latedfollowinginfectionwithSalmonellatyphimurium.
100ThephosphorylationsiteSer533isconservedamongtheNLRC4proteinsfromdifferentspecies,whichsuggeststhatitispotentiallyimportantforthestructureorfunctionofNLRC4.
Biochemicalcharacterizationofkinaseactivities,screeningofkinaseinhibitorsandinvitrophosphorylationassaysidentiedPKCδasthekinasethatphosphorylatestheSer533residueinmouseNLRC4.
100Importantly,theS533AmutantwasnotabletoactivatetheNLRC4inammasome,whereasthephospho-mimeticNLRC4S533Dmutantinducedrapidpyroptosisinmacrophagesintheabsenceofinfection.
However,PKCδ-decientmacrophagesretainedresidualinammasomeactiva-tionuponS.
typhimuriuminfection,whichsuggeststhatotherkinasesprovidedcompensatorymechanisms.
Matusiaketal.
101furtherdemonstratedthatphosphorylationofNLRC4primedtheinammasomeforactivation.
However,thedetectionofbacteriabyNAIP5wasindependentofNLRC4phosphoryla-tion,althoughbothrequiredtheD0domainofthebacterialagellin.
Incontrasttothestudiesdescribedabove,reconstitu-tionoftheNAIP–NLRC4complexesinthepresenceofbacterialagellinorneedleproteinsdemonstratedthatthephosphorylationofNLRC4isnotrequiredforassemblyoftheinammasomesignalingcomplex.
93Inaddition,crystallizationofanapparentlyinactiveformofNLRC4inwhichSer533wasalreadyphosphorylatedhasbeenachieved.
98Inathirdstudy,PKCδdidnotappeartoplayanyroleinNLRC4activationuponShigellaorSalmonellainfection.
94Thereasonsfortheseapparentcontradictoryresultsarenotclear,andfurtherstudiesarerequiredtoclarifythemechanismsofNLRC4phosphory-lationandtheroleofPKCδininammasomeactivation.
UBIQUITINATIONProteinubiquitinationanddeubiquitinationareversatileanddynamicprocessesthatregulatethedegradation,trafckingandsignalingpropertiesoftheproteomeinnumerousaspectsofthebiologicalprocesses.
102–105ConjugationoftheubiquitinchainsismediatedbyanE1–E2–E3cascadeinvolving~40E2and600E3enzymes,whereas~100deubiquitinatingenzymes(DUBs)catalyzetheremovaloreditingofubiquitinchains.
Thedistinctsurfacesofthevariousubiquitinchaintypesactasthree-dimensional'codes'thatare'read'bymorethan20ubiquitin-bindingdomainstofacilitatetheassemblyofdiverseproteininteractionnetworks.
106,107Thus,theubiquitination,deubiquitinationandubiquitinrecognitioneventsinvolvinginammasomecomponentsconstitutehighlydiverseprocessesthatsignicantlyexpandthecomplexityofinammatorysignaling.
ASCubiquitinationmodulatesinammasomeactivationInadditiontobeingphosphorylatedatmultiplesites,ASCisalsoubiquitinated.
Shietal.
43reportedthatactivationoftheAIM2inammasomewithpoly(dA:dT)resultedinK63-linkedpolyubiquitinationofASCinmacrophages,whichledtoitsrecruitmenttotheautophagypathwaythatdegradestheinammasome.
Thiswasproposedtobeamechanismtopreventexcessiveinammationbyeliminatingactivatedinammasomes.
Incontrast,Rodgersetal.
47showedthatthelinearubiquitinassemblycomplex(LUBAC)promotesASC-dependentinammasomeactivationthroughlinearubiquitina-tionofASC.
LUBACisanubiquitinligasecomplexcomposedofHOIL-1l,HOIPandSHARPIN.
HOIL-1l-decientbonemarrow-derivedmacrophagesdemonstratedreducedproductionofmatureIL-1βuponstimulationofNLRP3andAIM2,butNF-κBactivationwasnotaffected.
MultipledomainsofHOIL-1l,includingtheHOIP-binding,ubiquitin-bindingandubiquitina-tionligasedomains,arerequiredforrobustubiquitinationofASC,formationofASCspeck,andpromotionofNLRP3inammasomeactivation.
Ofnote,thesuppressiveeffectsonNLRP3weremoreseverethanthoseonAIM2,andtheIL-1βsecretionresultingfromNLRC4stimulationwasnotaffectedbytheHOIL-1ldeciency.
TheseresultssuggestaspecicroleforHOIL-1linNLRP3inammasomeregulation.
However,invitroubiquitinationexperimentsdemonstratedthatASCisdirectlylinearubiquitinatedbyLUBAC,whereasNLRP3wasnot.
ItislikelythatLUBACalsoregulatesotherfactorsspecicfortheNLRP3pathwaytomodulateitsactivation.
TheapparentcontradictionsbetweenthestudiesbyShiandRodgersregardingtheroles(suppressiveversusstimulatory)ofASCubiquitinationininammasomeregulationhavenotbeenresolved,althoughitwassuggestedthattheapparentdiffer-encesintheubiquitinationofASC(linearvsK63-linked)betweenthesestudiesmaybetheresultofcross-detectionoflinearubiquitinationbyK63-ubiquitinantibodies.
108ItremainstobedeterminedhowNLRP3-specicstimulitriggerlinearubiquitinationofASCtopromoteinammasomeactivation.
Importantly,theinvivoroleofLUBACininammasomeactivationwasdemonstratedthroughtheobservationthatHOIL-1lwasessentialforIL-1βsecretionandneutrophilrecruitmentinanMDP-inducedperitonitismodel,aswellasinanLPS-inducedlethalinammationmodelthatisdepen-dentontheNLRP3inammasome.
47Post-translationalmodicationsofinammasomesJYangetal70Cellular&MolecularImmunologyAnotherE3ubiquitinligase,TNFR-associatedfactor3(TRAF3),wassuggestedtotargetASCforK63-linkedubiqui-tinationuponinfectionwithRNAviruses,suchasvesicularstomatitisvirus(VSV)orinuenzavirus.
109Themitochondrialantiviralsignalingprotein(MAVS)servesasaplatformthatrecruitsbothTRAF3andASCtopromoteTRAF3-mediatedK63-linkedubiquitinationofASCatresidueLys174.
Infact,MAVSalsocolocalizeswithNLRP3within2hofVSVinfection,althoughtheMAVS–NLRP3interactionappearstobeindependentoftheMAVS–TRAF3association.
MAVS-mediatedubiquitinationofASCenhancesactivationoftheNLRP3inammasomeinresponsetoVSVinfection,butnotfollowingexposuretocrystallinematerials,suchasMSUcrystals.
Furthermore,MAVSdoesnotenhanceactivationoftheAIM2orNLRC4inammasomes.
Clearly,infectionsbyRNAvirusesthatactivatetheRIG-IpathwaymaystimulateMAVS-mediatedubiquitinationofASCandactivationoftheNLRP3inammasome.
WhetherRIG-IhasaroleinthisubiquitinationeventorwhetherproductionofinterferonbytheRIG-IpathwaypromotesNLRP3activationremainstobedetermined.
UbiquitinationofNLRP3negativelyregulatesinammasomeactivationThepotentialroleofubiquitinationininammasomeregula-tionwasinferredfromtheobservationthattheubiquitinligase-associatedproteinSGT1isassociatedwithanumberofNLRproteins,includingNLRP2,NLRP3,NLRP4,NLRP12,NLRC4,NOD1andNOD2.
110Inparticular,bindingofSGT1totheLRRdomainofNLRP3wasessentialformaintainingitsinactivebutsignaling-competentstate.
110ThisisanalogoustotheessentialroleofplantSGT1inactivationofthenucleotide-bindingsite/leucine-richrepeat(NBS–LRR)proteinsforhostprotection.
111However,thespecicrolesofubiquitinationinthefunctionsofanyoftheNLR/NBS–LRRproteinswerenotdemonstratedintheabovestudy.
Recently,oneoftheE3ubiquitinligasesforNLRP3wasidentiedastheSkp-Cullin-Fbox(SCF)familymember,F-boxL2(FBXL2),whichmediatestheubiquitinationandproteaso-maldegradationofNLRP3.
112FBXL2isinturnregulatedbyanotherF-boxprotein,F-boxO3(FBXO3),throughitsownubiquitinationanddegradation.
AsmallmoleculeFBXO3inhibitor,BC-1215,wasshowntoreleasetheinhibitionofFBXL2andreduceNLRP3-mediatedinammation.
FBXL2bindsTrp73inthePYDdomainandLys689intheLRRdomainofNLRP3,thelatterofwhichisaubiquitinationsite.
TheauthorsarguethatratherthanenhancingtheexpressionofNLRP3,theroleofLPSprimingistoreducetheubiquitin-mediatedproteasomaldegradationofNLRP3viatheelevatedexpressionofFBXO3.
Anotherubiquitination-mediatedprocessintheregulationoftheNLRP3inammasomewasidentiedthroughthedopamineD1receptorDRD1pathway.
113Theneurotransmit-terdopamineactivatestheDRD1signalingpathwaytoproducethesecondmessengercyclicadenosinemonophosphate(cAMP).
BindingofcAMPtoNLRP3,perhapsinconjunctionwithactivationofotherpathwaysbycAMP,promotesLys48-linkedpolyubiquitinationofNLRP3bytheE3ubiquitinligaseMARCH7.
TheubiquitinatedNLRP3issubsequentlydegradedthroughtheautophagypathway.
Thisubiquitination-mediatedregulationbydopamineanditsreceptorisspecicfortheNLRP3inammasomeanddoesnotaffecttheAIM2orNLRC4inammasomes.
Furthermore,invivoexperimentsdemonstratedthatthedopaminesignalingpathwaysuppressedneurotoxin-inducedinammation(amodelofParkinson'sdisease),lipopolysaccharide-inducedsystemicinammationandMSU-inducedperitonitis.
113ThissuggeststhatDRD1agonistsmayhavetherapeuticvalueinNLRP3-driveninam-matorydiseases.
TheroleofubiquitinationinregulatingtheNLRP3degradationwasfurtherdemonstratedthroughthestudyoftheautophagychaperonep62.
P62wasshowntocolocalizewithadetergent-insolublepolyubiquitinatedproteinaggregatescontainingNLRP3andtopromotethedegradationofNLRP3.
114P62alsopromotesthedegradationofdamagedmitochondriathroughmitophagy,thusreducingthereleaseofNLRP3-activatingstimulifrommitochondria.
115Inagreementwiththeseresults,p62deciencyinmiceresultsinanincreasedatheroscleroticplaqueburden114andelevatedinammationinalum-inducedperitonitisandfuminanthepatitis.
115InadditiontoFBXL2andMARCH7,anotherE3ubiquitinligase,X-linkedinhibitorofapoptosisprotein(XIAP),hasbeenimplicatedinregulationoftheNLRP3inammasome.
XIAPisamemberoftheIAPfamilythatinhibitscaspasesandsuppressapoptoticcelldeath.
116TheC-terminalRINGdomainofXIAPharborsanE3ubiquitinligaseactivitythatisrequiredforNOD2signaling.
117,118ItwasreportedthatthelossofXIAPoritsRINGdomainresultedinexcessivecelldeathorelevatedsecretionofIL-1β,althoughthecelldeathappearedtobeindependentofcaspase-1,-11or-8.
119Currently,itisnotclearwhetherXIAPdirectlyubiquitinatesNLRP3orwhetheritsRINGdomainmediatesubiquitinationofanyotherinamma-somecomponents.
SimilartotheGuanstudyoftheMAVS–ASCinteraction,MAVSwasreportedtopromoteNLRP3inammasomeactivationthroughtheassociationofMAVSwithNLRP3.
120Theresidues2-7(KMASTR)withinthePYDdomainofNLRP3areessentialforthisinteraction.
TherecruitmentofNLRP3tothemitochondriabyMAVSmaypromoteformationoftheASCspeck,120oligomerizationofNLRP3and/orstimulationofthemitochondrialformationofreactiveoxygenspecies.
121Theroleofubiquitinationwasnotexploredinthisstudy,although,presumably,TRAF3mayberecruitedbyMAVS,asshownintheGuanstudy,109inwhichTRAF3wasfoundtobelocatedincloseproximitytoNLRP3,whichharborsmultiplepotentialubiquitinationsites.
SimilartotheGuanstudy,109MAVSisrequiredforoptimalstimulationoftheNLRP3inammasomebypoly(I:C)120orSendaivirusinfection,121butnotbycrystallinesubstances,suchasalumorMSU.
120Thesesimilaritiessuggestpotentialcross-regulationoftheRIG-Iandinammasomepathways.
Inagreementwiththeseresults,associationofMAVSwithNLRP3suppressedthePost-translationalmodicationsofinammasomesJYangetal71Cellular&MolecularImmunologyMAVS-inducedtypeIinterferonresponsetoSendaivirusinfection.
121A20isanubiquitin-editingenzymethatisinvolvedinnegativefeedbackregulationofNF-κBsignaling.
122–125Inde-pendentofitsroleinNF-κBregulation,A20wasrecentlyreportedtobeaninhibitoroftheNLRP3,butnotNLRC4orAIM2,inammasome.
126,127Thesignicanceofthisinam-masomeregulatoryfunctionisborneoutbytheobservationsthatA20deciencyinmiceleadstopathologicalinammationthatresemblesrheumatoidarthritis126andthatheterozygousgermlinemutationsofA20inhumansleadtoearlyonsetsystemicinammation.
128Mechanistically,thesuppressionofNLRP3byA20wasdependentonTRIFandRIPK3.
Further-more,A20mayreducetheubiquitinationofpro-IL-1βatitsconservedresidueK133,thusimpactingitsmaturationbycaspasesorsecretionthroughtheautophagypathway.
127UbiquitinationofNLRC4activatestheinammasomeSug1isaregulatoryproteinwithubiquitinligaseactivitythatisassociatedwiththe26SproteasomeandhasbeenidentiedasabindingpartnerforNLRC4throughyeasttwo-hybridassays.
129Sug1bindsresidues91-253ofNLRC4,whichincludeitsCARD-NODlinkerregionandNODdomain.
TheSug1–NLRC4interactionreleasesitsautoinhibitedconformation,followedbyubiquitinationandaggregationofNLRC4thatinturnleadstorecruitmentandactivationofcaspase-8.
However,thesiteandtypeofubiquitinationofNLRC4havenotyetbeencharacterized.
Inaddition,itisnotclearhowtheubiquitinatedNLRC4recruitscaspase-8,althoughapotentialadaptermole-culethatbridgestheubiquitinatedNLRC4andcaspase-8hasbeenproposed.
129UbiquitinationanddegradationofIFI16mediatedbyviralE3-ubiquitinligaseIFI16belongstothep200familyofinterferon-inducibleproteinsandthePYHINfamily.
130,131AsaPRR,itfunctionsasaDNAsensorinthecytosolandnucleusthatinducesinterferonproduction.
Italsoinducestheassemblyofinam-masomecomplexesinresponsetoDNAviruses,suchasherpessimplexvirus1(HSV-1),31Kaposi'ssarcoma-associatedher-pesvirus(KSHV)32andEpstein–Barrvirus,132aswellasretro-virusessuchasHIV-1.
133,134InterferonproductiondownstreamofIFI16isdependentontheendoplasmicreticulumadaptermoleculestimulatorofinterferongenes(STRING),whichisalsoknownasmediatorofIRF3activation(MITA),endoplas-micreticulumIFNstimulator(ERIS)ormet-pro-tyr-ser(MPYS),135–138andanessentialtranscriptionfactorIRF-3.
139IFI16isknowntoformaninammasomecomplexwiththeadapterASCinthenucleusandtranslocatetothecytoplasmtoactivatecaspase-1.
32InadditiontodirectlybindingtoDNA,IFI16anditsmurineorthologp204wererecentlyshowntocooperatewithanotherDNAsensor,cyclicGMP–AMPsynthase,140torecognizeDNAfromHSV-1andFrancisellainfectioninhumanforeskinbroblastsandmacrophages,respectively.
141,142TocounterIFI16-mediatedimmuneresponses,HSV-1expressesanimmediate-earlyprotein,infectedcellprotein0(ICP0),whichcontainsaRINGngerdomainwithE3-ubiquitinligaseactivity.
WithinafewhoursafterHSV-1infection,ICP0inhibitsthenuclearlocalizationofactivatedIRF-3andpromotestheubiquitinationandproteasome-dependentdegradationofIFI16andp204.
143–145ThespecictypesofIFI16ubiquitinationhavenotbeencharacterized.
144ICP0-mediateddegradationisspecicforIFI16onthebasisthattheNLRP3andAIM2inammasomesarenotaffected.
144Incontrasttothestudiesdescribedabove,anotherreportdidnotobserveIFI16degradationuponexpres-sionofICP0intheabsenceofHSV-1infection,146whichsuggeststhatfactorsotherthanICP0mayberesponsibleforIFI16regulationfollowingHSV-1infection.
ThereasonsforsuchseeminglycontradictoryresultsarenotclearbutmaybeattributedtodifferentialdegradationofIF16invariouscelltypes.
Ubiquitinationofcaspase-1uponinammasomeactivationWhileinvestigatingactivationoftheNlrp1binammasomebytheanthraxlethaltoxin,Lamkanandcolleaguesnoticedthepresenceofhigh-molecular-weightspeciesinimmunoblotsofmacrophagecelllysatesforcaspase-1.
147Thiswasinterpretedasindicativeofcaspase-1post-translationalmodications,whichwasconrmedbyimmunoblotingforubiquitin.
Impor-tantly,high-molecular-weightcaspase-1specieswerealsopresentfollowingactivationoftheNLRP3inammasomebyATPornigericin,theAIM2inammasomebyFrancisellainfectionandtheNLRC4inammasomebySalmonellainfec-tion.
Theapparentpost-translationalmodicationofcaspase-1requireditsenzymaticactivityonthebasisthattreatmentwiththecaspase-1inhibitorYVAD-cmksignicantlyreducedtheformationofthesehigh-molecular-weightspecies.
Theseresultsalsosuggestthatcaspase-1ubiquitinationoccursdownstreamofcaspase-1activation.
Theauthorsproposedthatcaspase-1ubiquitinationmayrepresentanegativefeedbackmechanismthatdampensinammatorysignalingbytargetingactivatedcaspase-1fordegradation.
However,itiscurrentlynotclearwhetherubiquitinatedcaspase-1isdestinedfordegradationbecausetreatmentwiththeproteasomeinhibitorMG132suppressedratherthanenhancedcaspase-1activation.
147Futurestudiesarerequiredtoclarifythefateofubiquitinatedcaspase-1.
DeubiquitinationofNLRP3promotesinammasomeactivationObservationofNLRP3ubiquitinationsuggestedthatdeubiqui-tinationwasalikelymechanismforinammasomeregulation.
Lopez-Castejonetal.
148identiedaDUBinhibitorb-AP15thatinhibitedactivationoftheNLRP3inammasomeandpartiallyinhibitedactivationoftheAIM2inammasome.
Theauthorsconcludedthatmultipleas-yetuncharacterizedDUBsmayberequiredforIL-1βprocessingandrelease.
EmployinganNLRP3ubiquitinationassay,YuanandcolleaguesidentiedaDUB/isopeptidaseinhibitor,G5,thatsuppressedIL-1βsecre-tiondownstreamoftheNLRP3inammasomeinmacro-phages,butnotdownstreamoftheAIM2orNLRC4Post-translationalmodicationsofinammasomesJYangetal72Cellular&MolecularImmunologyinammasomes.
46G5treatmentenhancedpolyubiquitinationoftheNLRP3NODandLRRdomains,butnotthePYDdomain,andsuppressedtheformationofASColigomers.
Subsequently,thedeubiquitinationenzymeBRCC3(mouse)/BRCC36(human)wasidentied,whichcoimmunoprecipitatedwiththeNODandLRRdomainsofNLRP3anddeubiquiti-nateditsLRR.
46BRCC3isaJAMMdomain-containingZn2+metalloproteasethatspecicallycleavesLys63-linked,butnotLys48-linked,polyubiquitinchains.
149Currently,itisunclearhowNLRP3stimuliactivateBRCC3.
Nonetheless,deubiquitination-dependentactivationofNLRP3maybeapotentialtargetfortherapeuticmodulationofinammatorypathogenesesthatinvolveNLRP3.
PROTEOLYTICPROCESSINGOFINFLAMMASOMECOMPONENTSInadditiontophosphorylationandubiquitination,cleavageofinammasomesensorproteinsbyproteasesfrombacteriaorthehostandcomplexinteractionsamongproteases,theirsubstratesandproteaseinhibitorshavecrucialrolesinregulat-inginammasomeactivation.
Infact,serineproteaseinhibitorswereshowntosuppressactivationofbothNLRC4andNLRP3inammasomes.
Theseresultscontrastwiththoseforpan-cathepsininhibitors,whichwereineffective,suggestingthatproteolyticprocessingisanintegralcomponentofinamma-someregulation.
150,151Incontrasttophosphorylationandubiquitination,proteolyticprocessingisanirreversiblePTMprocessthatgeneratesnewproteinisoforms.
AnthraxproteaseactivatestheNlrp1binammasomeBothmouseNlrp1bandratNlrp1wereshowntobecleavedbytheanthraxlethalfactor(LF),azincmetalloproteasethatispartoftheanthraxlethaltoxin.
152–155ThecleavagesiteislocatedintheN-terminalregionoftheseproteins,outsideofthepredictedNODdomain.
ThiscleavageeventwassufcientforNlrp1bactivation,implyingthatNlrp1bcanserveasasensorforotherproteasesfromdiversepathogensorevenhostcells.
Mechanistically,proteasecleavagemayreleaseanauto-inhibitedconformationofNlrp1btofacilitateassemblyoftheinammasomecomplex.
156Alternatively,thecleavedNlrp1bmayrecruitunknownbindingpartnersthatmayinturnpromoteinammasomeactivation.
WhethersuchmechanismsapplytohumanNLRP1proteinremainstobedetermined.
ComparedwithmurineNlrp1proteins,humanNLRP1con-tainsanadditionalPYDdomainatitsNterminusthatmayendowitwithdistinctactivationmechanisms.
Inaddition,theLFcleavagesitesofmurineNlrp1proteinshavepoorsequenceconservation:murineNlrp1biscleavedataLys-Leusequence,whereasratNlrp1iscleavedataPro-Leusequence.
Therefore,itremainstobedeterminedwhetherthehumanNLRP1proteinisalsocleavedbytheanthraxLFproteaseorothermicrobialproteases.
Itisinterestingtonotethatsuchmam-malianimmuneresponsestoproteolyticeventsareanalogoustotherecognitionofmodicationsofhostproteinsbyplantresistance(R)proteins,themajorityofwhicharealsoNLRproteins.
Thissuggeststhatrecognitionofproteolyticeventsisanancientmechanismofimmuneprotection.
157–159EnterovirusproteasessuppresstheNLRP3inammasomeTheenterovirus71(EV71)isresponsibleformanyepidemicsintheworldandismanifestedbyhand,footandmouthdisease,andneurologicalcomplicationsinchildren.
160Following-upontheobservationthatEV71-infectedpatients,particularlythosewithneurologicalcomplications,showedelevatedlevelsofIL-1β,Wangetal.
161identiedNLRP3astheinammasomethatisactivateduponEV71infection.
Conversely,EV71encodestwoproteases,2Aand3C,thatspecicallycleaveNLRP3atG493-L494andQ225-G226,respectively,thusantagonizingtheNLRP3inammasome,butnototherinammasomes.
BothcleavagesitesarelocatedwithinthepredictedNODdomainofNLRP3,suggestingthatthiscleavagemaypreventNLRP3oligomerizationand/orassemblyoftheinammasomecomplex.
Infact,bothoftheEV71proteaseshavebeenshowntoantagonizeinnateimmuneresponsesthroughcleavageofantiviralsignalingproteins,suchasMAVS162andIRF7.
163MycobacterialZmp1proteasereducesinammasomeactivationInfectionofmacrophagesbyMycobacteriumtuberculosis(Mtb)leadstoformationofgranulomas,inwhichthebacteriabecomelatent,oftenforthelifetimesofinfectedindividuals.
164Reactivationoflatentinfectionscanbetriggeredunderimmunosuppressiveconditions,andunderthesecondi-tions,activetuberculosisensues.
IL-1andtheNLRP3165–173andAIM2174–177inammasomeshavebeenshowntohaveimpor-tantrolesinthehostresponsetomycobacterialinfection.
However,thereiscontroversyregardingtheprotectiveroleoftheNLRP3inammasome.
69,167,170,178,179Mycobacterialspeciesencodeazincmetalloprotease,Zmp1,thatwasshowntoinhibitactivationoftheNLRP3andNLRC4inammasomesonthebasisthatcomparedwiththewildtype,anMbovisBCGstraindecientinZmp1inducedsignicantlyhigherlevelsofIL-1βandphagolysosomebiogenesisinmurinemacrophages.
180However,whetherZmp1cleavescomponentsoftheinammasomeswasnotclaried.
Inaddition,suppres-sionoftheinammasomeswasnotreproduceduponinfectionofhumanmacrophageswithMycobacteriumtuberculosisstrainH37Rv.
69Itispossiblethattheproteasesencodedbydifferentmycobacterialstrainsmayantagonizeinammasomesthroughdistinctmechanisms.
OTHERPOST-TRANSLATIONALMODIFICATIONSOFNLRP3S-nitrosylationsuppressestheNLRP3inammasomeNitricoxide(NO)wasshowntoinhibitcaspases-1,2,3,4,6,7and8throughS-nitrosylationofthecaspasecatalyticcysteineresidue.
181–184Asaresult,NOoritsreactionproductssuppressedcaspase-1activityandinhibitedthereleaseofIL-1βandIL-18fromactivatedmacrophages.
185Inaddition,theNOdonorS-nitroso-N-acetylpenicillamine(SNAP)inhibitedPost-translationalmodicationsofinammasomesJYangetal73Cellular&MolecularImmunologycaspase-1activityinvitro,andtreatmentwiththeinducibleNOsynthase(iNOS)inhibitorNG-monomethyl-L-argininesigni-cantlyenhancedIL-1βsecretionfromwildtype,butnotiNOS-decient,macrophagesandmice.
Usingpuriedrecombinantcaspaseprotein,inhibitionofcaspase-1activitywasshowntobereversiblebytreatmentwithdithiothreitol(DTT),whichsug-gestedthatthecaspasecatalyticcysteineresidueisS-nitrosylatedbyNO.
However,DTTdidnotcompletelyreversetheeffectsofNOtreatmentwhenmacrophagecelllysateswereused.
ThissuggeststhatmodicationofthecaspasecatalyticcysteineresiduemaynotbethesolemechanismunderlyingtheinammasomeregulationmediatedbyNO.
185Inthiscontext,arecentstudyconrmedthatIFN-γ-stimulatedNOproductionmediatedthesuppressionofIL-1βsecretionbyMycobacteriumtuberculosis-infectedmacrophages.
186Thiswasproposedasaregulatorymechanismthatwouldreducethedestructiveinnateinammatoryresponseselicitedduringchronicmycobacterialinfection.
Mechanistically,NOorSNAPtreatmentleadstoS-nitrosylationofNLRP3,butnotAIM2.
TheresultingS-nitrosylatonresultsinsuppressionofASColigomerizationandIL-1βprocessing.
RelatedstudiesreportedthatsecretionofmatureIL-1βandIL-18byATPornigericin-stimulatedmacrophageswassignicantlyreducedupontreatmentwithSNAP,whereasonlypartialreductionwasobserveduponstimulationoftheAIM2orNLRC4inammasomes.
187–189SNAPtreatmentfor1hleadstoS-nitrosylationofbothcaspase-1andNLRP3,187whichimpli-catesdirectmodicationofNLRP3byS-nitrosylationasthemainregulatorymechanisminducedbyNO.
NLRP3isextremelyrichincysteineresidues.
ExactlywhichcysteineresiduesarethefunctionaltargetsofS-nitrosylationremainstobedetermined.
Interestingly,agroupofnaturallyoccurringcompounds,ginsenosides,wereshowntoreduceS-nitrosyla-tionofNLRP3byreducingiNOSexpression.
190ThiswasproposedasamechanismbywhichginsenosidescanprotectagainstLPS-inducedendotoxicshock.
ActivationofinammasomeshasbeenshowntopromoteNOproduction,perhapsasanegativefeedbackmechanism.
BothactivationoftheNLRP3inammasomeuponLeishmaniainfection191andstimulationoftheNLRC4inammasomebyintracellularagellin192wereshowntoactivateiNOSandthepotentialS-nitrosylationofinammasomecomponents.
Thismitigatethedetrimentaleffectsofuncontrolledinammation.
ADP-ribosylationofNLRP3triggersrobustinammationandcytopathologyMycoplasmapneumoniaeisacommonairwaypathogen.
Althoughinfectionbymycoplasmararelyresultsindeath,thehyperinammatoryconditionsthatmanifestinresponsetomycoplasmainfectionhavebeenassociatedwithchronicpulmonarydysfunction,suchasbronchialasthma,chronicobstructivepulmonarydiseaseandcysticbrosis.
193Therefore,immunomodulatorytherapeuticsincombinationwithconven-tionalantimicrobialtherapeuticsarerecommendedinthemanagementofmycoplasmapneumonia.
Amycoplasmatoxinobservedincommunity-acquiredrespiratorydistresssyndrome(CARDS)isanADP-ribosyltransferaseandwasshowntocolocalizewithNLRP3andtocatalyzeitsADP-ribosylation.
194Thispost-translationalmodicationisessentialforNLRP3activationuponmycoplasmainfectionofhumanandmousemacrophagesandmayaccountforthehyperinammatorylungpathologiesmanifestedintheclinic.
Importantly,theroleoftheCARDStoxinasaninammation-promotingsolublefactorisconsistentwithitsabilitytotriggerallergicreactionsintheairwaylongaftertheprimaryinfectionbymycoplasmasubsides.
195Assuch,theCARDStoxinrepresentsanimportanttherapeutictargetformodulatinglunginammationuponmycoplasmainfection.
ThesuppressiveeffectoftheCARDStoxinwasspecicfortheNLRP3,butnotNLRP1,inamma-someandwasdependentonboththeN-terminalcatalyticdomainoftheCARDStoxinanditsC-terminaldomain,whichisresponsibleforitsattachmenttohostcellsandsubsequentinternalization.
AlthoughthespecicdomainofNLRP3thatismodiedbytheCARDStoxinwasnotdened,itwassuggestedthattheCARDStoxinmayaltertheconformationofNLRP3oritsinteractionwithotherpartnerproteinsduringassemblyoftheinammasome.
194DetailedmechanismsregardingtheCARDStoxin–NLRP3interactionstillawaitfutureinvestigations.
DISCUSSIONHerewehavefocusedonourevolvingunderstandingofhowdiverseyethighlyspecicpost-translationalmodicationsregulateinammasomeactivation.
Becausethesepost-translationalmod-icationsaredependentonenzymaticactivities,theyrepresentattractivetherapeutictargetsformodulationofinammasomeactivitiesinabroadrangeofinammatorypathologies.
Itisclearthatarepertoireofpost-translationalmodicationsisinvolvedinthepreciseregulationofinammasomes.
Thesecombinatorialmodicationsregulateinammasomesbychangingtheconfor-mationsofinammasomeproteinsandbycontrollingtheirturnover,distributionandlocation,aswellasbyregulatingtheintricateprotein–proteininteractionsessentialforinammasomeassembly.
Variouspost-translationalmodicationsininammasomesignalingcanbecross-regulatedbyeachother,perhapswithinthecontextofdifferentcelltypes.
Themostprominentcrosstalkisbetweenphosphorylationandubiquitination,whichcanpositivelyornegativelyregulateeachother.
51,196Phosphoryla-tionandubiquitinationareamongthemostabundantPTMsinvolvinghundredsofenzymesthatcatalyzeboththeattach-mentandtheremovalofphosphate/ubiquitin.
Thishighlycomplexnetworkofinammasomeregulationmaycontributetotheabilityofinammasomestorespondtovariousstimuli.
HowthesemultiplePTMsspatiallyandtemporarilycoordinateduringtheprocessofspecicinammasomeactivationinaparticularcelltypewillbeanimportanttopicforfurtherstudies.
Forexample,thephosphorylationofASCbySykkinaseisrequiredforASCspeckformationandcaspase-1activationinmacrophages,butnotinbonemarrow-deriveddendriticcells.
57,58Inaddition,proteolyticcleavageofGSDMDandthesubsequentpyroptoticcelldeathofmacrophageswasPost-translationalmodicationsofinammasomesJYangetal74Cellular&MolecularImmunologynotobservedinneutrophilsuponactivationoftheNLRC4inammasome,althoughmatureIL-1βwasproduced.
40–42Itisclearthattoappreciatethefullscopeofinammasomeregulation,studiesofcell-type-specicPTMsandexplorationofadditionalPTMsareimportantfutureareasofinvestigation.
UnderstandingthemechanismsofPTMsininammasomeregulationmayoffertremendousopportunitiesforthedevel-opmentoftherapeuticagentstotargetinammatorycondi-tions,suchasautoinammatorydisorders,metabolicdisorders,neurodegenerativediseasesandcardiovasculardisorders.
Forexample,someofthespecickinaseinhibitorsthataredevelopedforcancertreatmentcanberepurposedasagentstotreatinammatorydisorders.
Thedopaminesignalingpathway,whichtriggersubiquitinationandinhibitionoftheNLRP3inammasome,amelioratedneuro-inammationinamousemodelofParkinson'sdiseaseandsystemicinamma-tioninamousemodelofsepsis.
113ThissuggeststhatanagonistofthedopamineD1receptororthedownstreamE3ubiquitinligaseMARCH7mayprovidetherapeuticbenetintheabovediseases.
Similarly,theG5andb-AP15inhibi-tors46,148ofDUBsthathavepreviouslybeenshowntoblockcaspase-1activationandIL-1βmaturationmaybedevelopedasagentstocombatdiseasesthatinvolveexcessiveactivationoftheNLRP3inammasome,suchascardiovasculardisorders.
197CONFLICTOFINTERESTTheauthorsdeclarenoconictofinterest.
ACKNOWLEDGEMENTSTheworkwassupportedbytheCaseResearchInstituteandUniversityHospitalsCaseMedicalCenterfundandtheNationalInstitutesofHealth(AR069908toT.
S.
X.
).
WethankDongniMaforassistancewithpreparingFigure1.
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