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The emerging role of human PYHIN proteins in innate immunity: Implications for health and disease

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Commentary

The

emerging

role

of

human

PYHIN

proteins

in

innate

immunity:

Implications

for

health

and

disease

Dympna

J.

Connolly,

Andrew

G.

Bowie

*

SchoolofBiochemistryandImmunology,TrinityBiomedicalSciencesInstitute,TrinityCollegeDublin,Dublin2,Ireland

1. Introduction

Theinnateimmunesystemiscomposedofgermline-encoded pattern recognition receptors (PRRs) that collectively serve as sensorsformonitoringtheextracellularandintracellular compart-mentsforsignsofinfectionortissueinjury.ThesePRRs,whichare responsiblefor the detectionof pathogen associated molecular

patterns(PAMPs)whichsignalthepresenceofa pathogen,and damageassociatedmolecularpatterns(DAMPs)whichsignalling tissueinjury, includetheToll-like receptors(TLRs), theretinoic acid inducible gene-like receptors (RLRs) and the nucleotide oligomerisation domain-likereceptors (NLRs) [1]. Recently the classificationofanewgroupofPRRshasbeenproposed.Theseare theAIM2-likereceptors(ALRs)thatcomprisesomemembersofthe PYHINfamily,namelyhumanAIM2andIFI16andmurinep204[2]. TheALRsareintracellularinnateimmunesensorsresponsible for thedetectionofDNA. Asa pathogen-derivedPAMP,DNA is capableofstimulatingprotectiveimmunitytoinfectionwhilethe erroneousdetectionofself-DNA,whereDNAlikelyactsasaDAMP, canprovokeexcessinflammationandautoimmunity.Thehuman ALRsAIM2andIFI16areencodedonaninterferon(IFN)-inducible geneclusterfoundonchromosome1q23.Thisregionalsoencodes threeotherPYHINproteins;MNDA,PYHIN1andPOP3[3–6].While thisregionencodesfiveproteinsinhumans,othermammalshave variablefamilyexpansions,withatleastthirteenfamilymembers presentinmice[7].Collectivelytheseproteinsarereferredtoas PYRIN and HIN domain-containing (PYHIN)proteins since they possessanN-terminalPYRIN(PYD)domainandinmostcasesat leastone C-terminalhematopoieticinterferon-induciblenuclear

ARTICLE INFO

Articlehistory:

Received31July2014 Accepted28August2014 Availableonlinexxx

Keywords:

Innateimmunity

Patternrecognitionreceptors DNAsensing

Signaltransduction Viralevasion

ABSTRACT

Theinnateimmuneresponsedependsontheabilityofimmunecellsto detectpathogensthrough germline-encoded pattern recognition receptors (PRRs). Recently discovered PRRs include some membersofthePyrinandHINdomain(PYHIN)family,whichareencodedonaninterferon-inducible geneclusterlocatedonchromosome1q23.TherearefivehumanPYHINproteins;Absentinmelanoma2 (AIM2),IFN-ginducibleprotein16(IFI16),Myeloidcellnucleardifferentiationantigen(MNDA),Pyrin andHINdomainfamilymember1(PYHIN1)andtherecentlyidentifiedPyrindomainonlyprotein3 (POP3).Earlystudiesreportedrolesfortheseproteinsincellcyclecontrol,tumoursuppressionand transcriptionalregulation.AIM2andIFI16havenowbeenshowntobeimmunesensorsofnon-selfDNA, suchasthatproducedbyvirusesininfectedcells.AIM2bindsDNAtoactivatetheinflammasome,while IFI16detectionofDNAcanleadtotheup-regulationoftypeIinterferonsorinflammasomeactivation. RecentstudieshaveshownhowIFI16sensesDNAviruses,andalsohowvirusesevadedetectionbyIFI16, whilestructuralstudieshavegreatlyadvancedourunderstandingofhowAIM2andIFI16bindDNAto activatetheseimmuneresponses.Furthermore,followingtheidentificationofPOP3,interplaybetween membersofthisgeneclusterhasbeenestablished,withPOP3actingasanegativeregulatoroftheAIM2 andIFI16inflammasomes.InthisreviewwediscussthecurrentunderstandingofhowPYHINproteins functionininnateimmunity,theirroleindiseaseandthetherapeuticpossibilitiesthatariseasaresult. ß2014ElsevierInc.Allrightsreserved.

Abbreviations:AIM2, absent in melanoma 2; ALR, AIM2-like receptor; ASC, apoptosis-associated speck-like protein containing a CARD; CARD, caspase activationand recruitment domain; cGAMP, cyclic GMP-AMP; cGAS, cGAMP synthase;DAI,DNA-dependentactivatorofIFN-regulatoryfactors;DAMP,damage associated molecular pattern; EBV, Epstein–Barr virus; HIN, hematopoietic interferon-induciblenuclearantigen;HIV-1,humanimmunodeficiencyvirus1; HSV-1,herpessimplexvirus1;IFI16,IFN-ginducibleprotein16;IFN,interferon;IL, interleukin;ISG,immunestimulatedgene;KSHV,Kaposi’ssarcoma-relatedherpes virus;MNDA,myeloidcellnucleardifferentiationantigen;MTB,Mycobacterium tuberculosis;NLR,nucleotideoligomerisationdomain-likereceptor;PAMP, patho-genassociatedmolecularpattern;PYD,pyrin;PYHIN,pyrinandHINdomainfamily member;POP3,pyrindomainonlyprotein3;PRR,patternrecognitionreceptor; RLR,retinoicacidinduciblegene-likereceptor;SLE,systemiclupuserythematosus; STING,stimulatorofinterferongenes;TLR,toll-likereceptor;YY1,yingyang1.

* Correspondingauthor.Tel.:+35318962435;fax:+35316772400.

E-mailaddress:agbowie@tcd.ie(A.G.Bowie).

ContentslistsavailableatScienceDirect

Biochemical

Pharmacology

j our na l ho me p a ge : w ww . e l se v i e r . com / l oc a te / b i och e mph a rm

http://dx.doi.org/10.1016/j.bcp.2014.08.031

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antigenwith200amino acidrepeats(HIN-200orHIN) domain (Fig.1).ThePYDdomain(alsoknownasDAPINorPAADdomain) belongstothedeathdomainsuperfamilyandisanalpha-helical motifthatformshomotypicinteractionswithotherPYD contain-ing proteins. The 200 amino acidDNA binding HIN domain is classified into three subtypes, termed A, B and C, based on consensusmotifs[8].POP3,themostrecentmemberofthisgene clusterto be identified, lacksthe HIN domains foundin other PYHINproteins[3].

ThePYHINproteins havebeenshowntolocalisetoboth the nucleusandcytosol.AIM2andPOP3arepredominantlycytosolic proteins,whileIFI16,MNDAandPYHIN1arepredominantlyfound inthenucleus owingtothepresence ofan N-terminalnuclear localisationsequence(NLS)[3,9].However,otherfactorssuchas acetylationor thepresenceof stimulatory DNA (in thecase of IFI16),ortheonsetofapoptosis(inthecaseofMNDA),canalso influencetheircellularlocalisation[10–12].

Duetorecentexcitingdiscoveriesdemonstratingrolesforsome PYHINproteinsasinnateimmunePRRs,inthisreviewwere-visit thefunctionsoftheseproteins.Herewewillfocusonthehuman PYHINs,discussingearlierstudiesonthisproteinfamily,theirnew rolesinDNA sensingand transcriptionalregulation,the mecha-nismsbywhich theseevents occur and possibleapproaches to targetingPYHINstherapeutically.

2. EarlyresearchonthehumanPYHINs

PriortodiscoveriesdemonstratingthatmembersofthePYHIN familyfunctioninpathogenrecognition,studiesfocusedonroles fortheseproteinsincellgrowth,cellcyclecontrol,cell differenti-ation, tumour suppression, apoptosis, and the DNA damage response.

MNDA,thefirsthumanPYHINfamilymemberdiscovered,was identified in HL-60 cells as a 55kDa proteinthat is expressed predominantly in the nucleus and specifically in cells of the myeloid lineage [13]. Early studies into the role of MNDA

suggested an involvement in myeloid differentiation. This idea wassupportedbystudiesrevealingthatMNDAcanbindnucleolin andnucleophosmin[14,15],bothofwhichhavebeenshowntobe involved in thematuration and biosynthesis of ribosomes. The noted nuclear localisation, and lineage- and stage- specific expressionofMNDAsuggestedthatthisPYHINproteinmayplay aroleinregulatinggenetranscription.MNDAhasbeenshownto bind to the transcription factor ying yang 1 (YY1), forming a ternarycomplexalongwiththeYY1targetDNA.Theinteraction betweenMNDAandYY1increasestheaffinityofYY1foritstarget DNAanddecreasesitsrateofdissociation[16].Thismeansthat cellular levels of MNDA could have a dramatic effect on the transcriptionofseveralgenes,highlightingitspotentialasakey regulatorofgeneexpression.Indeed,retroviralmediated expres-sionofMNDAinK592cells,whichnormallylackMNDA,correlated withinductionofdelta-like1(DLK1),whoseexpressionisessential for normal haematopoiesis, and specifically for myeloid cell development[17].MNDAitselfhasalsobeenproposedtoactas atranscriptionfactorinmonocytes[18]andthisissupportedbya morerecent studydemonstrating that MNDAcandirectly bind doublestrandedDNA(dsDNA)[19].ApossibleroleforMNDAin neutrophil apoptosis has also been proposed. Thus during apoptosis,MNDAiscleaved bycaspasesand thenrelocalisesto thecytosolwhere it accumulatesand associates withthe anti-apoptoticproteinmyeloidcellleukaemia1(MCL1).Thisleadsto MCL1degradation,resultinginapoptosiscausedbymitochondrial dysfunction [12]. Thisrelocalisation of MNDA tothe cytosolis thought to be required for proper execution of apoptosis and suggests that reduced cytoplasmic accumulation of MNDA contributestosuppressionofneutrophilapoptosis.Furthermore, insepticpatientscytoplasmicaccumulationandcleavageofMNDA inneutrophilswasdramaticallyimpaired[12].

ThesecondhumanPYHINfamilymemberidentifiedwasIFI16. IFI16migratesasthreedistinctproteinspecies(IFI16A,BandC)on SDS-PAGE,themostabundantlyexpressedandcommonlystudied isoformbeingtheBform.Thethreeisoformsaregeneratedasa

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resultofalternativeRNAsplicingandareclusteredat85–95kDa [20].EarlystudiesonIFI16focusedonitsroleincellcyclecontrol andtranscriptionalregulation.IFI16hasbeenshowntobindthe C-terminalregionofthetumoursuppressorproteinp53,augmenting p53-mediatedtranscriptionalactivation.Whilethemechanismof IFI16modulationofp53DNAbindingisstillnotcompletelyclear the data suggests that interaction of IFI16 with p53 may be importantforbindingofp53toDNA,whichthentranslatestoan increase in the transcriptional activity of p53 [21]. This is reminiscent ofMNDAs ability toenhanceYY1binding toDNA. Interestingly,IFI16canalsointeractdirectlyinvivowiththegene promoterregionofbothp53andc-myc[22].IFI16hasalsobeen showntobindBRCA1andthiscomplexhasbeenreportedtobe involvedinthep53-mediatedcelldeathpathway,suggestingthat IFI16 may play a role in DNA damage induced apoptosis [23].However, IFI16hasalsobeenshown toassociatewiththe retinoblastomatumour-suppressorproteinRbandthe transcrip-tionfactorE2F1,resultingintheinhibitionofRb-E2F1mediated transcriptional repression [24]. While accumulating evidence supportstheideathatPYHINscanactastranscriptionalregulators, it highlights that this regulationcan beindirectby influencing transcription factors, or direct resulting from a PYHIN:DNA association. By regulating transcription factors, IFI16 can also actasarestrictionfactorduringhumancytomegalovirus(HCMV) infection.Itdoes soby interactingwiththetranscriptionfactor Sp1,displacingitfrompromoters,therebyleadingtoadecreasein HCMV DNA synthesis, a decreasein viralgene expression and inhibitionofviralreplication[25].Furthermore,IFI16hasrecently beenshowntoberequiredforgeneinductioninresponsetoinnate immune RNA and DNA detection, since IFI16 is required for optimal RNA polymerase II occupancy on IFN-

a

and Immune StimulatedGene(ISG)promoters[26].Thisprovidesevidencefora roleforIFI16intranscriptionalregulationandrevealsadirect anti-viralroleforIFI16tobothDNAand RNAviruses.Inaddition,a prominentroleforIFI16inDNAsensingisnowappreciated(see below).

LikeIFI16,thethirdPYHINproteinidentifiednamelyAIM2hasa roleininnatedetectionofDNA.However,priortoitsidentification as a DNA sensor, a role for AIM2 in tumour suppression was proposed.IndeedAIM2wasactuallyfirstidentifiedintumourcells lineswhosetumorigenicityisintentionallysuppressed[27].Others supportedthisideathatAIM2isatumoursuppressorandthisrole hasbeennotedinmanysystems[28].MorerecentworkonAIM2 hasfocusedonitsroleasaDNAsensorandwillbediscussedin moredetailbelow.

ThenextPYHINfamilymemberthatwasidentifiedwasPYHIN1 (orIFIX asit is alsoknown). The PYHIN1 gene is predicted to encode six differentprotein isoforms as a result of alternative mRNAsplicing(

a

1,

a

2,

b

1,

b

2,

g

1,and

g

2).Eachoftheisoforms hasacommonN-terminalregion,whichcontainsaPYDandaNLS. Consequentlyeachoftheseproteinsarelocalisedinthenucleus. However,

a

2,

b

2and

g

2haveadeletionofnineaminoacidsinthe N-terminalregionwhencomparedto

a

1,

b

1and

g

1.Inaddition, isoforms

a

1,

a

2,

b

1and

b

2containaHINdomainwhichisabsent inboththe

g

1,and

g

2isoforms[6].LikethePYDofothernuclear expressedPYHINproteins, thatofPYHIN1 alsoappearstoform aggregates whenexpressed alone [29]. Furthermore,given that AIM2,IFI16andMNDAhavebeenshowntodimerise[15,20,30], thisraises thepossibility thattheir othercloselyrelated family memberPYHIN1candosoalso.Functionally,rolesforPYHIN1in cellgrowthandtumoursuppressionhavebeenreported.PYHIN1 expressionhasbeenreportedtobereducedinbreasttumoursand breastcancercelllines[6].ItisbelievedthatPYHIN1

a

1possesses tumoursuppressiveactivitymediatedbyitsabilitytointeractwith and destabilise the oncoprotein HDM2. This results in p53 stabilisationandactivationofp53responsivegenes,suchasthe

cyclindependent kinaseinhibitorp21CIP1. Consequently,CDK2 kinaseactivityis reducedand cellgrowthis inhibited[6,31]. A functional link between PYHIN1

a

and maspin has also been proposed.Maspinisaserineproteaseinhibitorandsuppressorof metastasis. In PYHIN1

a

expressing cells, histone deacetylase 1 (HDAC1)isdegraded,allowingmaspintranscriptionwhichresults inreducedmetastasis[32].

ThemostrecentadditiontothePYHINfamilyisPOP3[3].The gene encoding POP3 was recently shown to reside between PYHIN1andIFI16onthePYHINlocus(Fig.1A).POP3lacksaHIN domainresponsibleforDNAbinding,butpossessesaPYDdomain. Assuch,POP3canstronglyinteractwiththeALRsAIM2andIFI16, likelymodulatingtheirrolesininnateimmunity[3].

Asyet,thereislimitedinsightintothespecificmolecularroles ofthePYHINproteinsinmanyofthediverseprocessesdescribed abovewithwhichtheyhavebeenassociated.However,inrecent yearsresearchhasfocusedinparticularonAIM2andIFI16,given thatanimportantroleforthesePYHINsininnateimmunityhas been uncovered. These significant discoveries will be now discussedinmoredetail,withafocusonthelatestfindingswhich haveadvancedourunderstandingofhowcellssensethepresence ofpathogenandselfDNAleadingtoactivationofinnateimmunity.

3. IntracellularDNAsensing

The detection of intracellular DNA is a crucial event in the innate immune response to viruses and intracellular bacteria. However,aberrantDNAdetection(forexampleofselfDNA)can havenegativeconsequencesresultinginautoimmunity.Whileit hasbeenappreciatedforsomeyearsthatDNAcanbe immune-stimulatory, we have only recently begun to understand the molecularbasisofDNAsensing.TheendosomalTLR9wasthefirst describedPRRforDNA [33].However,followinginfectionswith intracellular pathogens, impairmentsin clearanceof exogenous DNAorimbalancedcontrolofendogenousDNAproducts,DNAcan also accumulate in the cytosol.This leads tocytokine and IFN induction, especially if and when cytoplasmic DNases may be defective. Therefore, numerous studies attempting to identify cytosolic DNAsensorswereundertaken.Thefirstcytosolic DNA sensoridentifiedwasDNA-dependentactivatorofIFN-regulatory factors (DAI) [34]. However, as the sensing ability of DAI is restricted to specific cell types, additional DNA sensors were believed toexist. Indeed, todate morethan ten cytosolic DNA sensorshavebeenproposed(Fig.2).SomeoftheseDNAsensors canalsofunctioninthenucleus.WhiletheotherDNAsensorshave beenreviewedelsewhere[35,36],AIM2andIFI16asmembersof thePYHINfamilyareofparticularinteresthere.

4. DiscoveryofAIM2asaDNAreceptor

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insightintotheroleofAIM2asaDNAsensor.Whilevalidatingits roleasa DNAsensorthesestudieshavealsodemonstratedthat AIM2 is involved in the detection of the cytosolic bacteria

Francisella tularensis, vaccinia virus and Listeria monocytogenes [38], pathogens which can also infect humans. Although the involvementofAIM2inMycobacteriumtuberculosis(MTB) detec-tioninhumanremainstobeinvestigated,AIM2knockoutmiceare alsohighlysusceptibletoMTBdemonstratingdefectivecaspase-1 activation,IL-1

b

andIL-18production[39].Furthermore,mouse p204,thefunctionalhomologueofIFI16contributestoIFN-

b

and IFIT1inductionfollowingMTBinfection[40],suggestingthatALRs mayalsoberesponsibleforMTBdetectioninhumans.

More recent work on AIM2 has sought to investigate the structure of theHIN domain in complex withdsDNA and the molecularmechanismsinvolvedinAIM2inflammasomeactivation followingDNAbinding.ThecrystalstructureofthedsDNAbound AIM2HINdomainhasrevealedthatAIM2recognisesDNAina non-sequence specific manner via electrostatic attraction between positivelychargedHIN domainresiduesand the dsDNA sugar-phosphatebackbone[41].Importantly,thesefindings providea rationale for the permissive nature of DNA binding by AIM2. Dockingandbinding studieshave revealedthatAIM2 can self-associate prior to dsDNA binding, via an AIM2HIN:AIM2PYD association [42]. This association maintains AIM2 in an auto-inhibitedstatepriortodsDNAbinding.Interestinglythenegatively

chargedsurfaceoftheAIM2PYDwhichcanassociatewiththeHIN domainofAIM2isthesameregionwhichbindsthePYDdomainof ASC following dsDNA binding.Such elegant mutually exclusive interactions arethought toensurethat AIM2interactswith the downstream adapter ASC only upon activation of AIM2 by the dsDNAligand.InadditiontoreleasingAIM2fromitsauto-inhibited state,dsDNAalsoactsasaplatformfortherecruitmentofmultiple AIM2molecules,allowingforthecloseapproximationofindividual AIM2PYDs.TheseAIM2oligomersinturnfacilitatethenucleationof multiple ASCPYDsleadingtothe formationof the multi-protein inflammasomecomplexrequiredforIL-1

b

processing[43].

5. ViralDNAsensingbyIFI16leadingtodownstreamsignalling

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allowsIFI16todetectdsDNAfrommanysourcesincludingKaposi’s sarcoma-relatedherpesvirus(KSHV)[11],Epstein–Barrvirus(EBV) [44],herpessimplexvirus1(HSV-1)[45]andhuman immunode-ficiencyvirus(HIV-1)[46].Onthecontrary,dsDNAsensingbyIFI16 islengthdependent[2],withdsDNAfragmentsof150basepairs reportedlybeingfavourablefortheformationofanoptimalbinding clusterofapproximatelytenIFI16protomers[47].Inordertobind stimulatorydsDNA,theHINdomainsofIFI16associatewithboth DNAstrandsacrossboththemajorandminorstrands,inkeeping withtherequirementofdsDNAratherthanssDNAfortheinitiation ofsignallingfromIFI16[2,41].WhilebothHINdomainsofIFI16can associatewithdsDNA,theHINbdomainpossessesastrongerDNA bindingaffinity[2,41].IncontrasttoAIM2,DNArecognitionbyIFI16 canoccur in both the cytosolic and nuclear compartments.For exampleDNAwhichhasbeentransfected,lentiviralDNAthathas accumulatedorherpesvirusDNAwhichhasbeenexposedinthe cytosolcanbedetectedbycytosolicIFI16[2,46,48]whileDNAfrom nuclearvirusessuchasHCMVisdetectedinthenucleus[10,49].As mentioned earlier, the NLS of IFI16 is required for the nuclear localisationofIFI16.Recently,twoacetylationsitesthatnegatively regulate NLS function have been identified. When these lysine residuesatposition99and128,whicharewithintwocriticalNLS motifs are acetylated, nuclear import is inhibited (Fig. 3). The acetylation status of IFI16 is thought to be regulated by the acetyltransferase p300 and the histone deacetylase HDAC and occursinvariouscelltypes[10].Thusanimportantfactorinthe regulationofIFI16localisationisitsacetylationstatus.

Whetherhost receptors such as IFI16 have any capacity to distinguishselfDNAfromnon-selfDNAatthemolecularlevelhas

beenalong-standingquestionininnateimmunity.Anearlydogma in theDNAsensing fieldwastheidea thatthenucleus wasan immune-privileged cellular compartment for DNA detection. However, we now know that some sensors such as IFI16 can specificallydetectnon-hostDNA inthenucleus.Arecentpaper examiningIFI16oligomerisationonDNAhasprovidedinsightsinto howthisdistinctionmightoccur.Firstly,Morroneetal.foundthat followingDNAbinding,IFI16continuestoassembleonthedsDNA strandinacooperativemannerviaPYD:PYDassociations,forming IFI16 foci from which downstream signal activation can be initiated [47]. For optimalpolymerisation to occur a region of approximately150bpofDNAisrequired.Thus,despiteexceeding thefootprintoftheHINdomains,thelengthoftheexposed linker-dsDNA between nucleosomes (10–20bp) or even that of the transcription bubble (approximately 17 bases) is too short to promote robust cooperative filament assembly of IFI16. This providesanelegantexplanationforhowthedistinctionismade beenselfandnon-selfDNAandidentifieslongnakedDNAasthe trueIFI16ligand.SecondlyMorroneetal.revealedanimportant rolefortheIFI16PYDdomain,demonstratingthatitiscrucialfor IFI16oligomerisation.

FollowingtheactivationofIFI16byimmunestimulatory non-selfDNA,thisreceptorrecruitstheendoplasmicresidentprotein STING (stimulator of interferon genes) [2]. The IFI16:STING association occurs in the cytosol following either cytosolic or nuclear DNA detection, as after herpesvirus DNA detection by nuclear IFI16this portion ofactivatedIFI16translocates tothe cytosol[11,49].HowexactlynuclearexportofIFI16isregulated after DNA sensing remains to be determined, but may involve

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regulatedalteration oftheacetylationstatus ofIFI16, since the acetylation status of IFI16 regulates its cellular location (as described above). Alternatively, in cells such as macrophages, whicharelesspermissivetoviralinfection,proteasomal degrada-tionofviralcapsidsallowsthereleaseofviralDNAintothecytosol priortonucleardeliveryofthegenome.ThisexposureofviralDNA activatescytosolicIFI16,whichalsoleadstoSTING/TBK1activation and IFN-

b

expression [48]. While STING and TBK1 are widely acceptedtobeactivatedfollowingIFI16stimulation,thedetailsof themoleculareventsinvolvedintheir activationhaveyettobe unravelled.STINGisknowntobeactivateduponbindingofcyclic GMP-AMP(cGAMP)whichisanovelsecondmessengerproduced bycGAMPsynthase(cGAS).LikeIFI16, cGAShasbeenshownto directlybinddsDNA,andinthiscasethisbindingeventstimulates thecatalyticactivityofcGAS,leadingtocGAMPproduction(Fig.2). HowIFI16‘activates’STING,andwhetherthisinvolvescGASand cGAMPiscurrentlyunclear,althoughwedoknowthatbothIFI16 andcGAScancontributetoDNAsensingincertaincelltypes[50]. Recently,IFI16hasbeenidentifiedasahostreceptor responsi-bleforthedetectionofHIVDNAinmacrophagesandCD4+Tcells

[46,51].Importantly,inmacrophagesIFI16wasshowntobindnot onlydsDNA,butalsossDNAcontaininglongstemstructuresand terminalloopregionsthatformaduplexstructure,bothwhichare produced during the HIV lentiviral life cycle [46]. As such, in macrophagesIFI16contributestothedetectionandearlycontrolof HIV in these cells. However, abortive infection of CD4+ T cells

withHIV,whichleadstoaccumulationofHIVDNA,causes IFI16-dependentpyroptosis[51],sincelikeAIM2,IFI16activationcanalso promoteinflammasomeactivationinsomecontexts. IFI16-depen-dentinflammasomeactivationhasbeenreportedtoalsooccurafter infection with herpes viruses such as KSHV, HSV-1 and EBV [11,44,45].However,HSV-1andHCMVinfectioncanalsoresultin IFI16dependentIFN-

b

inductionviatheSTING/TBK1pathway.As yetthemoleculardetailsofthedifferentialeventsinvolvedinIFI16 dependentASCandSTINGactivationremainunclear.

RecentstructuralstudiesonDNAbindingandoligomerisation asakeystepinpathwayactivationforbothAIM2andIFI16have provided some important answers to previously unresolved questionsinthefield,allowingusamorecompleteunderstanding ofhowtheseDNAsensorswork.However,thereisundoubtedlya greatdealmoretodecipher,withmanyquestionsstillremaining. Forexample,theexact cellularlocationof IFI16inducedSTING activationremainselusive.ItisalsounclearifIFI16polymerisation facilitatesinflammasomeformationaswellasSTINGactivation.In addition,whiletheimportanceofDNAlengthforIFI16bindinghas been explored the correlation between optimal binding and downstream pathway activation has not. An analysis of the optimalligand for AIM2 bindingand inflammasome formation wouldalsobeveryinteresting.

6. HostandpathogenregulationofAIM2andIFI16

Since AIM2 and IFI16 can drive inflammation via cytokine induction,andarealsocriticalinearlyviraldetectionasPRRs,itis to be expected that there are numerous host and pathogen mechanismstoregulatethem. Someofthesemechanismshave evolvedinhumans,whileothershaveco-evolvedinpathogensto counter ALR functionsin an attemptto evade detection,or to minimisetheextentoftheimmuneresponse.

An interesting mechanism of PYHIN regulation hasrecently beenuncoveredwiththediscoveryofthenewestmemberofthe PYHINfamily,POP3[3].POP3lackstheHINdomainfoundinother PYHINs(Fig.1),and solacksanyDNAbindingability.However, POP3candirectlybindbothAIM2andIFI16viaitsPYDdomain,and in doing so regulate downstream pathway activation. The associationofPOP3withAIM2diminishestheabilityofAIM2to

associatewithASCtherebypreventinginflammasomeformation. Anotherexampleofco-regulationwithinthePYHINfamilyisAIM2 regulationbyIFI16,whereIFI16canbindAIM2andinhibitAIM2 mediatedcaspase-1activation[5],possiblybyreducingthelevels offreeAIM2availableforASCassociation.Inmice,co-regulation within the PYHINfamily has alsobeen reported. Here, another PYHINfamilymemberp202iscapableofinhibitingAIM2[52].p202 lacksaPYDbutpossesstwoDNAbindingHINdomains,thefirstof whichiscapableofsequesteringcytosolicdsDNA.Interestingly,the secondHINdomainofp202bindstheHINdomainofAIM2,resulting in spatial separation of AIM2 PYD and the prevention of ASC clustering[53].WhilenoknownhumanPYHINpossessesasimilar domainarchitecturetomousep202,alternativelysplicedisoforms ofhumanAIM2havebeenpredicted[54].Perhapsthosewhichlack eitherthePYDortheHINdomaincanattenuateAIM2activationina manner similar to p202 inhibition of mouse AIM2. Finally, as mentionedearlierpriortodsDNAbindingAIM2isfoundinan auto-inhibitedstate,wherethePYDisinassociationwiththeHINdomain. Only following dsDNA binding is the AIM2PYD liberated and availableforASCPYDbinding[42],providinganotherexampleof regulationwithinthePYHINfamilyitself.

Asaresultofco-evolutionwiththeirhosts,viruseshavealso acquiredmechanismsfor blockinghostimmune signalling. The firstreportofviraltargetingofaPYHINproteincamefromherpes simplexvirus1(HSV-1).Ithadbeenknownforsometimethatthe HSV-1immediateearlyproteinICP0wascapableofinhibiting IRF-3signalling,howeveritsmechanismofinhibitionremainedelusive forsometime.TheabilityofICP0totargetIFI16forproteasomal degradation was proposed to be at least one key mechanism employedby ICP0 forimmune evasion [55].Expandingon this workJohnsonetal.demonstratethatICP0co-localiseswithIFI16 twoandfourhourspostHSV-1infection.Inaddition,thisstudy demonstrated that IFI16 is phosphorylated following HSV-1 infection,likelytargetingitforproteasomaldegradation[45]. How-ever,anotherstudywhichraisedconcernsaboutthedifficultiesin distinguishing between the direct and indirect effects of ICP0, reasonedthatICP0doeshaveanimpactonIFI16relatedactivities butthatIFI16isnotnecessarilyanICP0substrate[56]. Neverthe-less, this study agrees that following HSV-1 infection IFI16 is degradedbytheproteasome,whichisaveryeffectivemechanism tolimitprolongedviraldetection.MNDAmayalsobesubjectto targetingbyviralproteins,asLatency-associatednuclearantigen (LANA) from KSHV has been shown to associate with MNDA, howevertheimplicationsofthisinteractionareunknown[57].

Itisbecomingmoreapparentthatinnatehostsensorsfrequently utiliseaggregationdependentamplificationmechanismsforsignal activation[43,58].Indeed,theHCMVproteinpUL83hasbeenshown tointeractwiththePYDofIFI16,blockingitsoligomerisation[29], andthusantagonisingacriticalsteprequiredforreceptoractivation. UtilisingaHCMVstrainlackingpUL83,Lietal.demonstratedthat IFI16oligomerisationisrequiredforphosphorylationandnuclear translocationofIRF1andIRF3.However,asnodifferencein caspase-1cleavagewasnoted,theimportanceofIFI16oligomerisationfor inflammasome activation remains unclear. Interestingly, amino acidsintheIFI16PYDwhicharecriticalforIFI16oligomerisationare not well conserved in AIM2 [47], raising the possibility that AIM2PYD either oligomerises differently or not at all, although overexpressionoftheAIM2PYDdoesresultinfilamentformation [29].SinceAIM2cannucleateASCPYD,leadingtothepolymerisation ofASCintheAIM2inflammasome[43,58],itremainsapossibility thatAIM2PYDs whicharein veryclose proximitytoeachother ratherthanpolymerisedpersemaybesufficienttonucleateASCfor inflammasomeformation.

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co-expressionofpUL83withthePYDof MNDAorPYHIN1 also disruptedtheirabilitytoformnuclearoligomers.Thisraisesthe possibilitythatthisHCMVproteiniscapableofinterferingwiththe functionofallthreenuclearPYHINproteins.

7. DiseaserelatedrolesofthePYHINproteins

WhilethecurrentknownrolesofPYHIN proteinshaveplaced themattheforefrontofinnateimmunity,workingasprotectorsofthe hostbysensingdsDNAfrominvadingpathogens,thereisnowample evidenceshowingthatdysregulationofthesepathwayscanhavea negativeimpactonthehost.Forexample,dysregulated inflamma-someactivityresultinginexcessiveIL-1

b

production,or inappropri-aterecognitionofselfnucleicacidsleadingtoIFN-

b

induction,can bothpotentlypromoteparticularautoimmunediseases.

MuchoftheevidenceforrolesforthePYHINsindiseasecomes fromtheanalysisoftheirexpressioninvariousconditions.Ascan beseeninTable 1dysregulatedPYHIN expressioncanoccur in manydifferentconditionsrangingfromviralinfectionstocancer. However,inmanycaseswhiletheassociationisclearitmaybe difficulttodetermineifthesechangesinexpressionpatternsarea causeoreffectofthediseaseinquestion,particularlyduetothe factthatmanyPHYINproteinsareIFN-inducible.

Geneticfactorsarealsothoughttobeimportantindetermining diseaserisk,forexample,PYHINproteinshavebeenreportedtolie within a susceptibility locus for systemic lupus erythematosus (SLE)[59]and thePYHIN1 locushasbeenidentified asa novel susceptibility locus for asthma, specifically for individuals of Africandescent[60].

A protective role for AIM2 has been proposed in prostate diseases,withincreasedsteady-statelevels ofAIM2mRNAand protein in benign prostate hyperplasia but significantly lower levelsofAIM2expressioninprostatecancercellsascomparedto normalprostateepithelialcells[61].Furthermore,reducedAIM2 expressionincolorectalcancerisreportedlyassociatedwithpoor outcome,suggestingaprotectiveroleforAIM2herealso[62].In contrast elevated AIM2levels and inflammasome activityhave

beenreportedinpsoriaticlesions,possiblyprovokingthischronic autoimmunediseaseviasensingofselfDNA[63].AlteredPYHIN1 and MNDA levels in a number of conditions have also been reported(Table1).

IFI16is a targetfor autoantibodies andseveral studies have shown the presence of anti-IFI16 autoantibodies in chronic autoimmune diseases suchas SLE[64],systemic sclerosis[65], rheumatoid arthritis [66]and Sjo¨gren’s syndrome [67].IFI16 is knowntorelocalisefromthenucleustothecytosolfollowingUV treatmentorviralDNAdetection.ItishypothesisedthatIFI16can alsobereleased intotheextracellularmilieu.This extracellular IFI16caninturnbindtotheplasmamembraneofendothelialcells, and down regulate both their migratory activities and tube morphogenesis [66]. In this context IFI16 maybe acting as an alarmin,propagatingthestresssignalandcausingfurtherdamage. AsmentionedearlierIFI16isanimportantsensorforHIVDNAin multiplecelltypes[46,51].Thisworkwasfurthersupportedwith findingsfromHIVpatientsdemonstratingthattheyhaveelevated IFI16levels,possiblyarisingasaresultofchronicimmuneactivation. WhileelevatedIFI16levelsdidcorrelatewithahighviralloadand lowCD4+cellcounts,IFI16mRNAratherthanproteinlevelswere

analysed[68].ThismaynotreflectproteinexpressionlevelsofIFI16 andalsodoesnottakeintoaccountotherfactorssuchassubcellular localisationandproteintrafficking.Thislimitationisalsomirrored in some other studies and highlightsthe importance of further studiesrelatingtotheexactrolesofPYHINsindisease.

While many studies are suggestive of a role of the human PYHINsinanumberofconditions,mechanisticstudiesimplicating theseproteinsinthepathogenesisofthesediseasesarestilllacking and willneedtobecarried outifthePYHINproteinsaretobe utilisedasbio-markersorconsideredastherapeutictargets.Also, increasedPYHINexpressionduringdiseasecouldbeaconsequence ofincreasedIFNexpression,presentasaresultofthediseaserather than beinga causative factorin the onsetof thedisease itself. Howeverifthisisthecase,elevatedPYHINproteinlevelsorthe presence of anti-PYHIN antibodies may still be useful as biomarkersincertaindiseases.

Table 1

ExpressionofPYHINsindisease.

Protein Disease Comment Reference

AIM2 Abdominalaortic aneurysm(AAA)

IncreasedAIM2proteinexpressioninbothsporadicinfiltratinglymphoidcellsandlymphoid aggregateslocatedintheoutermediaandadventitiaofbloodvessels

[76]

Colorectalcancer ReducedexpressionofAIM2proteinassociatedwithpooroutcome [62]

HepatitisBvirus(HBV) HigherAIM2proteinexpressioninHBVassociatedglomerulonephritistissue [77]

Prostatecancer ReducedAIM2mRNAexpressionintumourspecimens [61]

Psoriasis IncreasedamountsofAIM2mRNAandproteininlesionalskin [63]

Systemiclupus erythematosus(SLE)

IncreasedAIM2mRNAexpressioninSLEpatientsasaresultofthereducedDNAmethylationstatusof theAIM2gene

[78]

IFI16 HIV ElevatedIFI16mRNAlevelsinperipheralbloodmononuclearcellsfromuntreatedHIVpatients [68]

Rheumatoidarthritis(RA) AutoantibodiesagainstIFI16presentinseraofpatients [67,79]

Sjo¨gren’ssyndrome(SjS) AutoantibodiesagainstIFI16presentinseraofpatients [66]

Systemiclupus erythematosus(SLE)

AutoantibodiesagainstIFI16presentinserapatients [64,79]

IncreasedIFI16proteinexpressioninallepidermallayersandinthedermalinflammatoryinfiltratesof lesionalskin

Systemicsclerosis(SSc) AutoantibodiesagainstIFI16presentinseraofpatients [65,79]

IncreasedIFI16proteinexpressioninallepidermallayersandinthedermalinflammatoryinfiltratesof lesionalskin

MNDA Lymphoma HighMNDAproteinexpressioninmarginalzonelymphomas [80]

Myelodysplastic syndrome(MDS)

DecreasedMNDAproteinexpressioninmarrowsamplesfrompatientswithMDS [81]

Sepsis CytoplasmicaccumulationofandcleavageofMNDAinneutrophilsisimpairedresultinginless neutrophilapoptosis

[12]

Sjo¨gren’ssyndrome(SjS) ElevatedMNDAmRNAinpatientswithprimarySjS [82]

PYHIN1/IFIX Asthma SNPsinthePYHIN1locuslinkedtosusceptibilityforindividualsofAfricandescent [60]

Breastcancer DownregulatedPYHIN1mRNAexpressioninbreastcancertissues [6]

Systolicblood pressure(SBP)

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8. TherapeuticapproachestotargetingPYHINproteins

WhileitisclearthatthePYHINfamilyplaysacentralrolein innateimmunity,theevidencetodatefortheinvolvementofthe PYHINsindiseasemainlycomesfromalteredexpressionpatterns indisease,thepresenceofauto-antibodiesagainsttheseproteins inseraof patients and singlenucleotide polymorphismsin the genomicregionsencodingthePYHINs(Table1).

Since aberrant and excessive DNA sensing contributes to autoimmuneandinflammatorydiseases[69]theinvolvementof the ALRsAIM2 and IFI16 at an early point in innate immune activationwithregardtotheirroleinDNAsensing,positionsthem atan ideal point fortherapeutic targeting. Caspase-1,which is activatedfollowingHIVDNArecognitionbyIFI16[51]hasbeen targetedusingacaspase-1inhibitorthatissafeforuseinhumans. Thisinhibitorhasbeenshowntoblockcaspase-1cleavage,IL-1

b

secretionandCD4+Tcelldeath[70],demonstratingthatinhibiting

IFI16 induced signalling in HIV patients has great therapeutic promise.

However,asdiscussedearliertheDNAbindingcapabilitiesof thehumanPYHINsarenotsolelyutilisedinviraldetection,asthe human PYHINs can also act as transcriptional regulators. In addition, most ALRs bind dsDNA in a non-sequence specific manner, so the use of synthetic oligodeoxynucleotides (ODN) whichcanbindDNAsensorsinamannerthatiscompetitivewith immunestimulatoryDNAmustbehighlyspecifictoavoidaffecting thefunctionsofotherALRs.ForexampleODN-A151hasbeenshown toinhibitdsDNAinducedtypeIIFN,TNF

a

,ISGinductionandcaspase 1 dependent IL-1

b

and IL-18 maturation by binding to AIM2. However,biotinylatedODN-A151 alsoimmunoprecipitatedwith IFI16,furthersuggestingthatmultipledsDNAinducedsignalling eventsarelikelytobeeffectedbyODN-A151[71].LL-37isahuman cathelicidin antimicrobial peptide that forms a complex with extracellularself-DNA,sequesteringitandinhibitingtheabilityof DNA to induce IL-1

b

production via the AIM2 inflammasome. However,LL-37promotescellularuptakeofself-DNAandwhile LL-37/self-DNAcomplexesdo notappeartoactivate AIM2, theydo initiateinflammatorycascades viaTLR9[63].Thishighlightsthe difficultyintargetingamoleculewhichcanexerteffectsviamultiple PRRs.

Alternatively,targetingamorespecificassociationsuchasthose initiated directly after DNA:ALR binding for instance PYD:PYD interactionsmaybemorepromising.Itisbecomingapparentthat oligomerisationisaunifyingmechanismcriticalfordownstream signalactivation[29,43,47,58].DsDNAbindingtoIFI16andAIM2 inducesthepolymerisationofIFI16PYDdomains,orthe polymeri-sationofAIM2PYDandASCPYDrespectively.Targetingthesekey associations,whichdeterminethestabilityofthesignalactivation complexes,could encourage their disruptionthereby alleviating autoimmunedisorders. Alternatively,complex stability could be improved, resulting in enhanced immunity. The elucidation of crystalstructuresforIFI16PYD:PYDassociationsAIM2PYD:ASCPYD wouldbeextremelyadvantageousinthisregard.

Viral inhibitorsofthePYHINssuchaspUL83mayalsoprove very informative. Understanding how this and other viral inhibitorsworkmayaidthedesignofspecificpeptideinhibitors ofhumanPYHINs.Thisapproachhasprovedsuccessfulinother contexts,forexamplea peptidederivedfromthevacciniavirus proteinA46(knownasVIPER)iscapableofinhibitingTLRinduced signalling by interfering with downstream adapter protein associationswhicharerequiredforsignalactivation[72].In the contextofDNAsensing,PYHINsoperatewithinthecelltherefore intracellular delivery of therapeutics will be a crucial step to overcome.However,targetingcirculatingIFI16forexamplemay beanotherpromisingwaytotreatautoimmuneandinflammatory disorders.

Anotherchallengeinthedevelopmentoftherapeuticstargeting proteinsofthePYHINfamilywillbetousefullyinterpretdatafrom mouse models. Given the complexity of the PYHIN family in mouse,whichhaveatleast13members[7,73]itmaybedifficultto determinethecorrelationifany,withdatafromhumansstudies. Although human IFI16 and mouse p204 have been shown to functionsimilarly,andhaveasimilardomainstructurecontaining aPYRINdomainandtwoHINdomains,humanandmouseAIM2 aretheonlydirectorthologuesidentifiedbetweenthesespecies basedonthephylogeneticanalysesofbothHINandpyrindomains [7,73].WhilehumanandmousePYHINssuchasIFI16andp204 appear to be functional orthologues, it will nonetheless be importanttobearin mindthemanydifferencesbetween these twosystems.TheregulationofthePYHINsmayalsodifferbetween the mouse and human systems, and further complicate the interpretation of data. For example, POP3, which negatively regulatesAIM2andIFI16,isabsentinmouse[3].

9. Concludingremarks

Inrecentyearssignificantprogresshasbeenmaderegarding thePYHINproteins,particularlyconsideringtherolesofALRsin DNAsensing.WhiletheIFNinduciblegeneclusterhasbeenknown for many years, the very recent discovery of a novel protein encoded in this region (POP3) emphasises that there may be numerousexcitingdiscoveriesstilltobemade.Forexample,the existenceofnon-codingRNAsinthisregionwithpotentiallynovel regulatoryroleshasyettobeinvestigated.Furthermorerolesfor MNDAandPYHIN1ininnateimmunityremaintobedetermined. Inthecomingyearswehopetogainadeeperunderstandingofthe diverse rolesof theseproteins. In addition, understanding how exactlytheirvariousfunctionsareregulatedbothbythehostand byviruseswillbeextremelyinformative.Interestingly,thePYHINs arenottheonlyproteinfamilywithdualrolesasinnateimmune sensors and transcriptional regulators. For example, the NLRs NLRC5andNLRP12haveadditionalrolesactingastranscriptional regulatorsofMHCclassI,andtheassociatedgenes

b

2M,TAP1,and LMP2 [74,75]. Future work will likely reveal further gene regulatoryrolesforthePYHINsbothasnucleicacidsensorsand asdownstreamcomponentsofsignaltransductionpathways.In addition,investigationintothedifferentialmolecularandcellular events involved in ALR-stimulated STING or inflammasome activationwillgreatlyadvanceourunderstandingofdownstream pathway activation and reveal novel therapeutic opportunities. Whilegreatprogresshasundoubtedlybeenmadeinrecentyears withregardtotherolesofhumanPYHINsandtheirmechanismsof action, weare somewayfromreaping thebenefitsof targeted PYHINcontrolby therapeuticmeans. Furtherresearchon these proteinswillcertainlyyieldimportantinformationthatwerequire foramorecompleteunderstandingoftheirroles,particularlyin disease. In turn this will greatly aid the development of therapeuticstargetingPYHINproteins.

Acknowledgements

WorkonPYHINsintheauthors’labisfundedbygrantsfrom ScienceFoundationIreland (11/PI/1056) andtheNational Insti-tutesofHealth(AI093752).

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