Actin Post-translational Modifications: The Cinderella of Cytoskeletal Control

Review

Actin Post-translational Modifications: The Cinderella of Cytoskeletal Control

Sylvia Varland,

Joël Vandekerckhove,

and Adrian Drazic

*

Actin is oneof themostabundant proteins in eukaryoticcells andthe main componentofthemicrofilamentsystem.Itplaysessentialrolesin numerous cellularactivities,includingmusclecontraction,maintenanceofcellintegrity, and motility, as well as transcriptional regulation. Besides interacting with various actin-binding proteins (ABPs), proper actin function is regulated by post-translationalmodifications(PTMs),suchasacetylation,arginylation,oxi- dation,andothers.Here,weexplainhowactinPTMscancontributetofilament formation and stability, and may have additional actin regulatory functions, whichpotentiallycontributetodisease development.

TheFundamentalsofActinFunctionality

Actin(seeGlossary)accountsforupto15%ofthetotalproteinlevelinmusclecellsand1– 3%innonmusclecells.Itexistsinbothamonomericglobularstate(G-actin)andpolymerized filamentousstate(F-actin;Figure1A),andtheswitchbetweenthetwostatesishighlydynamic.

Theactinfilamentsplaycrucialrolesincountlesscellularfunctions,includingmusclecontrac- tion,cellsignaling,aswellascellintegrityandmotility[1].Themultifunctionalityofactinisbased onthreepillars(Figure1B):chaperonin-assistedfolding[2],interactionswithactin-binding proteins(ABPs)[1],andpost-translationalmodifications(PTMs;Figure1C)[3].Numer- ousstudiesandreviewsdescribetheinfluenceABPshaveontheactincytoskeleton.Inthis review,however, wedescribethe mostrecent findingsonactinPTMsshedding lightona crucial,butoftenoverlooked,aspectofactinbiology.

Actinsrepresentafamily ofisoformswhich arehighlysimilarinsequence(93%sequence identity)andeachconservedthroughoutevolution.Basedontheiraminoacidsequences,six isoformsweredescribedandclassifiedaccordingtothetissuesinwhichtheywerefoundin mammalsandbirds:fourmuscleforms;a-skeletal,a-cardiac,a-smooth,g-smooth,andtwo nonmusclecytoplasmicactins:b-cytoplasmicandg-cytoplasmic[4].a,b,andgrefertotheir respectivemobilityduringisoelectricfocusing,whichisexclusivelyduetothenumber(3/4)and nature(Asp/Glu)oftheN-terminalacidicresidues.Forexample,theNterminusofb-cytoplasmic actinisAc-DDDIAALVV-whilethatofg-cytoplasmicactinisAc-EEEIAALVI-.Thefourunderlined residuesconstitutetheonlydifferencesinatotalof375residuespresentinthesetwoisoforms, emphasizingtheirconservednature.Despite theirsequenceandstructuralsimilarities, actin isoformsdisplaybothoverlappinganduniquecellularroles(reviewedin[5]).Thishasbeenclearly demonstratedinmicewhereknockoutofb-actinresultsinembryoniclethality[6,7],whileg-actin- deficient miceshowdevelopmentaldefects,butareviable [8,9].Althoughtheseremarkably differenteffectsarenotyetfullyunderstood,itisknownthatthesetwoisoactinsdisplaydistinct intracellularlocalizationpatterns[5].Furtherinvitroexperimentsrevealthatmixturesofisoactinsin filaments couldaffect polymerizationdynamics,stability,andinteractions withABPs[5,10].Ontop ofthesesubtledifferences,PTMscouldcontributebyaffectingactinstructure,localization,and function.Most PTMs willaffect the isoactinsina similarmanner, giventhe actinsequence

Highlights

Post-translational modifications of actinaffectitsfoldingandstructure, aswellasinteractionwithactin-bind- ing proteins,and thus interferewith cytoskeletondynamics.

TheactinN-terminalacetyltransferase, NAA80,wasrecentlyidentified,thus solvinga30-year-oldmysteryonthe finalstepofactin’suniqueandcon- servedN-terminalmaturationprocess.

Acetylationandarginylationcompete foractin’sNterminus,bothaffecting filament formation, interaction with actin-binding proteins, and cell motility.

ActinoxidationofMet44andMet47by the MICAL enzymes promotes, in synergywithcofilin,thedisassembly ofactinfilamentsandislinkedtocan- cerdevelopment.

Toxin-mediatedmodificationsofactin mayleadtoactinfilamentaggregation, andinsomecasescelldeath.

1DepartmentofBiomedicine, UniversityofBergen,JonasLiesvei 91,N-5020Bergen,Norway

2DepartmentofBiologicalSciences, UniversityofBergen,

Thormøhlensgate53A,N-5020 Bergen,Norway

3DonnellyCentreforCellularand BiomolecularResearch,Universityof Toronto,160CollegeStreet,Toronto, ONM5S3E1,Canada

4DepartmentofBiochemistry,UGent CenterforMedicalBiotechnology, GhentUniversity,AlbertBaertsoenkaai 3,9000Gent,Belgium

*Correspondence:

adrian.drazic@uib.no(A.Drazic).

502 TrendsinBiochemicalSciences,June2019,Vol.44,No.6 https://doi.org/10.1016/j.tibs.2018.11.010

Glossary

Actin:familyofmultifunctional globularproteinsthatareableto polymerizeintofilamentsandinteract withamultitudeofactin-binding proteins.Thefamilyconsistsofat leastsixisoformsinhumans (a-skeletal,a-cardiac,a-smooth muscle,g-smoothmuscle, b-cytoplasmic,andg-cytoplasmic).

Actin-bindingproteins(ABPs):

ABPsaresignalingpathway- controlledactininteractors,which regulatethepolymerizationand depolymerizationofactinfilamentsas wellastheirorganizationinthe cytoskeletonnetwork.KnownABPs include:theArp2/3complex, profilins,gelsolin,formins,andcofilin.

ATE1:arginyl-tRNA-protein transferase1catalyzesthe attachmentofargininetotheN terminusofanacceptorproteinorto internalaminoacidsidechains.

Cytoskeleton:highlyorganized proteinnetworkinalldomainsoflife (prokaryotes,archaea,and eukaryotes),consistingofhundreds ofproteinswhichareinterconnected byfilaments(actins),tubules (tubulins),andthecytokeratin network.

F-actin:polymeric,filamentousform ofactin.

G-actin:monomeric,globularform ofactin.

MICAL:MICALs(molecule interactingwithCasL)arecytosolic, multidomainenzymesthatbelongto afamilyfeaturingmonooxygenase activity.Theyreversiblyoxidize Met44andMet47ofF-actin.

NAA80/NatH:Na-acetyltransferase protein80/N-terminalacetylation complexHbelongtotheN-terminal acetyltransferase(NAT)familythat, togetherwiththeNatBcomplex,are involvedintheuniqueN-terminal maturationprocessofactinby acetylatingitsNterminus.TheNAT enzymefamilyprovidesNt- acetylationforabout80%ofthe humanproteome.

Nt-acetylation:additionofanacetyl group(Ac)totheNterminusofa protein.Nt-acetylationiscatalyzedby N-terminalacetyltransferasesusing acetyl-CoAasdonor.

Nt-arginylation:additionofan arginineresidue(Arg/R)totheN- terminusofaprotein.Nt-arginylation

similarities.However,asdescribedlaterinthisreview,thereareclearcasesofisoform-specific PTMscontributingtodifferentiatedfunctions.

Post-translationalModifications:TheUnderratedPlayersofActin CytoskeletonDynamics

ThefirstactinPTM,N-terminal(Nt)acetylation,wasreportedforskeletalmuscleactinin1966 byGaetjensandBárány[11],andlateridentifiedinallotheractinisoforms.Today,morethan 140 PTMs have been described in eukaryotic actin sequences ([3] and http://www.

phosphosite.org). Some actin PTMs are quantitative and reversible, whereas others are rare,affectingonlyaminorityofthemoleculesthatmakeupthe cellularactinpool.Thus, manyactinPTMsshouldbeconsideredaspartialmodifications.ActinPTMsarefoundon94 differentsidechains(Table1,KeyTable)whichconstituteabout45%oftheresiduesthatcan bemodified.Specifically,newphosphorylation,ubiquitination,andSUMOylationsiteshave beenidentifiedbyglobalproteomicsanalysesinrecentyears[12–19].Interestingly,wehave noticedregionswherethefrequencyofPTMsissignificantlylowerthanaverage(regions:95– 145,240–256,and331–354).Thisfollowstheoverallaccessibilityofthesidechainresidues intheactinstructure,thoughlossofATP/ADPorinternalcleavagescouldalsoinducepartial denaturation,resultinginunspecificlow-levelmodifications.Itiscurrentlynotcleartowhich extentthelattercontributetoactin’scellularrole,orwhethertheyshouldbeconsideredas structural noise. Furthermore, our knowledge about the regulation, reversibility, and the interplaybetweenindividualPTMsremainslimited.Giventhehighnumberofreportedactin PTMsandtheabsenceofdetailedstudiesformostofthem,wefocusherepredominantlyon recentreportscoveringNt-acetylation,Nt-arginylation,andoxidationofactin.Wedis- cusstheir molecular and physiologicalconsequences, and theirpotential roleindisease development.

StructuralandRegulatoryImplicationsofActinPTMs

AlthoughnotallactinPTMsappearatthesametimeonthesamemolecule,andsomePTMs haveonlybeenreportedinparticularorganisms,theirsheernumberposesaseriouschallenge foraglobalunderstandingoftheirregulatory mechanisms.Forinstance,howcanan actin molecule,whose primaryroleis togeneratedynamicfilaments composedofgeometrically conservedbuildingblocksrepeatedoverseveralthousandtimes,giverisetothesestructures whendecorated withpotentiallystructuredisturbing PTMs?Howcanboth G-and F-actin interactinadynamicandrigorouslycontrolledmannerwithaplethoraofABPswhencarrying thislargenumberofmodifications?PTMscanhoweverparticipateinthestructuralarchitecture ofactinandmodifytheirfilaments.Oneofthebestknownexamplesisthestructureofarthrin,a 55-kDaheavyformofactinfirstobservedininsectmusclethinfilaments[20].Thisinsectactin, whichismonoubiquitinatedatLys118(Table1),appearsateveryseventhsubunitalongthe filamentlongpitchhelices.Itwassuggestedthatarthrinregulatesmusclecontractileactivity [20].AmorerecentreportonstructuralregulationoftheactinfilamentnetworkreferstoNt- arginylationofb-actinbyarginyl-tRNAproteintransferase1(ATE1).Inthiscase,Nt-arginylated actinsformnormalfilamentstructures.Non-Nt-arginylatedactinisolatedfromATE1knockout (KO)cells,ontheotherhand,formsbundlesandaggregates,resultinginshorterfilaments.Ona cellular level this leads to disorganization of lamellipodia and filopodia, an effect which is attributedtoalteredinteractionswithABPs[21].

Giventhemultifunctionalnatureofactin,onecanexpectthatthefinaloutcomeofthishigh numberofPTMscouldbeextremelycomplex.SomePTMswillaffectsteady-statefilament growthbyblockingoneofthefilamentendsorreducingtheconcentrationofpolymerization competentmonomers.SomePTMsmayinterferewiththeactin-ABPequilibriumordriveactin

TrendsinBiochemicalSciences,June2019,Vol.44,No.6 503

moleculestowardsdegradationpathways.Andifthisisnotyetsufficientlycomplex,PTMsmay enhanceorswitchoffeachother’seffectsbycrosstalkingmechanisms.Thecircuitsthatare producedcouldfunctionvialoopsthatontheirturnactivatenovelcircuits.Thesequantumbits ofmodificationsaremostlikelynotsimplynoise,butcouldpushthecellfollowingstochastic mechanismstowardsareversibleorirreversibledestiny.Forinstance,Tyr53canbeatargetfor phosphorylation, but also for nitration during oxidative stress. Similarly, Cys374 is highly reactiveandcanacceptdifferenttypesofmodifications(Table1).Itisnotclearwhetherthese modificationswillresultinthesameeffectbecausetheydisplayadifferentchemicalnature.An interestingexampleofthecomplexityinvolvessomeprominentABPslikeADF/cofilin,gelsolin (Figure1D),profilin,andDNaseI(Figure1E).Profilinbindstotworegionsinactin(Figure1E), whilecofilininteractswithactinviathreesites(Figure1D)[1,22].Partofthesesitesoverlapwith eachother.Thus,modificationsinactincouldtiltthebalancebywhichthesetwoABPsexert theircontrolonactinassembly.N-terminalmaturationofactinisanelegantexamplewherea particularproteinmodificationdependsonthepreviousone.Here,thesuccessiveactionsof methionine aminopeptidases,N-terminal acetyltransferases,and ATE1 result inmost actin moleculesbeingNt-acetylated, whereasaminorityisNt-arginylated (discussedlaterinthis review).

N-TerminalProcessingofActin:AUniqueMaturationMechanism

ActinsarefirstsynthesizedasprecursormoleculeswhicharefurtherNterminallyprocessedby successiveactionsofN-terminalacetyltransferasesandaminopeptidases.Thisprocesswas firstdescribed byRedmanandRubensteinintheearly1980s [23],andonlyrecently more detailsontheplayershavebecomeavailable.Thesixexpressedmammalianactinisoformsare divided intotwo categories basedprimarily onthe natureof theirunprocessed N-terminal sequences(Figure2A)[4].ForclassIactins(nonmuscleb-andg-actin)theinitiatormethionine (Met1)isdirectlyfollowedbythreeacidicaminoacids(MDDD-/MEEE-).Theactinmaturation processbeginswhenthenascentNterminusiscotranslationallyNt-acetylatedbyNatB,which alsoacetylatesothereukaryoticproteinsbeginningwithMD-/ME-[26].Normally,acetylationof acidicNterminiensuresthatMet1isretained,butinanunusualtwistfromnature’ssidetheNt- acetylatedMet1isremovedbyastillunidentifiedaminopeptidase.Theneo-Nterminus(DDD-/

EEE-)isthenNt-acetylatedbytherecentlyidentifiedNAA80/NatHgeneratingthematureactin protein[27–29].ForclassIIactins(striatedandsmoothmuscleactins)anadditionalcysteine residue(MCD/E-)complicatestheN-terminalprocessing.Inthiscase,Met1iscotranslationally removedbymethionineaminopeptidasefollowedbyNt-acetylationoftheexposedcysteine, presumablybyNatA.Finally,anunknownaminopeptidaseremovestheacetylatedcysteine and the processed acidicN terminusis then reacetylated, most likely by NAA80 [27,28], therebycompletingthematurationprocess.

N-terminalactinmaturationgainednewattentionwhenitwasdiscoveredthattheprocessedN terminusofb-actin(DDD-)caneitherbeacetylatedbyNAA80orarginylatedbyATE1[30].Nt- arginylationofb-actinisfoundtooccuronAsp3aftertheproteinhasundergonesequential removalofboththefirstandsecondaminoacid(RDD-)(Figure2A)[30].Thismodificationprofile has notbeen observed on any other actinisoforms. However, it would beinteresting to understandwhyAsp3isnotfurtherNt-acetylated,whichshouldbethermodynamicallyamore favorablereactionoverthearginylationstep.A recentstructuralanalysisindicatesthatDD- startingactinformsapoorsubstrateforNAA80[29].Alternatively,subcellularvariationsinthe substrateconcentrations, aswellas theenzymeamountsandactivities,couldleadtolocal competitions.Indeed,arecentstudysuggeststhatNt-arginylatedb-actininmouseembryonic fibroblast(MEF)cellsisconcentratedattheleadingedgeoflamellipodia,andisthusmainly linked to active migration [31]. Moreover, non-Nt-arginylated actin forms filamentous

iscatalyzedbythearginyltransferase ATE1usingarginyl-tRNAasdonor.

Nterminus:startofaproteinora polypeptide,whichhasafreeamino group(-NH2).Theaminogroupis usuallypositivelychargedat physiologicalpH(7.4).N-terminal modificationswillmaskorchange thischarge.

Post-translationalmodifications (PTMs):proteinmodificationsthat areaddedaftertheproteinhasbeen fullytranslatedand/orfolded.

Modificationsolecules,suchas oxidationandacetylation,tothe additionofpolypeptidessuchas SUMOylationandubiquitination.

ROS:reactiveoxygenspeciesare highlyreactivemoleculesandfree radicalsderivedfromoxygenthat contributestooxidativestress,which leadstovariousdiseases.

N terminus N terminus

(A) (B)

(C)

(D) (E)

PolymerizaƟon DepolymerizaƟon

Barbed

end (+) Pointed

end (-)

MethylaƟon AcetylaƟon ArginylaƟon SUMOylaƟon UbiquiƟnaƟon

ADP-ribosylaƟon Met-oxidaƟon Cys-oxidaƟon PhosphorylaƟon Tyr-nitraƟon

Subdomain 2 Subdomain 2

Subdomain 1 Subdomain 1

Subdomain 3 Subdomain 3

Subdomain 4 Gelsolin G3 interacƟon site Subdomain 4

Gelsolin G1 interacƟon site Profilin interacƟon site

Cofilin interacƟon site

Cofilin interacƟon site

DNase I interacƟon site

AcƟn

Chaperone assisted folding AcƟn binding proteins AcƟn modificaƟons

SUMO

ADP-ribose

UbiquiƟn

Figure1.Post-translationalModificationsofActin.(A)ActincanbepresentasfreemonomerscalledG-actin(redcircles),orpolymerizeintomicrofilamentsknown asF-actin(redchains).Theswitchbetweenthetwostatesishighlydynamicandpartlyregulatedbypost-translationalmodifications(PTMs).(B)Thethreepillars supportingthemultifunctionalityofactin:chaperone-assistedfolding,actinbindingproteins,andPTMs.(C)StructuralformulaeofmajoractinPTMs.Actinmolecules canbepost-translationallymodifiedby,forexample:methylation,acetylation,arginylation,SUMOylation,ubiquitination,ADP-ribosylation,methionineoxidation, cysteineoxidation,phosphorylation,andtyrosinenitration(attachmentshowninred).(D)and(E)b-actinstructure(PDB:2BTF)[85]showingATP(magenta)and selectedaminoacidresidues(colorcodeforatoms:carbon,green;nitrogen,blue;oxygen,red;cysteines,yellow)carryingPTMs.Interactioninterfacesfor(D)the gelsolinsubunitsG1andG3(greenbox)andcofilin(burgundybox),aswellas(E)DNaseI(pinkbox)andprofilin(bluebox)arehighlighted,demonstratingthatmany aminoacidresiduesthatarepartoftheseinterfacesaresubjectsofmodifications,andthusPTMscaninterferewithABPbinding.

TrendsinBiochemicalSciences,June2019,Vol.44,No.6 505

aggregatesinvitro,whileATE1KOcellsshowimpairedlamellaformationandcellmigration (Figure2B)[30].AcetylationenhancesthenegativenatureoftheNterminus,byneutralizingthe free a-amino group, while arginylation on the other hand decreases the negative charge density. It is therefore not surprising that both modifications play a role in cytoskeleton morphologyandaffectactin’spolymerizationkinetics[27,30].NAA80specificallyNt-acetylates b- andg-actin, and presumably also acetylates the N terminus of class II actins [27–29].

Furthermore,NAA80’sactivityregulatesactincytoskeletondynamicsandcellmorphologyby reducing actin filament assembly as well as filopodia and lamellipodia formation, which ultimatelydeceleratescellmigration(Figure2B)[27,32].Mostofthedataonactin’sstructure reveallargefluctuationsintheNterminus(aminoacidresidues1–6),indicatingdisorder.The Nt-acetylationeffectobservedonactinfilamentelongationisthereforedifficulttoexplain.TheN terminusisalsonotpositionedincloseproximitytothemonomer–monomerinterface,makinga direct effect less likely. However, the introduction of conformational changes cannot be excluded.Mostlikely theeffectisinducedbycontactswithABPs,sincetheN terminusof bothmonomericandfilamentousactinisexposedonthesurfacewhereitcaninteractwitha numberofregulatoryproteins,suchasmyosin[33],andpotentiallyformins[27,34].Indeed, earlystudiesongeneticallyengineeredyeastactindemonstratedthatthenegativenatureof actin’sNterminusenhancestheactivationofmyosin’sATPaseactivity[35].

KeyTable

Table 1. Major Post-translational Modi fi cations of Actin Where Modi fi ed Residues Are Numbered According to Class I Actins (b/g-Actin)

ActinPTM Modifiedresidues^a

Acetylation Met1,Asp2,Glu2,Cys2^d,Asp3^d,Lys50^b,Lys52^d,Lys61,Lys68,Lys113^b,Lys191^b,Lys193^d,Lys213^b,Lys315^b,Lys326^b,Lys328^b ADP-ribosylation Arg28^c,Arg95^c,Thr148^b,Arg177^b,Arg206^c,Arg372^c

Arginylation Asp3,Ser52^b,Ser54^d,Ile87^b,d,Phe90,Gly152^d,Leu295^d,Asn299^b,d Carbonylation His40,His87^b,His173,Cys374^b

Crosslinking Lys50/Glu270^b Disulfidebond Cys285^b,Cys374^b Glutathionylation Cys217^b,Cys374^b

Methylation Lys18^b,Lys68^b,His73^b,Lys84,Ile87^b,d,Asn299^b,d,Lys326^b,c Tyrosinenitration Tyr53^b,Tyr69^b,Tyr91^b,Tyr198^b,Tyr218^b,Tyr240^b,Tyr294^b,Tyr362^b S-nitrosylation Cys217^b,Cys257^b,Cys285^b,Cys374^b

Oxidation Cys17,Met44,Met47,Trp81^d,Met82,Trp88^d,Met178,Met190,Cys217^b,Met227,Cys257^b,Met269,Cys272^b,Cys285^b,Met235, Trp342^d,Met355,Trp358^d,Cys374^b

Phosphorylation Ser14,Ser33,Ser52^b,Tyr53^b,Ser60,Thr66,Tyr69^b,Thr77,Thr89,Tyr91^b,Tyr143,Thr148^b,S155,Thr160,Thr162,Tyr166,Tyr169, Thr186,Tyr198^b,Ser199^b,Thr201,Ser201^d,Thr202,Thr203,Tyr218^b,Thr229,Ser233,S235,Ser239,Tyr240^b,Thr249,Thr262^d,S265, S271,Tyr294^b,Thr297,S300,Tyr306,Thr318,Ser323,Thr324,Ser324^cTyr362^b,Ser365

SUMOylation Lys61^b,Lys68^b,Lys84^b,Lys113^b,Lys284^b,Lys291^b,Lys315^b,Lys326^b,Lys328^b

Ubiquitination Lys18^b,Lys50^b,Lys61^b,Lys68^b,Lys84^b,Lys113^b,Lys118^cLys191^b,Lys213^b,Lys215,Lys238,Lys284^b,Lys291^b,Lys315^b,Lys326^b, Lys328^b,Lys359

aHighlightedinbold:aminoacidmodificationsdescribedinthisreview.

bAminoacidresidesknowntobemodifiedbytwoormorePTMs.

cOnlydescribedinnon-mammalianactins.

dModifiedresiduesthatareobservedinclassIIactins(a-cardiac,a-smooth,a-skeletal,andg-smooth)wheretheNterminusstartswithMC-.

(A)

(B)

Class I acƟn

Class II acƟn

α-cardiac α-skeletal α-smooth γ-smooth β-cyto γ-cyto

↓ Cell size

↓ Lamella forma on

↓ Cell spreading

↑ Filament forma on

↓ Reduced G/F ra o

↑ Nt-arginyla on?

↑ Lamellipodia and filopodia

↑ Cell mo lity

Filament aggrega on

↑ Nt-acetyla on?

NAA80 NAA80

NAA80 NAA80

ATE1 ATE1

ATE1

Ac R

R

Ac

MetAP NatA? AP?

AP?

AP?

NatB

Figure2.Actin’sUniqueN-TerminalMaturationProcessandFunctionalConsequencesofNt-ModificationsontheActinCytoskeleton.(A)Actinsare firstsynthesizedasprecursormoleculeswhicharerarelydetectedintheirnativestateowingtoauniqueN-terminalmaturationprocess.ForclassIactins(b/g-actin)the nascentNterminiarecotranslationallyacetylatedbyNatB,followedbyremovaloftheacetylatedMet1byastillunidentifiedaminopeptidase(AP).Finally,thenewly exposedacidicNtermini(DDD-/EEE-)areacetylatedbyNAA80/NatH(Ac-DDD-/Ac-EEE-).Afewb-actinNterminiwillnotbeNt-acetylated,insteadtheyundergo furtherproteolyticprocessing,andthenewNtermini(DD-)arethenNt-arginylatedbyarginyl-tRNAproteintransferase1(ATE1)(RDD-).InthecaseofclassIIactins (a-actinsandg-smoothmuscleactin),methionineaminopeptidase(MetAP)removesMet1attheribosomefollowedbyacetylationofCys2presumablybyNatA(Ac-CD/

E-).Subsequently,theacetylatedCysresidueisremovedbyanunknownaminopeptidaseandtheresultingacidicNterminusisfinallyacetylatedbyNAA80(Ac-D/E-).

(B)Acetylationandarginylation(top)changestheN-terminalchargedensityandaffectsactinstructureandfunction.IntheabsenceofNAA80-mediatedacetylationof actin’sNterminus(middle),actinfilamentelongationanddepolymerizationareaccelerated.Moreover,NAA80HAP1knockoutcellsshowincreasedlamellipodiaand filopodiaformation,andcomparedtocontrolcellshaveincreasedcellmotility,asshownbyscratchwoundassayandchemotaxismigration.Consequently,NAA80acts asanaturalbrakeforcellmovement.Nt-arginylationpreventsactinfromaggregatinginvitro(bottom).ATE1knockoutmouseembryonicfibroblasts(MEFs)appear smallerthancontrolcells,andfailtoformnormallamella,causingimpairedcellmovement.ATE1isthoughttoregulateactivemigrationattheleadingedge.Ac,acetyl;

R,arginine.

TrendsinBiochemicalSciences,June2019,Vol.44,No.6 507

StressedActin:ARegulatoryPathway

For a long time, actin modifications caused by oxidative stress were considered to be exclusively destructive. Oxidative stress is caused by reactive oxygen species (ROS;

Figure3A),including:hydrogenperoxide(H₂O₂),hypochlorousacid(HOCl),andreactivenitrogen species(RNS),suchasnitricoxide(NO).ROSarebyproductsofphysiologicalredoxregulation, butarealsoactivelyproducedbyneutrophilsoftheinnateimmunesystem[36].AtlowROS concentrationsthethiolgroups(SH)ofcysteinescanbeoxidizedtosulfenicacidorbegluta- thionylated(Figure 3B,C). Thesemodificationscan bereversed byredox proteins,suchas thioredoxinandglutaredoxin.Moreover,theaccessiblemethionineresiduescanbereversibly oxidizedtotwomethioninesulfoxidediastereomers(Met-S-SOandMet-R-SO;Figure3C).Met- (S/R)-SOcanbereducedinhumansbyfourstereoselectivemethioninesulfoxidereductases:one MsrA,andthreeMsrBs(B1,B2,B3)[37].ItshouldbenotedthattheoxidationofMettoMet–SO convertsits hydrophobicside chainintoa hydrophilic moiety.Consequently, theoxidation/

reductionprocesscouldhavea profoundeffectonactin’s structuresandABP-interactions.

Oxidativestressfurtherleadstotheformationofdisulfidebridgesormixeddisulfidebondswith glutathione(Figure 3C). Themost vulnerabletarget in actin is Cys374,which canform an intramoleculardisulfidebondwithCys285(Figure3B),thelattercausingdelayeddissociation betweenactinandspectrin[38,39],andreducedactin filament dynamics[40].However,S- glutathionylationofCys374appearscrucialforthedisassemblyoftheactomyosincomplex,thus promotingcontractionofthecytoskeletonduringcellspreadingandtheformationofstressfibers [41].H2O2/HOClmainlytargetaccessiblecysteineandmethionineresiduesinG-actin,(Cys272, Cys285,Cys374, Met44, Met47, Met190,Met227, Met269,andMet355), which are more solvent-exposed than others (Figure 3B), especially when notburied inside actin filaments [42].WhenapplyinghighROSconcentrations,cysteineandmethionineresiduescanbecome irreversiblymodified(sulfinicandsulfonicacid,andmethioninesulfone).Inaddition,newmod- ificationsoccur,suchastyrosinenitration(Tyr294)[43]andhistidinecarbonylation(His40,His87, andHis173)[38,44,45].ThesemodificationsusuallyimpairactinpolymerizationanddestabilizeF- actin bundles [40,46]. Especially inthe case ofsevere oxidative stress, actin carbonylation accumulatesandleads toaggregationof actin[47].Consequently, theseirreversible modifications inhibitcellproliferation,motility,andreducecellviability.Itisnoteworthythatthemanystudiesthat identifiedactinmodificationsuponROStreatmentwereperformedinvitro,andthusthephysio- logicalrelevanceisnotalwaysobvious.

Despitetheirdestructivenature,ROS haveinrecent yearsbeenshownto actassignaling moleculesunderphysiologicalconditions,andtheirinducedmodificationsarekeyregulatorsin certaincellularpathways.MICAL-mediatedmethionineoxidationwasdiscoveredtoinitiateF- actindepolymerization[48].TheMICAL enzymesbelongtotheclass offlavoproteinmono- oxygenases,usingNADPHandH2O2tostereoselectivelyoxidizeMet44andMet47(Met44/47) ofactintoMet-R-SO[48–50].MICALsbinddirectlytoF-actin,enhancingitscatalyticactivity [48,51].TheoxidationofMet44/47,whichdependsontheADP/ATPnucleotide-bindingstate ofF-actin,destabilizesactinfilamentsandinitiatestheirdisassembly.Itfurthercausesconfor- mationalchangesofF-actin,whichincreasesthesusceptibilityforcofilin(anF-actindepoly- merizingfactor),andthusacceleratesfilamentdisassembly(Figure3D)[52].

Anothersignalingmolecule,NO,whichisenzymaticallygeneratedbytheendothelialnitricoxide synthase (eNOS), has important functions in T-cell regulation and activation [53]. eNOS colocalizeswithF-actinneartheGolgi,andmodifiesCys374byS-nitrosylation.Thisimpairs bindingtoprofilin-1,resultinginreducedactinpolymerizationandrelocalizationinsidethecell (Figure3E)[54].Theseexamplesdemonstratetheimportanceofactinoxidationasaregulatory factorandnotonlyasa‘killer’modification.

(A) (B)

(C) (D)

(E)

Oxygen

Superoxide

Hydrogen peroxide

Hydroxyl radical

Water

Neutrophil

Myeloperoxidase

Hypochlorite acid

Suscep bility towards ROS

Intra- and intermolecular

disulfide bond Sulfinic

acid

Sulfenic

acid Sulfonic

acid Free thiol

S-nitrosothiol

Methionine

Methionine sulfone

Methionine-(R) sulfoxide Methionine-(S)

sulfoxide Trx / Grx

Trx / Grx Oxida on

Oxida on Oxida

on Oxidaon

Oxida on

Oxida on Oxida

on

Glutathionyla on Glut

athion yla

on GSH

MsrA MsrB

Polymeriza on Cofilin

T-cell regula on T-cell regula on via eNOS

Ac n Ac n Ac n Ac n

Profilin-1

Profilin-1

Profilin-1 eNOS

Low High

His40 His173

Tyr294 His87

Cys374

Met190 Met227

Cys217 Cys257

Met44 Met47

Met355

Cys272

Cys285 180°

ROS concentraƟon

→ Reduced polymeriza on

→ Normal polymeriza on

L-citrulline

Figure3.

(Figurelegendcontinuedonthebottomofthenextpage.) ROS-MediatedActinModificationsandRegulationbyMICALEnzymes.(A)Reactiveoxygenspecies(ROS)areproductsofredoxreactionsandare generatedwhenmolecularoxygen(O2)isnotcompletelyreducedtowater(H2O),resultinginsuperoxide(O2 ),hydrogenperoxide(H2O2),andhydroxylradicals(∙OH).

TrendsinBiochemicalSciences,June2019,Vol.44,No.6 509

PhysiologicalConsequences ofActin PTMs

ActinPTMsplayacentralroleinmanybiologicalprocesses,includingneurodevelopment.Neurons haveanelaborate networkofactinfilaments,especiallyindendritic spinesandgrowthcones.

Dynamic phosphorylation of Tyr53regulates F-actin turnover rates by destabilizinglong actin filaments.Moreover,itpromotes thestabilityofshorteractinfilaments,whichfacilitatesafaster reorganizationofthecytoskeletonindendriticspinematuration[55],aprocesscrucialforlearningand memoryformation.NeuronaldevelopmentalsodependsontheactivityofNADPHoxidase2(Nox2), whichregulatesthedistributionofH2O2inneurons[56].Nox2colocalizeswithF-actinbundlesinthe periphery of neuronal growth cones where its H2O2-producing activity regulates F-actin dynamics and neuriteoutgrowth[57].Furthermore,ATE1wasrecentlyshowntobecrucialfornormalneuronal outgrowthandmigrationinmice[58].ItwassuggestedthatATE1’sroleinbraindevelopmentarises fromcotargetingofATE1andb-actinmRNAstothegrowthcones,resultinginalocalsynthesisof arginylatedb-actinthatregulatesneuriteoutgrowth.Moreover,ATE1^-/-micedieduringembryogen- esis,mostlikelyduetodefectiveheartandvasculardevelopment[59].Theexactunderlyingmolecular mechanism(s)fortheroleofarginylationincellmotility[30],embryogenesis[59],andtissuedevelop- ment[58,60]isnotcompletelyunderstood,giventhatATE1hasmorethanoneproteintarget.

ActinPTMsarealsoessentialforeffectivecytokinesisandpropercelldivision.Thedioxygenase ALKBH4localizestothecontractileringwhereitdemethylatesK84me1ofactin,thuscreatinga bindingsitefornonmusclemyosinII.ALKBH4-deficientcellsdisplaydefectivecleavagefurrow organization,resulting incytokinesis failure andformation of multinucleatedcells [61]. After cleavagefurrowingressionandmidbodyformation,actinmustbeclearedfromtheabscission sitetoenablemembranescissionbytheESCRTmachinery.ThisisachievedbyGTPaseRab35 activationofMICAL1,which isthenrecruitedtotheabscissionsitewhereit promotesrapid depolymerizationof F-actin from both ends,leading to an efficient clearing ofF-actin [62].

CytoskeletalreorganizationcanbealsoinitiatedbytheAblkinase,whichphosphorylatesthe Tyr500of MICAL1,enhancingitsactivity[63].SincetheAblkinaseresponds toanumber of stimuli, suchasthesemaphorin/plexincomplex,orthegrowthfactorsEFGandPDGF,MICALactivation andsubsequentactinoxidationhasabroadspectrumofphysiologicalconsequences[64,65].

Actin shuttles between the cytoplasm and nucleus in an ABP-dependent manner. In the nucleus,actinisthoughttofacilitatechromatinremodelingandgenetranscription.MICAL2 inducesF-actindepolymerizationinthenucleus,enablingnewlyrestoredG-actintoactasa transcriptionalregulatorinserumresponsefactorsignaling[66].Moreover,nuclearactincanbe SUMOylatedinaprocessthatrequiresLys68andLys284[67,68].Ithasbeenspeculatedthat SUMOylationregulatesnucleartraffickingandactinstructure[67].

Duetoactin’sabundanceandroleinmusclecells,itisnotsurprisingthatactinPTMsparticipatein thecontractilemachinerybymodulatingtheelectrostaticinteractionsbetweenF-actin,tropomy- osin,andmyosin.Forexample,acetylationofLys326andLys328maskspositivechargesthatare crucialforproperthinfilamentregulation[69,70].Expressionofpseudo-acetylatedcardiacactin

Hypochloriteacidisactivelyproducedbytheenzymemyeloperoxidaseinneutrophilsoftheinnateimmunesystemasdefensemechanismstowardsinvading pathogens.(B)b-actinstructure(PDB:2BTF)[85]highlightingsurfaceexposedCys,Met,His,andTyrresiduesthataresusceptibletowardsROS-mediated modifications.(C)ROStargetmainlyCysandMetresiduesofactininaconcentration-dependentmanner,resultinginavarietyofPTMs(disulfidebondformation, nitrosylation,glutathionylation,multilevelcysteineoxidation,andstereoselectivemethionineoxidation).MostofthesePTMsarereversedbyredoxenzymes(Trx, thioredoxin;Grx,glutaredoxin;MsrA,methioninesulfoxidereductaseA;MsrB,methioninesulfoxidereductaseB).HighROSconcentrationsleadtoirreversible modifications(sulfinicandsulfonicCysoxidation,Metsulfone).(D)MICALenzymesbindtoF-actinandcatalyzeinanNADPH-dependentreactiontheoxidationof Met44andMet47toMet-R-SO,initiatingdepolymerization.Inaddition,thisattractstheF-actinseveringproteincofilin,therebyacceleratingthedepolymerizationeffect.

Met-R-SOcanbereducedtoMetbyMsrB,whichallowsactintoenteranewpolymerizationcycle.(E)Nitricoxide(NO)isinvolvedinT-cellregulationandactivation.

eNOSS-nitrosylatesCys374ofactin,impairingprofilin-1binding,andthusreducingactinpolymerizationrates.

(K326Q,K328Q,andK326Q/K328Q)inindirectflightmusclesofDrosophilamelanogasterleads toperturbedmusclestructureandfunctionaswellasdisruptingflightperformance[71].Masking ofLys326andLys328isthoughttoaltertheelectrostaticinteractionswithtropomyosin(Glu181) and/or myosin (Glu286), destabilize the inhibitory positioning of tropomyosin, and thereby enhanceactomyosinformationcausingmusclehypercontractility[71].Actinacetylationmight thereforebe crucial for proper musclefunction. Indeed, the K328Q actin mutation causes nemalinemyopathywithmusclestiffnessandhypertonia[72].

TheRole ofActin PTMsin Diseases

Theactincytoskeletonhasanindisputableroleinhumandevelopmentandfailuretoorches- tratethedynamicinterplaybetweenactinandABPscouldleadto actin-relateddiseases,a conceptwhichwasfurtherelaboratedbyRubensteinandWen(Box1)[73].Theydescribeda regulatoryallostericsysteminhumanactinsthatappearpronetodisease-causingmutations.

SomeofthemosteffectivemutationscolocalizewithaPTMhotspotinanotherwisepoorly modifiedregion.Wethereforesuggestsimilarmolecularpathophysiologyupondysfunctional actinmodifications,especiallywithregardtoABPinteractions(Box1).

Abnormalcellinvasionandmetastasisisahallmarkofcancer,twoprocessesinwhichtheactin cytoskeletonplaysadominantrole.Therefore,itisnotsurprisingthatactinPTMshavebeenlinked tocancerdevelopmentandtumorigenesis.BothNAA80andATE1KOcellsdisplaydefectivecell motility[27,30],whichisacommonfeatureamongcancercellsandcontributestoinvasionand metastasis. Reduced ATE1 expression has been reported in varioushuman cancers [74].

Moreover,ATE1KOMEFsexhibitdefectivecontactinhibitionwhichisthoughttosupportthe uncontrolledgrowthindenseculturesandinvasivebehaviorinMatrigels[74].Thedirecttumori- genicpotentialofNAA80-deficientcellshasyettobeinvestigated.Nevertheless,severalsomatic mutationsinNAA80andATE1arereportedintheCOSMICcancerdatabase(v86,released14 Aug2018)[75].Forexample,the mutationprofile ofNAA80inhumancancersincludes 45 missensemutations andtwo frameshiftmutations that,as faras we know,have notbeen characterized.Theframeshiftdeletionmutationp.E92fs*5(resultingin92outof308aminoacid residues)should,intheory,giverisetoacatalytic-inactiveformofNAA80.Furthermore,severalof theresiduesthatareaffectedbymissensemutations(W105R,R107H,R112H,F123S,G190D, L194Q, P258A, P266L,P267S, P283L,G298W, andI308M) are evolutionarily conserved, implyingthatthese residuesmaybeimportantforNAA80’sstructureandfunction[29].Itis currentlynotknownwhetheranyoftheseNAA80mutationshavedisease-causingeffect(s).But onecouldspeculatethatsomeofthemaffectNAA80’sactivityandactinNt-acetylation,thus alteringcytoskeletondynamicsandpromotingtumorprogression.

TheemergingroleoftheMICALenzymefamilyinF-actindisassembly,akeyelementofcell motilityandmigration,hasplacedtheMICALsatthenewhorizonofcancerresearch.MICAL1 expressionwasdirectlylinkedtoincreasedcellmigrationandinvasivenessinvariousmelanoma andbreastcancermodels[76–78].ROSproductionbyMICAL1,whichpromotesepithelial– mesenchymaltransition (EMT),and thusmetastasis formation, was linkedto typical EMT- dependentsignalingcascades,suchassemaphorin/plexin[76]andRab35signaling,aswellas thePI3K/AKTpathway[77]andtheEGF-inducedMAPK/ERKpathway[78].RegulationofEMT was also linked to MICAL2 expression. Gastric and renal epithelial cancer cells show an increaseinEMTuponMICAL2expression,andareducedviability,motility,andinvasiveness whenMICAL2isdepletedfromthesecells[79].

ActinPTMs also play exceptionalrolesin the developmentof infectiousdiseases. Several bacterialpathogensrelease toxins that induceADP-ribosylation and crosslinking, of actin,

TrendsinBiochemicalSciences,June2019,Vol.44,No.6 511

whichinterferewiththe hostcells’ability to polymerizeactin(Box 2). Althoughothertoxic effectorsaresecretedbypathogensintothehostcell,theactin-modifyingtoxinstakeakeyrole inalteringthehostcellcytoskeletontotheadvantageofthepathogen(Box2).

CompetitionforActin’sNTerminus:Nt-Acetylationversus Nt-Arginylation Nt-arginylationwasonlyreportedforb-,butnotg-actin,inwhichtheDDD-actinN-terminal sequencewasconvertedintoan RDD-actinsequence,turninga 3chargedNterminus

Box1.PathogenicActinAllostericRegulatorySystem:ANewConcept

Bartlett,Rubenstein,andtheircolleagues,hypothesizedtheexistenceofapathogenicactinallostericregulatorysystem wherethebindingofABPsinitiatesconformationalchangesinstructuralnetworks,affectingactinfilamentformationand stability[73,86,87].Centraltothehypothesisisactin’spathogenichelix(Lys113–Thr126),whichextendsfromthe filamentsurfacetothestrand–strandinterphase,andtheC-terminalhelix(Val370–Phe375;FigureI).Thetwohelixesare interconnectedviainteractionsbetweenGlu117andHis371.Moreover,Lys113inthepathogenichelixextendstowards theactin–actininterfacewhereitformsanionicbridgewithGlu195ofanactinsubunitintheopposingstrand.This filament-stabilizinginteractionismodulatedbyArg256onthecross-strandmonomer,givingrisetoatriangularunit.

Together,theseinteractingstructuralelementsarethoughttoconstituteanallostericsystemwheresurfacebindingof ABPsmayinduceconformationalchangesthatpropagatethroughouttheactinmoleculeandaffectfilamentdynamics.

Thepathogenichelixisamutationalhotspotandimplicatedinseveralactinopathies,includingnemalinemyopathy, Baraitser–Wintersyndrome,anddeafness(reviewedin[73]).Forexample,twomissensemutationsing-actin(K118M andK118N)cangiverisetononsyndromicdeafness.Astudyusingyeastactinrevealedthatbothmutationsaffectthe structureandfunctionoftheDNaseIbindingloop,andinthecaseofK118Nresultedinfasterfilamentformation[88].

TheK113Emutationina-actinisassociatedwithnemalinemyopathy[89]andwasrecentlyreportedtosuppressactin catch-slipbonds[90].Inyeast,expressionofK113Eactinleadstogrowthdefectsanddefectiveactinpolymerization [91].WithinthebroadrepertoireofactinPTMswenoticethatbothLys113andLys118,whicharemembersofthe pathogenichelix,arelocatedwithinaregionthatishighlyPTMsilent.Residues96–142donotcarryPTMs,exceptfor Lys113whichcanundergoacetylation,SUMOylation,orubiquitination.WeassumethatunintendedLys113modifica- tionscouldinduceeffectssimilartothedifferentphenotypesdescribedforthemutationsatthissite.Anotherexampleis Cys257,whichishighlysusceptibletoROS-inducedmodifications[3].Missensemutationsintheneighboringresidue Arg256areimplicatedinseveraldiseases[73].Interestingly,differentmutationsofArg256causevarioussymptoms whereR256CandR256HareassociatedwithTAADaneurysms,whileonlytheR256Cmutationcausescerebral aneurysms.Finally,wenotethatseveralresidueswithintheC-terminalhelixaremodified,includingCys374whichis highlyreactive.Together,theseeffectsemphasizethefunctionalimportanceofthisregionforproperactinfunction.

Monomer 2

Monomer 1

FigureI.Actin’sPathogenicHelix.Shownisamodelstructureoftwoa-skeletalactinmonomers(PDB:2ZWH)[92].

Theinteractionbetweentwoprotomersinanactinfilamentisdependentonthepathogenichelix(magenta,Lys113– Thr126)andtheC-terminalhelix(orange,Ala365–Phe375)ofmonomer1(cyan),andtheresiduesGlu195(redspheres) andArg256(pinkspheres)ofmonomer2(green).