Review
First Things First:
Vital Protein Marks by
N-Terminal Acetyltransferases
Henriette Aksnes,
1Adrian Drazic,
1Michaël Marie,
1and Thomas Arnesen
1,2,*
N-terminal(Nt) acetylationis known tobe a highly abundant co-translational proteinmodification,buttherecentdiscoveryofGolgi-andchloroplast-resident N-terminalacetyltransferases (NATs)revealedthat itcanalsobe addedpost- translationally.Nt-acetylationmayactasadegradationsignalinanovelbranch of theN-end rule pathway, whose functions includetheregulation of human bloodpressure. Nt-acetylationalso modulates proteininteractions, targeting, andfolding.Inplants,Nt-acetylationplaysaroleinthecontrolofresistanceto droughtandinregulation ofimmuneresponses.Mutations ofspecifichuman NATsthatdecreasetheiractivitycancauseeitherthelethalOgdensyndromeor severeintellectualdisabilityandcardiovasculardefects.Insum,recentadvan- ceshighlightNt-acetylationasakeyfactorinmanybiologicalpathways.
YourFavoriteProteinIsProbablyAcetylatedatitsN-Terminus
N-terminalacetylation(Nt-acetylation),alsocalledN/-acetylation(seeGlossaryandFigure1), isaproteinmodificationthatispoorlyrepresentedintextbooksand,assuch,manyscientistsare unawarethat80%ofallhumanproteinsreceiveanacetylgroupattheirN-terminus.Owingto severalrecentadvancesinthefield,however,thereisnowagrowinginterestinthisprotein modification. Severalrecent contributionsshed light onthemolecular mechanisms through whichtheN-terminalacetyltransferases (NATs)exerttheir importantbiologicalfunctions.
Thisreviewaimsatadvancingourcurrentunderstandingoftheprotein,cellular,andphysiologi- calconsequencesofNt-acetylationbasedonamultitudeofrecentreports.Consequently,italso highlights the fact that Nt-acetylation can no longer be viewed as an automatic negligible modification,butinsteademergesasacrucialcomponentinmanybiologicalpathways.
HowManyandWhichProteinsAreN-TerminallyAcetylated?
Nt-acetylationhasbeenestablishedasahighlyabundantproteinmodificationineukaryoticcells [1–4].Infact,itissocommonthatitisreasonablysafetopresumethatyourfavoriteproteinis probablysubjecttothismodification.ThedeterminantforundergoingNt-acetylationismainly theidentityofthefirsttwoaminoacidsattheN-terminus.SeveralNATs(NatAtoNatFinhumans) collectivelycatalyzeNt-acetylationofamajority(80%)ofthedifferenttypesofN-terminioccurring intheproteome,resultingintheNt-acetylome(Figure2A).NatAhasspecificitytowardsA-,S-, T-,V-, C-, andsometimes G-starting N-termini, whoseinitiatormethionine (iMet) has been removed by methionine amino peptidases (MetAPs) [2,5]. NatD also acts within the iMet- processed group;however,it is far moreselectivebecauseit has only beenshownto Nt- acetylatehistonesH2A andH4, makingitscontributiontotheNt-acetylomenegligible[6,7].
ThoseN-terminithatretaintheiMetaremodifiedbyNatB,NatC,NatE,andNatF.Inthisgroup NatBacetylatesMet-’Asx/Glx’-typeN-termini(MD-,ME-,MN-,andMQ-starting)[8,9]whereas
Trends
The identification of the first mem- brane-associated NAT, Naa60/NatF, andthefirstchloroplastNAT,Naa70/
NatG,establishednewmodesofthe NAT machinery in their capacity to acetylatetransmembraneandlumenal chloroplastproteins,respectively.
The structure of the NatA complex revealed molecular determinants for substrate-specific acetylation, includ- ingthesignificantimpactoftheauxiliary Naa15onthespecificityofthecatalytic Naa10.
Nt-acetylationhasbeenshowntoreg- ulate protein complex stoichiometry throughtheAc/N-end rule pathway, which has also been connected to hypertension.Further,alinkwasestab- lishedbetweenNt-acetylationandglo- balproteinfolding.
Nt-acetylationhasbeenfoundtoplay essentialrolesinA.thalianadrought- stressandimmune responses,in C.
elegansdevelopmentandmetabolism, aswellasinhumandiseases.
1DepartmentofMolecularBiology, UniversityofBergen,5020Bergen, Norway
2DepartmentofSurgery,Haukeland UniversityHospital,5021Bergen, Norway
*Correspondence:
[email protected](T.Arnesen).
Glossary
GCN5-relatedN-acetyltransferase (GNAT)superfamily:protein superfamilyrelatedtogeneralcontrol nondepressed5(GCN5)comprising, amongothers,thecatalyticN/- acetyltransferases.
N-terminalacetyltransferase (NAT):enzymethatcatalyzesNt- acetylation.
N-terminalacetylation(Nt- acetylation):alsoknownasN/- acetylation,thisisaprotein modificationinvolvingtheadditionof anacetylgrouptothefreeamino group(N/-group)ofasubstrate protein(Figure1).
NAA/Naa:N/-acetyltransferase gene/protein,respectively.Names giventohumanN-terminal acetyltransferasesarenumerically organizedbyacetyltransferaseactivity type:NatAactivityismediatedbythe catalyticsubunitNaa10andthe auxiliarysubunitNaa15,whileNatB activityismediatedbythecatalytic Naa20andauxiliaryNaa25subunits, andsoon.
Nt-acetylome:thepartofthe proteomethatissubjectedtoNt- acetylation.
NatC,NatE,andNatFactonMet-’hydrophobic/amphipathic’-typeN-termini(ML-,MI-,MF-, MY-, and MK-starting) [10–15]. The recently identified plant NatG acetylates M-, A-, S-, T-startingN-termini,howeveritlacksahumanortholog[16].
AlthoughproteomictechnologiesareincapableofdirectlydefiningtheNt-acetylationstatusof theentireproteome,anestimateofthetotalNt-acetylationeventsispossiblebyextrapolationof proteomicsdatatoallSwiss-Protentries(Figure2,basedon[17]).Throughthisapproach,itis calculatedthatthe NATfamily Nt-acetylates 80%ofthehuman proteome (Figure 2A). This amountrelatestobothfullandpartialNt-acetylationbecausemanyproteinsexistasbothNt- acetylatedandunacetylatedvariants.Henceitisverylikelythatagivenprotein,forexampleyour favoriteprotein,issubjecttoNt-acetylation.Indeterminingthelikelihoodofaparticularprotein beingNt-acetylatedbasedonitstwofirstaminoacidsonecanmakeuseofthechartinFigure2B wherethesizeofsubgroupswithineachNATsubstrateclassisshownaswellasthefrequency ofNt-acetylationeventswithinthesubgroups.IftheN-terminusoftheproteininquestionstarts withalanine,whichisintheNatAsubstrateclass,thereisa95%chancethatitisNt-acetylated, butifitstartswithvalinethereisa80%chancethatitsN-terminus isunacetylated. NatBis distinguishedinthissensebecauseithasnear100%coverageoftheMet-’Asx/Glx’-typeN- termini.ThecombinedactionofNatC,EandFwithintheMet-’hydrophobic/amphipathic’-group willprobablyundergofurtherrefinementinsubstratespecificity-profilingbecauseitactsonlyon 75% of thesequence-predicted substrates. A recent study on NatF revealed that it has selectivityformembraneproteinsandthattheNatFsubstratecategoryisenrichedfortrans- membraneproteins[17].Thisstudywasalsothefirsttoinvestigatethemembrane-boundpartof theNt-acetylome,whichwasfoundtobeequallylargeintheamountofproteinsNt-acetylated, althoughtheseweretypicallyNt-acetylatedtoalesserdegree,meaningthatthereweremore partialNt-acetylationevents.
TheEnzymesCatalyzingN-TerminalAcetylation TheNATCompilation
The eukaryotic NAT machinery known to date is composed of seven NATs (NatA–NatG) (Figure 3) ofwhich five (NatA–NatE) are present in Saccharomyces cerevisiae (reviewed in [18,19]),whereasmulticellulareukaryoteshaveasixthNAT,NatF[15,17],andinadditioninthe plantArabidopsisthalianaaseventhNAT,NatG[16]hasbeenidentified.TheeukaryoticNAT machineryismorecomplexthanthatofprokaryotes,whichonlyhavethreeknownNATs.Itisnot wellunderstoodwhy eukaryoticcellshaveevolveddifferentNATs. NATdiversificationcould
N-terminal amino group
Polypepde
H H
H H
N+ N
CH CH
C
C
C O
O
O
NH N
H
R1 R1
H3C
H3C C O
S CoA SH CoA
α α
Acetyl-Coenzyme A N-terminal acetyltransferase
(NAT) Polypepde
Figure 1. The Addition of an Acetyl Group to a Protein N-Terminus Is Catalyzed by an N-Terminal Acetyltransferase(NAT).N-terminalacetylationisaproteinmodificationthatchangesthechemicalpropertiesofthe N-terminusbyneutralizingitspositivechargethroughtheadditionofanacetylgroup.Thereactionisenzymaticallycatalyzed byN-terminalacetyltransferases(NATs)thattransfertheacetylgroup(-COCH3,red)fromacetyl-CoA(Ac-CoA)ontothe N/-group(NH3+
,yellow)oftheveryfirstaminoacidresidueofthesubstrateprotein.Thismodificationisoftenreferredtoas N/-acetylationtodistinguishitfromtheacetylationofNe-groupsonlysinesidechains.
conceivablyhaveco-evolvedwiththeincreasingcomplexityoftheproteome.However,based onthecurrentlyidentifiedenzymes,NATsdidnotdivergeduringtheevolutionofeukaryotesin whichproteomecomplexityvastlyincreased[19].ThemorecomplexeukaryoticNATmachinery entailsaspecializationtowards differentsubstrategroups thatcould provideamoreflexible systeminterms ofregulation.Infact,someexamplesofupstreamregulationofNATshave recentlybeenuncovered(discussedinfollowingsections).TherecentlydiscoveredNATs,NatF targetingtransmembraneproteinsandNatGtargetingchloroplastproteins,areclearexamples ofspecialization,andlikelyexistowingtoaneedforparticularsubgroupsofthesesubstrate typestobeNt-acetylated.
(A)
Proteome
(B)divided into Nt-acetylome and non-Nt-acetylome
Proteome
divided into NAT-substrate classes
Nt-acetylome 80%
T- (82%) C- (50%) G- (33%) V- (20%) S-
(99%)
Nt-Ac 38%
[67%]G- Un-Nt-Ac 8%
P- [100%]
Various
MV-, MA-, MG-
and other Nt-Ac
21%
ML- (88%)
MN- (100%) Un-Nt-Ac0.2% ME-
(99%)
MD- (100%)
Nt-Ac 20.8%
Un-Nt-Ac 7%
Un-Nt-Ac 5%
A- (95%)
V- [80%]
A-, T-, C-, S-
MQ- (95%) MQ- [5%]
MI- (71%) MF- (67%) MY- (75%) MK- (54%) MM- (100%)
NatA 46%
NatB NatC/E/F 21%
28%
Non-matching 5%
NatA 38%
NatB 21%
NatC/E/F 21%
Non-Nt- acetylome
20%
5%
8%
0.2%
7%
Figure2. Occurrence ofN-TerminalAcetylation in the HumanProteome.(A)Tovisualizethecommonoccurrenceof Nt-acetylationandN-terminal acetyltransferase(NAT)activity,thehumanproteomeisdividedintotheNt-acetylome(80%)andthenon-Nt-acetylome(20%),andsubdividedbasedonNAT-type substratecategoriestoillustratethecontributionbythedifferentNATs.NotethattheNATsubstrateclassesdescribedtodatealsocontainsomepartofthenon-Nt- acetylome,asindicatedbythecolor-coding.(B)ToprovideatoolfordeterminingthelikelihoodthataparticularproteinN-terminalsequenceundergoesNt-acetylation, thehumanproteomewasdividedintoNATsubstrateclasseswiththecontributionofdifferentN-terminiindicated.TheinnerpieshowsthesizeoftheNATsubstrateclass;
themiddlecirclematchesthepiein(A)andindicatestheamountofNt-acetylatedversusunacetylatedproteinswithineachsubstrategroup;andtheoutercircleprovides amoredetailedoverviewofthedifferentN-terminiconstitutingasubstrateclassthatcanbeusedtopredicttheNt-acetylationstatusofagivenprotein.Forexample, alanineA-startingproteinsconstitutethelargestsubgroupwithintheNatA-typesubstrateclass,and95%ofA-startingproteinsareNt-acetylated.ForNatB,thesubstrate categoryaccountsfor21%oftheproteomewithonly0.2%beingunacetylated,allowingtheassumptionthatanyMD-,ME-,MN-,MQ-startingproteinisNt-acetylated.
NatC/E/F-typesubstratesaremoredifficulttopredictbasedoncurrentsubstrateprofilesbecausethisgroupaccountsfor28%oftheproteome,butwith7%unmodified.
ML-isthemostcommonsubstratewithintheNatC/E/Fclass,and88%ofML-N-terminiareNt-acetylated.NumbersarebasedonanestimateoftotalNt-acetylation eventsmadebyextrapolationofthemostrecentproteomicsdata(alsoincludingorganellarproteins)[17]toallSwiss-Protentriesbasedontheoccurrenceofthefirsttwo aminoacids.PartialNt-acetylationisconsideredasanNt-acetylationevent,meaningthatagivenproteincanexistinbothNt-acetylatedandnon-Nt-acetylatedforms, referredtoasapartiallyNt-acetylatedprotein.ProteomeoccurrencesofpartialiMetretentionforMA-,MS-,MT-,MG-,andMV-werecalculatedbasedontheirdetection asiMet-processed/unprocessed.DatawerelackingforMW-andMR-startingproteins,andthesenumberswereinferredbasedonstructuralsimilarity.
(A)
NatA
NatE NatF NatG
NatB NatC NatD
Ac
Ac
Ac
Ac
Ac Ac Ac
Naa10
Naa10
Naa15 Naa50 Naa60 Naa70
Membrane
Nucleus
Yeast Mammalian Plant
Nucleus
Nucleus
Chloroplast
Golgi Golgi
A A
A
B B
B
C
C
C
D
D
D
E
E
E
F
F
G
? Chloroplast
Amino acids Π Small α Asx/Glx φ Hydrophobic Ω Aromac Ψ Aliphac [+] Posively charged
x Undetermined HYPK
Naa15 Naa50
Naa20 Naa30
Naa35 Naa40
Naa38 Naa25
Ribosome mRNA
HYPK
Π
M
M
M M S
α φ/Ω/Ψ/[+] G
Π/φ/Ω/Ψ/[+]
Π/φ/Ω/Ψ/[+]
Π/Mx
(B)
Figure3.TheN-TerminalAcetyltransferase(NAT)MachineryinEukaryoticCells.(A)SubunitcompositionofthesevencurrentlyknowneukaryoticNATs,NatA–
NatG.Thecatalyticsubunits(Naa10,Naa20,Naa30,Naa40,Naa50,Naa60,andNaa70)typicallyassociatewithuptotwoauxiliarysubunits(Naa15,Naa25,Naa35,and Naa38)thatcontributetoactivityoftheNATcomplexthroughribosomeanchoringand/orsubstratespecificity-modulation.Aminoacidsarecodedasfollows:p,small;/, Asx/Glx;f,hydrophobic;W,aromatic;C,aliphatic;[+],positivelycharged;andx,undetermined.SpecificN-terminiofthedifferentNATclassesareshowninFigure2.(B) IntracellulardistributionofthedifferentNATcomplexesfromunicellular(yeast)tomulticellular(mammalsandplants)eukaryotes.WhileNatA–NatEresideinthecytosol, thetwomostrecentlyidentifiedNATsareorganellar.NatFisfoundinbothmammalsandplantswhereit,atleastinhumancells,associateswiththeGolgisurface.NatGis foundinsideplantchloroplasts.Inaddition,someoftheNATcatalyticsubunitsappeartolocalizetothenucleus(e.g.,Naa10andNaa50;notshown).
NATactivitytypicallyrequiresaNATcomplexinwhichthecatalytictransferasesubunitjoinsup totwoauxiliarysubunitsthatmaymediateribosomeanchoringandinsomecasescontributeto substratespecificity(Figure3A)[20–22].TheNatAcomplexisformedbythecatalyticsubunit Naa10andtheauxiliarysubunitNaa15[5,23].Naa50isalsoattachedtotheNatAcomplex togetherwithHYPK(Huntingtin-interactingproteinK)[20,24,25].Naa50alsoformspartofthe NatEcomplex(togetherwithNaa15andNaa10),whichdisplaysdistinctsubstratespecificity andadistinctdepletionphenotypecomparedtoNatA[12,14,20,21,26].NatBiscomposedof thecatalyticsubunitNaa20andtheauxiliarysubunitNaa25[27,28].The catalyticsubunitof NatCisNaa30,whichassociateswithtwononcatalyticsubunits,Naa35andNaa38,ofwhich Naa35 mediates ribosomal association, whereas the role of Naa38 is less well described [11,13,22].ForNatD(Naa40),NatF(Naa60),andNatG(Naa70)noauxiliarysubunithasbeen identifiedsofar(Figure3A).Interestingly,arecentstudyrevealedthatanN-terminalsegmentof Naa40,whichisuniquecomparedtotheothercatalyticNaas,mightplayarolethatisanalogous totheribosomal-bindingauxiliarysubunitsfoundintheotherNATs[29].
TheWorkplaceofNATs
MostNATsarelocalizedinthecytosol(Figure3B).NatA–NatEareassociatedwithribosomes, wheretheyperformco-translationalNt-acetylation(Figure3A)[20,22].Inaddition,somecatalytic subunitsmaylocalizetothenucleus[7,17,23]andNaa10andNaa50alsodisplayNATactivityin theabsenceoftheribosome-anchoringsubunit,Naa15,butwithalteredsubstratespecificities [12,14,17,21].GiventhattheseNATsalsoexistinnonribosomalforms,thissuggeststhatNATs mightalsoactpost-translationally[14].In fact,severalinternal peptides,producedbypost- translationalcleavage invivo,areNt-acetylated[30,31] andaspecific exampleofa protein undergoingsuchpost-translationalprocessingisactin[32].
OfparticularinterestaretwonewlyidentifiedNATswithorganellarlocalization(Figure3B).NatFis associatedwiththeGolgiapparatusfacingthecytosolicsidewhereitNt-acetylatestransmem- braneproteins[17].NatGwasidentifiedasthefirstNATlocalizedinsideanorganelle,inthiscase inchloroplastsoftheplantA.thaliana[16].Theserecentstudiesunderpinthepost-translational natureofNt-acetylationandfinallyconnectthismodificationtocellularorganelles.
Nt-AcetylationfromaStructuralViewpoint:ABlueprint
StructuralinformationontheNATsiscurrentlyemergingasthecrystalstructuresofseveralN/- acetyltransferasesandNATcomplexeshavebeensolved.Therecentlydevelopedbisubstrate analog-basedN/-acetyltransferaseinhibitors[33]havebeenusefulinsomeofthesestructural studies.
AcrystalstructurewithbothsubunitsoftheNatAcomplex(Naa10andNaa15)hasbeensolved (Figure4A)[21].TheNaa15subunitconsistsof37/-helicesarranged into13TPR(tetratri- copeptiderepeat)motifs[21].Theseareconservedmotifscomposedofsequencesof34amino acidsthatgenerallyserveasprotein–proteininteractionmotifs[34].SomeoftheTPRmotifsin Naa15are involved in Naa10binding [21], andit islikely that theyare also crucial forthe associationsofNaa15with Naa50,HYPK,andtheribosome(Figure3A). Theoveralltertiary structureofNaa15formsaring-likestructurewithacavityintowhichNaa10binds(Figure4A).
TheNaa15–Naa10bindingismainlyconductedbyalargehydrophobicinterfaceaswellasbya fewhydrogenbonds[21].
Thecatalytic subunitsofNATsbelongtotheGCN5-related N-acetyltransferase(GNAT) superfamilytogetherwithsomeofthelysineacetyltransferases(KATs).AlltheNATssharethe structuralGNAT-domain(Figure4B),whichisanevolutionarilyconservedcharacteristicofNATs, asshownbystructuralanalysesofthebacterialNATRimI[35]andtheNATfromthearchaea Sulfolobussolfataricus[36].TheGNATdomainconsistsofacentralacetyl-CoAbindingmotif
(Q/RxxGxG/A)flankedbyfour/-helicesandsevenb-sheetsegments[16,21,29,37,38].The acetyl-CoAbindingdomainisformedbyseveralsecondaryelements(/3andb2,b3,andb4) [37].Substratebindingandthecatalyticbi-bireactionoccurinasemi-opencavitythatharbors both acetyl-CoA and the first 4–5 amino acids of the N-terminal peptide (Figure 4B,C).
(A)
Naa15
Ala - Glu - Ser - Ala - Ser Ac - Coenzyme A
Naa10
(B)
(D)
HO O
O O
O O
O O O
S N
HO HO
OH
OH
OH OH OH OH O
O O
O O
N N N
N NH2
O O O P P
P O
HN H
N H
N N
H N
H HN
(C)
Figure4.StructuralModelsoftheNatAComplex.(A)CrystalstructureoftheSchizosaccharomycespombeyeast NatAcomplexshowingtheinteractionbetweenthecatalyticsubunitNaa10(cyan)andtheauxiliaryunitNaa15(pink).The semi-transparencyofNaa15showstheinsertionofNaa10intotheNaa15bindingcavity.(B)X-raystructureoftheGNAT- fold(cartoon)depictedonNaa10(surface)showingthepositionsoftheacetyl-CoAbindingdomain-forming/-helix/3 (yellow)andb-sheetsb2(blue),b3,andb4(greentones)aswellasthepeptidesubstrate-bindingpocket-formingN-terminal /1-loop-/2(blue/cyan,lowerarrow)andtheC-terminalb-hairpinofb6-loop-b7(orange/red,upperarrow).(C)Association ofabisubstrateanalog-basedinhibitorwiththepeptide-acetyl-CoAsubstrate-bindingpocketsoftheNaa10monomer enzymeinitsNatAcomplexconfiguration.(D)Chemicalstructureofthebisubstrateconjugateshownin(C),consistingof acetyl-CoA(blue)covalentlylinkedtoaNatAsubstratepeptidefragmentSer-Ala-Ser-Glu-Ala.Bracketsrepresentthe segmentofthesubstratepeptidenotcrystallizedandnotshownin(C).TheX-raydiffractionstructuresin(A–C)aremodified fromproteindatabank(PDB)entry:4KVM[21].
SubstratespecificityisassuredbyanN-terminalhelix-to-helixloopandaC-terminalb-hairpin loop(Figure4B)[21,37].ThisC-terminalb-hairpininNATshasanextendedloopcompared toKATs ofthe samesuperfamily, restricting the substratespecificity to /-amino groups [39,40].Thus, theserecentstructural advancesandenzymaticinvestigationsrevealthat it isimprobablethatNATscanacetylatee-aminogroupsonlysineresidues[39],assuggested forNaa10andotherNATsbypreviousinvitrostudies[41,42].However, NATsmayactas N-terminal propionyltransferases, transferring slightly largerchemical groups onto protein N-termini[43].
Thestructureofthe highlyselectiveNaa40differsfrom Naa10andNaa50.Inthiscase the substratespecificity-providingb-hairpinisflippedawayfromthesubstrate-bindingsite,andin compensationthe/1-loop-/2adoptsanewconformationowingtoanextendedN-terminal segment[29].Thisuniqueconformationoftheextendedloopisalsothereasonfortheselectivity ofNaa40.In contrasttoNaa10,Naa50,andNaa60,whichhave thehighestpreferencefor peptide residue one and decreasing selectivity towards the succeeding residues, Naa40 requiresthefirstfourresiduesforsubstratebinding[21,29,37,38].
IntheNatAcomplex,bindingto Naa15affectstheconformationoftheNaa10/1-loop-/2, whichaltersthesubstratespecificityofNaa10fromacidicN-terminitowardsS-orA-starting (NatA-type)N-termini [14,21].Itis possible thatfuturestructural studiesof NatB andNatC complexeswillshowsimilarmodulatingeffectsbythelargeauxiliarysubunits(Naa25,Naa35)on theirrespectivecatalyticsubunits.
Tosummarize,amajorityofproteinsreceiveanacetylgroupattheirN-terminusthroughthe enzymaticactivityofseveralNATs(NatAtoNatFinhumans)actingondifferentsubtypesofN- termini.TheNATsareoftencomplexesinwhichacatalyticGNATfold-containingenzyme(e.g., Naa10) is paired with a ribosome-binding subunit (e.g., Naa15), and the recently solved structuresrevealhowauxiliarysubunitsmayalsomodulatesubstratespecificity.Nt-acetylation wasrecentlyconnectedtotheorganellesthroughtheidentificationofGolgi-andchloroplast- residentNATs.ThefollowingsectionfocusesonhowtheproteinssubjectedtoNt-acetylation areaffectedatthemolecularlevel.
SubstrateResponsestotheNt-AcetylGroup
WiththewidearrayofproteinsundergoingNt-acetylation,itisnotsurprisingthatthesubstrate proteinsare affectedvery differently (Figure 5). There areseveralexamples ofproteins that cruciallydependontheNt-acetylgroupforsomepartoftheirfunctioning.However,theoverall rolesofNt-acetylationareonlyrecentlybeginningtoemerge.
Nt-AcetylationofCellularProteinsAffectsProteasomalHalf-LifeRegulation
Afundamental function ofNt-acetylation inpromotingproteindegradation wasproposed in 2010whenitwasshownthattheacetylgroupattheN-terminusofaproteincanactasaspecific degradationsignal(termedAc/N-degron)thatistargetedbytheAc/N-endrulepathway,anovel branch of the previously characterized N-end rule pathway [44]. In this case, acetylated N-termini starting with M,S, A, T, orV were identified as Ac/N-degrons, and Doa10 [44]
and Not4 [45] E3 ubiquitin ligases were described as specific recognition components (Ac/N-recognins) of theAc/N-end rulepathway (Figure 5-1). Recently,Ac/N-degrons were describedasconditionalinthattheymaybeshieldedfromrecognitionthroughparticipationina proteincomplex(Figure5-2)[45].ThisworkalsoidentifiedtwoAc/N-degron-containingsub- strates,Hcn1and Cog1,which wereeachfoundto havetheir Ac/N-degronshielded upon complexformationwithCut9andCog2/3,respectively.TheacetylatedN-terminalMetresidue ofHcn1isenclosedwithinaTPRsuperhelixchamberinCut9[46].This wasthefirstcrystal structureshowinganNt-acetylatedMetataprotein–proteininteractionsite.
Continued work on the Ac/N-end rule pathway demonstrated that it is complemented by anotherbranchof theN-end rule, theArg/N-end rulepathway [47].In thiscase Ubr1,the ubiquitinligaseoftheArg/N-endrulepathway,wasshownto recognizeunacetylatedMetif followed by a bulky hydrophobic residue; that is, unacetylated NatC/E/F-type N-termini (Figures2and 3).This meansthat bothNt-acetylationandlack thereofcantargetproteins fordegradationvia theN-endrulepathway.A physiologicallyrelevant exampleofsuchdual targetingwaslaterprovidedbyastudyonRgs2,aregulatorofmammalianG-proteinsignaling [48]. The wild-type MQ-Rgs2 is subjected to degradation by the Ac/N-end rule pathway subsequentto fullNatB-mediated Nt-acetylation(i.e.,allRgs2molecules areNt-acetylated).
Bycontrast,anaturallyoccurringN-terminalmutantassociatedwithhypertension,ML-Rgs2,is onlypartiallyNt-acetylatedbyNatC(i.e.,bothNt-acetylatedandun-Nt-acetylatedvariantsof Rgs2areproduced),andthusmaybedegradedviaeitherofthetwobranchesoftheN-endrule.
Overall,thisleadstothemutanthavingashorterhalf-life.Theresultisanimbalanceinsignaling governingbloodpressurecontrolinpatientsbearingthatmutant,thusconnectingtheN-endrule pathwayandNt-acetylationtohumanphysiology[48,49].
Protein degradaon
Complex formaon
NAT
Subcellular localizaon
Post-translaonal ER import Protein
aggregaon Protein folding
Proteasome
Protein–protein interacon
Protein–membrane interacon
Ac/N-recognin
N-terminal acetylaon
Sec62
E3 Ub
Ac
Ac
Ac
Ac
Ac
Ac Ac
1
5 4
2
3
Figure5.N-TerminalAcetylationMayAffectProteinsinSeveralDifferentways.SummaryofdescribedexamplesofproteinfunctionaleffectsofNt-acetylation.
NATactivityleadstoanNt-acetylatedprotein.ThismodifiedproteinmayberecognizedbyanAc/N-recogninE3ligase(e.g.,Doa10)andtargetedforproteasomal degradationthroughtheAc/N-endrulepathway,inwhichcasetheacetylatedN-terminusactsasanAc/N-degron(1).AnotherfateofanNt-acetylatedproteinmaybe participationinacomplexinwhichtheNt-acetylgrouphasaparticularroleintheprotein–proteininteractionsite,actingasaninteractionmediator(2).Complexformation mayalsorepresentawayfornewlycorrectlyfoldedproteinstoshieldtheirAc/N-degronandpreventAc/N-endruletargeting.Nt-acetylation-dependenttargetingtothe correctsubcellularlocalization(3)mayoccurthroughprotein–proteininteractionsordirectprotein–membraneinteractions;inthelatter,theNt-acetylgrouppossibly stabilizesamembrane-interactingsecondarystructure,suchasanN-terminal/-helixlikeinthecaseof/-synuclein.AnotherexampleofNt-acetylation-dependent targetingisthefindingthatacetylatedN-terminiprohibitpost-translationalimportofproteinsintotheERviatheSRP-independentpathwayusingtheSec62translocation channel(4).Finally,NAT-depletionisalsoassociatedwithproteinaggregation,suggestingthatNt-acetylationisinvolvedinglobalproteinfolding(5).Abbreviations:Ac, acetylgroup;ER,endoplasmicreticulum;NAT,N-terminalacetyltransferase;SRP,signalrecognitionparticle;Ub,ubiquitin.
ThegeneralizabilityoftheAc/N-endruleischallengedbythefactthatfewerthan10proteins havebeendemonstratedtofollowthisdegradationpathway[44,45,47,48].Inaddition,several previouslyknownsubstratesofanAc/N-recognin,suchasDoa10,donotappeartorequireNt- acetylationfortheirdegradation[50],suggestingthatDoa10,similarlytoUbr1(theN-recogninof theArg/N-endrulepathway),may,inadditiontotheAc/N-degronrecognitionsite,alsocontain other substrate-binding sites. Moreover, a more complex picture is suggested by studies describingincreasedproteinhalf-livesduetoNt-acetylation,suchastheDrosophilaHyx [3]
andthehumanTHOC7[51].
Nt-AcetylationasaMediatorofProteinInteractionsandComplexFormation
ComplexformationmaynotonlyshieldproteinsfromNt/Ac-mediateddegradationbutmayalso beinvolvedinenhancingprotein complexformationperse (Figure5-2).Forexample,NatC- mediatedNt-acetylationincreasestheaffinityofUbc12forDcn1,andyeastandhumancrystal structuresrevealedthatMet-1oftheN-terminusofUbc12isburiedinahydrophobicpocketin Dcn1[52].Later,thisconceptwasexpandedbecausethisdistinctiveNt-acetylation-dependent interactionisstructurallyconservedacrossafamilyofmammalianNEDD8ligationenzymes[53].
X-raystructuresofSir3bindingtothenucleosomerevealedanNt-acetylation-dependentinter- actioninwhichtheNt-acetylgroupdoesnotdirectlyparticipateattheinteractionsite,butinstead stabilizesabindingloop[54,55].AnotherNt-acetylation-dependentinteractionisexemplifiedby phosducin-like3(PDCL3),achaperoneinvolvedintheregulationofvascularendothelialgrowth factorreceptor-2(VEGFR-2).InteractionbetweenthesetoproteinsdependsonNatB-mediated Nt-acetylationofPDCL3andresultsinVEGFR-2protectionfrommisfoldingandaggregation[56].
Nt-AcetylationasaDeterminantforProteinSubcellularLocalization
SeveralexamplesinwhichNt-acetylationactsasalocalizationdeterminantinyeasthavebeen known for some time [18,57,58]. However, despite more recent efforts [59,60], potential additionalyeastsubstratesdepending ontheNt-Acgroup forlocalizationremain unknown.
SomeoftheexamplesofNt-acetylation-dependentproteinsubcellular targeting(Figure5-3) occurthroughprotein–proteininteraction.However,adirectinteractionbetweenanNt-acety- latedproteinandthe membranecanberationalized bythe increasedhydrophobicity ofan acetylatedN-terminus(Figure1)contributingtotheoverallhydrophobicityorstabilizationofa membrane-interactingregion.ArecentclinicallyrelevantexampleistheParkinson'sdisease- implicatedprotein/-synuclein.Nt-acetylationstabilizesanN-terminal/-helixin/-synuclein, whichincreasesitsaffinityformembranes[61].Structuralcharacterizationfurtherrevealedan Nt-Ac-/-synuclein–Cu(I)complex,inwhichNt-acetylationaswellascopperbindingtotheN- terminalregioncontributetostabilizationofthe/-helicalstructure[62].TheNt-acetylgrouplikely stabilizesthehelixmacrodipoleandcausemorefavorableH-bondsasaresultoftheabsenceof the/-aminopositivecharge.Theserecentreportson/-synucleinnotonlysubstantiatethelink betweenNt-acetylationandtargetingtomembranesbutalsosuggestapossible roleofNt- acetylationinmetallobiology.
Another mode of Nt-acetylation-dependent subcellular targeting was indicated by a study demonstrating thatthe absenceofNt-acetyl groups ispart ofan early determiningstep in the cellular sorting of nascent polypeptides following the signalrecognition particle (SRP)- independent pathway for post-translational translocation across the endoplasmicreticulum (ER)membrane(Figure5-4)[63].InthiscasetheN-terminiofcytosolicproteinswerereportedto typicallybeamongthehighlyNt-acetylatedNATsubstrateclasses,NatAorNatB,ascompared totheN-terminiofsecretoryproteins,whichweretypicallyenrichedforNatC/E/F-typesequen- ces.Moreover,mutatingsuchsecretoryproteinsintomoreNt-acetylatablevariantsinhibited theirtargetingtotheER.However,notethatmanyoftheproteinsharboringsecretorysignal sequencesmatch theNatC/E/F-targetclass (Figure 2) meaningthattheycannot perse be consideredasnon-Nt-acetylated.
Nt-AcetylationAffectingProteinFoldingandAggregation
ThepresenceorabsenceofchargeattheN-terminusmaybeacentralfactorinproteinfolding.
Indeed,a potential roleof Nt-acetylation inglobal protein folding (Figure 5-5) was recently uncovered[64].ItwasshownherethatNt-acetylationdeficiencyinNatA-deletedyeastleadsto theaccumulationofmisfoldedproteins andincreased levelsofchaperones.This studyalso suggestedthatNatAmightbeimplicatedintheSup35/PSI+prioncycleinyeast.Prionsare heritableelementstransmittedviaprotein,typicallyorderedintofilamentousaggregates(amy- loids).In[PSI+]yeastcellstheSup35proteinformsamyloids.Sup35isaNatAsubstrate,and deletionofNatAin[PSI+]phenotypiccellsdecreasesthestabilityofunacetylatedSup35amyloid andrelievesthephenotypeofthe[PSI+]cells[64].
NatAdepletioninhumancellswasalsoshowntocauseaggregationofHuntingtin(Htt).HYPK (Htt-interactingproteinK)stablyinteractswithNatA(Figure3A),whichisessentialforitsoptimal Nt-acetylationactivity[25];HYPKalsoactsasachaperonethatpreventsaggregationofHtt[65].
Knockdown of HYPK, but interestingly also Naa10 or Naa15, increases the aggregation tendencyofmutantpolyQHtt,whichisfoundinpatientswithHuntington'sdisease[25].Thus, itappearsthattheNatA–HYPKcomplexisessentialforproperHttfolding,possiblyasaresultof thedirectNt-acetylationofHtt.Anotherexampleofdisease-relatedaggregationthatiscon- nectedtoNt-acetylationis/-synucleininParkinson'sdisease,describedabove.
InterplayBetweenNt-AcetylationandOtherProteinModifications
ItwasrecentlyfoundthatUbe2w,anE2enzyme,canubiquitylatethe/-aminogroupsofprotein N-termini [66,67],whereas ubiquitylationmore commonly occurs onthe e-amino group of lysines.ThesereportsindicatethepossibilityofaninterplaybetweenE2sandNATsthatbothact onproteinN-termini,inwhichNt-acetylationmaypotentiallyblockE2sandubiquityl-mediated proteasomaldegradation,orinhibitsignalingeventsmediatedbyN-terminalmonoubiquitylation.
AnotherexampleofsuchinterplaywithothermodificationsisthefindingthatNatD-mediatedNt- acetylationofhistoneH4blocksthemethylationofH4Arg3,whichinturnregulatesribosomal DNA silencing [68].A recent study also revealed kinetic competition between Naa50 and MetAPs, and proposed that Naa50-mediated Nt-acetylation may act to retain the iMet of otherwiseMetAP-susceptibleN-termini[69].
Severalstudieshavenowdemonstratedtheimportance ofNt-acetylationformany different proteins.ThemolecularmechanismsinwhichNt-acetylationtakepartarediverseandencom- passhalf-liferegulation,protein–proteinandprotein–membraneinteractions,subcellularlocali- zation,folding,andaggregation.Thefollowingsectionsdiscussthephysiologicalconsequences ofNt-acetylationasrevealedbyrecentstudiesonmodelorganismsandhumandiseases.
PhysiologicalFunctionsofNATs inModelOrganisms
TheconsequencesofNt-acetylationarealsomanifestedinbiologicalsystems,andstudiesusing different model organisms demonstrate NATs to beessential for a varietyof physiological processes(Figure 6).Specificlinksbetween organismphenotypesandmolecular regulation oforbyNATsarenowintheprocessofbeinguncovered.
In Arabidopsis, Nt-acetylation was recently shown to impact ondrought-stress adaptation becauseNatA-depleted plants haveincreased drought-tolerance (Figure 6A-1) [70]. In that study,oneoftheveryfewknownexamplesofupstreamregulationofNATactivitywasalso revealed. Wild-typeplants subjected to droughtstress wereshown to havedecreased Nt- acetylationlevelscausedbyarapidtranscriptionaldownregulationofNatAsubunitsbyabscisic acid,aplantstresshormone.The findingthatNatAfunctionisindispensableinArabidopsis supportsitsphysiologicalimportance,andoverallthisreportsuggeststhatdecreasesinNatA- mediatedNt-acetylationisanimportantswitchincellularstressresponses[70].
AnotherrecentplantstudyidentifiesNt-acetylationasakeyswitchinimmuneregulationand reportstheantagonisticregulationoftwodifferentN-terminalvariantsofthesameproteinbytwo NATs(Figure6A-2)[71].Thekeyplantimmunityprotein,SNC1,existsastwoNt-variants,MD- SNC1(NatBsubstrate)andMMD-SNC1(thereinreportedasaNatAsubstrate).Peculiarly,these
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Figure6.RegulatoryConsequencesofNATActivityattheOrganismLevel.(A)ImpactofregulatedNt-acetylationinArabidopsis.(A1)Underdrought,plants producethephytohormoneabscisicacid(ABA)thatinducepartofthedrought-stressresponsethroughtranscriptionalregulation,allowingtheplanttoadapttothe droughtenvironment.Aspartofthisprocess,ABAnegativelyaffectstheactivityofNatA,causingadrought-responsivereductioninNt-acetylationlevels.Experimental inductionofNatA-depletionwiththeconcomitantreductioninglobalNt-acetylationtriggersconstitutiveinductionofthedrought-stressresponse,thuspre-adapting theseplantstodroughtandincreasingtoleranceandsurvival,althoughNatA-depletionundernormalwatersupplyconditionscausesvegetativegrowthretardation[70].
(A2)SNC1isaNod-likereceptorservingasaimmunereceptor,andthusplaysanimportantroleindefenseagainstpathogensinplants.Differentialtranslationinitiation generatestwoNt-variants:MMD-(SNC1)andMD-(SNC1),whichareeachsubjecttoNt-acetylation,butbydifferentNATsandwithdifferentconsequences.FortheNatA substrate,MMD-(SNC1),theAc-MetlikelyactsasanAc/N-degron,destabilizingSNC1andtherebycausingdecreasedpathogentolerance.Bycontrast,theshorterMD- SNC1formisNt-acetylatedbyNatBandstable,conferringincreasedimmuneresistance[71](*indicatessomeuncertaintyaboutwhichNATactuallyperformsNt-Acof theMMDvariant;seetext).(B)InfluenceofNATsinC.elegansmetabolismanddevelopment.(B1)TheC.eleganslifespanandreproductivecycleconsistsoffourlarval stages(L1–L4),adulthood,andadormantlarvaedauerstagethatisinducedbyanunfavorableenvironment.daf-31/NAA10-mutatedlarvaedonotadoptaproperdauer stageunderstarvedconditions.Inaddition,thesedaf-31/NAA10-mutated,starvedlarvaedonotresumedevelopmentuponexposuretonutrients[75].(B2)Molecular modelofhowNATsaffectthepathwaybalancingdauerstageformationandgrowth,startingwiththetransmembranereceptorDAF-2.Naa10/NatAstimulatestheactivity ofDAF-16,atranscriptionfactorregulatingstress-responsegenesandwhoseinhibitionbyDAF-2controlsdauerformation.NatCactsfurtherdownstreaminthis pathwaybecauseitisrepressedbyDAF-16.UnderrichenvironmentalconditionsNatCnegativelyimpactsonstressresistanceanddauerformation[76].
twovariantsrespondverydifferentlytoNt-acetylation:Ac-MMD-SNC1isdestabilizedandAc- MD-SNC1isstabilized,leadingtoadecreasedorenhancedimmuneresponse,respectively.
Hence,thisstudyconnectsproteinhalf-liferegulationbyNt-acetylation(discussedearlier)to biologicalregulation,andalsodemonstrateshowthismayinterplaywithalternativeNt-variants toprovidehomeostaticregulation.Itcanbespeculatedthatsuchatwo-facedregulationmight applytootherMM-startingproteinsaswell[71,72].NotethatMMD-SNC1doesnotmatchthe canonicalNatAspecificity,andmodificationcouldconceivablyinvolveNaa50/NatE(Figure3A).
PreviousstudiesinArabidopsishaveidentifiedphenotypesassociatedwithNATloss-of-func- tion.PleiotropicdevelopmentaldefectswereassociatedwithlossofNatBactivityasobservedin tcu2/NAA25-mutatedplants[73].Inaddition, NatC-mediatedNt-acetylationisnecessaryfor normalgrowthandefficientphotosynthesis,asrevealedbythemak3-1/NAA30mutantinwhich thesynthesisoftwokeyphotosyntheticproteins,D1andCP47,isaffected[74].
InCaenorhabditiselegans,Nt-acetylationhasbeenlinkedtodevelopmentandmetabolismas daf-31/NAA10mutantsfailedtoproperlyenterandexitthedauerstage,whichisessentialfor C.eleganssurvivalduringstarvation(Figure6B-1)[75].ThesefindingsindicatethatNatAplays an important role in regulating adaptive metabolic behavior through an insulin-like IGF1 signalingpathwaybyinducing theactivityofthe transcriptionfactor DAF-16.Interestingly, thesamepathwaywasfound toinvolve NatC,acting as amodulatorof stressresistance downstream of DAF-16 [76]. In this case, loss-of-function mutations in natc-1/NAA35 increasedresistancetoseveralstressors,includingoxidativestress.Thisstudythusrepresents anotherexamplewhereaNATisbothregulatedbyandakeyregulatoryfactorinimportant(in thiscase,metabolic)physiologicalprocesses.SuchametabolicinvolvementbyNatChasalso previouslybeensuggestedbyyeastnutrientstressstudies(reviewedin[18]).NatCcouldalso possiblybelinkedtoagingbecausearecentyeaststudyfoundthisNATtoaffectproteasome distribution(anage-dependentprocess) andshowed thatloss ofNatCspecifically affects growthofreplicativeoldcells,inotherwords,those thathaveproducedahighnumberof daughtercells[77].
TheinvolvementofNaa10indevelopmentwasalsosupportedbyaDrosophilamelanogaster studyinwhichreducedNaa10levelscausedpleiotropicoogenesisdefects[78].Further,naa10- nullmutationswereshowntoaffectcellgrowthanddivision,andcausedlethality.Drosophila fliesmutatedinNaa50(NatE)[24]andflycellsdepletedforNaa60(NatF)[15]areviable,buthave phenotypesrelatedtocellularchromatidcohesion.
ThezebrafishDanioreriowas recentlyadopted inNAT studies,andhere theessentialityof Naa10fordevelopmentwasagaindemonstrated.Developmentalabnormalitiessuchasabent axis,abnormaleyes,andbenttailswereobservedaftermorpholino-mediatedknockdownof naa10[79].Thesemorphantswerefurtherdescribedtodisplayincreasedlethalityandgrowth retardation.Anearlierzebrafishstudy alsoconnectedNatCtodevelopment,andembryonic growthcontrol andvessel development wereaffected, possibly through changesinmTOR signaling[80].TheNt-acetylation-dependentproteininteractionbetweenAc-MQ-PDCL3and VEGFR-2describedaboveisassociatedwithangiogenesisinzebrafishandmice[56].Thesame reportshowedthatamutantPDCL3thatisunabletoundergoNt-acetylationwasunresponsive to hypoxia,aknown stimulatorof angiogenesiswhich normallyregulates theexpression of PDCL3.
Overall,theseorganism-basedstudiesundoubtedlytestifythatNATsplayimportantphysiolog- icalrolesindevelopment,stresssignaling,andmetabolicadaptation,andtheywillnodoubt continuetoprovideimportantcontributionstoourunderstandingofthefunctionofNt-acetyla- tioninhumans.
