PAPTi: A Peptide Aptamer Interference Toolkit for Perturbation of Protein-Protein Interaction Networks.

10.5 MB·English

by  Yeh, Johannes T. -H.

PAPTi: A Peptide Aptamer Interference
Toolkit for Perturbation of Protein-Protein
SUBJECTAREAS: Interaction Networks
INTRACELLULAR
SIGNALLINGPEPTIDES
ANDPROTEINS
JohannesT.-H.Yeh1,RichardBinari1,TenzinGocha2,RamanujDasgupta2&NorbertPerrimon1
PROTEINENGINEERING
PROTEIN-PROTEININTERACTION
NETWORKS 1DepartmentofGenetics,HowardHughesMedicalInstitute,HarvardMedicalSchool,77AvenueLouisPasteur,BostonMA02115,
USA,2DepartmentofBiochemistryandMolecularPharmacology,andtheNYUCancerInstitute,NewYorkUniversityLangone
BIOLOGICALTECHNIQUES
MedicalCenter.
Received Signalingproteinsoftenformdynamicprotein-proteininteraction(PPI)complexestoachieve
19September2012 multi-functionality.MethodstoabrogateasubsetofPPIinterfaceswithoutdepletingthefull-lengthprotein
willbevaluableforstructure-functionrelationshipannotations.Here,wedescribetheuseofPeptide
Accepted
AptamerInterference(PAPTi)approachforstructure-functionnetworkstudies.Weidentifiedpeptide
11December2012 aptamersagainstDishevelled(Dsh)andb-catenin(b-cat)totargettheWntsignalingpathwayand
demonstratethattheseFN3-basedMONOBODYs(FNDYs)canbeusedtoperturbproteinactivitiesbothin
Published
vitroandinvivo.Further,toinvestigatethecrosstalkbetweentheWntandNotchpathways,weisolated
29January2013
FNDYsagainsttheNotchAnkyrin(ANK)regionanddemonstratethatperturbingtheANKdomainof
NotchincreasestheinhibitoryactivityofNotchtowardsWntsignaling.Altogether,thesestudies
demonstratethepowerofthePAPTiapproachtodissectspecificPPIinteractionswithinsignaling
Correspondenceand networks.
requestsformaterials
shouldbeaddressedto Statistical analyses have revealed that Protein-Protein Interaction (PPI) networks follow the ‘‘scale-free’’
N.P.(perrimon@ highlyconnectedrulewitheachprotein(node)interactingonaveragewith3.6partnersandwithsomehub
receptor.med.harvard. proteinshavingmorethan61.Withthislevelofinterconnectivity,cellularproteinscanformrobustPPI
edu);J.T.H.Y.(jeyh@ networks tomaintain cellularfunctions. Strategies thatabrogateasubset ofinteractions (edges)fromanode
withoutdepletingthewholeproteinfromthePPInetworkarethusvaluablefortheanalysisofsignalingprotein
genetics.med.harvard.
multi-functionalityandforthestructure-functionrelationshipannotationsofspecificPPI2(Fig.1A).Themost
edu)orR.D.(Ramanuj.
commonly used approaches for such ‘‘edgetic’’ analyses are mutagenesis screens2,3 and chemical genetics4.
[email protected])
Mutagenesis screens, whereby proteins generated from random mutagenesis are tested functionally (see for
example3),aretimeconsumingandoftenproblematic,astheperturbationcanresultinprematurelytruncated
or misfolded proteins that behave as loss-of-function null alleles. Chemical genetics on the other hand uses
bioactive small molecules as perturbation tools to mimic the effect of loss-of-interaction alleles4. Although a
powerfulapproach,chemicalgeneticsislimited,asmostbioactivecompoundsavailableareenzymeinhibitors
(mostly kinases) rather than protein-protein interface disruptors5, reflecting the fact that PPI interfaces are
generally wideand shallow andtherefore lackthedeep hydrophobic pocketsrequired for smallmolecules to
beengulfedwithhighaffinitiesandspecificities6.
Peptideaptamers7,aclassofaffinitybindersthatconsistofshortstretchesofvariablepeptideregionscon-
strainedwithinaproteinscaffold7–9,provideapowerfulalternativeapproachtotargetPPIinterfaces.Whilethey
havebeenshowntobeeffectiveatinhibitingcellsignalingbothinvitroandinvivo10,11,12,thegenerationofpeptide
aptamers forfunctional studiesin cellsignalingstill hasnotbeenextensively explored.Thus,weemployed a
PeptideAptamerinterference(PAPTi)approachtoselectforaptamersthattargetPPIinterfaces.OurPAPTi
strategy (Fig. 1B, 1C) entails the engineering of a stable small protein scaffold, the human FN3 domain
(Monobody)9,13,thatcandisplayvariableproteinbindingsequenceswithhighstabilityandaffinityanalogous
toantibody-antigeninteractions7,12.Byconstructingarandomizedlibrarywithdiversifiedaminoacidsequences,
targetspecificmonobodypeptideaptamerscanbeselectedforfunctionalPPIinterference.Here,wedescribethe
use of such a diversified PAPTi library to identify and validate inhibitory peptide aptamers toward the Wnt
signaling network. The Wnt pathway is an evolutionarily conserved signaling pathway essential to animal
development,stemcellself-renewal,andadulttissuehomeostasis14.Conversely,dysregulationofWntsignaling
iscommonlyassociatedwithhumandiseasessuchascancer14.Consequently,peptideaptamersthatperturbWnt
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Figure1|SchematicrepresentationsofPAPTiplatform. (A)PPInetworkfunctionalanalysis.(B)FN3monobodyscaffold.StructureoftheFN3
Monobodyscaffold9,13(FNDY,left)usedinthisstudy.ThescaffoldhasanIgGfoldsimilartotheantibodyIgG(right).Therandomizedloopsinthisstudy
(BCloopandFGloop)withintheFNDYscaffoldarecoloredinyellow.Asacomparison,thethreeantigenrecognitionvariableregionswithinthe
antibodyIgGscaffoldarecoloredinred.(C)PAPTiflowchart:(1)Targetproteinexpression:Targetproteinsaredividedintofoldeddomainsandeach
individualdomainisexpressed,purifiedandbiotinylated.(2)Phagelibraryconstruction:DiversifiedFN3domainmonobodypeptideaptamers(FNDY)
areclonedinfusionwithM13phagepIIIcoatprotein,resultinginaphagedisplaylibrarywith231010complexity.(3)Selectionoftargetbindingphages.
(4)ELISAvalidation:Individualphageclonesareamplifiedandvalidatedfortargetbindingin96-wellELISAplates.Positiveclonesareselectedforfurther
characterization.(5)PositiveindividualFNDYclonescanbesubclonedintovariousdestinationvectorsforsubsequentapplications.(6)Cell-based
reporterassays:Toselectfunctionalpeptideaptamers,candidateFNDYsaretransfectedandexpressedinreportercells,andtestedusingluciferase-based
reporterassays.(7)Invivofunctionalvalidation:FNDYsareselectedtogeneratetransgenicanimals.
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Figure2|PAPTiofthecanonicalWntsignalingpathway. (A)TheWntsignalingpathway.ThecanonicalWntsignalingconsistsofWnt(ligand),
Frizzled(Fz)andLRP(receptor),Dsh(effector),GSK3/Axin/APC(destructioncomplex)andb-cat(transcriptionalactivator).(B–E)Cell-basedactivity
assaysofFNDYs.(B)FNDY-DXandFNDY-DPagainstDshinhibitTOPFLASHluciferaseactivityinHEK293Tcells.(C)TOPFLASHvalidationof
FNDYsagainstb-catinHEK293cells,anddominantnegative(dn)TCF4positivecontrol.(D)FNDY-B8inhibitsTOPFLASHreporteractivityas
effectivelyasthednTCF4constructinthehumancolorectalcancercelllineDLD1.(D)FNDYsdonotinhibitHedgehog,TGF-b,BMP,andNotch
pathways,asindicatedbytheGli-Luc,TGFb-Luc,BMP-Luc,andCSL-Lucreporters,respectively.(Graybar:stimulatedreportercellsco-transfectedwith
emptyvector,Blackbar:stimulatedreportercellsco-transfectedwithFNDYs).(E)Invivomisexpressionofb-catFNDYpeptideaptamersinthe
Drosophilawingresultsinseveregrowthdefects,andblisteringphenotypes.(A:WildTypecontroladultwing;B:FNDY-B8-NLSexpressingadultwing).
SimilarobservationsweremadeintransgenicfliesexpressingFNDY-B8-NLSunderMS1096-GAL4wingpouchdriver(datanotshown).(F)FNDY-B8
alsoinhibitsflyArmadillo(Arm).TOPFLASHreporteractivitywasinhibitedinHEK293Tcellsco-transfectedwithpCMV-Arm(15ng/well)and
FNDY-B8(100ng/well&200ng/well).(Y-Axis:Relativeluciferaseactivity.Basal:CellsnotactivatedbyArm).
signalingwillbevaluabletoolsinbiologyandtranslationalmedicine. the PAPTi approach led us to discover that targeting Notch ANK
Inthisreport,wedescribetheidentificationofpeptideaptamersthat repeats can be utilized as a mean to inhibit Wnt signaling. All
inhibit the canonical Wnt signaling effector proteins Dishevelled together, this study provides a proof-of-concept for using PAPTi
(Dsh) and b-catenin (b-cat). Furthermore, we demonstrate that forfunctionalPPIstudies.
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Figure3|EfficacyofFNDYinvivo. (A–D)ControlimaginaldiscsdisplayingthenormalexpressionoftheSenseless(Sens)protein(red,panelC),in
tworowsofcellsatthedorsal/ventral(D/V)boundaryof3rdinstarlarva.(E–H)MisexpressionofUAS:FNDY-B8-NLSunderthecontrolof
apterous-GAL4(ApGAL4),whichisexpressedonlyinthedorsalcompartment,asshownbytheco-expressionofGFP(UAS-GFP)(E)resultsinamarked
reductionofSensexpressionspecificallyinthedorsalcompartment(yellowarrowheadsinG;alsocompareexpressionofSensindorsalversusventral
compartmentinH).(I-I99)QuantitationofSensexpressionindorsalversusventralcompartmentalongaconfocalZ-stackacrosstheD/Vboundary
(denotedbytheyellowdashedlineintheX-YimageinpanelI,andtheZ-stackshowninI9).NotethesignificantlossofSensstaining(red)inthecells
expressingGFP(green)inpanelI9,andasquantifiedusingfluorescenceintensityprofileinpanelI9,whichshowsareciprocalrelationshipbetweenthe
expressionofGFP(greenline)andexpressionofSens(redline).TheseresultsindicatethattheectopicexpressionofUAS:FNDY-B8-NLSinvivois
sufficienttoabrogateendogenousWingless/Wntsignaling,mostlikelybyactingonnuclearArm/b-cat.
Results thethreewelldefineddomainsofDrosophilaDsh(DIX,PDXand
PAPTiscreenagainstWntsignalingpathway.SinceDshandb-cat DEPdomains,respectively)astargetsforapeptideaptamerscreen.
arethetwomajoreffectorsofthecanonicalWntsignalingpathway Fromaphagedisplayscreenofalibrarywith231010complexity,we
(Fig.2A),wesoughttosetupaphagedisplayPAPTiselectionsystem obtainedacollectionofpeptideaptamers[referredtoasFNDYs(for
(Fig.1C,seeMethods)toscreenforpeptideaptamersagainstthese FN3-based MONOBODY)]: FNDY-DX targeting the Dsh DIX
proteins.Weexpressedandpurifiedthefull-lengthhumanb-catand domain, FNDY-DP against Dsh DEP, and FNDY-B against b-cat.
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Figure4|FNDY-FBoxchimeraasatoolforsyntheticallytargetingproteindegradation. (A)Schematicrepresentationofcanonicalb-catdegradation
viatheSCFb-TrCPcomplex.Phosphorylatedb-catisrecruitedtotheSCFb-TrCPE3ligaseviatheF-Boxproteinb2TrCP14.Illustrationsofproteinstructuresbased
onPDB2GL7(b-cat)28,PDB1LDJ(Cul1)29,andPDB1P22(b-TrCP-Skp1)30.(B)FNDY-FBoxSCFE3ligasecomplex(SCFFNDY)restoresb-cat
degradation.(Upper)ReplacingtheWD-40repeatsdomainofb-TrCPwithvariablepeptideaptamermoietiestodegradedesiredtargetproteinsvia
proteinubiquitinationpathway.ThemethodisreminiscenttotheuseofnanobodiestodegradeGFP18.(Lower)HEK293Tcellsoverexpressing
non-phosphorylatableb-cat(S33Y,S37A,S41A,T45A)weretransfectedwitheithertheFNDYpeptideaptamerorSCFFNDY.24hposttransfection,
significantdepletionofb-catwasachieved.
Interestingly,ourscreenagainstDshPDZfailedtoyieldhighaffinity in vivo. Because FNDY-B8-NLS exhibits strong inhibitory activity
binders, possibly due to the strong binding preference of PDZ against Wnt signaling, we generated transgenic flies carrying
domains for free carboxyl terminus peptides rather than internal inducible FNDY-B8-NLS (to enhance higher nuclear targeting in
peptidemotifs. vivo, a 3X SV40 NLS sequence was added), using the Gal4/UAS
Next,weusedtheWntpathwayreporterTOPFLASH(dTF12),a system16. UAS:FNDY-B8-NLS flies crossed with the apterous-Gal4
constructwith12multimerizedLEF/TCFbindingsitesupstreamofa driver showed a disorganized wing phenotype: crumpled wing
heatshockminimumpromoterdrivingtheexpressionofluciferase,as phenotype which likely results from differential growth in the
afunctionalreadouttoselectforFNDYsthatcanperturbWntsignal- dorsal versus ventral compartment, smaller wing size and large
ing4.FromtheDshandb-catscreens,weidentifiedbioactiveclones bubbles or blisters that appear on the wing blade (Fig. 2E),
thatshowedvariousdegreesofinhibitoryeffectsagainsteitherDshor suggesting perturbed Wg signaling during wing development. A
b-cat(Fig.2B,2C;TableS1).AmongtheFNDYstestedinHEK293T similar phenotype was observed with UAS:FNDY-B5-NLS, albeit
reporter cells, FNDY-B8 exhibited the strongest activity and was as less profound. In addition, consistent with human b-cat and
effective as dominant negative Tcf4 (Fig. 2C). Similar result was Drosophila Armadillo (Arm) being highly homologous, FNDY-B8
observed in the human colorectal cancer cells DLD1, a cancer cell also inhibited Arm in the TOPFLASH assay (Fig. 2E). To further
linewith truncatedAPC (Fig. S1). The effect ofthese peptideapta- demonstrate that the disorganized wing phenotype is Arm
mers are Dsh or b-cat associated as the inhibitory effect of either associated, we stained the wing disc and found that the
FNDY-DP4,FNDY-B5orFNDY-B8wasreversibleupontransfection endogenousWgtarget,senseless(sens)wasrepresseduponFNDY-
ofincreasingamountofDshorb-cat,respectively(Fig.S2).Further, B8-NLSexpressioninthewingdisc(Fig.3).Furthermore,expression
thepeptide aptamersare specifictowardstheWnt pathwayas they ofB8-NLSdidnotinfluencecutstainingoneithersideofthedroso-
had no significant effects on Bone Morphogenetic Protein (BMP), ventral (D/V) boundary (Fig S3), even though there is reduced
Hedgehog (Hh), TGF-b and Notch pathway activities (Fig. 2D), senseless expression in the dorsal compartment, suggesting a
whereas Notch reporter activity was increased (,50%) by FNDY- specificeffectofB8-FNDYininfluencingexpressionofWgtargets
DP4 (Fig. 2D), reminiscent to previous findings that Dsh act as a ratherthanNotchtargets.Collectively,thesefindingsindicatethat
negativeregulatorofNotch15(seebelowforfurtherdiscussion). FNDY-B8-NLSiseffectiveatblockingWntsignalinginvivo.
FNDY-B8perturbs Drosophilawingdiscdevelopment. Wenext SCFFNDY as modules for synthetically targeting protein
askedwhetherFNDYsaresuitableforfunctionalPPIperturbation degradation. To further expand the applications of peptide
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Figure5|TargetingNotchANKrepeatscaninhibitWntsignaling. (A)CrosstalkbetweenWntandNotchpathways.ThecanonicalNotchsignaling
consistsofDelta(ligand),Notch(effector)andSu(H)/CSL(transcriptionfactor).DshhasbeendshowntonegativelyregulateNotch15,andNotchcan
negativelyregulateWntsignaling20,23.(B)SelectionofNotchANKrepeatsaptamers(FNDY-NAK1andFNDY-NAK2).(Upper)Notchintracellular
domains.(Lower)ELISAvalidationofindividualFNDYphageclones.EnrichedIndividualFNDYclonesweretestedonELISAplatescoatedwithNotch
ANKrepeatsorcontrol(streptavidin)proteins.Bindingactivitieswerequantitatedcolorimetricallybyabsorbanceat450nm.(Graybars:Streptavidin
controlprotein;Darkbars:NotchANK).Inthisassay,falsepositiveclones(clone5&clone13;clone16iswild-type,non-displayingM13phage)were
discarded.AllthepositivehitsweresequencedandtwodistinctFNDYsequences(FNDY-NAK1andFNDY-NAK2)wereidentified.(C)Both
FNDY-NAK1andFNDY-NAK2antagonizeDshactivatedTOPFLASH.HEK293TcellsweretransfectedwithDshtoactivateTOPFLASHluciferase
activity.Thisluciferaseactivityissuppressedbyco-expressingeitherFNDY-NAK1orFNDY-NAK2.(D)TransfectingDECNotch1inHEK293Tcells
inhibitsDshactivatedTOPFLASHreporterexpression.FNDY-NAK2showsasimilarinhibitoryeffectinthisassay.(E)Westernblot(WB)ofcytoplasmic
Notch.OverexpressingFNDY-NAK2stabilizesthecytoplasmicproteinlevelofNotch1.ThecytoplasmicfractionofHEK293celllysatewascollectedand
blottedwithNotch1antibody(b-tubulinasaloadingcontrol).(F)c-secretaseinhibitorinhibitsTOPFLASHactivity.HEK293Tcellsweretransfected
withDshandtreatedwiththec-secretaseinhibitorDAPT(5mMand25mM).TheinhibitionofTOPFLASHcausedbyFNDY-NAK1andFNDY-NAK2
areshownasacomparision.(Mock:cellstransfectedwithDsh;FNDY-NAK1/2:cellstransfectedwithDsh1FNDY-NAK1/NAK2;DAPT:cells
transfectedwithDshandtreatedwithDAPT).(G)Dshoverexpressionleadtonuclearb-cataccumulation,whichwasdecreaseduponFNDY-NAK2
overexpression.(Note:Inordertoseesignificantb-cataccumulationinthenuclearfractionbywesternblot,higheramountofDshplasmidwas
transfectedinthecellsthanthatwasusedinTOPFLASHassays.FNDY-NAK2wasabletoslightlyreducetheratioofnuclearb-cat).
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aptamersinmanipulatingcellularsignaling,wenextusedFNDYsas proposed that cytoplasmic, but not nuclear, Notch antagonizes
toolkit modules for protein modification such as ubiquitinylation. Wnt signaling19,20, and that the ANK repeats interact with the
Since E3 ligases facilitate protein ubiquitinylation by placing the RING type E3 ubiquitin ligase Deltex22, it is possible that FNDY-
substrate targets and the activated ubiquitin into proximity NAK1andFNDY-NAK2stabilizecytoplasmicNotchbyinterfering
(Fig. 4A), peptide aptamers can be used as a substrate recruiting withtheinteractionbetweenNotchandDeltex.Consistentwiththis
module to create an artificial E3 ligase17 (Fig. 4B). A similar hypothesis,WntpathwayactivitywasdecreasedinHEK293Tcells
approach,wherasaGFPnanobodyinfusionwithDrosophilaSlimb treated with DAPT, a c-secretase inhibitor that prevents nuclear
F-Box (the deGradeFP system)18, has been shown to effectively Notchaccumulation20(Fig.5F).Alternatively,asmembraneassoc-
deplete GFP-tagged fusion proteins in vitro and in vivo. Thus, we iatedNotchcanalsosequesterb-cataccumulation23,FNDY-NAK2-
designedFNDYsinfusionwiththehumanb-TrCPF-Box(residues mediatedstabilizationofNotchcouldmoreefficientlypreventb-cat
139-255) of the SCFb-TrCP complex (termed SCFFNDY thereafter) to to signal in the nucleus. In this regard, we observed that FNDY-
promote target protein degradation. We generated a number of NAK2 did not promote b-cat protein degradation (Fig. 5G).
SCFFNDY-B constructs and tested their activities toward b-cat However, in the presence of high-level Dsh overexpression,
degradation. Under physiologicalconditions when Wntstimulation FNDY-NAK2couldstillslightlyreducethenuclearratioofaccumu-
is absent, b-cat is undergoing constant degradation. However, in lated b-cat (Fig. 5G), suggesting that b-cat is sequestered but not
many cancer cells, b-cat protein accumulation is observed due to degradedbythestabilizedNotch.Altogether,theseresultsillustrate
impaired degradation complex formation. To demonstrate that the use of FNDYs to annotate the function of protein domains
SCFFNDY-B can restore b-cat degradation, we transfected the cells involvedinsignaling.
with the non-phosphorylatable form of b-cat (S33Y, S37A, S41A,
T45A b-cat), in which the phosphorylation by GSK3b is abolished Discussion
and thereby impaired for b-TrCP WD40 domain binding. In these Altogether,ourstudiesdemonstratethepowerofFNDYsforfunc-
cells,bothSCFFNDY-B2andSCFFNDY-B5wereeffectiveatpromotingb-cat tionalPPIperturbationandinparticulartodissectspecificPPIinter-
degradation,albeitSCFFNDY-B2wasmoreeffective(Fig.4C)likelydue actions within signaling networks. Specific PPI-mediated pathway
to the favored orientation of the substrate being recruited to the crosstalkisessentialtocellularsignaling.Consequently,thereexist
SCFFNDY-B2 E3 ligase complex. This result indicates that synthetic particular interests in annotating the structure-function relation-
SCFFNDY-B2 can be used to restore b-cat degradation for oncogenic ships of multi-domain signaling proteins in the PPI network. In
Wntsignaling(Fig.4C,4D). particular, we demonstrated that the PAPTi approach led us to
discoverthattargetingNotchANKrepeatsmaybefurtherutilized
FNDYs that targeting Notch ANK repeats domain can inhibit as a mean to inhibit Wnt signaling in some types of cells. This
Wntsignaling.TofurtherexploretheuseofFNDYstointerrogate providesaproof-of-conceptforusingPAPTifor‘‘edgetic’’functional
thefunctionofproteindomains,wetestedtheinteractionbetween PPIstudies.
the Wnt and Notch pathways. Previous studies have shown that Compared to transgenic RNAi24, which allows perturbation of
Notch synergizes or antagonizes Wnt signaling, arguing that geneactivityinaspatialandtemporalmanner,PAPTiallowspecific
Notch modulates Wnt signaling in a context dependent manner. proteindomainstobetargeted25.Further,incontrasttosmallmole-
Importantly, Notch can form direct PPI with Wnt pathway cules,theexpressionofpeptideaptamerscanbespatiallyandtem-
components to regulate Wnt signaling (Fig. 5A)15,19. In particular, porally controlled by conditional gene expression methods25. Not
theANK repeats containingdomain ofNotch1(Fig. 5B)hasbeen limited to PPI network biology, structural elucidation of Peptide
showntoplayapivotalroleinNotchactivity,buthasnotyetbeen Aptamer-Targetinteractionscanalsoserveasatemplateforstruc-
describedasaregulatoryregionforWntpathwaycrosstalk.Thus,we ture guided drug discovery25. For future extension of PAPTi
generatedFNDYsthattargettheANKdomainandaskedwhether coverage,itwouldbedesirabletogeneratealargecollectionofbioac-
theycanaffectanyaspectofNotchandWntcrosstalk(Fig.5B).We tive, renewable FNDY peptide aptamers. Recently, an antibody
identifiedtwoindependentclones,FNDY-NAK1andFNDY-NAK2 consortiumhasreporteditssuccessingeneratingalargecollection
(Fig. 5B), which significantly inhibited TOPFLASH activity in ofrenewableSH2domainantibodies26.Similarly,owingtocurrent
HEK293T cells activated by either Dsh or b-cat (Fig. 5C, 5D). To availableresourcessuchascompleteORFeomecollectionsofmodel
validate the binding specificity of the peptide aptamers, we organisms,highthroughputscreeningplatformsandsensitivepro-
performed GST-pull down assays by incubating GST tag fusion teomicsmethods,itshouldbefeasibletoscaleupFNDYproduction
proteins with lysates from HEK293 cells expressing individual toreachproteome-widecoverageforPPIstructure-functionanalysis.
FNDYconstructs.FNDY-BconstructswerefoundtobeGST-b-cat Well-characterized FNDY clones from such a collection would
specific whereas FNDY-NAK1 and FNDY-NAK2 only recognize enableedgeticanalysisorPPIstudies.Asaresourceplatform,one
GST-Notch RAMANK (Notch1 1761-2127 which comprises the oftheparameterstoensurequalitycontrolofsuchreagentsistheir
RAMandANKdomains)(FigS4),indicatingtheNotchassociated binding specificity, which could be addressed by incorporating a
suppressionofWntsignalingbyFNDY-NAK1andFNDY-NAK2. proteome-wide high density protein array27 into the PAPTi work
In addition, because FNDY-NAK1 and FNDY-NAK2 are two flow(Fig.1C).
independent clones with distinct variable sequences selected
againsttheNotchANK repeatregion, it isunlikely thatthey both
Methods
inhibitWntsignalingduetononspecific,off-targetcross-reactivity
Phagedisplayscreen.Toconstructthelibrary,degenerateoligonucleotidepools
withWntpathwaycomponents.Thus,thisresultsuggeststhatNotch
encoding5randomizedaminoacidresidueswereligatedintotheparentalFN3
acts as a direct regulator of the Wnt/b-cat signaling via its ANK scaffoldinthephagemidvectortoreplacethecorrespondentwild-typeBCandFG
repeatsdomain. loopsequences9,13(SeeSupplementaryInformationfordetails).TheresultingFNDY
librarythuscontained10randomizedresidues(5randomresiduesineachloop)that
ArecentstudyhassuggestedthatNotchactsasadirectnegative
couldbeselectedfortargetbinding.Theresultingplasmidswereelectroporatedinto
regulatorofb-catbypromotingitslysosomaldegradation20.Totest XL1BlueE.Colicompetentcellstocreateaphagedisplaylibrarywith231010
whetherNotchnegativelyregulatesWntsignaling,weoverexpressed complexity.Targetproteinswerebiotinylated(usingbiotinylationkitfrom
DECNotch121,anactivatedformofNotch1,intheNotch-responsive Sigma-Aldrich)andimmobilizedonstreptavidincoatedELISAplates(Nunc),and
HEK293Tcells(FigS5,S6).Inthesecells,Notch1negativelyregu- thephagelibrarywasthenappliedintothewellsforcapturingthetargetbinding
phages.Afterincubationat4uCfor8h,unboundphageswereremovedbyextensive
lates Wnt signaling20 (Fig. 5D) and FNDY-NAK2 could increase
washingwithTBST.TheremainingtargetboundphageswereabsorbedbyXL1Blue
cytoplasmic Notch1 protein level (Fig. 5E). Since it has been E.Coliandamplified.Afterthreeroundsofpanningandenrichment,individual
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phagecloneswereobtainedandthephagemidDNAwassequencedtodeterminethe 15.Axelrod,J.D.,Matsuno,K.,Artavanis-Tsakonas,S.&Perrimon,N.Interaction
selectedsequences. betweenWinglessandNotchsignalingpathwaysmediatedbydishevelled.
Science271,1826–32(1996).
FNDYexpressionconstructs.ToexpressFNDYpeptideaptamersinmammalian 16.Brand,A.H.,Manoukian,A.S.&Perrimon,N.EctopicexpressioninDrosophila.
cells,selectedFNDYORFcDNAswereinsertedintopEF-mycvectors(Invitrogen)by MethodsCellBiol44,635–54(1994).
PCRcloning.ForDrosophilaexpression,FNDYORFcDNAswereclonedintothe 17.Colas,P.,Cohen,B.,KoFerrigno,P.,Silver,P.A.&Brent,R.Targeted
pUASTexpressionvector,whichcontainsa53Gal4bindingsiteinthepromoter modificationandtransportationofcellularproteins.ProcNatlAcadSciUSA97,
region.Togeneratetransgenicflies,thepUAST-FNDYconstructswereinjectedinto 13720–5(2000).
flyembryosandthepositivetransformantswereselectedfollowingstandard 18.Caussinus,E.,Kanca,O.&Affolter,M.Fluorescentfusionproteinknockout
procedure. mediatedbyanti-GFPnanobody.NatStructMolBiol19,117–21(2011).
19.Sanders,P.G.etal.Ligand-independenttrafficofNotchbuffersactivated
LuciferaseReporterAssays.FortheWntTOPFLASHreporterassay,HEK293cells ArmadilloinDrosophila.PLoSBiol7,e1000169(2009).
culturedin96-wellplatesweretransfectedwith100ng/wellTOPFLASHplasmidand 20.Kwon,C.etal.Notchpost-translationallyregulatesbeta-cateninproteininstem
2.5ng/wellpCMV-RLplasmidtogetherwithorwithout100ng/wellpEF-FNDY andprogenitorcells.NatCellBiol13,1244–51(2011).
peptideaptamerplasmidsand10,25ng/wellpCMV-DshorpCMV-b-catenin
21.Kopan,R.,Schroeter,E.H.,Weintraub,H.&Nye,J.S.Signaltransductionby
plasmids.FortheNotchreporterassay,cellsweretransfectedwith100ng/wellof
activatedmNotch:importanceofproteolyticprocessinganditsregulationbythe
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