Zones d’i te a tio - Principaux résultats II

Principaux résultats II

2. Zones d’i te a tio

L’ide tifi atio des zo es d’i te a tio à pa ti des spe t es M“ et M“/M“ des esp es oss-linkées a pu t e alis e de a i e auto atis e a e l’utilisatio du logi iel Xli k-Identifier développé par nos collaborateurs [150] et adapté pour notre système.

Les es zo es d’i te a tio s o t t ide tifi es pou les CPPs R/W9 et (R/W)₁₆ en complexes avec les protéines actine et albumine.

Pour les systèmes (R/W)9 ou (R/W)16-a ti e zo es d’i te a tio s de la p ot i e o t t ises e évidence : La zone [35–57] comprenant les lysines cross-linkées K45 et K56, la zone [91–111]

comprenant la lysine cross-linkée K108, la zone [202–210] comprenant la lysine cross-linkée K208 et

la zone [308–333] comprenant les lysines cross-linkées K310, K321 et K323. O peut s’ to e de e

pas t ou e u e plus g a de sp ifi it d’i te a tio a e u ou deu sites p i il gi s. Il se peut ue la nature à la fois chargée positivement et hydrophobe confère à la fois la possibilité aux peptides (R/W)9 et (R/W)16 d’i te agi de a i e sp ifi ue ais aussi de se lie ia des i teractions non spécifiques simplement du fait de leur composition chimique. Cependant, parmi ces quatre zones deux, [46–57] et [202–210], pou aie t a oi u i t t iologi ue puis u’elles sont connues pour leu i pli atio da s la odulatio de la d a i ue de l’a ti e [276]. De plus, la zone d’i te a tio

[46–57] fait pa tie du do ai e de liaiso à l’a ti e de la th osi e β et pou ait pa o s ue t

expliquer le déplacement de la thymosine β par (R/W)16 observé lors des expériences de compétition en RMN [2].

Pour les systèmes (R/W)9 ou (R/W)16-al u i e i zo es d’i te a tio o t té identifiées [24–44],

[210–218], [233–248], [452–459] et [548–557] quel que soit le ratio protéine : CPP utilisé (1:1 ou

10: i di ua t ue pa i es diff e ts sites au u ’est olo is de a i e p f e tielle. L’al u i e se le t e apa le de fi e un grand nombre de CPPs, dans des zones multiples et réparties sur sa surface exposée, sans spécificité apparente, ce qui va dans le sens du rôle de transport/protection que cette protéine exerce dans le sang.

ScienceDirect

j o u r n a l ho me p ag e :h t t p : / / w w w . e l s e v i e r . c o m / l o c a t e / e u p r o t

An integrated cross-linking-MS approach to

investigate cell penetrating peptides interacting

partners

SéverineClaviera,b, Xiuxia Duc,Sandrine Sagana,Gérard Bolbacha,b, EmmanuelleSachona,b,∗

aLaboratoiredesBiomolécules,UMR7203UPMC-ENS-CNRS-INSERMERL1157,cc182,4placeJussieu, 75252ParisCedex05,France

bPlateformedespectrométriedemasseetprotéomique,IBPS-UPMC,cc41,7-9quaisaintBernard, 75252ParisCedex05,France

cDepartmentofBioinformaticsandGenomics,UniversityofNorthCarolinaatCharlotte,USA

a r t i c l e i n f o

Articlehistory:

Received25November2013 Receivedinrevisedform 7March2014

Accepted11March2014 Availableonline24March2014

Keywords:

Chemicalcross-linking Cellpenetratingpeptides Interactingpartners Massspectrometry Actin

Albumin

a bs t r a c t

Cellpenetratingpeptides(CPPs)areattractingattentionbecauseoftheirabilitytodeliver biologicallyactivemoleculesintocells.Ontheirwaytheycaninteractwithmembraneand intracellularproteins.TofullyunderstandandimproveCPPefficiencyasdrugdeliverytools, theirpartnersneedtobeidentified.ToinvestigateCPP-proteincomplexes,chemical cross-linkingcoupledtomassspectrometryisarelevantmethod.Withthisaim,wedevelopedan originalapproachbasedontwoparallelstrategies,anintactcomplexanalysisanda bottom-upone,tohaveaglobalcharacterizationofthecross-linkedcomplexescompositionaswell asadetailedmappingoftheinteractionzones.

Biologicalsignificance:Therobustandefficientcross-linking-MSworkflowpresentedherecan easilybeadaptedtoanyCPP-proteininteractingsystemandcouldthuscontributetoabetter understandingofCPPsactivityascell-specificdrugdeliverytools.Wevalidatedtherelevancy ofthiscross-linking-MSapproachwithtwobiologicallyactiveCPPs,(R/W)9and(R/W)16,and twointeractingproteinpartners,actinandalbumin,previouslyreportedusingisothermal titrationcalorimetry(ITC)andNMR.Cross-linking-MSresultsobtainedontheseprevious studiesallowedustogofurtherbyprovidingadetailedmappingoftheinteractionzones. TheidentifiedinteractionzonesbetweenactinandCPPs(R/W)9and(R/W)16arebiologically meaningful.Twocross-linkedzones[46–57]and[202–210]ofactinareindeedinvolvedin themodulationofitsdynamics.Moreover,[46–57]domainhasalsobeendescribedasone interactiondomainforthymosin␤4whoseactinbindingcanbedisplacedbycompetition with(R/W)16(NMRexperiments).

∗ Correspondingauthorat:LaboratoiredesBiomolécules,UMR7203UPMC-ENS-CNRS-INSERMERL1157Plateformedespectrométriede masseetprotéomique,IBPS-UPMC,France.Tel.:+33144273234.

E-mailaddress:emmanuelle.sachon@upmc.fr(E.Sachon).

http://dx.doi.org/10.1016/j.euprot.2014.03.002

Twenty years ago, the discovery of peptides able to ubiq-uitously cross cellular membranes commonly named cell penetrating peptides (CPPs), with very limited toxicity, launchedanovelfieldinmoleculardeliverybasedonthese non-invasivevectors.MostCPPsarepositivelycharged pep-tidesthoughthepresenceoffewanionicorhydrophobicCPPs wasalsodemonstrated.Afteradecadeofdebateonthe traf-fickingroutes ofCPPstothe heart ofcells,it isnow more orlessacceptedthat thesepeptides useconcomitantly dif-ferent internalization pathways, including pinocytosis and directmembranetranslocationprocesses[1].CPPsare gener-allyconsideredasbiologicallyinertintracellulardeliverytools. However,someCPPshaveintrinsicallybiologicalactivityand arepartofarecentlydescribedclassofCPPsbaptized biopor-tide[2].

Forinstance,previousstudiesshowed, thatonlythetwo CPPs (R/W)9 and (R/W)16 (RRWWRRWRR and RRWRRWWR-RWWRRWRRrespectively)amongotherCPPstested,areable toremodel the actin cytoskeleton in oncogentransformed NIH3T3/EWS-FlicellsoncetheseCPPshadcrossedthe plasma-membrane [3]. In order to explain the actin-remodeling activityofthetwoCPPs,thehypothesis ofadirect interac-tionwithactinwastested.(R/W)9and(R/W)16peptideswere actuallyfoundtodirectlyinteractinvitrowithG-actinbyNMR andITCexperiments[3](Kd≈10␮MandKd=0.4␮M, respec-tively).Inaddition,competitivebindingexperimentsbyNMR showedthat(R/W)16wasabletodisplacetheactin sequester-ingproteinthymosin␤4fromG-actin[3].

Itwasalsorecentlyreportedthatarginine-richCPPs inter-actwithserumproteinslikealbumin,modifyingtheirability tointernalizeincells[4].Thereforeouraiminthisstudywas toanalyzefurthertheinteractionof(R/W)9and(R/W)16with actinandalbumin.

Forthispurpose,chemicalcross-linkingwaschosen. Cross-linking reactions are conventionally based on the use of abifunctional cross-linker, whichis acarbon chainspacer bearing reactive sites at both ends that can either be identical(homobifunctional)ordifferent(heterobifunctional). Reactive sites are mainly activated esters targeting either lysine residues (e.g. N-hydroxysuccinimide (NHS) ester) or cysteine residues(e.g.maleimide ester)althoughside reac-tionswithtyrosine,threonineandserinehavebeenreported

[5].

Bycreatingacovalentbondbetweentwoormore inter-actingpartners,chemicalcross-linkinggivesasnapshotofa molecule’senvironmentand,combinedwithMS,constitutes a powerful tool to map protein-protein or peptide–protein interactions(distanceconstraintsandinteractingdomains). Howeverchemicalcross-linkingisoftencharacterizedbylow reaction yields. In addition, a wide variety of cross-linked productsare usuallycreated. Therefore, cross-linkers bear-inganaffinitytag[6,7]allowingselectiveenrichmentofthe sampleincross-linkedspecieshavebeendeveloped. Cleav-ablecross-linkers [8,9], isotopelabeled cross-linkers [10]or cross-linkersbearingaCHCAmatrixmoietyforMALDI analy-sis[11]improvedetectionandidentification/characterization of the cross-linked species. Affinity purification using a

chromatographicmethodslikestrongcation-exchange(SCX)

[13,14]andsizeexclusionchromatography(SEC)[15] consti-tutepromisingapproachestoenrichsamplesincross-linked species.

Analysisoflowabundantcross-linkedpeptides requires high sensitivityfortheirdetection andhigh massaccuracy (masserror<10ppm)fortheiridentificationsincethe num-ber ofcombination oftwopeptides isenormous.Moreover tandemMSofcross-linkedpeptidesisrequiredforthe char-acterizationoftheinteractionzones.

A number oftailoredsoftwares havebeen developedto dealwiththeselectiveacquisitionorinterpretationofthese MS/MSspectra.Amongothers,FINDX[16]hasbeendesigned toselectivelyfragmentinter-proteincross-linksby LC–MALDI-TOF/TOF using 14N/15N mixedisotope strategy,xQuest [13]

isdedicatedtothesearchofisotopicallytaggedcross-linked peptidesandCrossWork [17]orXlink-Identifier[18]support label-freeanalysesofchemicalcross-linkingsamples.

Inthisstudy,weset-upageneralinvitroanalyticalworkflow couplingcross-linkingandmassspectrometry (cross-linking-MS), involving enrichment steps as well as manual or automatedMSandMS/MSdataprocessingtotestpotential interactingpartnersofanyCPPsequence.

Tovalidateourcross-linking-MSworkflow,westudiedthe systemsdescribedabove:(R/W)9or(R/W)16 interactingwith actin oralbumin.Forthispurpose,CPPanalogssuitablefor chemical cross-linkingexperiments, weresynthesized with the following sequences: Biot(O₂)-G4-K-RRWWRRWRR-NH₂ and Biot(O₂)-G₄-K-RRWRRWWRRWWRRWRR-NH₂, respec-tively.TheKresidueaddedattheN-terminusofthepeptides sequences allowed the cross-linking reaction, the biotin tag (Biot(O2)) was added for purification purpose and was separated from the biologically active motif by a four G residuesspacer.This{Biot(O)₂-Gn-K-}groupiseasytoaddat theN-terminusofpeptideseithermanuallyorautomatically duringpeptidesynthesiswhateverthepeptidesequence.

The originality ofour approach resides in the compre-hensive study of the cross-linking reaction mixture from twoangles:aglobalviewoftheinteractingsystemwithan intact complexanalysiscombinedtothe precise character-ization of interacting zones by a bottom-up analysis. The intact complex analysis is based on the MALDI-TOF anal-ysis in linear mode ofthe cross-linking reaction mixtures and onthe modeling ofthespectraobtainedusing the in-house SIMUL-XLprogram.Thebottom-upanalysisconsists in the tryptic digestion of the cross-linking reaction mix-turesfollowedbytheaffinitypurification(biotin/streptavidin) ofthe biotinylatedcross-linked peptidesand theiranalysis bytandemMS(MALDI-TOF/TOFand/ornanoLC–ESI-Orbitrap). The MS/MS data are either manually interpreted with the help ofGPMAW software [19] (MALDI-TOF/TOF spectra) or automatically searchedusing Xlink-Identifier software [18]

(nanoLC–ESI-MS/MSspectra).

For practical reasons the intact complex analysis was developedonlywith(R/W)₉andbothactinandalbumin pro-teins. In contrast, the bottom-up analysis was performed using both(R/W)9 and(R/W)16 andbothactinand albumin proteins.

2.1. Materials

Bovin serum albumin and actin from rabbit muscle were purchasedfrom Sigma(St.Louis,Missouri,USA).Promix1, proteinsstandardmixture,wasfrom LaserBioLabs(Sophia Antipolis, France). Bis[sulfosuccinimidyl] suberate (BS3) was from Thermo Scientific (Waltham Massachussetts, USA) and the K100 stabilization kit from CovalX (mix of three cross-linkers 1,1′-(suberoyldioxy)bisazabenzotriazole (SBAT), 1,1′-(suberoyldioxy)bisbenzotriazole (SBBT) and 1,1′-(glutaroyldioxy)bisazabenzotriazole (GBAT)) (Schlieren, Switzerland). Trypsin Gold, Mass spectrometry Grade, was from Promega (Fitchburg, Wisconsin, USA). Dynabeads M280 streptavidin-coated magnetic beads were from Invi-trogen (Carlsbad, New-Mexico, USA). The CPPs (R/W)9: Biot(O₂)-G4-K-RRWWRRWRR-NH₂ (m/z2127.12) and (R/W)₁₆: Biot(O₂)-G₄-K-RRWRRWWRRWWRRWRR-NH₂ (m/z 3309.76) were synthesized (Fmoc strategy) and purified in-house. Ziptip®

C4 pipette tips were from Millipore (Darmstadt, Germany).

2.2. Cross-linkingreactions

Monomericactin(G-actin)purchasedlyophilizedinTris,ATP, andCaCl₂wasdialysedagainsta20mMHEPES,150mMNaCl, 0.5mM DTT, 200␮M CaCl₂, 200␮M ATP and 0.005% NaN₃ pH 8 buffer (Actin non-denaturing buffer).Stock solutions ofCPPand actin were dilutedin the actin non-denaturing buffertoobtainthedesiredconcentrations(typically10␮M). For CPP and albumin a 20mM HEPES, 150mM NaCl buffer was used. For all experiments, after 15minpre-incubation ofthe CPPand proteinpartners, 50mM cross-linkers solu-tionspreparedextemporanelyindimethylformamide(DMF)for theK100 kit,inwaterforBS3, were addedtohave a2mM final concentration of cross-linkers (200 folds higher than proteinsconcentration) and the reactions were allowed to proceed for120min at room temperature (RT) under gen-tlestirring.Typicalcross-linkingreactions(XL)volumeswere 50␮L.Forexperimentswithlowpeptidesconcentrations(1or 0.1␮M),reactionvolumeswereadapted(200␮Land2000␮L respectively)toallowpurifyingenoughcross-linkedpeptides. Controlreactionmixtures notedCT wereperformed inthe sameconditionsbutwithoutcross-linkers.Cross-linking reac-tionswerequenchedbyaddingTris-base(finalconcentration 15mM).

2.3. Enzymatictrypsindigestion

25␮Lofsamplesweresubmittedtoin-solutiontryptic diges-tion.Disulfidebridgeswerereduced(5mMdithiothreitol)and cysteinesalkylated(20mMiodoacetamide).Trypsindigestion was conducted at 37◦C overnight (1:30 (w:w) protease-to-proteinratio).

purification

Avolumeoftrypticdigestcorrespondingto80pmolofCPPwas incubatedwith200␮gofstreptavidin-coatedmagneticbeads for60minatRTundergentlestirring.Beadswereconditioned beforeuseandwashedafterincubation[12].Finally,peptides wereelutedfromthebeadswith5␮L0.1Mhydrochloricacid (HCl)undergentlestirring(30min).

2.5. MSanalysisanddatatreatment

MALDI-TOF spectra were obtained with a MALDI-TOF/TOF

AB4700 Proteomics Analyzer mass spectrometer (Applied Biosystems) in positive ions linear or reflector mode and delayedextraction.CHCA(Sigma)wasusedasthematrixand solubilizedat5mg/mLin1/1ACN/0.1%TFA.

IntactcomplexesanalysisinlinearmodeMALDI-TOF:0.5␮Lof non-digested reactionmixturespurifiedbyC₄ Ziptip®

were mixedwithanequalvolumeofmatrixanddepositedonthe sampleholder.Atotalof20,000lasershotspersamplewere acquiredinthem/zrange10,000–100,000(focusmass60,000). Modelingsofthespectraobtainedforintactcross-linked complexesandcorrespondingcontrolswereachievedusing SIMUL-XL,aprogramdevelopedin-house(VisualBasicV6.0).

Bottom-up analysis in reflectormode MALDI-TOF/TOF:After affinitypurification,0.5␮Loftheelutionmixturewasmixed with0.5␮Lmatrixand0.5␮Lweredepositedonthesample holder.Atotalof10,000lasershotsperspotwere acquired in them/z range 500–5000.Externalcalibrations were real-ized using the peptidecalibration standardPepmix4 (Laser BioLabs). Amanual interpretation ofthe spectrawas done comparing the cross-linkingreaction mixture(XL) and the peptideandproteinmixturewithoutcross-linker(CT)spectra. DifferentialpeaksidentifiedinXLspectrumwereconfronted withthecross-linkedpeptideslistsgeneratedinsilicousing GeneralProteinMassAnalysisforWindows(GPMAW)version 6.1[19](LighthouseData,Odense,Denmark)withanumberof missed-cleavagesfortrypsinof2.

NanoLC–ESI-Orbitrap:Thecompletecharacterizationofthe cross-linkedpeptideswasperformedusinganUltimate3000 Nano-HPLCsystem(Dionex)coupledwithaLTQ-Orbitrap-XL massspectrometer(ThermoScientific).Sampleswereinjected bytheautosamplerandconcentratedonatrappingcolumn (Pepmap,C18,300␮m×5mm,5␮m,100 ˚A,Dionex)withwater containing 2%ACNand 0.1%formicacid (solventA).After 10min,thepeptideswereelutedontotheseparationcolumn (Pepmap, C18, 75␮m×150mm, 2␮m 100 ˚A, Dionex) equili-bratedwith98%solventA.Peptideswereseparatedusingthe gradient 0–50min 2–40% solvent B(98% ACN+0.1%formic acid), 50–60min 40–60%solvent B, and 60–70min 60% sol-ventBataflowrateof200nL/min.TheLTQ-Orbitrapmass spectrometerisoutfittedwithananoESIinterface. Electro-spray emitters were 360/20␮mo.d.×10␮m i.d.fused-silica tips(PicoTipEmitter,StandardCoatedSilicaTip,New Objec-tive).Theheatedcapillarytemperatureandsprayvoltagewere 200◦C and 1.5kV,respectively.Orbitrap spectra(automated gaincontrol(AGC)2×105)werecollectedfromm/z300–2000 ataresolutionof30,000intheprofilemodefollowedbydata dependentsequentialCIDandHCDMS/MSspectraofthethree

Fig.1–Generalanalyticalworkflowtoinvitroidentifyandcharacterizeproteinpartner(s)ofaknownCPP.Thechemical cross-linkingreactionbetweenabiotinylatedCPP,theBS3cross-linkerandapotentialproteinpartnerispresented(top scheme).Thecross-linkedcomplexeswerecharacterizedusinganintactcomplexanalysis(A)andabottom-upone(B).

mostintenseionswithanormalizedenergyof35forboth frag-mentationmodes.Adynamicexclusiontimeof60swasused todiscriminateagainstpreviouslyanalyzedions.

MS/MS spectra were automatically searched using the Xlink-Identifier software [18]. Xlink-Identifier is a search enginefor identifyingand characterizing cross-linked pep-tidesfrom label-free experiments (neither the peptide nor the cross-linkeris isotopicallylabeled). Ittakes the MS/MS spectrain.dtaor.mgfformatandtheproteinsequencesin FASTAformat.Searchparametersincludethefragmentation technique, the nature of the dynamic modifications (car-bamidomethylation(C),oxidation(M)),themaximumnumber ofmissed-cleavages,theprecursorandfragmentmass toler-ance(respectively10ppmand0.6Da)andfinallythedefinition ofthecross-linkerspacerarmmassinvolvedintheformation ofthecross-linkingproducts(96.0211DaforGBAT,138.0618Da forSBAT,SBBTorBS3).

Asmentioned,Xlink-Identifierisafullyautomatedsearch enginespecificallyforcross-linkinganalysis. Itisequipped withavisualization moduleallowing researchersto exam-inetheannotatedMS/MSspectraanddetailsofeachmatched peak.

3. Resultsanddiscussion

In this study, we synthesized analogs of (R/W)9 and (R/W)16 CPPs (Biot(O2)-G4-K-RRWWRRWRR-NH2 and

Biot(O2)-G4-K-RRWRRWWRRWWRRWRR-NH2, respectively) adaptedforcross-linkingexperiments.Tostudythe interac-tionoftheseCPPswithproteinpartners,across-linking-MS approachintegratingtwoparallelstrategies,anintact com-plex analysis and a bottom-up one, was developed and optimized(Fig.1).

3.1. Chemicalcross-linkingreaction

Actinisaglobular42kDaproteinthatbindsanATPmolecule andadivalentcation(Mg2+orCa2+).Thesecofactorsare neces-sarytomaintaintheintegrityoftheprotein.Commercialactin wasavailableasalyophilizedpowdercontainingTrisbuffer, whichisnotcompatiblewithchemicalcross-linkingbecause ofits reactive primary amine group.The samplewas thus dialysedagainstanHEPESbufferalsocontainingappropriate concentrationsofsaltsandactincofactors.Initial concentra-tionof10␮Mwaschosenforthetwopartnersasthespecificity ofchemicalcross-linkingwithactivatedesterswas demon-stratedinthelow␮Mrange[20].

We worked witha mixture of three cross-linkers (K100 stabilizing kit): SBAT, SBBT and GBAT (Fig. S-1). This mix-tureofcross-linkersisrecommendedtostabilizecomplexes withamolecularweightbelow100kDaandallowstestingtwo spacerlengthsatthesametime(11.4 ˚AforSBATandSBBTand 7.7 ˚AforGBAT).TheK100kitcross-linkersweresolubilizedin DMF.Tomakesurethatthesmallproportionofthisorganic

Fig.2–PositiveionslinearmodeMALDI-TOFofintactcontrolsandchemicalcross-linkingreactionmixture.Thecontrols (REF,CLandCT)arepresentedonthethreetopspectra,thecross-linkingexperiment(XL)inthelowerspectrum.Mass spectraweremodeledusingSIMUL-XLprogram.Modelings(black)thatbestfittheexperimentaldata(gray)arepresentedin abox(doublychargedspecies).:standarddeviation,:averagenumberof(R/W)9,N:averagenumberofcross-linker.

solventisnotaffectingtheproteinorpeptideconformation and their interactions and thus the cross-linkingreactions results,experimentswererepeated withthe BS3 water sol-ublecross-linkerwhichhasthesamespacerarmasSBATor SBBT.

SupplementaryFig.1canbefound,intheonlineversion, atdoi:10.1016/j.euprot.2014.03.002.

3.2. Intactcomplexanalysis:determinationof(R/W)9 CPP–proteinratioinintactcross-linkedcomplexes

LinearmodeMALDI-TOF:MALDI-TOFisusuallynotamethod ofchoicetostudynon-covalentcomplexesduetoimperfect preservation of complexes during target preparation and unspecificnon-covalentmultimersdesorptionduetospatial proximity and high concentration in the matrix crystals. Howeveritiswelladaptedtothestudyofcovalentcomplexes formed using chemical cross-linking. Linear mode MALDI-TOFanalysesoftheintactcomplexeswerecarriedoutafter asimpledesaltingstepofthesamplesonaC4ZipTip®

.The aimwastoassesstheefficiencyofthecross-linkingreaction

bydeterminingtheaveragenumberof(R/W)9CPPscovalently attachedperprotein,fordifferentreactionconditions.

Toassessthecontributionofthedifferentcomponentsto theobservedsignalforacross-linkingreaction(XL),controls are necessary: theproteinalone(REF), the proteinplus the cross-linkeronly(CL)(toassessthemono-links,intraandinter cross-linked proteins),and the (R/W)9 CPPand the protein mixturewithoutthecross-linker(CT)(toassessnon-covalent interactionsthatcouldremainevenwiththeacidicCHCA dis-sociativematrix).Manyprecautionsneedtobetakeninthe preparationof thesecontrolsand in massspectra

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