Cell Penetrating Peptides and Cationic Antibacterial Peptides

10 Pages·2014·1.26 MB·Spanish

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.289,NO.21,pp.14448–14457,May23,2014
©2014byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Cell Penetrating Peptides and Cationic Antibacterial Peptides
TWOSIDESOFTHESAMECOIN*□S
Receivedforpublication,April1,2014Published,JBCPapersinPress,April5,2014,DOI10.1074/jbc.M113.515023
JonathanG.RodriguezPlaza‡,RosmarbelMorales-Nava§,ChristianDiener‡,GabrieleSchreiber¶,
ZyanyaD.Gonzalez‡,MariaTeresaLaraOrtiz‡,IvanOrtegaBlake§,OmarPantoja¶,RudolfVolkmer(cid:2),EddaKlipp**,
AndreasHerrmann**,andGabrielDelRio‡1
Fromthe‡BiochemistryandStructuralBiologyDepartment,InstitutodeFisiologíaCelular,UniversidadNacionalAuto´nomade
Me´xico,CircuitoExteriorS/NCiudadUniversitaria,04510Me´xicoD.F.,Me´xico,§Materialsscienceandbiophysicsdepartment,
InstitutodeCienciasFisicas,UniversidadNacionalAuto´nomadeMe´xico,Av.UniversidadS/N,Col.Chamilpa,62210Cuernavaca,
Morelos,Me´xico,¶InstitutodeBiotecnología,UniversidadNacionalAuto´nomadeMe´xico,A.P.510-3,ColoniaMiraval,Cuernavaca,
Morelos,Me´xico62250,(cid:2)Institutfu¨rMedizinischeImmunologie,Charite´-Universita¨tsmedizinBerlin,HessischeStrasse3-4,10117
BerlinandLeibniz-Institutfu¨rMolekularePharmakologie,Robert-RoessleStrasse10,13125Berlin,Germany,and**Theoretische
undMolekulareBiophysik,Humboldt-Universita¨tzuBerlin,Invalidenstrasse42,10115Berlin,Germany
Background:Iztlipeptides(IP)areanewclassofantibacterialpeptidesthatcouldbeinternalizedbyreceptor-mediated
endocytosisinyeast.
Results:IP-1penetratescellsindependentlyofendocytosisandmakesporesinmembraneswithlargeelectricpotential.
Conclusion:AntibacterialpeptideslikeIP-1makeporesorpenetratemembranesdependingonthemembranepotential.
Significance:Antibacterialpeptideswithpenetratingactivityarerobusttocontrolbacterialinfections.
Cell penetrating peptides (CPP) and cationic antibacterial thekillersequence.Insidethecell,thekillersequencetargets
peptides(CAP)havesimilarphysicochemicalpropertiesandyet mitochondria, which share similar structure with bacteria, to
itisnotunderstoodhowsuchsimilarpeptidesdisplaydifferent destabilize their membrane and ultimately induce apoptosis
activities.Toaddressthisquestion,weusedIztlipeptide1(IP-1) (4).
becauseithasbothCPPandCAPactivities.Combiningexperi- Theabilitytodelivermacromoleculesinsidecellsisimpor-
mental and computational modeling of the internalization of tant both in academic research as well as in biotechnological
IP-1,weshowitisnotinternalizedbyreceptor-mediatedendo- applicationssuchasdrugdeliveryandgenetherapy(5).Chem-
cytosis,yetitpermeatesintomanydifferentcelltypes,including icalandphysicalmethodshavebeendevelopedtodelivercargo
fungiandhumancells.WealsoshowthatIP-1makesporesin moleculesinsidecells.Thesemethods,e.g.electroporationand
thepresenceofhighelectricalpotentialatthemembrane,such cationic lipids/liposomes, have been shown to deliver hydro-
asthosefoundinbacteriaandmitochondria.Theseresultspro- phobic macromolecules, yet these induce cellular side effects
videthebasistounderstandthefunctionalredundancyofCPPs and are limited to in vitro applications (6). Cell penetrating
andCAPs. peptides(CPPs)overcomesomeoftheselimitationsandHIV-
Tat, penetratin, transportan, and octa-arginine among others
representextensivelystudiedCPPs(7).ManyCPPsareableto
Hunter-killer peptides (HKPs)2 combine two activities: a deliverthemselves,smallcargo,andrelativelylargemacromol-
ligand peptide (hunter sequence) and a cationic antibacterial eculesdirectlyacrossthecellularplasmamembrane(8).
peptide (killer sequence) (1). Such design aims to selectively Understanding the penetrating mechanism of CPPs is
inducecelldeathoftargeteukaryoticcells.Forinstance,HKPs importanttocontrolthedeliveryofmacromoleculesbyCPPs.
havebeenappliedsuccessfullytotreatcancer(2)andobesity(3) In this sense HIV-Tat and nona-arginine peptides have been
inanimalmodels.Themechanismofactionofthesepeptides showntobeinternalizedbyendocytosis(9),yetotherCPPsuse
proposesthatthehuntersequencerecognizesareceptoronthe an energy-independent mechanism of internalization (5).
cell surface and upon binding the hunter sequence may be Despitesuchdifferences,commonfeaturesexistamongCPPs;
internalizedbyreceptor-mediatedendocytosiscarryingwithit for instance, most CPPs do not show any cell specificity, are
amphipathic,andpresentanetpositivecharge(10).Relevantto
thisworkisthatcationicantibacterialpeptides(CAPs),likethe
*ThisworkwassupportedinpartbytheAlexandervonHumboldtFounda-
killerpeptidesusedinHKPs,arealsoamphipaticandcationic
tion,ConsejoNacionaldeCienciayTecnologia(82308and128575),Pro-
gramadeApoyoaProyectosdeInvestigacióneInnovaciónTecnológica (11).Itisnotclearthough,howpeptideswithsimilarphysico-
Grants IN205911 and IG100513 (to G.D.R. and I.O.B.), and German chemical features such as CPPs and CAPs can perform two
ResearchCouncilGrantCRC740(toE.K.andA.H.). distinctfunctions;CPPspenetrateeukaryoticcellswithoutany
□S ThisarticlecontainssupplementalTableS1.
1To whom correspondence should be addressed. E-mail: gdelrio@ifc. apparent toxicity, and CAPs kill bacteria. Given their similar
unam.mx. physicochemicalproperties,theexplanationtothisfunctional
2Theabbreviationsusedare:HKP,Hunter-killerpeptide;IP-1,Iztlipeptide1; differencemayresideinthetargetcellandnotinthepeptide
CPP,cellpenetratingpeptide;CAP,cationicantibacterialpeptide;MATa,
itself.Ifso,CPPsmayworkasCAPsinthepresenceofbacterial
matingtypeA;POPC,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine;
TAMRA,6-carboxytetramethylrhodamine. cells. In agreement with this idea, several CPPs have been
This is an open access article under the CC BY license.
14448 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER21•MAY23,2014
FunctionalRedundancyofCPPsandCAPs
Nacional Auto´noma de Me´xico, Mexico). See Table 1 for a
detailedlistofthecelllinesusedinthisstudy.
Fortheelectrophysiologicalexperiments1-palmitoyl-2-ole-
oyl-sn-glycero-3-phosphocholine(POPC)dissolvedinchloro-
form and powdered cholesterol were purchased from Avanti
FIGURE1.Iztlipeptide1.Embeddingofthe(cid:2)-pheromonesequenceintoa
cationic antibacterial peptide sequence is shown. Note that the residues PolarLipids(Alabaster,AL)andstoredinthedarkat(cid:3)20°C.
addedtothe(cid:2)-pheromone(boldletters)donotpresentanyantibacterialor Potassium chloride (KCl, ACS grade) and calcium chloride
antifungalactivityandwereaddedtothepheromonetomatchthephysico-
(CaCl , ACS grade) were purchased from Merck. All organic
chemicalpropertiesofcationicantibacterialpeptides(11). 2
solventswereACSgradeandwerepurchasedfromMallinck-
rodt Baker. Borosilicate glass capillaries were obtained from
shown to have antibacterial activity (12, 13). Furthermore,
WorldPrecisionInstruments(Sarasota,FL).Praxair(Cuerna-
giventhatCPPsandCAPsactoncellularmembranes,itcanbe
vaca,Me´xico)suppliedhighpuritynitrogengas.
assumed that these membranes modulate their functions. In Thematingpheromone(cid:2)-factorfromS.cerevisiaewaspur-
termsofthemechanismofactionproposedfortheinteraction
chased from Sigma (catalogue number T6901). Anaspec, Inc.
withcellularmembranes,CAPsareabletomakeporesby“bar-
producedtheIP-1andIP-labeledwithHilytefluor488,whereas
rel-stave,” “carpet” or “toroidal-pore” mechanisms (14); such
wesynthesizedTAMRA-IP-1.SeeTable2forthesequencesof
pores ultimately end up killing bacterial cells. On the other
thepeptidesusedinthisstudy.
hand,CPPscrosseukaryoticcellularmembraneswithoutmak-
Measuring the Cell Death Induced by Iztli Peptide 1—An
ing pores. To the best of our knowledge there is no evidence
early stationary culture for every assay was obtained from a
thatmayexplainhowacellularmembranemayswitchaCPPto
single colony of BY4741, and every endocytosis null mutant
becomeaCAP.
grewfor12hinYPDmedium(yeastextract1%,peptone2%,
We have recently described a new class of HKPs (15): Iztli
glucose2%).ThesecultureswereusedtoinoculateafreshYPD
peptides (IPs). IPs are CAPs that harbor a hunter sequence
within;suchdesigndiffersfromtypicalHKPsthatfuseahunter mediumtoreacha0.03opticaldensityinatotalvolumeof200
sequence next to a CAP sequence (see Fig. 1). To study the (cid:3)l.BY4741strainwastestedagainstTAMRA-IP-1(62.9(cid:3)M)or
mechanism of action of these peptides, IPs were designed to Hilytefluor 488-IP-1 (26.9 (cid:3)M), and every endocytosis null
targetSaccharomycescerevisiaeusingthepheromone(cid:2)-factor mutantwastestedagainstthepheromone(cid:2)-factor(10(cid:3)M)and
as the hunter sequence; only mating type A (MATa) cells PI-1(10(cid:3)M)ina100-wellhoneycombplate,eachcontaining
expressing the receptor for the pheromone (Ste2p) would be 200 (cid:3)l, incubating at 30°C with shaking using a Bioscreen C
killedbytheactionofIPs.Indeed,fourIPs(IP-1,IP-2,IP-3,and (OyGrowthCurvesAbLtd).Theactivityofthesepeptideson
IP-4)sharingthesamepheromonesequencebutdifferinginsix each strain was determined by measuring the A every
600nm
residues at the N terminus were shown to have antibacterial hourfor24h.
activity,toswellmitochondria,andtoinducethepheromone Modeling the Internalization of Iztli Peptides—The models
response pathway, and only MATa cells expressing the Ste2p wereimplementedbasedontheprocessesdepictedinFig.3,A
receptorwerekilledbyIPs(15).Thus,Ste2pisrequiredforthe andB.Thebiochemicalreactionswereassumedtofollowmass-
killingasexpectedfromtheHKPmodelofaction,yetthiscan- action kinetics and resulted in the ordinary differential equa-
notbetakenasevidenceforcellularuptakeviaSte2p. tionsprovidedbelow.Modelsimulationswereperformedusing
Inthecurrentworkwestudiedthemechanismofinternal-
theLSODAsolverandanevolutionaryoptimizationalgorithm
ization of IP-1 at concentrations required to kill cells. Our
performed fitting to the measured time course of dead cells.
resultsindicatethatIP-1isinternalizedintomanycelltypesin
Here,weusedtheimplementationswithinthemodelingsoft-
the absence of receptor-mediated endocytosis, yet binding to
ware Copasi. For each model only two parameters had to be
thereceptorisrelevantforthekilling.WealsoshowthatIP-1
fitted (denoted as k /k and k /k in Fig. 3, A and B). The
formsporesonlyinthepresenceofhighelectricpotentialval- E D In D
remaining parameters were obtained from previously pub-
ues.Therelevanceofourfindingsisdiscussedintermsofthe
lishedmeasurements.
biological and biotechnological advantage of multifunctional
Modelswereconsideredsignificantwhentheycouldrepro-
peptides,suchasIPs.
ducethemeasuredtimecoursesignificantlybetterthanazero-
EXPERIMENTALPROCEDURES interceptlinearreferencemodel.Thus,weobtainedpvaluesfor
theentiretimecourse,orpartsofit,fromFtestscomparingthe
Strains and Peptides—The strains used in this study were:
S.cerevisiaeBY4741(MATahis3(cid:2)1leu2(cid:2)0met15(cid:2)0ura3(cid:2)0), linearmodeltothefitprovidedbytheoptimizationofthemod-
(cid:2)did4::kanMX,(cid:2)ypt7::kanMX,(cid:2)rvs161::kanMX,(cid:2)pep5::kanMX, els. Those analyses were performed in R (The R Project for
(cid:2)yck1::kanMX, and (cid:2)yck2::kanMX; BY4742 (MAT(cid:2) his3(cid:2)1 StatisticalComputing).
leu2(cid:2)0 lys2(cid:2)0 ura3(cid:2)0) purchased from OpenBiosystems Can- Endocytosis Model—The endocytosis model comprised the
dida glabrata CGM99 (Ura3D::Tn903(G418R), Trp 1D), and reactions from Fig. 3A. This resulted in the following set of
AspergillusnidulansCLK43;theselasttwowerekindlyprovided, ordinarydifferentialequations(squarebracketsdenoteconcen-
respectively,byDra.IreneCastan˜o(InstitutoPotosinodeInvesti- trations,andtheindicese,m,v,andcdenotelocationsinthe
gaciónCientíficayTecnológica,SanLuisPotosi,Mexico)andDr. extracellularmedium,themembrane,(pre)vacuole,andcyto-
JesusAguirreLinares(InstitutodeFisiologíaCelular,Universidad sol,respectively).
MAY23,2014•VOLUME289•NUMBER21 JOURNALOFBIOLOGICALCHEMISTRY 14449
FunctionalRedundancyofCPPsandCAPs
TABLE1
Celllines
Organism Strain ORF Genotype Source
S.cerevisiae BY4741 WT MATahis3(cid:2)1leu2(cid:2)0met15(cid:2)0ura3(cid:2)0 OpenBiosystemsYKOMATaStrain
YKL002W MATa(cid:2)did4 Collection-GlycerolStocks
YML001W MATa(cid:2)ypt7
YCR009C MATa(cid:2)rvs161
YMR231W MATa(cid:2)pep5
YHR135C MATa(cid:2)yck1
Endocytosisnullmutants MATa(cid:2)gene::kanMX
(seesupplementalTableS1)
BY4742 YNL154C MATa(cid:2)yck2 OpenBiosystemsYKOMAT(cid:2)Strain
Collection-GlycerolStocks
C.glabrata CGM99 WT MAT(cid:2)his3(cid:2)1leu2(cid:2)0lys2(cid:2)0ura3(cid:2)0 DomergueR.,etal.(52)
A.nidulans CLK43 WT Ura3D::Tn903(G418R),Trp1D Kawasaki,L,etal.(53)
WT pabaA1yA2;veA1
TABLE2
Syntheticpeptides
Name Sequence Measuredmolecularweight
g/mol
(cid:2)-Pheromone NH-WHWLQLKPGQPMY-COOH 1684
2
IP-1 NH-KFLNRFWHWLQLKPGQPMY-COOH 2490.8
2
TAMRA-IP-1 TAMRA-KFLNRFWHWLQLKPGQPMY-COOH 2903.2
Hilytefluor488-IP-1 NH-KFLNRFWHWLQLK(GGG-CO-(CH)S-maleimidoHilytefluor488)-PGQPMY-COOH 3335.4
2 22
d[Ste2 ] ments(16–18).k wasthencalculatedfromk andthemeank
dt m (cid:4)k2[Ste2mIztlim](cid:3)k1(cid:4)Ste2m][Iztlie](cid:5)k5 (Eq.1) forSte2pbinding1to(cid:2)-factorfrom9experim2ents,resultingiDn
k (cid:8)0.185(cid:3)M(cid:3)1s(cid:3)1(18–26).Weassumedthatdissociationof
1
d[Ste2 Iztli ] (cid:2)-factorfromSte2phasthesamekineticsinthe(pre)vacuole
m e (cid:4)k (cid:4)Ste2 ][Iztli ](cid:3)(k (cid:5)k (cid:6)(cid:4)Ste2 Iztli (cid:7)
dt 1 m m 2 3 m m before degradation. The rate of endocytosis of Ste2p in pres-
enceof(cid:2)-factor,k ,wasderivedfromthepublishedhalf-timeof
3
(Eq.2) 5minas2.3(cid:3)10(cid:3)3s(cid:3)1(27).Thedegradationrateofinternalized
Ste2p,k ,wasobtainedfromthehalf-lifeoftotalSte2pinthe
d[Ste2vIztliv](cid:4)k (cid:4)Ste2 Iztli ](cid:3)(k (cid:5)k(cid:6)(cid:4)Ste2Iztli(cid:7) presence4 of (cid:2)-factor in cycloheximide-treated cultures as
dt 3 m m 4 E v v
5.8(cid:3)10(cid:3)4s(cid:3)1(24).Because(cid:2)-factorisnormallydegradedalong
(Eq.3) withSte2p,weassumedthatthedegradationrateofIP-1within
thecellshappenswiththesamerateasforSte2p.Thesynthesis
d[Ste2v](cid:4)k(cid:4)Ste2Iztli](cid:3)k (cid:4)Ste2(cid:7) (Eq.4) rateofSte2p,k5,waschosenas7(cid:3)10(cid:3)5(cid:3)M(cid:3)s(cid:3)1tocounteractthe
dt E v v 4 v degradedSte2pafterabout90minasreportedearlier(20).
TherateofIP-1escapingfromtheendosome,k ,aswellas
d[Iztli ] E
dt c (cid:4)kE(cid:4)Ste2vIztliv(cid:7)(cid:6)k4(cid:4)Iztlic(cid:7) (Eq.5) thelethalIP-1concentration,kD,wereobtainedbyfittingthe
modeltothemeasuredtimecourseofdeadcellsbyanevolu-
Thecellvolumewaschosenas4.9(cid:3)10(cid:3)15liters.Duetothelarge tionary optimization algorithm. We required the parameter
values to stay in biologically reasonable intervals. k was
volumeoftheextracellularmedium,theextracellularconcen- E
requiredtostayintherangeof0to1s(cid:3)1andk within0to10
trationofIP-1wastreatedasaconstantreferringtotheamount D
usedintheexperiments.Thenumberofdeadcells,n ,inthe (cid:3)M. The best fit estimated kE and kD as 1 s(cid:3)1 and 0.13 (cid:3)M,
D respectively.Oneshouldnotethatthefitforcesthemodelto
populationwasexpressedbytheminimalconcentrationofIP-1
realizeanalmostimmediateescapeofIP-1fromthevacuole.
withinthecellstokillacell,k .n wasevaluatedateachtime-
D D See Table 3 for a list of parameters and values used in these
stepoftheordinarydifferentialequations,yieldinganalgebraic
models.
systemwheren wasgivenbytheformula,
D DirectModel—Thedirectmodelcomprisedonlyaninternal-
Iztli izationofIP-1,whichdependsonitsaffinityforSte2pasindi-
n (cid:4)n c (Eq.6)
D tot k catedinFig.3B.Theresultingordinarydifferentialequationis,
D
Here,n denotesthetotalnumberofcellsinthepopulationand d[Iztli ]
wasobttaoitnedbyfittingafourth-orderpolynomialintimetothe dt c (cid:4)kIn[Iztlie](cid:3)k4(cid:4)Iztlic] (Eq.7)
colony-forming unit measurements of untreated cells. Iztli
c
denotedtheconcentrationofIP-1insidethecellsthathadescaped The same assumptions as in the endocytosis model were
the (pre)vacuole. All simulations and fits were performed using made, and we fitted k and k as before. Here k was again
In D D
Copasi. boundbetween0and10(cid:3)M,andk waskeptbetween0and
In
Theparametersk throughk wereobtainedfromliterature. infinity.Thefittedparameterswerek (cid:9)2.5(cid:3)10(cid:3)4s(cid:3)1andk (cid:9)
1 5 In D
k wasobtainedas10(cid:3)3s(cid:3)1fromameanof3directmeasure- 8.9(cid:3)M.
2
14450 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER21•MAY23,2014
FunctionalRedundancyofCPPsandCAPs
TABLE3 ation (4–6°C) before the bilayer formation and peptide
Data for modeling MATa killing by IP-1 dependent on cellular incorporation.
internalization
Micropipette Fabrication—Glass capillaries with filament
Model Parameter Value
(1.00-mm external diameter and 0.58-mm internal diameter)
Endocytosis/direct Cellvolume 4.9(cid:10)10(cid:3)15l fromWorldPrecisionInstrumentswerepulledusingtheP2000
EEnnddooccyyttoossiiss//ddiirreecctt kk1 01.01(cid:3)835s(cid:3)(cid:3)1M(cid:3)1s(cid:3)1 instrument from Sutter Instruments (Novato, CA). Micropi-
EEnnddooccyyttoossiiss//ddiirreecctt kk23 25..38(cid:10)(cid:10)1100(cid:3)(cid:3)34ss(cid:3)(cid:3)11 pettes were then silanized and filled with the same working
Endocytosis/direct k4 7(cid:10)10(cid:3)5(cid:3)Ms(cid:3)1 solutionusedforthepreparationofsmallunilamellarvesicles
EEDnnirddeooccctyyttoossiiss kkk5DE 012.s.15(cid:3)3(cid:10)1(cid:3)1M0(cid:3)4s(cid:3)1 aanndceuosfedmwicirtohpiniptehtetefsolilnowthinegw6o0rk–i1n2g0smoliunt.ioTnhewaavse3r.a4g6e(cid:11)res2i.s8t0-
Direct kIDn 8.9(cid:3)M gigaohms.
Solvent-free Tip-dip Lipid Bilayer Formation—The con-
trolled-temperaturechamberwaselectricallyinsulatedwithin
ConfocalMicroscopyofLabeledIztliPeptide—Fortheinter-
a Faraday cage and suspended in elastic bands to reduce the
nalization images presented in this study, S.cerevisiae MATa
mechanicalvibration.Asampleofthesmallunilamellarvesicle
(BY4741),MAT(cid:2)(BY4742),orC.glabratacellsweregrownat
wasputinthechamber.Thesamplewassettledfor10minat
30°Cfor24hinsyntheticcomplete(SC)media(yeastnitrogen thedesiredtemperature,andthenabilayerwasformedatthe
basew/oaminoacids0.67%,potassiumphosphatemonobasic tip of the micropipette by consecutive immersion in small
0.1%, glucose 2%, amino acids 0.079%); afterward they were unilamellarvesiclesuspensionuntilacapacitiveresponsetothe
dilutedtoafinalabsorbanceof0.4at600nmandtreatedwith square potential applied was observed (30, 31). The standard
68.9(cid:3)MTAMRA-IP-1or89.9(cid:3)MHilytefluor488-IP-1for1hat seals obtained in this manner were of (cid:12)400 gigaohms with a
30°C.InthecaseofA.nidulanstheseweregrownaspreviously 2-kHz low pass filter and a current root mean square (I )
r.m.s.
reported (28); briefly, cells were grown in supplemented glu- (cid:12)0.12pA.Whenthebilayerwasformed,theproperamountof
cose minimal nitrate medium, and 2 (cid:10) 105 spores were sus- Iztli peptide stock solution was added to obtain the desired
pendedin2mlofwaterandincubated60minat37°Cat300 concentration,andaftertheequilibriumconditionsthe5-min
rpm with TAMRA-IP-1 (13.77 (cid:3)M). In every case 80 (cid:3)l from experimentsweredone.
thesesampleswerecentrifugedat300rpmfor5min,thesuper- Pore Formation of Iztli Peptide 1 on Artificial Membranes—
natantwasremoved,andthesampleswereobservedunderthe Smallunilamellarvesicles,micropipettes,andsolvent-freetip-
confocal microscope (Olympus FluoView FV1000) at 60(cid:10) diplipidbilayerswerepreparedasdescribedabove.Toobserve
amplification.HEK239andNL20humancelllinesweregrown thepossibleformationofporesinthesemembranes,theelec-
to80%confluenceinDMEMmediumsupplementedwith2mM tricalmeasurementsofthecurrentsignalwereamplifiedwith
Glutamax,50units/mlpenicillin,50(cid:3)g/mlstreptomycin,and anAxopatch200Bsettovoltage-clampmode,2-kHzlowpass
15%fetalbovineserumat30°Cand5%CO .Cellsweretreated filter, and maximal scaling output, digitally converted with a
2
withtrypsinandwashedtwicewithPBSandrecoveredbycen- Digidata1200A,acquiredwiththeaidofpClamp8.2softwareat
trifugation. The pellet was resuspended in Opti-MEM(cid:4) to afrequencyof10kHz(allfromMolecularDevicesAnalytical
reduceserumfluorescence,and2(cid:10)104cellswereculturedina Technologies),andstoredinapersonalcomputer.Thebilayer
ChamberSlide(cid:4)system(NUNC(cid:4)Lab-Tek(cid:4)II-C2TM)andincu- seal was measured by applying a square 5-mV step potential.
batedfor1htoallowthe adhesionofcellstothebottomofthe Thesamplingratewassetto100(cid:3)s.Oncethesealwasformed,
slide to form a monolayer. Afterward HEK239 cells where the I obtained was consistently 0.12 (cid:11) 0.03pA. The tem-
r.m.s.
treatedwith13(cid:3)M,andNL20cellswheretreatedwith24(cid:3)M perature of the experiments was 25 (cid:11) 2°C. The Iztli peptide
TAMRA-IP-1 and visualized using confocal microscopy wasaddedinsolutiontohaveaconcentrationof5,10,15,20,
(OlympusFLUOVIEWFV-1000)atanemissionwavelength and25(cid:3)M.Thepotentialsappliedinallofthereportedexper-
of570nmfordifferenttimes.ForsuchpurposeaCO cham- imentswere150and200mV.Caremustbetakentobesurethat
2
ber was used during the analysis. All images were recon- experimentsareperformedunderequilibriumconditions.We
structedusingthesoftwareFVviewerprovidedbytheman- determinedtheaverageconductanceasafunctionoftimeand
ufacturerofthemicroscope. discoveredthatthispropertyneeds15–20mintoattainequi-
SmallUnilamellarVesiclePreparation—POPCandcholes- librium.Allrecordswereaposterioribase-line-correctedusing
terolwerestoredat-20°Candusedwithoutfurtherpurifica- aFortranprogram.Theoccupanciesobtainedweredefinedas
tion.Stockchloroformsolutionsforeverylipidwereprepared theratioofthetimeexpendedinonestatetothetotaltimeof
onceaweekandstoredat(cid:3)20°C.CholesterolandPOPCsolu- experiment.Theaveragecurrentsreportedcorrespondtothe
tionsweremixedtoobtainthedesiredmolefractionofsterol averagevalueofallpointsregisteredinagiventime.Theaver-
(30%).Evaporationofthesolventwasdoneinarotativeevapo- age currents reported correspond to an average current in a
rator (B-177; Bu¨chi Labortechnik, Flawil, Switzerland). The 5-min experiment obtained from an all points histogram. All
suspensionwaspreparedbyaddingtheworkingsolution(5mM graphics were done with Grace software, a two-dimensional
KCl,1mMCaCl ,10mMHEPES,pH8.0)tothefilmdeposited graph-plottingtool,forUnix-likeoperatingsystems.
2
intheflaskinanN -enrichedatmosphereandthentreatedin GOTermEnrichmentEstimation—Todeterminethepartic-
2
anultrasonicbathtoproducedispersionandobtainunilamellar ipationofendocytosisonthekillingmediatedbyIP-1,weana-
vesicles(29).Thesuspensionwasstoredfor2hunderrefriger- lyzedtheenrichmentofGOtermsinthe291genesknownto
MAY23,2014•VOLUME289•NUMBER21 JOURNALOFBIOLOGICALCHEMISTRY 14451
FunctionalRedundancyofCPPsandCAPs
cytosis rate of the pheromone; all those rates have been
reportedandwereusedformodeling(see“ExperimentalPro-
cedures”). On the other hand, the rate of escape from endo-
somesandthenumberofIPmoleculesrequiredtokillcellsare
notknown;thesetwoquantitieswereestimatedbyfittingthe
modeltotheobservednumberofdeadcells(quantifiedfrom
thenumberofcolonyformingunits;see“ExperimentalProce-
dures”).Theadjustmentbetweentheobservedandcalculated
valuesforthenumberofdeadcellsispresentedinFig.3C.We
observedthatthemodelassumingreceptor-mediatedendocy-
tosis could not reproduce the observed time course of dead
cells, as it predicts a plateau phase due to depletion of free
receptorsatthemembranebyendocytosis;suchaplateauwas
notobservedintheexperimentaldata(p(cid:13)0.14,seeFig.3C).In
contrast,whenwedidnotconsiderendocytosisofthepeptide
FIGURE2.Celldeatheffectofsixnullmutantspreventinginternalization but still binding of IP-1 to Ste2p (see Fig. 3B), the model fit
ofSte2p.Sixstrainscarryinggenenullmutants(YKL002W(www.ncbi.nlm. reproducesthenumberofdeadcellsovertime(p(cid:14)4.2e(cid:3)3;see
nih.gov),YML001W,YCR009C,YMR231W,YHR135C,andYNL154C)reported
toreducetheinternalizationofSte2pwerecomparedintheirsensitivitytobe Fig.3C).Theseresultsareinagreementwithourobservation
killedbyIP-1withthewild-typestrain(BY4741).Toestimatethenumberof aboutamechanismofinternalizationdifferentfromSte2p-me-
cellsremaininginthepresenceofIP-1,thefigurepresentstheareaunderthe
growthcurves(summationofA valuesrecordedeveryhourispresentedas diatedendocytosisyetrequiringSte2pbindingforkillingactiv-
600
thepercentage)ofthesesixmutantsplusthewild-typestraininthepresence ity.Notethattheuseofall-Denantiomerstodiscardreceptor-
(ORFname(cid:5)IP-1;graybars)andintheabsence(ORFname;blackbars)ofIP-1.
mediated endocytosis does not apply to the study of IP-1
Thefigurespresenttheaveragedataobtainedfromfourindependentexper-
iments;thelinesontopofeachbarrepresenttheS.D.calculatedforeachdata consideringtherequirementofSte2pbindingforkillingMATa
set.TheopticaldensitywasfollowedintheSynergyMXplate-reader(BioTek) cells.
incubatedat30°C.
Tofurtheranalyzetheinvolvementofendocytosisinthekill-
ingbyIP-1(beyondreceptor-mediatedendocytosis),wecarried
affectendocytosis(seesupplementalTableS1).Forthisanalysis outageneticscreeningon291outof441nullmutantsanno-
weusedasacontrolsetthesetofgenesinyeast.Weusedthe tated on SGD (Saccharomyces Genome Database) to affect
serviceprovidedbyGO. endocytosisinyeast(seesupplementalTableS1).Fouroutof
these291ORFs(YHR161C,YKL081W,YKL119C,YOR141C)
RESULTS
fullyprotectedMATacellsfromthekillingbyIP-1(seesupple-
IztliPeptide1IsNotInternalizedthroughReceptor-mediated mentalTableS1).ToevaluateiftheeffectofthesefourORFs
Endocytosis—Basedontheproposedmechanismofactionfor could implicate the functioning of the core endocytosis
HKPs(4),itwouldbeexpectedthattherateofkillingyeastcells machinery for the killing activity of IP-1, we conducted two
byIPswouldbeproportionaltotherateofinternalization.Ifso, complementary analyses. First, we identified those mutants
reduction or elimination of Ste2p endocytosis may affect the with the largest annotated effect on endocytosis (YDR017C,
rateofkillingbyIP-1.Totestthiswecomparedtheestimated YDR173C,YJL204C,YOR089C,YKL126W;seesupplemental
overallnumberofgrowingcellsintheabsenceandpresenceof TableS1)andobservedthatnoneprotectedagainstthekilling
IP-1 for wild-type and six strains reported to fail endocytic byIP-1;theseresultssuggestthatthecoremachineryofendo-
internalizationofSte2pinMATacells(seeFig.2).Thefigure cytosis is not participating in the killing by IP-1. Second, we
showstheeffectoncellgrowthexpressedastheareaunderthe wereabletogroup30outofthe291ORFsbytheendocytosis
growthcurverecordedat600nm.Ourresultsshowthatthereis GO term and noticed that only one of these fully protected
nodifferenceinthecellgrowthinthepresenceofIP-1forthe againstIP-1killingactivity(YHR161C,involvedinclathrincage
wild-type and the mutants; consequently we conclude that assembly;seesupplementalTableS1),furthersupportingthat
Ste2p-mediatedendocytosisdoesnotplayaroleinthekillingof thecoremachineryofendocytosisdoesnotplayaroleonthe
MATacells.NotethateventhoughSte2p-mediatedendocyto- killingofMATabyIP-1.Thus,eventhoughfourgenesrelated
sis is discarded, Ste2p signaling is required for the killing of toendocytosisprotectagainstthekillingofIP-1,overallendo-
MATa by IP-1 (15); null mutants for STE2 or other critical cytosisdoesnotseemtoplayaroleonthekilling.
genesinvolvedinthepheromonesignalingcascadeinaMATa Iztli Peptides Are Cell-penetrating Peptides—To determine
genotype are resistant to killing by IP-1, and thus binding to thepresenceofIP-1insidecells,thepeptidewasmodifiedwith
Ste2pmayplayaroleinthekillingmechanismofactionofIP-1. twodifferentfluorophores:TAMRAandHilytefluor488.The
To test whether endocytosis might explain the observed Hilytefluor derivative was based on previous results on the
kineticsofcelldeathinducedbyIPs,webuiltakineticmodelfor (cid:2)-pheromone(33);notethattheuseofthesetwofluorophores
internalizationoftheIPsusingthereportedratesrelatedtothe was to discard that any possible observed internalization was
internalization of the pheromone (32) (see Fig. 3A). In this dependent on the nature of the fluorophores. Both peptides
modelthenumberofIPsinsidecellsdependsontheaffinityof maintainedantifungalactivity(seeFig.4A)andwereinternal-
thepheromonetoitsreceptor(Ste2poranyotherwithsimilar izedbyMATacells(seeFig.4B).AlthoughIP-1fullyinhibited
affinity),thedegradationrateofthepheromone,andtheendo- thegrowthofMATacellsat10(cid:3)M(15),theTAMRAderivateof
14452 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER21•MAY23,2014
FunctionalRedundancyofCPPsandCAPs
FIGURE3.ModelsforIztlipeptidesinternalizationonyeastcells.A,themodelassuminganendocyticimportofIztlipeptideviaSte2p((cid:10)denotes
degradation).B,themodelassumingpermeationofIztlipeptidewherethepeptidemaycrossthemembraneindependentofSte2p-mediatedendocytosis.C,
comparisonofthetwomodelsfittedtothemeasuredcolony-formingunittimecourseofdeadcells(n(cid:8)8,barsdenoteS.D.).Themodelassumingdirectimport
generallyperformedbetter,noticeableforearlytimepoints.Mio,millionsofcells.
FIGURE4.AnalysisoftwofluorogenicderivatesofIztlipeptide1.Theantifungalactivity(A)andtheabilitytogetinternalizedbyS.cerevisiaeMATacells(B)
areshownfortheTAMRA-IP-1(leftside,red)andHilytefluor488-IP-1(rightside,green).Thechangeincelldensityovertimeispresentedinthepresence(black
symbols)orintheabsenceofthesepeptides(whitesymbols):squares,TAMRA-IP-1;triangles,Hilytefluor488-IP-1.ScalebarsinBare5(cid:3)m.
IP-1 (TAMRA-IP-1) had almost half the activity (minimal IztliPeptidesMakeChannelsatHighMembranePotentials—
inhibitoryconcentration(cid:8)40(cid:3)M)oftheHilytefluor488deriv- WedeterminedtheabilityofIP-1toinduceiontransportacross
ative(minimalinhibitoryconcentration(cid:8)27(cid:3)M).Weusedthe lipidicbilayers.ForthisweconsidereditsactiononPOPCplus
TAMRA derivative for the rest of our studies. MATa cells 30%cholesterolartificialmembranes.Wedidnotobserveany
exposedtoTAMRA-IP-1for15minshowedstrongstainingof electricalconductanceinducedbythepeptideat50or100mV
thecellmembrane,andafter30minthepeptidewasalsolocal- (datanotshown).Yetat150and200mVtherewasobservable
izedinsidethecells(seeFig.5,A–D).Toevaluatewhetherthe activity.Thedependenceoftheionophoreticactivityonpep-
internalization was dependent on the Ste2p receptor, the tideconcentrationispresentedinFig.7,AandB.Atlowcon-
MAT(cid:2)cellswerealsoexposedtothislabeledpeptide(seeFig. centration (5 (cid:3)M) there are mainly small channels that can
6A).Finally,otherfungi(seeFig.6B)andhumancells(seeFig. barelybedistinguishedfromthenoiseofthebaseline(seeFig.
6C) were exposed to the TAMRA-IP-1, and in all cases the 7B). At medium concentration (10 (cid:3)M) there is clear activity
peptidewasinternalizedwithoutanyobservabletoxicity(data (seeFig.7A)mainlyofachannelof4.5picosiemensevenifthere
notshown).TheseresultsfurthersupportthenotionthatIP-1 isamultitudeoflargerchannelswithsmalloccurrence(seeFig.
internalizationisindependentofSte2p-mediatedendocytosis. 7B).Atlargeconcentrations(15(cid:3)M)theactivityisincreased,
MAY23,2014•VOLUME289•NUMBER21 JOURNALOFBIOLOGICALCHEMISTRY 14453
FunctionalRedundancyofCPPsandCAPs
Activity(cid:8)totalcurrent(cid:3)
(numberofsamplingpoints(cid:10)appliedvoltage) (Eq.8)
where total current is the sum of the current value at each
samplingpoint.Thisyieldsanaverageconductance,i.e.avir-
tualchannelthatwouldbeopenallthetime.Thecorrespond-
ingactivitiesarethen0.64,1.63,and3.31picosiemensfor5,10,
and15(cid:3)M,respectively.
These results raised the question that even for eukaryotic-
likemembranes,IP-1couldmakeporesprovidedalargeelec-
tricpotential.Plants,likebacteriaandmitochondria,maintain
a large transmembrane potential due to the action of plasma
membraneH(cid:5)pumpsfuelledbyATP(34).IP-1didnotalterthe
FIGURE5.Time-dependentinternalizationofIztlipeptide1inyeastcells.
restingmembranepotentialofsoybeanrootswithlowpoten-
TAMRA-IP1wasusedtofollowtheinternalizationofthepeptideinMATacells.
Thesiteswherethepeptideisfoundnearorwithincellsappearasreddots. tials ((cid:14)100 mV); however, we could not find cells with high
Scalebarsare5(cid:3)m.DIC,differentialinterferencecontrast. potentials((cid:13)100mV)(datanotshown).
DISCUSSION
HKPshavebeenshowntohavethepotentialtotreatcancer
andobesityamongothers(1).Thebasisfortheiruseasthera-
peuticsistheirselectivity;onlythecellsexpressingthereceptor
forthehunterpeptideshouldinternalizethekillerpeptide,and
consequentlyonlythosecellsshouldbeeliminated.Evenwhen
the results in animal models have shown the effectiveness of
thesepeptides,sometoxicityhasbeenreportedforothertissues
besidesthosetargetedinanimals(3,4).Thistoxicitycouldbe
explained considering the presence of the receptor for the
hunterpeptideinothertissuesthanthetargetone.Ourresults
suggestanalternativeexplanationfortheobservedtoxicityof
HKPs.Thatis,weshowedthatwhenakillerpeptidepresents
CPP activity (e.g. IP-1, see Fig. 1) it may loose the specificity
providedbythehunterpeptide(seeFigs.4,A–C,5,A–D,and6,
A–C).Accordingly,weshowedthatIP-1isnotinternalizedvia
receptor-mediatedendocytosis(seeFigs.2and3)orendocyto-
sisatall(seesupplementalTableS1).However,IP-1isstillable
to kill MATa cells as long as the pheromone receptor is acti-
vated by the peptide (15). Thus, the observed specificity dis-
playedbyIP-1(orotherHKPs)maybeexplainedbytherequire-
ment to activate the hunter receptor (e.g. Ste2p) and/or by
increasingtheavidityforIP-1inMATacells.Inthislastcase
oursimulationresultsshowthattheattractionofIP-1bySte2p
accountsforthekineticsofcelldeathinducedbyIP-1inMATa
cells. Additional experiments are required to validate these
observations, which may provide the basis to explain the
FIGURE6.Iztlipeptide1getsinternalizedbydifferentcelltypes.S.cerevi-
observedkillingspecificityofHKPs.
siaeMAT(cid:2)cells(A),C.glabrata(leftinpanelB),A.nidulans(rightinpanelB),
HEK239(leftinpanelC),andNL20(rightinpanelC)humancellswereexposed The design of HKPs combines CAP and CPP in separate
toTAMRA-IP-1(red)totestforitsinternalization. domains,yetsuchdesignmaysometimesberedundantconsid-
eringthatsomeCPPs(35)bythemselvesmayhaveCAPactiv-
andamultitudeofchannelsintherangefrom3to20picosi- ity.Insuchacase,theseCPPswheninternalizedcarryalonga
emenswasobserved;largechannelswerealsoobservedbutless killer peptide (CAPs) into cells. If so, those CPPs may affect
frequentlythanat10(cid:3)M.At20(cid:3)Mfrequentsealrupturesand mitochondria function. In agreement with this idea, a recent
closingsappeared,andat25(cid:3)Mtherewasanimmediaterup- metabolomic analysis comparing the toxic potential of four
ture of the membrane patch (data not shown). This latter CPPs (transportan, penetratin, HIV Tat derived peptide, and
behavior could be explained by the peptide entering the seal nona-arginine) showed the toxicity of transportan (36), sug-
regiontodisturbit,orelseitaffectedthemembraneintegrity. gestingthepotentialCAPactivityoftransportan.Thetoxicity
We can quantify the activity at each concentration with the was estimated by the effect on cellular redox potential and
followingexpression, energydepletion,bothrelatedtomitochondriafunction.Ina
14454 JOURNALOFBIOLOGICALCHEMISTRY VOLUME289•NUMBER21•MAY23,2014
FunctionalRedundancyofCPPsandCAPs
FIGURE7.Iztlipeptide1makespores.A,exampleofa5-minrecordofelectricalactivityforIP-1(10(cid:3)M)inPOPCmembranewith30%molcholesterolina
transmembraneelectricpotentialof200mV.Theinsetcorrespondstoa10-ssectionshowinganexampleofthevariabilityoftheopeningtimesofthepores
formed.B,averageallpointhistogramsforfiveexperimentsattheconcentrationsof5(cid:3)M(black),10(cid:3)M(red),and15(cid:3)M(blue)onthesamebilayerandat200
mVofappliedpotential;thedurationofeachexperimentwas5min.Theallpointshistogramisconstructedbyfillingthehistogrambucketswiththenumber
ofpoints,i.e.thenumberoftimesthataparticularcurrentwasregisteredduringtheexperimentwitha100-(cid:3)ssampling.Thishistogramisnormalizedby
thetimedurationoftheexperiment,andtherefore,the(cid:8)valuecorrespondstothenumberofcountsineachbucketdividedbythetotalnumberofcounts.The
recordswerebase-line-correctedandfilteredforpeakswithdurationof(cid:14)30(cid:3)s(tosuppresselectricnoiseofthebath)withahomemadesuiteofprograms.The
insetcorrespondstolargeconductancechannelsthatpresentsmalloccurrence,whichdependsonpeptideconcentration.
similar fashion, some CAPs may as well display CPP activity. Considering the complexity of biological systems (46), our
Indeed,magainin2hasbeenshowntotranslocateacrossphos- observations about the overlapping roles of CPPs and CAPs
pholipidbilayers(37).Thus,IPdesignmayavoidredundancyof represent a special case of functional redundancy. Functional
activities,renderingshorterHKPs. redundancy in genes occurs when one gene has the ability to
BeyondthedesignofHKPs,itisinterestingtostudythebasis performthefunctionofanothergene.Acommonexampleof
ofthisredundantfunctionofCPPsandCAPs.Wehypothesized suchgeneticredundancyisfoundingeneduplications(47).A
that the membrane potential may play a role on switching consequenceforasystemwiththeseredundantcapabilitiesis
betweentheseactivities;particularly,itisnoticeablethatmito- the emergence of robustness (48). In the case of antibacterial
chondria and bacteria maintain larger membrane potentials peptides,therobustnessthisredundancyconferstoCAPscan
thanmammaliancells,andsuchadifferencemayexplainthe be appreciated; by acquiring the ability to get internalized by
toxicityofCAPstobacteriaandnottomammaliancells.Inthe
eukaryoticcells,CAPscanbecomemoreeffectiveantibioticsby
case of IPs, we have previously observed that these alter
killingbacteriacellsresidingbothattheextracellularandintra-
themembranestabilityofmitochondriaandkillbacteriabutdo
cellularspacesofeukaryoticcells.Additionally,undercertain
notaffecttheviabilityofmammaliancells(15).Hereweshowed
circumstances some bacteria may reduce their membrane
that IP-1 is capable of making pores in artificial membranes
potential (e.g. sporulation), thus preventing CAPs to disrupt
onlywhenalargeelectricpotential((cid:13)100mV)ispresent(see
theirmembrane(49).Insuchcases,thepossibilitythatCAPs
Fig. 7, A and B). These results are in agreement with recent
maypermeatecellularmembraneswithlowelectricpotential
reportsshowingthatsomeCPPsmayswitchfrompenetrating
andactuponanintracellulartargetmayprovideanalternative
mammaliancellswithnoapparenttoxicitytopeptidescapable
mechanismtomaintaintheirantibioticactivity.
of killing/swelling bacterial cells (12, 13). Understanding the
ThemultifunctionalityofCAPshasbeenrecognizedaswell
mechanism involved in this functional switch may assist in
inthecontextofneurodegenerativediseases;forinstance,the
designingCPPs.
(cid:7)-amyloidpeptidethatiscommonlyassociatedwithAlzheimer
In that sense it has been observed that natural antibiotics,
diseaseexertsantimicrobialactivityagainstcommoninfectious
peptides,andpolyenescanformdifferentmembranestructures
bacteria(50);anotherneuropeptide,substanceP,displaysfea-
thatallowfortransmembranetransport,detergent-likecarpet
tures of CAP and has antibacterial activity (11). Yet neither
disruption, pores, and toroidal pores independently of other
everypeptideinvolvedinneurodegenerationcanbeexpected
intracellularactions(seeforexampleRefs.38and39).Itisalso
tohaveanantibacterialactivity(51)noreveryCPPmayhavea
clearthattheiractivitiesarestronglydependentontheenviron-
CAPactivity.IdentifyingthedifferentfunctionalitiesofCAPs
mental conditions (40). For example, the dependence on the
transmembrane electric potential to form pores has been may have an impact on hunter-killer peptide design as dis-
observedforpolyenes(41)andforAlamethicin(42).Hence,itis cussedbeforeorintheetiologyofdifferentdiseaseswherepep-
not surprising that IP-1 presents this behavior. IP-1 forms tides (e.g. amyloid, penetrating) may be present, such as in
transmembrane channels of different conductivity, similar to Alzheimerdisease.
AmphotericinB(43)andAlamethicin(44),andalsodetergent- Finally, the present work provides an experimental system
likeactivity,similartoNystatin(41)andAlamethicin(42).In (IPtargetedagainstS.cerevisiae)andmathematicalmodel(see
eithercase,theenvironmentalconditionscanaffecthelixfor- Fig. 3 and “Experimental Procedures”) that may be useful to
mationforpeptides(39)and/ortheaggregationofmonomers furthercharacterizethemechanismofinternalizationofCPPs
forpolyenes(45).SuchasituationmayprevailforIP-1andmay andtoexplorealternativestocontroltheselectivityofCPPsand
explainthedualroleobservedinthisstudy. HKPs.
MAY23,2014•VOLUME289•NUMBER21 JOURNALOFBIOLOGICALCHEMISTRY 14455
FunctionalRedundancyofCPPsandCAPs
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