Rev. bras. farmacogn. vol.26 número3

(1)

w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Effects

of

(

₋

)-6,6

′

_{-dinitrohinokinin}

_on

_adult

_worms

_of

_Schistosoma

mansoni:

a

proteomic

analyses

Lizandra

G. Magalhães

a,∗

_,

_Thais

_C.

_Lima

a

_,

_Renato

_G.

_de

_Paula

b

_,

_Enyara

_R.

_Morais

c

_,

_Daniela

_P.

_Aguiar

a

_,

Luiz

G. Gardinassi

b

_,

_Gustavo

_R.

_Garcia

b

_,

_Rosangela

_S.

_Laurentiz

d

_,

_Vanderlei

_Rodrigues

b

_,

Jairo

K. Bastos

e

_,

_Ademar

_A.S.

_Filho

f

_,

_Ana

_P.

_Yatsuda

e

_,

_Wilson

_R.

_Cunha

a

_,

_Márcio

_L.A.

_Silva

a

a_Grupo_de_Pesquisa_em_Ciências_Exatas_e_{Tecnológicas,}_Universidade_de_Franca,_Franca,_SP,_Brazil b_Faculdade_de_Medicina_de_Ribeirão_Preto,_Universidade_de_São_Paulo,_Ribeirão_Preto,_SP,_Brazil c_Instituto_de_Genética_e_Bioquímica,_Universidade_Federal_de_Uberlândia,_Patos_de_Minas,_MG,_Brazil d_Faculdade_de_Engenharia,_Universidade_do_Estado_de_São_Paulo,_Ilha_Solteira,_SP,_Brazil

e_Faculdade_de_Ciências_{Farmacêuticas,}_Universidade_de_São_Paulo,_Ribeirão_Preto,_SP,_Brazil f_Faculdade_de_Ciências_{Farmacêuticas,}_Universidade_de_Juiz_de_Fora,_Juiz_de_Fora,_MG,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18September2015 Accepted3February2016 Availableonline3March2016

Keywords:

(−)-6;6′_{-dinitrohinokinin}

Lignan

Massspectrometry Proteome

Schistosomamansoni

Two-dimensionalgelelectrophoresis

a

b

s

t

r

a

c

t

Schistosomiasis,achronicdiseasethataffectsmillionpeopleworldwide,iscaused bytrematode flukesofthegenusSchistosoma.Thelackofananti-schistosomiasisvaccineandmassive monother-apywithpraziquantel reinforces theneed forsearch and developmentof new therapeutic drugs. Recently,wedemonstratedthattheessentialoilofPipercubebaL.,Piperaceae,andtheirderivative diben-zylbutyrolactolic(−)-6,6′_{-dinitrohinokinin,}_presents_in_vitro_and_in_vivo_activities_against_Schistosoma

mansoni.Here,weidentifiedchangesintheproteinexpressionafterexposureto dibenzylbutyrolac-tolic(−)-6,6′_{-dinitrohinokinin.}_We_applied_{two-dimensional}_gel_{electrophoresis}_(2-DE)_to_S._mansoni

solubleproteinextractsandobservedatleast38spotstobeaffectedbydibenzylbutyrolactolic(− )-6,6′_{-dinitrohinokinin.}_We_further_identified₂₅_{differentially}_expressed_proteins_by_mass_{spectrometry.}

Enrichmentforbiologicalprocessesandpredictiveanalysesofprotein-proteininteractionssuggestthat dibenzylbutyrolactolic(−)-6,6′_{-dinitrohinokinin}_targets_proteins_involved_mainly_in_metabolic_processes,

especiallycarbohydratemetabolism.Insummary,thisstudyprovidesaninterestingapproachto under-standtheanti-parasiticactivityofsemi-synthetic(−)-6,6′_{-dinitrohinokinin}_a_derivative_compound_from

lignanandforthedevelopmentofnewtherapystrategies.

Introduction

Schistosomiasisiscausedbyaparasiticinfectionwith trema-todeflukesof thegenusSchistosomaand affectsmorethan200 millionpeopleworldwide (Rollinsonet al.,2013).Currently, an antischistosomalvaccine is not available, whereas Praziquantel (PZQ)isthedrugofchoiceforthetreatmentofpatients(Caffrey, 2015).AlthoughPZQhavebeeneffectiveinthetreatmentofthe disease,thepossibilityofdrugresistancecausedbyrepeatedand massivemonotherapyreinforcestheneedtodevelopnewsafeand effectivedrugsforthepreventionandtreatmentagainst schistoso-miasis(Ciolietal.,2014;Colleyetal.,2014).

∗ Correspondingauthor.

E-mail:lizandra.magalhaes@unifran.edu.br(L.G.Magalhães).

Pipercubeba L.,Piperaceae, knownasthe tailed pepper, has

been used in traditional medicine for the treatment of dysen-tery,syphilis,abdominalpain,diarrhea,enteritisandasthma(Usia

etal.,2005;Gutierrezetal.,2013).Wedemonstratedpreviously

thatextractsandisolatedcompoundsfromPiperspeciespresent anti-parasitic activity (Saraiva et al., 2007, 2010; Esperandim et al., 2013). Furthermore,we showed that the essential oilof P. cubeba and their derivative dibenzylbutyrolactolic (−)-6,6′

-dinitrohinokinin(1,DNH),presentsinvitroandinvivoactivities againstSchistosomamansoni(Magalhãesetal.,2012;Pereiraetal.,

2015).

High-throughput strategies including genomics, transcrip-tomics, proteomics and metabolomics have become important approachestounderstandschistosomebiologyandpathogenesis, but also constituteimportant tools tothe exploration of novel drugs, vaccines or diagnosis (De Marco and Verjovski-Almeida,

2009; Chuan etal., 2010).In viewof thesefacts,we identified

http://dx.doi.org/10.1016/j.bjp.2016.02.001

(2)

changesintheproteinexpressioninducedbyexposuretoDNH. Weappliedastrategybasedontwo-dimensionalgel electrophore-sis(2-DE)followedbymassspectrometry,whichprovidedinsights intokeyproteins,molecularfunctionsandbiologicalprocesses tar-getedbyDNH.

Materialsandmethods

ObtainmentofDNH

Prior to the DNH synthesis, (−)-cubebin (2) was isolated fromPipercubebaL.,Piperaceae,seedsand(−)-hinokinin(3)was obtainedbyoxidationof(−)-cubebin(DaSilvaetal.,2005).(− )-Hinokinin was then submitted to a nitration reaction in the presenceoffumingHNO3.Thereactionwasprocessedunder

mag-neticstirringat0◦_C_for₂_h._The_obtained_DNH₍₁₎_was_crystallized_as

yellowishpowderfromMeOH.ThepurityofthesynthesizedDNH wasestimatedtobehigherthan95%bybothHPLCand1_H_and13_C

NMRanalyses,aswellasbyitsmeltingpoint(Setchelletal.,1981;

Costa,2000).

Yield 97.6%; [␣]D25 −29.07◦ (c 0.008, CHCl3, ee>99%); mp

191–193◦_C;1_H_NMR₍₃₀₀_MHz,_CDCl

3)ı7.5(s,1H);ı7.48(s,1H);

ı6.8(s,1H);ı6.6(s,1H);ı6.1(m,2H);ı4.35(dd,1H,J=7.1Hz andJ=9.1Hz);ı4.0(dd,1H,J=7.3HzandJ=9.1Hz);ı3.26(d,2H, J=7.1Hz);ı3.2(dd,1H,J=6.3HzandJ=13.6Hz); ı3.0(dd,1H, J=7.8HzandJ=13.6Hz);ı2,8(m,2H);13_C_NMR₍₇₅_MHz,_CDCl

3):

178.0,152.3,152.2,147.6,143.1,142.9,130.9,130.7,112.5,111.2, 106.6,106.2,103.6,103.5,71.4,45.7,41.7,37.2,34.21.

Parasitestrain

TheLE(LuizEvangelista)strainofS.mansoniwasmaintained bypassagethroughBiomphalariaglabratasnailsandBALB/cmice. After49±2days,S.mansoniadultwormswererecoveredunder aseptic conditionsfrom micepreviouslyinfected with200 cer-cariaebyperfusionoftheirliversandmesentericveins(Smithers

andTerry,1965).ExperimentswereapprovedbyAnimalResearch

EthicsCommitteefromRibeirãoPretoMedicalSchool–University ofSãoPaulo,undertheprotocol021/2009.

S.mansonisolubleproteinextractionandtwo-dimensional polyacrylamidegelelectrophoresis(2-DE)

Parasitesexposedto25␮MDNHorRPMI1640medium(0.1% DMSO)weretransferredto0.5mlofhomogenizationbuffer[25mM Tris–HCl,pH7.5,1mMphenylmethanesulfonylfluorideand1mM dithiothreitol] and worms were lysed by sonication (5×10s, 21kHzat7␮mamplitude)at10sinterval,onice.Cellulardebris wasremovedfromthelysatebycentrifugationfor1hat15,000×g, 4◦_C._The_supernatant_containing_soluble_proteins_was_collected_and

quantifiedusingtheCoomassieProteinAssayKit(Pierce Biotech-nology).

Solubleproteins(200␮g)weredissolvedinanisoelectric focus-ingbuffer[7Murea,2Mthiourea,4%(w/v)CHAPS,0.5%(v/v)carrier ampholytes(IPGbuffer3–10l,GEHealthcare),40mMdithiotreitol (DTT)and 0.002% (v/v)bromophenolblue] andrehydrated into

2

3

1

linear13-cmImmobilinedrystripspH3–10(GEHealthcare)for 16hat20◦_C._Isoelectric_focusing_was_carried_out_on_Ettan_IPGphor

(GEHealthcare),byapplyingacurrentof50mA/strip,following the steps:1hat 500V (step), 1hat 1000V (gradient), 2.5hat 8000V(gradient)and0.5hat8000V(step),totaling16kVh.The focusedstripswereequilibratedfor15minin15mlequilibration buffer[50mMTris–HClpH8.8,6Murea,30% (v/v)glycerol,2% (w/v)SDS], supplementedwith1%(w/v) DTTandsubsequently for15minin15mlequilibration bufferwith2.5%(w/v) iodoac-etamide.Proteinswereseparatedin12.5%(w/v)sodiumdodecyl sulfatepolyacrylamidegelelectrophoresis(SDS-PAGE)usingthe Ruby600apparatus(GEHealthcare)andSDSelectrophoresisbuffer (25mMTris,192mMglycineand0.1%(w/v)SDS).Eachgelwas performedintriplicate.

Proteinvisualizationandimageanalysis

2-DEgelswerefixedfor1hwith3%(v/v)phosphoricacidand 50%(v/v)ethanol,followedbystainingwithCoomassieBrilliant BlueG250(GEHealthcare)during24h.2-DEproteinprofileswere obtainedaftergeldistaininginMilliQwater.Gelsweredigitized at300dpiand16bitsdepthresolutionwithanImageScanner(GE Healthcare)operatedbytheLabScansoftware(GEHealthcare).The softwareImageMaster2DPlatinum6.0(GEHealthcare)wasusedto mergetheimageofeachexperimentalreplicateontoamaster2-DE gelimage,usingthesixwell-definedlandmarkasreferencetospot detection.Differentialproteinexpressionwasanalyzedby compar-isonofspotintensityingelscontainingproteinsfromS.mansoni exposedtoDNHandunexposedcontrols.Spotsthatpresentedat least1.5-foldchangeinintensitygivenbyStudent’sttest(p<0.05) weremanuallyexcisedandanalyzedbymassspectrometry.

Ingeldigestionandidentificationofproteinsbyliquid

chromatography-tandemmassspectrometry(LC-MS/MS)

Spots obtained from 2-DE gels were digested with grade-modified trypsin (Promega), as described by Shevchenko et al.

(1996).Afterdigestion,peptideswereextractedwith50%(v/v)

ace-tonitrileplus5%(v/v)formicacidsolution.Theresultingpeptides weredriedagainandre-suspendedin12␮lof0.1%(v/v)formicacid. Analiquotof4.5␮lofthedigestedproteinwasthenseparatedona C18RP-UPLCcolumn(100mm×100␮mI.D.×1.7␮mparticlesize, nanoAcquityUPLC,Waters)usingalineargradientof15–90%(v/v) acetonitrilein0.1%(v/v)formicacidfor10minat0.6␮l/min.The HPLCsystemwascoupledtoaQ-TofUltimaAPImass spectrom-eter(MicroMass/Waters)fittedwithanano-electrospraysource. Theparameterswere35Vconevoltage,nano-electrospray volt-agewas3.5kV,and100◦_C_source_temperature._The_equipment_was

(3)

250

A

₃ _pH ₁₀

B

₃ _pH ₁₀

Molecular mass (kDa)

150 100

75

50

37

25

20

15

Fig.1.Effectof(−)-6,6′_{-dinitrohinokinin}_on_Schistosoma_mansoni_adult_protein_profile._Comparative_{bidimensional}_gels_analyses_of_S._mansoni_soluble_proteins_before_(A)

andafterexposureto25␮MDNH(B).Numbersrepresent38spotswhoseintensitywassignificantlyaffected(p<0.05)byDNH.

The data obtained were processed using Mascot Distiller v.2.3.2.0 (Matrix Science Ltd.) and proteins were identified by correlationoftandemmassspectraagainstpublicavailableS. man-soni(27,729 sequencesand12,093,356 residues) depositedinto NCBIdatabase.SearchparametersforMASCOTwereasfollows:S.

mansonitaxonomicrestriction;trypsincleavage,onemissed

cleav-age; carbamidomethyl–cys as fixedmodification (monoisotopic mass57.0215Da),methionineoxidationasvariablemodification (monoisotopicmass15.9949)carbamidomethyl(C);variable mod-ification,oxidation(M);peptidetolerance,0.1Da;MS/MSfragment iontolerance.Resultswereregardedassignificantwithanallowed likelihood for a random hit of p<0.05, according to the MAS-COT.

Bioinformaticanalysis

Identified proteins were analyzed for Gene Ontology (GO) enrichmentandproteinclassusingthebioinformaticstools PAN-THER (Mi et al., 2013) and UniProt (Consortium, 2014). The networksofinteractionfortheidentifiedproteinswereperformed using STRING database v 9.05, employing prediction meth-odsasneighborhood, genefusion,co-occurrence,co-expression, experiments, database and textmining sources (Jensen et al., 2009). We performed analysis in both Cluster of Orthologous Groups/EukaryoticOrthologousGroups(COG/KOG) (withahigh confidenceof0.700) andPROTEIN-modes(witha medium con-fidence of 0.400). The identity of the putative proteins were consideredsignificantiftheypresentedatleast75%ofhomology withsequencesfromSchistosomajaponicum.

Results

Differentialproteinanalysesrevealedapproximately38spots (i.e., proteins) that were significantly affected by 25␮M DNH (Fig.1Aand B).Most oftheseparated proteinshavemolecular weightsgreaterthan25kDaandaredistributedamongthe isoelec-tricpointsfrom4to10.Basicproteins(i.e.,proteinsthatpresent anisoelectricpointfrom8to10)weredirectlyaffectedby expo-suretoDNH,whichresultedintheirabsenceorlowerexpression. Although,weobservedthatspotsnumberedas4,8,9,19,35,36, 37and38arepresentinproteinprofileofS.mansoniunexposedto DNH,thesespotswerenotidentifiedafterdrugexposure(Fig.1A

andB).AfterexposuretoDNH,thespots2,5,10,11,17,18and21 werepresentorhadatleasta2-foldincreaseinexpression,while nineteenproteinshadatleasta2-foldreductionornoexpression (Fig.1AandB).

Togain furtherknowledge onchanges ofproteinexpression

byS.mansoniafterexposuretoDNH, weperformedLC-MS/MS.

Foreachspectrum,asearchforproteinsequencesfromS.mansoni wasperformedwithMASCOTatNCBInrproteindatabase(27,729 sequencesand12,093,356residues).Wefurtheridentified25 dif-ferentially expressed proteins by mass spectrometry, however, twentyfouridentifiedproteinshadMASCOTscoreshigherthan28 (p<0.05)andonlyoneproteinspot(spotn◦₁₉₎_had_MASCOT_score

below28(p>0.05)(Table1).Ofinterest,thetegumentalantigenSm 20.8andphosphoglyceratemutase(spots10and11,respectively) wereexpressedfollowingexposuretoDNH,whereasthe peroxire-doxin,gelsolinandamajoreggantigen(spotsnumberedas5,17 and18,respectively)weretheproteinswiththehighest expres-sionafterexposuretoDNH(Fig.1AandB,andTable1).Notably, exposuretoDNHdecreasedtheexpressionofthepeptidyl-prolyl cis-transisomerase,glutathione-S-transferase(GST), 14-3-3 pro-tein,14-3-3epsilon,calreticulinandparamyosin(spotsnumbered as1,12,13,14,28and32,respectively)(Fig.1AandB,andTable1). MASCOTdidnotretrievesignificantmatchestospots3,4,16,20, 21,24,25,26,27,30,33,34and36,thereforewewereunableto identifytheirproteincontent(Fig.1).

(4)

Table1

SchistosomamansoniadultproteinsidentifiedbyLC-MS/MS.

Spotn◦ _Protein_name _Accession_numbera _MW_(kDa)/pI _MW_(kDa)

experimental

Coverage%c _Mascot_Scored _Expression_after exposureDNHe

1 Peptidyl-prolylcis-trans

isomerase

Smp040130 18/8.26 12 46 390 Down

2 Nucleosidediphosphatekinase Smp092750 17/7.74 10 79 453 Up

5 Peroxiredoxin,Prx1 Smp059480 21/6.10 18 36 231 Up

6 Calponin-related Smp086330 21/8.68 17 55 453 Down

7 Putativeadenylatekinase Smp071390 22/8.52 19 45 331 Down

8 Triosephosphateisomerase Smp 003990 28/4.63 22 54 481 Down

9 Putativecarbonylreductase Smp033540 31/7.64 24 2 37 Down

10 Putativetegumentalantigen

Sm20.8

Smp086530 21/6.90 23 56 196 Up

11 Phosphoglyceratemutase Smp096760 28/7.71 22 50 234 Up

12 GlutathioneS-transferase Smp054160 24/6.56 22 16 132 Down

13 14–3-3protein,putative Smp 009760 28/4.74 23 46 553 Down

14 14–3-3epsilon Smp034840 28/4.85 25 52 354 Down

15 Camp-dependentprotein

kinasetypeII-alpharegulatory subunit

Smp079010 43/5.12 32 33 286 Down

17 Putativegelsolin Smp008660 42/5.57 34 46 433 Up

18 Putativemajoreggantigen Smp049300 39/6.23 33 18 287 Up

19 PutativefourandAhalflim domain

Smp048560 32/7.61 26 14 20 Down

22 Malatedehydrogenase Smp047370 37/8.70 34 31 746 Down

23 Glyceraldehyde-3-phosphate

dehydrogenase

Smp056970 37/8.16 36 32 305 Down

28 Calreticulinautoantigen

homolog,putative

Smp030370 45/4.69 49 39 231 Down

29 Putativealphatubulin Smp090120 51/4.97 49 53 1416 Down

31 Putativeheatshockprotein Smp072330 81/4.92 75 25 623 Down

32 Putativeparamyosin Smp 021920 90/5.3 98 14 142 Down

35 Taurocyaminekinase Smp194770 77/7.87 72 11 91 Down

37 Aconitatehydratase Smp063090 66/8.45 74 18 88 Down

38 Putativeuncharacterized

protein

Smp042170 40/7.63 38 20 167 Down

a_S._mansoni_Genedb_database_identifier.

b_Predicted_molecular_weight_(MW)_and_isoelectric_point_(pI)_recovered_in_the_NCBI_protein_database_and_Protparam_tool. c _Coverage_%:_The_Sequence_Coverage_is_the_percentage_of_the_database_protein_sequence_covered_by_matching_peptides.

d _Score:_Relative_for_the_probability_that_the_observed_match_between_the_experimental_data_and_the_database_sequence_is_a_random_event. e_Spots_that_changed_at_least_1.5-fold_in_intensity_(down_or_up)_after_exposure_to_DNH_compared_to_unexposed_control.

Next,tounderstandwhethertheidentified proteinsinteract witheachotherand thereforecontributetocommonbiological processes, we performed predictive protein-protein interaction analysesusingString.Analysisofdifferentiallyexpressedproteins

of S.mansoni assigned toclustersof orthologous groups (COG)

oreukaryoticorthologousgroups(KOG)showsthatallproteins, exceptforglutathione-S-transferase(GST)(KOG1695),might inter-actwithoneormorenodesinthepredictednetwork(Fig.3A).As expected,thenetworkdepictsanupperclusterofproteinsassigned withCOGs,whileinasecondcluster,themajorityofnodeswere assignedwithKOG.Furtheranalysisresultedina morespecific networkofpotentialtargetsinducedorrepressedbyexposureto DNH,whichpossesssignificantidentitywithproteinsofS. japon-icum(Fig.3B).Ofnote,fiveproteinsenrichedinthisnetworkwere reduced after exposureto DNH, while only nucleoside diphos-phatekinaseandphosphoglyceratemutasepresentedasignificant increaseinintensitywhencomparedtounexposedcontrols(Fig.1

andTable1).

Discussion

Here,weshowthatDNHmodulatestheprofileofaproteinset

fromS.mansoniadultworms.Severalofthoseproteins,affected

byexposuretoDNH,participateofimportantmetabolicprocesses andhavealreadybeenexploitedaspotentialvaccinecandidates

(Pearce,2003;Kohamaetal.,2010;Fonsecaetal.,2012;Beaumier

etal.,2013)orforimmunodiagnosis(ElAswadetal.,2011;Qian

etal.,2012;Yuetal.,2014).Forexample,reducedexpressionof

paramyosin,amajorstructuralcomponentthathasbeenassociated aspotentmucosalimmunogenagainstSchistosomassp.(Lanaretal.,

1986;Jizetal.,2009;Kohamaetal.,2010),mightreflectsimilar

activitywiththeessentialoilofP.cubebaandDNHbyaffecting themotilityofadultworms(Magalhãesetal.,2012;Pereiraetal.,

2015).

Analysisofprotein–proteininteractionsoperatedonCOG/KOG mode resulted in two major clusters of interactions, whereas proteins assigned to COG were mainly enriched in metabolic processes,suchasadenosine triphosphate(ATP)generationand carbohydrate metabolism(Fig.3A andTable 2).Forexample,S.

mansoninucleosidediphosphatekinase(COG0105)andadenylate

kinase(COG0563)catalyzereactionsthatgenerateATP(Senftand

Crabtree,1983).Ourresultsdemonstratedthatadenylatekinase

(5)

Table2

EnrichmentanalysisbyGeneOntologyandPANTHERterms.

IDsa _Protein_(COG/KOG_IDs)b _GO_molecular_function _GO_biological_process _PANTHER_protein_class

Smp040130 Peptidyl-prolylcis-trans isomerase(COG0652)

Isomeraseactivity Proteinfolding,Intracellular

proteintransport,Nuclear transport

Isomerase

Smp092750 Nucleosidediphosphatekinase

(COG0105)

ATPbinding CTPbiosyntheticprocess,UTP

biosyntheticprocess

–

Smp059480 Peroxiredoxin,Prx1(COG0450) Oxidoreductaseactivity, Peroxidaseactivity

Metabolicprocess Peroxidase

Smp086330 Calponin-related(COG5199) – – –

Smp 071390 Putativeadenylatekinase

(COG0563)

Nucleotidekinaseactivity Purinenucleobasemetabolic process,Pyrimidinenucleobase metabolicprocess

Nucleotidekinase

Smp003990 Triosephosphateisomerase

(COG0149)

Isomeraseactivity Glycolysis Isomerase

Smp033540 Putativecarbonylreductase

(COG1028)

Oxidoreductaseactivity Steroidmetabolicprocess Dehydrogenase

reductase

Smp086530 Putativetegumentalprotein

Sm20.8

Catalyticactivity,Structural constituentofcytoskeleton, Proteinbinding,Enzyme inhibitoractivity

Nitricoxidebiosyntheticprocess, Nucleobase-containingcompound metabolicprocess,Cellcycle,RNA localization,Intracellularprotein transport,Vesicle-mediated transport,Regulationofcatalytic activity,Cellularcomponent organization

Enzymemodulator, Microtubulefamily, Cytoskeletalprotein

Smp096760 Phosphoglyceratemutase

(COG0588)

Bisphosphoglycerate 2-phosphataseactivity, Bisphosphoglyceratemutase activity,Phosphoglycerate mutaseactivity

Glycolyticprocess –

Smp054160 Glutathione-S-transferase (KOG1695)

Glutathionetransferase activity

Transferaseactivity –

Smp009760 14-3-3protein,putative

(COG5040)

Proteinbinding,ATPbinding, Proteindomainspecific binding

Cellcycle Chaperone

Smp034840 14-3-3epsilon(KOG0841) Proteinbinding,ATPbinding,

Proteindomainspecific binding

Cellcycle Chaperone

Smp 079010 Camp-dependentprotein

kinasetypeII-alpharegulatory subunit,putative(KOG0616)

Kinaseactivity,Protein binding,Kinaseregulator activity

Proteinphosphorylation Cellularprocess

Regulationofcatalyticactivity

Kinasemodulator

Smp008660 Putativegelsolin(KOG0443) Structuralconstituentof

cytoskeleton,Cytoskeletal proteinbinding

Cellularprocess,Cellular componentorganizationor biogenesis

Cytoskeletalprotein

Smp049300 Putativemajoreggantigen

(KOG3591)

– – –

Smp048560 PutativefourandAhalflim domains(KOG1704)

Zincionbinding,Metalion binding

– –

Smp047370 Malatedehydrogenase

(COG0039)

Oxidoreductaseactivity Generationofprecursor metabolitesandenergy, Carbohydratemetabolicprocess, Tricarboxylicacidcycle

Dehydrogenase

Smp056970 Glyceraldehyde-3-phosphate

dehydrogenase(COG00570)

Glyceraldehyde-3-phosphate dehydrogenase(NAD+) (phosphorylating)activity, NADandNADPbinding

Glucosemetabolicprocess Glycolysis

Oxidoreductase Dehydrogenase

Smp030370 Calreticulinautoantigen

homolog,putative(KOG0674)

Calciumionbinding Proteinfolding Calcium-binding

protein

Smp090120 Putativealphatubulin

(COG5023)

Structuralconstituentof cytoskeleton

Cellularcomponentmovement, Mitosis,Intracellularprotein transport,Cellularcomponent organization

Tubulin

Smp072330 Putativeheatshockprotein (COG0071)

ATPbinding Proteinfolding Chaperone

Smp021920 Putativeparamyosin

(KOG0161)

Motoractivity,Structural constituentofcytoskeleton, Proteinbinding,Enzyme regulatoractivity

Metabolicprocess,Cytokinesis, Cellularcomponentmovement, Mitosis,Cellcommunication, Musclecontraction,Sensory perceptionofsound,Sensory perception,Mesoderm development,Anatomical structuremorphogenesis, Intracellularproteintransport, Vesicle-mediatedtransport, Regulationofcatalyticactivity, Cellularcomponentorganization

(6)

Table2(Continued)

IDsa _Protein_(COG/KOG_IDs)b _GO_molecular_function _GO_biological_process _PANTHER_protein_class

Smp194770 Taurocyaminekinase

(KOG3581)

ATPbinding,Metalionbinding, Oxidoreductaseactivity, Taurocyaminekinaseactivity

Catalyzes –

Smp063090 Aconitatehydratase

(COG1048)

Hydrolyaseactivity Generationofprecursor

metabolitesandenergy, Carbohydratemetabolicprocess, Tricarboxylicacidcycle,Cellular aminoacidbiosyntheticprocess

Dehydratase, Hydratase

Smp 042170 Putativeuncharacterized

protein(KOG1557)

– – –

a_S._mansoni_Genedb_database_identifier.

b_COG,_cluster_of_orthologous_groups;_KOG,_eukaryotic_orthologous_groups;_GO,_Gene_Ontology_terms;_PANTHER_{classification}_resultant_from_enrichment_in_UNIPROT_and PATHERtools.

10

9

8

7

6

5

4

4 3

Proteins

A

B

C

Fig.2.ClassificationofSchistosomamansoniadultproteinsaccordingtobiologicalprocessesandmolecularfunction.EnrichmentofGObiologicalprocesses(A),GOmolecular function(B)andPANTHERproteinclass(C)retrievedbyPANTHERclassificationsystem.

parasitichelminthesdependheavilyoncarbohydratemetabolism tosupplytheirenergydemand,whereasonequartertoonethirdof theirenergyderivesfromaerobicrespiration(Coles,1973;Barrett,

2009).

Evaluationof putativeinteractions onSTRING protein mode resultedinadistinctandsmallernetwork(Fig.3B).Paramyosin, GST,triosephosphateisomeraseandglyceraldehyde-3-phosphate dehydrogenaseandtheirreducedexpressionafterexposuretoDNH indicatesthatthoseproteinsparticipateofcommonbiological pro-cesses.Indeed,allofthemwereenrichedinmetabolicprocesses, whiletriosephosphateisomeraseandglyceraldehyde-3-phosphate dehydrogenasewerespecificallyassociatedtoglycolysis(Fig.3B

andTable2).PreviousstudydemonstratedthatS.mansoniGSThasa

lowlevelofhomologywithGSTfoundinmammalians,which rein-forcesitspotentialasatargetfordevelopmentofnewdrugsand vaccines(Tayloretal.,1988).Inaddition,severalanthelminticscan

alsobindtoglutathionetransferases,especiallythosecontaininga phenolicring(Barrett,2009).

Previousstudyrevealedthatperoxiredoxinissignificantlymore abundant after exposure to PZQ (Aragon et al., 2009), thus as expected,weobservedanincreaseinexpressionofthisenzyme afterexposuretoDNH.Overall,ourdatasuggestthatschistosomes exposedtoDNHmayincreasetheproductionofperoxiredoxinsasa defensemechanismagainstthestressandoxidativeprocess,which mightbeacommonprocessbetweenparasiticworms.

Acidicproteinsofthe14-3-3family arewidelyexpressedby

S.mansoni.Theseproteinsself-assemblespontaneouslyandform

phosphoserine–threonine-bindingdimersthat caninteractwith over 70 different proteins (Tzivion and Avruch, 2002). Studies haveshownthattheseproteinscouldbeinvolvedinthe regula-tionofPKCfunctionsduringthewormdevelopment(Wiestetal.,

(7)

A

B

Glutathione S-transferase

Nucleoside diphosphate kinase

Adenylate kinase

Phosphoglycerate mutase

Triosephosphate isomerase Paramyosin

Glycer aldeh

yde-3 -phosphate

dehydrogenase

Fig.3. NetworkofinteractionsfromSchistosomamansoniproteinsaffectedby exposuretoDNH.IdentifiedS.mansoniproteinswerematchedtoS.japonicum

onSTRING databasev9.05.(A)NetworkofinteractionsonClusterof ortholo-gousgroups/Eukaryotic orthologousgroups (COG/KOG)mode. (B) Networkof interactionsonproteinmode;nucleosidediphosphatekinase,adenylatekinase, glu-tathioneS-transferase,glyceraldehyde-3-phosphatedehydrogenase,paramyosin, triosephosphateisomerase,phosphoglyceratemutase.

foundthat 14-3-3 epsilon interacts with the intra-cytoplasmic phosphorylated S.mansoni receptor kinase-1, but alsobinds to and modulatetheactivityof humantrans-forming growth fac-tor␤receptor-1(T␤RI)(McGonigleetal.,2001;Siles-Lucasand

Gottstein,2003).

Tegumentalantigensmediateseveralsignalingandtransport processes,suchastheregulationofcalciumlevelsandcontrolof musclecontraction(Mohamedetal.,1998).Therefore,these pro-teinsrepresentpotentialtargetstonewtherapycompoundsand vaccines(Fonsecaetal.,2012).DNHmayalsoactonparasite struc-ture,sinceweobservedahighexpressionofthetegumentalantigen Sm20.8inresponsetoDNH.Thishypothesisisfurthercorroborated bytheincreasedexpressionofgelsolinthathasbeenassociated withinhibitionofcellapoptosisbyblockingmitochondrial mem-branelossandpreventingthereleaseofcytochromeC(Koyaetal., 2000).Indeed,tegumentalantigenSm20.8,gelsolin,calponin,alpha tubulinand paramyosin areproteins particularlyimportant for theorganization ofthesyncytialtegumentofschistosomes and haveasignificantroleinhost-parasiteinteractions(Jonesetal.,

2004).

Overall,ourstudyaddsnewknowledgeintothe understand-ing of anti-schistosomicidal activity of semi-synthetic (−)-6,6′

-dinitrohinokininaderivativecompoundfromlignanandraisesnew perspectivesforfuturetherapeuticapproaches.

Authorcontributions

TCL, MLAS, RSL and JKB contributed by synthesis of DNH anddraftingthepaper.LGM,RGP,DPGandERMcontributedby proteomeanalysisanddraftingthepaper.LGM,LGGandGRG con-tributedbyanalyzingthedata.APY,VR,AASFandWRCsupervised theproteomeanalysisandcriticallyreadthemanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

The authors thank the Laboratório Nacional de Biociências, Brasil,CNPEM-ABTLUS,Campinas,Brazil,forsupportwiththemass spectrometricanalysis.ThisprojectwassupportedbyFundac¸ãode AmparoàPesquisadoEstadodeSãoPaulo,Brasil(grantnumbers 1998/14956-7and2010/17378-8andscholarships2009/15207-4, 2013/00382-0and2011/23819).

References

Aragon,A.D.,Imani,R.A.,Blackburn,V.R.,Cupit,P.M.,Melman,S.D.,Goronga,T., Webb,T.,Loker,E.S.,Cunningham,C.,2009.Towardsanunderstandingofthe mechanismofactionofpraziquantel.Mol.Biochem.Parasitol.164,57–65. Barrett,J.,2009.Fortyyearsofhelminthbiochemistry.Parasitology136,1633–1642. Beaumier,C.M.,Gillespie,P.M.,Hotez,P.J.,Bottazzi,M.E.,2013.Newvaccinesfor neglectedparasiticdiseasesanddengue.Transl.Res.J.Lab.Clin.Med.162, 144–155.

Caffrey,C.R.,2015.Schistosomiasisanditstreatment.FutureMed.Chem.7,675–676. Chuan,J.,Feng,Z.,Brindley,P.J.,McManus,D.P.,Han,Z.,Jianxin,P.,Hu,W.,2010.Our wormyworldgenomics,proteomicsandtranscriptomicsinEastandsoutheast Asia.Adv.Parasitol.73,327–371.

Cioli,D.,Pica-Mattoccia,L.,Basso,A.,Guidi,A.,2014.Schistosomiasiscontrol: praz-iquantelforever?Mol.Biochem.Parasitol.195,23–29.

Coles,G.C.,1973.Furtherstudiesonthecarbohydratemetabolismofimmature

Schistosomamansoni.Int.J.Parasitol.3,783–787.

Colley,D.G.,Bustinduy,A.L.,Secor,W.E.,King,C.H.,2014.Humanschistosomiasis. Lancet383,2253–2264.

Consortium,T.U.,2014.ActivitiesattheUniversalProteinResource(UniProt).Nucl. AcidsRes.42,D191–D198.

Costa,P.R.R.,2000.Safroleandeugenol:studyofthechemicalreactivityandusein thesynthesisofbiologicallyactivenaturalproductsanditsderivative.Quim. Nova23,357–369.

DaSilva,R.,deSouza,G.H.B.,DaSilva,A.A.,DeSouza,V.A.,Pereira,A.C.,Royo,V.D.A., ESilva,M.L.,Donate,P.M.,DeMatosAraújo,A.L.,Carvalho,J.C.,Bastos,J.K.,2005. Synthesisandbiologicalactivityevaluationoflignanlactonesderivedfrom(− )-cubebin.Bioorg.Med.Chem.Lett.15,1033–1037.

DeMarco,R.,Verjovski-Almeida,S.,2009.Schistosomes–proteomicsstudiesfor potentialnovelvaccinesanddrugtargets.DrugDiscov.Today14,472–478. ElAswad,B.E.D.W.,Doenhoff,M.J.,ElHadidi,A.S.,Schwaeble,W.J.,Lynch,N.J.,2011.

Useofrecombinantcalreticulinandcercarialtransformationfluid(CTF)inthe serodiagnosisofSchistosomamansoni.Immunobiology216,379–385. Esperandim,V.R.,DaSilvaFerreira,D.,SousaRezende,K.C.,Magalhães,L.G.,Medeiros

Souza,J.,Pauletti,P.M.,Januário,A.H.,daSilvadeLaurentz,R.,Bastos,J.K.,Símaro, G.V.,Cunha,W.R.,Andrade,E.,Silva,M.L.,2013.Invitroantiparasiticactivityand chemicalcompositionoftheessentialoilobtainedfromthefruitsofPipercubeba. PlantaMed.79,1653–1655.

Fonseca,C.T.,BrazFigueiredoCarvalho,G.,CarvalhoAlves,C.,deMelo,T.T.,2012. Schistosomategumentproteinsinvaccine anddiagnosisdevelopment:an update.J.Parasitol.Res.2012,541268.

Gutierrez,R.M.P.,Gonzalez,A.M.N.,Hoyo-Vadillo,C.,2013.Alkaloidsfrompiper: areviewofitsphytochemistryandpharmacology.MiniRev.Med.Chem.13, 163–193.

Jensen,L.J.,Kuhn,M.,Stark,M.,Chaffron,S.,Creevey,C.,Muller,J.,Doerks,T.,Julien, P.,Roth,A.,Simonovic,M.,Bork,P.,vonMering,C.,2009.STRING8–aglobal viewonproteinsandtheirfunctionalinteractionsin630organisms.Nucl.Acids Res.37,D412–D416.

(8)

(IgE)responsestoparamyosinpredictresistancetoreinfectionwithSchistosoma japonicumandareattenuatedbyIgG4.Infect.Immun.77,2051–2058. Jones,M.K.,Gobert,G.N., Zhang,L.,Sunderland, P.,McManus, D.P.,2004.The

cytoskeletonandmotorproteinsofhumanschistosomesandtheirrolesin sur-facemaintenanceandhost-parasiteinteractions.BioEssaysNewsRev.Mol.Cell. Dev.Biol.26,752–765.

Kohama,H.,Harakuni,T.,Kikuchi,M.,Nara,T.,Takemura,Y.,Miyata,T.,Sato,Y., Hirayama,K.,Arakawa,T.,2010.IntranasaladministrationofSchistosoma japon-icumparamyosininducedrobustlong-lastingsystemicandlocalantibodyas wellasdelayed-typehypersensitivityresponses,butfailedtoconferprotection inamouseinfectionmodel.Jpn.J.Infect.Dis.63,166–172.

Koya,R.C.,Fujita,H.,Shimizu,S.,Ohtsu,M.,Takimoto,M.,Tsujimoto,Y.,Kuzumaki,N., 2000.Gelsolininhibitsapoptosisbyblockingmitochondrialmembrane poten-tiallossandcytochromecrelease.J.Biol.Chem.275,15343–15349.

Lanar,D.E.,Pearce,E.J.,James,S.L.,Sher,A.,1986.Identificationofparamyosinas schistosomeantigenrecognizedbyintradermallyvaccinatedmice.Science234, 593–596.

Magalhães,L.G.,DeSouza,J.M.,Wakabayashi,K.A.L.,Laurentiz,R.,da,S.,Vinhólis, A.H.C.,Rezende,K.C.S.,Simaro,G.V.,Bastos,J.K.,Rodrigues,V.,Esperandim,V.R., Ferreira,D.S.,Crotti,A.E.M.,Cunha,W.R.,eSilva,M.L.A.,2012.Invitroefficacy oftheessentialoilofPipercubebaL.(Piperaceae)againstSchistosomamansoni. Parasitol.Res.110,1747–1754.

McGonigle,S.,Beall,M.J.,Feeney,E.L.,Pearce,E.J.,2001.Conservedrolefor 14-3-3epsilondownstreamoftypeITGFbetareceptors.FEBSLett.490,65–69. Mi,H.,Muruganujan,A.,Casagrande,J.T.,Thomas,P.D.,2013.Large-scalegene

function analysiswith the PANTHER classification system.Nat. Protoc.8, 1551–1566.

Mohamed,M.M.,Shalaby,K.A.,LoVerde,P.T.,Karim,A.M.,1998.Characterization ofSm20.8,amemberofafamilyofschistosometegumentalantigens.Mol. Biochem.Parasitol.96,15–25.

Pearce,E.J.,2003.Progresstowardsavaccineforschistosomiasis.ActaTrop.86, 309–313.

Pereira,A.C.,Silva,M.L.,Souza,J.M.,Laurentiz,R.S.,Rodrigues,V.,Januário,A.H., Pauletti,P.M.,Tavares,D.C.,Filho,A.A.,Cunha,W.R.,Bastos,J.K.,Magalhães, L.G.,2015.Invitroandinvivoanthelminticactivityof(−)-6,6′_{-dinitrohinokinin}

againstschistosomulaandjuvenileandadultwormsofSchistosomamansoni. ActaTrop.149,195–201.

Qian,C.Y.,Wang,J.,Yu,C.X.,Yin,X.R.,Song,L.J.,Zhang,W.,Jin,Y.,Ke,X.D.,2012. Char-acterizationofIgGresponsesofrabbitstoSj14-3-3proteinafterexperimental infectionwithSchistosomajaponicum.Parasitol.Res.111,2209–2211.

Rollinson,D.,Knopp,S.,Levitz,S.,Stothard,J.R.,Tchuenté,L.A.,Garba,A.,Mohammed, K.A.,Schur,N.,Person,B.,Colley,D.G.,Utzinger,J.,2013.Timetosettheagenda forschistosomiasiselimination.ActaTrop.128,423–440.

Saraiva,J.,Veja,C.,Rolon,M.,daSilva,R.E.,Silva,M.L.A.,Donate,P.M.,Bastos,J.K., Gomez-Barrio,A.,deAlbuquerque,S.,2007.Invitroandinvivoactivityoflignan lactonesderivativesagainstTrypanosomacruzi.Parasitol.Res.100,791–795. Saraiva,J.,Lira,A.A.M.,Esperandim,V.R.,daSilvaFerreira,D.,Ferraudo,A.S.,

Bas-tos,J.K.,Silva,M.L.A.,deGaitani,C.M.,deAlbuquerque,S.,Marchetti,J.M.,2010. (−)-Hinokinin-loadedpoly(d,-lactide-co-glycolide)microparticlesforChagas disease.Parasitol.Res.106,703–708.

Senft,A.W.,Crabtree,G.W.,1983.Purinemetabolismintheschistosomes:potential targetsforchemotherapy.Pharmacol.Ther.20,341–356.

Setchell,K.D.R.,Borriello,S.P.,Gordon,H.,Lawson,A.M.,Harkness,R.,Morgan,D.M., Kirk,D.N.,Adlercreutz,H.,Axelson,M.,1981.Lignanformationinman–microbial involvementandpossiblerolesinrelationtocancer.Lancet2,4–7.

Shevchenko,A.,Wilm,M.,Vorm,O.,Mann,M.,1996.Massspectrometricsequencing ofproteinssilver-stainedpolyacrylamidegels.Anal.Chem.68,850–858. Siles-Lucas,M.D.M.,Gottstein,B.,2003.The14-3-3protein:akeymoleculein

para-sitesasinotherorganisms.TrendsParasitol.19,575–581.

Smithers,S.R.,Terry,R.J.,1965.Theinfectionoflaboratoryhostswithcercariae ofSchistosomamansoniandtherecoveryoftheadultworms.Parasitology55, 695–700.

Taylor,J.B.,Vidal,A.,Torpier,G.,Meyer,D.J.,Roitsch,C.,Balloul,J.M.,Southan,C., Sondermeyer,P.,Pemble,S.,Lecocq,J.P.,1988.Theglutathionetransferase activ-ityandtissuedistributionofaclonedMr28KprotectiveantigenofSchistosoma mansoni.EMBOJ.7,465–472.

Tzivion,G.,Avruch,J.,2002.14-3-3proteins:activecofactorsincellularregulation byserine/threoninephosphorylation.J.Biol.Chem.277,3061–3064. Usia,T.,Watabe,T.,Kadota,S.,Tezuka,Y.,2005.PotentCYP3A4inhibitory

con-stituentsofPipercubeba.J.Nat.Prod.68,64–68.

Wiest,P.M.,Burnham,D.C.,Olds,G.R.,Bowen,W.D.,1992.Developmentalexpression ofproteinkinaseCactivityinSchistosomamansoni.Am.J.Trop.Med.Hyg.46, 358–365.

Yu,Q.,Yang,H.,Guan,F.,Feng,Y.,Yang,X.,Zhu,Y.,2014.DetectionofIgGinseraof patientswithSchistosomiasisjaponicabydevelopingmagneticaffinity enzyme-linkedimmunoassaybasedonrecombinant14-3-3protein.Trans.R.Soc.Trop. Med.Hyg.108,37–41.