Cell-penetratingpeptideandionchannelsblocker
Crotamine isa basicpeptide presentinthe venom ofthe SouthAmerican rattlesnakeCrotalusdurissusterrificus.MultipleBIOLOGicalfunctionshavebeenattributedtoCrotamine.Itisanaturalcell-penetratingpeptidewithselectivebiologicalactiontowardsactivelyproliferatingcelltypes.Moreover,ithasbeenreportedthatcrotamineisablockerofKv1.3(IC50around300nM)aswellasKv1.1andKv1.2.Ithasanalgesicpropertiesandmyonecroticeffects.Inaddition,crotaminebelongstothebeta-defensinpeptidesandassuchdemonstratesantibacterialpropertiesbyinteractingwithlipidmembranes.
Description:
AAsequence: YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG-OH
Disulfidebridges: Cys4-Cys36;Cys11-Cys30;Cys18-Cys37
Length(aa):41
Formula:C214H326N64O54S7
MolecularWeight:4883.82Da
Appearance:whitelyophilizedsolid
Solubility:waterorsalinebuffer
CASnumber:
Source:synthetic
Purityrate:>95%
Reference:
CharacterizationofribonucleicacidsfromthevenomglandsofCrotalusdurissusterrifucus
DeLuccaFL.,etal.(1974)CharacterizationofribonucleicacidsfromthevenomglandsofCrotalusdurissusterrifucus(Ophidia,Reptilia)aftermanualextractionofthevenom.Studiesontemplateactivityandbasecomposition.BiochemJ.PMID:4463939
Theanalgesicactivityofcrotamine,aneurotoxinfromCrotalusdurissusterrificus
Crotamine,a4.88kDaneurotoxicprotein,hasbeenpurifiedtoapparenthomogeneityfromCrotalusdurissusvenombygelfiltrationonSephadexG-75.Wheninjected(i.p.ors.c.)inadultmaleSwissmice(20-25g),itinducedatime-dosedependentanalgesiceffectwhichwasinhibitedbynaloxone,thussuggestinganopioidactionmechanism.Whencomparedwithmorphine(4mg/kg),crotamine,eveninextremelylowdoses(133.4microg/kg,i.p.,about0.4%ofaLD50isapproximately30-foldmorepotentthanmorphine(w/w)asananalgesic.Onamolarbasisitismorethan500-foldmorepotentthanmorphine.Itisalsomuchmorepotentthanthelowermolecularweightcrudefractionsofthesamevenom.Theantinociceptiveeffectsofcrotamineandmorphinewereassayedbythehotplatetestandbytheaceticacid-inducedwrithingmethod.Therefore,bothcentralandperipheralmechanismsshouldbeinvolved.Histopathologicalanalysisofthebrain,liver,skeletalmuscles,stomach,lungs,spleen,heart,kidneysandsmallintestineofthecrotamineinjectedmicedidnotshowanyvisiblelesioninanyoftheseorgansbylightmicroscopy.Sincecrotamineaccountedfor22%(w/w)ofthedesiccatedvenom,itwasidentifiedasitsmajorantinociceptivelowmolecularweightpeptidecomponent.
MancinAC.,etal.(1998) Theanalgesicactivityofcrotamine,aneurotoxinfromCrotalusdurissusterrificus(SouthAmericanrattlesnake)venom:abiochemicalandpharmacologicalstudy.Toxicon.PMID:9839677
Crotamineisanovelcell-penetratingproteinfromthevenomofrattlesnakeCrotalusdurissusterrificus
Hereinwereportthatcrotamine,asmalllysine-andcysteine-richproteinfromthevenomoftheSouthAmericanrattlesnake,canrapidlypenetrateintodifferentcelltypesandmouseblastocystsinvitro.Invivocrotaminestronglylabelscellsfrommousebonemarrowandspleenandfromperitonealliquid,asshownbyfluorescentconfocallaser-scanningmicroscopy.Nuclearlocalizationofcrotaminewasobservedinbothfixedandunfixedcells.Inthecytoplasm,crotaminespecificallyassociateswithcentrosomesandthusallowsustofollowtheprocessofcentrioleduplicationandseparation.Inthenucleus,itbindstothechromosomesatS/G2phase,whencentriolesstartdividing.Moreover,crotamineappearsasaMarkerofactivelyproliferatingcells,asshownby5-BrdUcell-proliferationassay.CrotamineinthemicromolarrangeprovednontoxictoanyofthecellculturestestedanddidnotaffectthepluripotencyofEScellsorthedevelopmentofmouseembryos.
KerkisA.,etal.(2004)Crotamineisanovelcell-penetratingproteinfromthevenomofrattlesnakeCrotalusdurissusterrificus.FASEBJ.PMID:15231729
BiologicalversatilityofcrotamineacationicpeptidefromthevenomofaSouthAmericanrattlesnake
Moleculesisolatedfromanimals,insects,plantsormicroorganismscanprovideprototypesfordesignofbiopharmaceuticalproducts.Somevenomtoxinsandtheirderivativesareusedinmedicine,whileothersprovidetemplatesfordevelopmentofnewdrugs.Themildtoxin,crotamine,asmallbasiclow-molecular-weightpolypeptidepurifiedfromthevenomofaSouthAmericanrattlesnake,Crotalusdurissusterrificus.Crotaminewasdiscoveredmorethan50yearsagoandonlyinthepastsixyearshasitsexceptionalbiologicalversatilitybeendemonstrated.Particularly,itscell-penetratingABIlity,whichallowscrotaminetocrosscellmembranesandtoaccumulateinthenucleus;itsuseforintracellularvesicletrackingandasacellcyclemarkeranditscapabilityfordeliveringDNAintoreplicatingmammaliancells.Bothantimicrobialactionandpotentialselectiveantitumoractivityofcrotaminehavealsobeenfound.Multidisciplinaryapproachesandpathwaysofdiscoveryplacedcrotamineinararecategoryofversatilebiomolecules,inwhichconcentration,moleculartargetpreference,structuralancestryandspecificitytowardbiologicalmembranesplayanintegralrole.Crotamineisadruggablepeptidewithhighpotentialforuseasanimagingagentfordetectingdividingcells,forintracellulardeliveryofhydrophilicbiomolecules,andasanalternativechemotherapeuticcompoundagainstaggressivetypesofcancer.
KerkisI.,etal.(2010)BiologicalversatilityofcrotamineacationicpeptidefromthevenomofaSouthAmericanrattlesnake.ExpertOpinInvestigDrugs.PMID:21062230
Stateoftheartinthestudiesoncrotamine,acellpenetratingpeptidefromSouthAmericanrattlesnake
Animalvenomscompriseanaturallyselectedcocktailofbioactivepeptides/proteinsandothermolecules,eachofwhichplayingadefinedrolethankstothehighlyspecificinteractionswithdiversemoleculartargetsfoundintheprey.Researchfocusedonisolation,structural,andfunctionalcharacterizationsofnovelnaturalbiologics(bioactivepeptides/proteinsfromnaturalsources)hasalongwaytogothroughfromthebasicsciencetoclinicalapplications.Herein,weoverviewthestructuralandfunctionalcharacteristicsofthemyoneurotoxincrotamine,firstlyisolatedfromtheSouthAmericanrattlesnakevenom.Crotamineisthefirstvenompeptideclassifiedasanaturalcellpenetratingandantimicrobialpeptide(CPPandAMP)withamorepronouncedantifungalactivity.IncontrasttootherknownnaturalCPPsandAMPs,crotaminedemonstratesawidespectrumofbiologicalactivitieswithpotentialbiotechnologicalandtherapeuticvalues.Morerecentstudieshavedemonstratedtheselectiveinvitroanticanceractivityofcrotamine.Invivo,usingamurinemelanomamodel,itwasshownthatcrotaminedelaystumorimplantation,inhibitstumorcellsproliferation,andalsoincreasesthesurvivalofmiceengraftedwithsubcutaneousmelanoma.Thestructuralandfunctionalpropertiesandalsothepossiblebiotechnologicalapplicationsofminimizedmoleculesderivedfromcrotaminearealsodiscussed.
KerkisI.,etal.(2014)Stateoftheartinthestudiesoncrotamine,acellpenetratingpeptidefromSouthAmericanrattlesnake.BiomedResInt.PMID:24551848
Thenaturalcell-penetratingpeptidecrotaminetargetstumortissueinvivoandtriggersalethalcalcium-dependentpathwayinculturedcells.
Ourgoalwastodemonstratetheinvivotumorspecificaccumulationofcrotamine,anaturalpeptidefromthevenomoftheSouthAmericanrattlesnakeCrotalusdurissusterrificus,whichhasbeencharacterizedbyourgroupasacellpenetratingpeptidewithahighspecificityforactivelyproliferatingcellsandwithaconcentration-dependentcytotoxiceffect.Crotaminecytotoxicityhasbeenshowntobedependentonthedisruptionoflysosomesandsubsequentactivationofintracellularproteases.Inthiswork,weshowthatthecytotoxiceffectofcrotaminealsoinvolvesrapidintracellularcalciumreleaseandlossofmitochondrialmembranepotentialasobservedinrealtimebyconfocalmicroscopy.Theintracellularcalciumoverloadinducedbycrotaminewasalmostcompletelyblockedbythapsigargin.Microfluorimetryassaysconfirmedtheimportanceofinternalorganelles,suchaslysosomesandtheendoplasmicreticulum,ascontributorsfortheintracellularcalciumincrease,aswellastheextracellularmedium.Finally,wedemonstrateherethatcrotamineinjectedintraperitoneallycanefficientlytargetremotesubcutaneoustumorsengraftedinnudemice,asdemonstratedbyanoninvasiveopticalimagingprocedurethatpermitsinvivoreal-timemonitoringofcrotamineuptakeintotumortissue.Takentogether,ourdataindicatethatthecytotoxicpeptidecrotaminecanbeusedpotentiallyforadualpurpose:totargetanddetectgrowingtumortissuesandtoselectivelytriggertumorcelldeath.
NascimentoFD(2012)Thenaturalcell-penetratingpeptidecrotaminetargetstumortissueinvivoandtriggersalethalcalcium-dependentpathwayinculturedcells.MolPharm.PMID:22142367
Crotamine:anovelcell-penetratingpolypeptidenanocarrierwithpotentialanti-cancerandbiotechnologicalapplications.
Crotamineisabasic,42-residuepolypeptidederivedfromsnakevenomthathasbeenshowntopossesscell-penetratingproperties.CrotamineformsnanoparticleswithavarietyofDNAandRNAmolecules,andcrotamine-plasmidDNAnanoparticlesareselectivelydeliveredintoactivelyproliferatingcellsincultureorinmice.Assuch,thesenanoparticlescouldformthebasisforanucleicaciddrug-deliverysystem.Herewedescribethepreparation,purification,andbiochemicalandbiophysicalanalysisofvenom-derived,recombinant,chemicallysynthesized,andfluorescent-labeledcrotamine;theformationandcharacterizationofcrotamine-DNAand-RNAnanoparticles;andthedeliveryofthesenanoparticlesintocellsandanimals.
HayashiMA.,etal.(2012)Crotamine:anovelcell-penetratingpolypeptidenanocarrierwithpotentialanti-cancerandbiotechnologicalapplications.MethodsMolBiol.PMID:22791447
