gruppo italiano di biomembrane e bioenergetica · 2 gruppo italiano di biomembrane e bioenergetica...

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GRUPPOITALIANODIBIOMEMBRANEEBIOENERGETICA

GIBBExecutiveBoard

PaoloBernardi(Padova),AlessandroGiuffrè(Roma),GiovannaLippe(Udine),

VitoDePinto(Catania),FrancescoFrancia(Bologna),CesareIndiveri(Calabria),

LuigiPalmieri(Bari),ClaudiaPiccoli(Foggia).

ExhibitorsandSponsors

GraphicDesign:AndreaMagrì

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Organizingcommitteemembers

Secretariat:VitoDePinto,FrancescaGuarino,AngelaMessina,Tel.+390957384244-4231.E-mail:[email protected];[email protected];[email protected];[email protected]

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Venue

AuditoriumGiancarloDeCarlo

MonasterodeiBenedettinidiSanNicolòL’arenaPiazzaDante,32–Catania

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Program

Wednesday14th

13:00 Registration

15:00 WelcomeAddressAuthorities,GIBBPresidentandtheOrganizingCommittee

15:30 OpeningPlenaryLectureChair:PaoloBernardi(UniversityofPadua,Italy)SirJohnWalker,Nobellaureate(UniversityofCambridge,UnitedKingdom)Therespiratorychaininmitochondria:atriumphforbiochemistryandbiophysics

16:30 SessionI:ATPsynthaseandthecomplexesofrespiratorychainOralPresentationsChair:GiovannaLippe(UniversityofUdine,Italy),NazarenoCapitanio(UniversityofFoggia)

16:30 KarinB.Busch(WestfälischeWilhelms-UniversityofMünster,Germany)RespiratorysupercomplexassemblywithCIVmonitoredbyfluorescencelifetimeimagingmicroscopy

16:45 ClaraFerreiraPereira(UniversityofPorto,Portugal)PhosphoproteomicscreeningidentifiesATPsynthasebetasubunitasatargetoftheyeasttype2A-likeproteinphosphataseSit4p

17:00 DaniloFaccenda(RoyalVeterinaryCollege,London,UnitedKingdom)ControlofmitochondrialbioenergeticsandstructurebytheATPaseinhibitoryfactor1:apro-survivalrelayviaOPA1

17:15 AndreaUrbani(UniversityofPadua,Italy)ElectrophysiologicalpropertiesofchannelformedbybovineFOF1ATPsynthaseinplanarlipidbilayer

17:30 LishuGuo(UniversityofPaduaandCNRInstituteofNeuroscience,Italy)MatrixCa2+isessentialforopeningofthemitochondrialpermeabilitytransitionpore

17:45 MarcoMalferrari(UniversityofBologna,Italy)ThecytochromebH291Lmutationstronglyimpairsubiquinoloxidationandprotontranslocationatthebacterialbc1QOsite

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18:00 KarenTrchounian(YerevanStateUniversity,Armenia)

InputofhydrogenasesinH2cyclingandprotonmotiveforcegenerationinEscherichiacoliduringfermentation

18:15 EndofSession

18:30 WelcomecocktailGiardinodeiNovizi

20:00 GuidedtourofMonasterodeiBenedettini

Thursday15th

9:00 PlenarylectureChair:VitoDePinto(UniversityofCatania,Italy)Prof.DoronRapaport(UniversityofTübingen,Germany)Biogenesisofmitochondrialoutermembraneproteinsinevolutionarycontext

9:45 SessionII:Specializedtransportersandpores,structure&functionOralpresentationsChair:CesareIndiveri(UniversityofCalabria,Italy)

9:45 VanessaChecchetto(UniversityofPadua,Italy)Anewmitochondrialpotassiumchannelinvolvedincardioprotection

10:00 SimonaReina(UniversityofCatania,Italy)VDAC3cysteineoxidationstateandchannelactivity:invitroandincellulostudies

10:15 MariafrancescaScalise(UniversityofCalabria,Italy)Structure/functionrelationshipsofhumanLAT1aminoacidstransporterandscreeningofactive-sitetargetedinhibitors

10:30 LaraConsole(UniversityofCalabria,Italy)Regulationofthemitochondrialcarnitine/acylcarnitinecarrierbyS-nitrosylation

10:45 MariaCarmelaDiRosa(UniversityofCatania,Italy)ThesecondVDACisoforminS.cerevisiae,anunknownprotein:structural-functionalcharacterization

11:00 AngelaOstuni(UniversityofBasilicata,Italy)Structuralandfunctionalstudiesonmultidrugresistanceprotein6(MRP6):newinsights

11:15 Coffeebreak

ChiostrodiLevante

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12:00 SessionIII:Specializedtransportersandpores,physiologicalandpathologicalroles

OralpresentationsChair:FrancescoFrancia(UniversityofBologna)

12:00 LuigiLeanza(UniversityofPadua,Italy)Directpharmacologicaltargetingofamitochondrialionchannelselectivelykillstumorcellsinvivo

12.15 DariaGrobys(AdamMickiewiczUniversityinPoznan,Poland)VDACinviabilityandenergeticcouplingstatusoftheinduciblePC12cellmodelofHuntington'sdisease

12:30 SimonaBarbato(UniversityofBologna,Italy)InvolvmenteofmiRNAsintheacquiredBRAF-Iresistanceofmetastaticmelanomacells

12:45 RobertaPeruzzo(UniversityofPadua,Italy)Novelpsoralen-derivativeswithincreasedsolubilityincancertreatment

13:00 BMRGENOMICS:SponsorTalk

13:30 LunchChiostrodiLevante

15:00 PlenarylectureChair:AlessandroGiuffrè(UniversityofRomeLaSapienza,Italy)Dr.ChristianFrezza(UniversityofCambridge,UnitedKingdom)Mitochondrialdysfunctionandcancer:beyondmetabolism

15:45 SessionIV:BioenergeticsmetabolismandmitochondrialdysfunctionOralpresentationsChair:GiancarloSolaini(UniversityofBologna,Italy)

15:45 FerdinandoChiaradonna(UniversityofMilano-Bicocca,Italy)ProteinkinaseAactivationpromotescancercellresistancetoglucosestarvationandanoikis

16:00 LaraGibellini(UniversityofModenaandReggioEmilia,Italy)LonP1differentlymodulatesmitochondrialfunctionandbioenergeticsofprimaryversusmetastaticcoloncancercells

16:15 GiacomoLazzarino(CatholicUniversityofRome,Italy)Theresponseofthemitochondrialqualitycontrolsystemtogradedtraumaticbraininjury

16:30 FrancescaMalagrinò(CNRInstituteofMolecularBiologyandPathology,Rome,Italy)Novelpathogenicmechanismsfrominvestigationofhumanhydrogensulfidemetabolism

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16:45 MarcoSchiavone(UniversityofPaduaandCNRInstituteofNeuroscience,Italy)

AlisporivirrescuesmitochondrialrespiratorydysfunctioninDuchennemusculardystrophymodels

17:00 CoffeebreakChiostrodiLevante

17:30 SessionV:ReactiveOxygenSpeciesandmitochondriaOralpresentationsChair:ClaudiaPiccoli(UniversityofFoggia,Italy)

17:30 GinevraNannelli(UniversityofSiena,Italy)Theroleofmitochondriainthemaintenanceofendothelialfunctionafterexposuretoredoxinjury

17:45 GiuliaGorini(UniversityofBologna,Italy)Reactiveoxygenspeciesdecreaseinhumancellsadaptedtohypoxia

18:00 MariannaTomasello(CNRInstituteofBiostructuresandBioimaging,Catania,Italy)MisfoldedSOD1mutantaltersVDAC1-Hexokinase1complexespromotingmitochondrialdysfunctioninanAmyotrophicLateralSclerosismodel

18:15 AndreaMagrì(UniversityofCatania,Italy)Hexokinases1peptideprotectsVDAC1fromSOD1G93AmutanttoxicinteractioninanALSmodel:implicationsfortheneurodegenerativedisease’streatment

18:30 GloriaScattolin(UniversityofPadua,Italy)MitochondrialreactiveoxygenspeciessensitizeT-ALLcellstoapoptosisbyengagingtheOMA1-OPA1axis

18:45 GIBBMembersAnnualMeeting

20:00 BusMeetingPointatpiazzaDante(infrontofMonastero)

20:30 SocialDinnerRistoranteFeudoDelizia,MountEtna

Friday16th

9:15 PlenarylectureChair:AngelaMessina(UniversityofCatania,Italy)Prof.LaszloTretter(SemmelweisUniversity,Budapest,Hungary)CyclophylinDKOprotectsagainstbioenergeticchangesdetectedinAlzheimer'smodel(Tg(APPSwe)/Tg(PSEN1))mice

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10:00 SessionVI:NewandnotablethemesinBioenergeticsandBiomembranes

OralpresentationsChair:MarcelloPinti(UniversityofModena&ReggioEmilia,Italy),AlexanderRuban(UniversityofLondon,UnitedKingdom)

10:00 AlexanderRuban(UniversityofLondon,UnitedKingdom)Dynamicphotosyntheticmembrane

10:15 KylieA.Minor(UniversityofAlberta,Canada)Light-DrivenEnergyProductionforCell-FreeMetabolicSystems

10:30 WolfgangBuckel(PhilippsUniversityofMarburg,Germany)Flavin-basedelectronbifurcationinanaerobicBacteriaandArchaea

10:45 NunzioIraci(UniversityofCatania,ItalyandofCambridge,UnitedKingdom)Extracellularvesiclesareindependentmetabolicunitswithasparaginaseactivity

11:00 AndonisKarachitos(AdamMickiewiczUniversityinPoznan,Poland)Mitochondriamonitoringinstudiesofsuccessfulanhydrobiosis

11:15 CoffeebreakChiostrodiLevante

11:45 MaryF.Rooney(TrinityCollegeDublin,Ireland)GenotypevariationattheMSTNlocusisassociatedwithskeletalmusclemitochondrialabundanceandfibrecompositioninuntrainedThoroughbredhorses

12:00 ClaudioLaquatra(UniversityofPadua,Italy)Zebrafish(Daniorerio)asamodeltostudythepathophysiologicalroleofthemitochondrialchaperoneTRAP1

12:15 SarfarazhussainFarooqui(UniversityofVienna,Austria)AnalysisoftheroleofLetm1inthedevelopmentofzebrafish

12:30 MartinaLaSpina(UniversityofPaduaandCNRInstituteofNeuroscience,Italy)InductionofautophagyandmitochondrialbiogenesisbythenaturalcompoundPterostilbene:mechanismsandbiomedicalpotential

12:45 AntonioFilippi(UniversityofUdine,Italy)Flavonoidtransportinplantmicrosomes:characterizationofquercetinuptake

13:00 Concludingremarksandawards

13:30 LunchBoxChiostrodiLevante

14:30 Endofthemeeting

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ABSTRACTBOOK

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ProfessorSirJohnWalkerUniversityofCambridge,UnitedKingdomProf.Walkerstudiesthestructure,function,regulationandassemblyofthe ATP synthase in mitochondria, and increasingly in bacteria. ForprovidingthemolecularbasisoftherotarymechanismoftheF1-catalyticdomain,hewasawardedtheNobelPrizeinChemistryin1997.In2008,he became the Founding Director of the Medical Research Council’sMitochondrial Biology Unit, in Cambridge, where he continues hisresearchasDirectorEmeritus.

Therespiratorychaininmitochondria:atriumphforbiochemistryandbiophysics

PlenaryLecture

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ProfessorDoronRapaportUniversityofTuebingen,GermanyProf.Rapaport is studyingmitochondrialbiogenesisanddynamics.HegraduatedinChemistryin1988atTelAvivUniversityandobtainedhisPhDinBiophysicsin1995atTheWeizmannInstituteofScience.Currently, he heads a research group in the Interfaculty Institute ofBiochemistry,UniversityofTuebingen.

BiogenesisofmitochondrialoutermembraneproteinsinevolutionarycontextThemitochondrialoutermembrane(MOM)mediatesmultipleinteractionsbetweenthemitochondrialmetabolicandgeneticsystemsandtherestoftheeukaryoticcell.Biogenesisofthismembraneinvolvesintegrationofnewlysynthesizedproteins intothe lipidbilayer.Amongtheseprecursorproteinsarethosethatspanthemembraneonce,twiceorwithmultiplesegments.Ouraimistofullydefinethebiological processes and molecular mechanisms that underlie the biogenesis of MOM proteins.Employinginvitro,inorganelloandinvivoexperimentswecouldcharacterizeuniqueimportpathwaysforthevariousfamiliesofMOMproteins.Remarkably,thebiogenesisofβ-barrelproteinsisconservedfromGram–negativebacteriatomitochondriaandthisconservationallowstheorganelletorecognizedandprocessbacterialsubstrates.

PlenaryLecture

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DoctorChristianFrezzaUniversityofCambridge,UnitedKingdomDr. Christian Frezza is working on the role of altered mitochondrialmetabolismincancer.HegainedhisMScinMedicinalChemistryattheUniversity of Padova in 2002. He then got a PhD on mitochondrialdynamicsandapoptosisinPadova.In2008,hemovedtoGlasgow,wherehe investigated the roleofmitochondrial defects in tumorigenesis. In2012 he took up his current position as Group Leader in CancerMetabolismattheMRCCancerUnitinCambridge,UK.

Mitochondrialdysfunctionandcancer:beyondmetabolism

Mutations of the tricarboxylic acid cycle (TCA cycle) enzyme fumarate hydratase (FH) cause thehereditarycancersyndromeHereditaryLeiomyomatosisandRenalCellCancer(HLRCC).FH-deficientrenalcancersarehighlyaggressiveandmetastasizeevenwhensmall, leadingtoanabysmalclinicaloutcome.ThelinkbetweenlossofFHandtumorformationisstillunderintenseinvestigation.Evidencesuggests that fumarate, a small molecule metabolite that accumulates in FH-deficient cells, maycontributetotumorigenesisinHLRCC.Forinstance,accumulationoffumaratehasbeenassociatedwiththestabilizationoftheHypoxiaInducibleFactorsHIFsandtotheactivationoftheantioxidantmasterregulatorNRF2,via succinationof itsnegative regulatorKeap1.However, thecontributionof thesesignalingcascadestotumorigenesisofHLRCChasbeendebatedandtheoncogenicroleoffumaratestillunclear.HereweusedamultidisciplinaryapproachtoinvestigatetheconsequencesofthelossofFH and present evidence that FH-deficient cells undergo a fumarate-dependent epithelial-to-mesenchymal-transition, a phenotypic switch associated with cancer initiation, invasion, andmetastasis.WeproposethatthisphenotypicswitchmightprimecelltotransformationandcontributetothetumorigenesisandmetastatizationofFH-deficientcancers.

PlenaryLecture

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ProfessorLaszloTretterSemmelweisUniversity,Budapest,HungaryProf.Tretterisinterestedonmitochondrialphysiology,ROShomeostasisand metabolic biochemistry. He obtained M.D. and PhD degree atSemmelweisUniversitywhereheisatpresenttheheadofDepartmentofMedicalBiochemisty.

CyclophylinDKOprotectsagainstbioenergeticchangesdetectedinAlzheimer'smodel(Tg(APPSwe)/Tg(PSEN1))mice

PlenaryLecture

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RespiratorysupercomplexassemblywithCIVmonitoredbyfluorescencelifetimeimagingmicroscopyBettinaRieger1,2,DariaShalaeva3,Anna-CarinaSöhnel1,2,WladislawKohl2,PatrickDuwe1,ArmenY.Mulkidjanian3,4,5andKarinB.Busch1,2

1DepartmentofBiology,WestfälischeWilhelms-UniversityofMünster,Münster(Germany);2Schoolof Biology, University of Osnabrueck, Osnabrueck (Germany); 3 School of Physics, University ofOsnabrueck,Osnabrueck(Germany);4A.N.BelozerskyInstituteofPhysico-ChemicalBiology;5SchoolofBioengineeringandBioinformatics,M.V.LomonosovMoscowStateUniversity,Moscow(Russia)[email protected],CIIIandCIVarefoundinvariousstablesupercomplexcompositionswhenextracted from membranes, leading to the further assumption that these assemblies also have aspecificfunctionalrole.Ensuing,itisofhighinteresttostudyrespiratorysupercomplexesalsoinsitu.Here,wedemonstratethatfluorescencelifetimeimagingmicroscopy(FLIM)withasinglefluorescentsensorisaversatilemethodfornon-invasivelivecellimagingofdynamicsupercomplexformation.Thesensorhastobelocalizedatapositionthatisburiedinanassembledsupercomplexbutexposedtotheaqueousphaseinadisassembledcomplex.Accordingtostructuralinformation[1],Cox8aandCox7care subunitsofCIV that fulfill this condition. Indeed,we founda significantly reduced fluorescencelifetimeofa sensorplacedat thesesubunitsbutnot fora sensoratCox4,where theC-terminus islocalizedmoreat thesurfaceofCIVat thesamesideof themembrane[2]. Introductionofa linkerbetween Cox8a and the fluorescent protein to lift the sensor out of the supercomplex resulted inprolonged fluorescence lifetime. The same effect was observed when the supercomplex assemblyfactorsweredownregulated.FRETbetweensubunitsofCIIIandCIVconfirmedtheresults[2].Thus,respiratorysupercomplexassemblyinlivecellscanbemonitoredbyuseoffluorescentprobesatcriticalpositions. This complements the important biochemical and physiological studies performed withisolated or re-constituted supercomplexes and allows for following the dynamic supercomplexassemblyinlivecells.[1]LettsJAetal,Nature537(2016)644-648.[2]RiegerBetal,SciRep7(2017)46055.

SessionI:ATPsynthaseandthecomplexesofrespiratorychain

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PhosphoproteomicscreeningidentifiesATPsynthasebetasubunitasatargetoftheyeasttype2A-likeproteinphosphataseSit4p

Clara Pereira1,2, Telma Martins1,2, Andreia T. Pereira2, Hugo Osório1,4, PedroMoradas-Ferreira1,2,3andVitorCosta1,2,3

1InstitutodeInvestigaçãoeInovaçãoemSaúde,UniversidadedoPorto(Portugal);2IBMCInstitutodeBiologiaCelulareMolecular,UniversidadedoPorto(Portugal);3DepartamentodeBiologiaMolecular,InstitutodeCiênciasBiomédicasAbelSalazar,UniversidadedoPorto(Portugal);4IPATIMUPInstitutodePatologiaeImunologiaMoleculardaUniversidadedoPorto(Portugal).clara.pereira@ibmc.up.ptSit4pisthecatalyticsubunitofatype2A-relatedproteinphosphataseinSaccharomycescerevisiae.Itis involved in a wide spectrum of cellular functions, including the transcriptional repression ofmitochondrialgenes,whichstronglyimpactonmitochondrialfunction[1].Inthisstudy,thehypothesisthatSit4pmayalsoregulatemitochondrialproteinspost-translationallywasinvestigated.Forthat,abidimensionalgelelectrophoresisandimmunoblottingprocedurewasusedtoidentifymitochondrialproteinswithincreasedphosphorylationinSit4pdeficientcells.Among9proteins identified,Atp2p,theβsubunitofF1FoATPsynthasecomplex,exhibitedthemostevidentincreaseinphosphorylation.Usingco-immunoprecipitationassays,wefoundthatSit4pandAtp2pinteractphysically.Usingmassspectrometry,twoAtp2pphosphorylationsites(Thr124andThr317)wereidentifiedinSit4pdeficientcells.ExpressingAtp2-T124Dor-T317DphosphomimeticsresultedinhigherAtp2plevels,accompaniedbyincreasedabundanceandactivityoftheF1FoATPsynthasecomplex.Italsoresultedinanincreaseinmitochondrialrespiration,highercellularATPlevelsandimprovedgrowthinrespiratoryconditions.OurresultsrevealaroleforSit4ponthepost-translationalregulationofAtp2pwhichmayplayaroleinthemetabolicadaptationtodistinctenergeticdemands.[1]JinCetal,FEBSLett,581(2007)5658-63.Acknowledgments:fundingprojectNorte-01-0145-FEDER-000008PortoNeurosciencesandNeurologicDisease Research Initiative at I3S supported by Norte Portugal Regional Operational Programme(NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European RegionalDevelopmentFund(FEDER).CPissupportedbyFSE/POPHthroughiFCTgrantIF/00889/2015.


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ControlofmitochondrialbioenergeticsandstructurebytheATPaseinhibitoryfactor1:apro-survivalrelayviaOPA1Danilo Faccenda1,2, Junji Nakamura3, Giulia Gorini1, Gurtej K Dhoot1, RebecaMartín-Jiménez1, Caterina Ferraina4, Daniela Strobbe4, Mauro Piacentini4,5,MasasukeYoshida3,ClaireRussell1andMichelangeloCampanella1,2,4

1 Department of Comparative Biomedical Sciences, Royal Veterinary College, London (UK); 2 UCLConsortium for Mitochondrial Research, University College London, London (UK); 3 Department ofMolecularBioscience,KyotoSangyoUniversity,Kamigamo-Motoyama,Kyoto(Japan);4DepartmentofBiological Sciences,UniversityofRomeTorVergata,Rome (Italy); 5National Institute for InfectiousDiseases,IRCCSLazzaroSpallanzani,Rome(Italy)[email protected](IF1)isaubiquitouslyexpressed,mitochondrialproteinthatisparticularlyabundantinhigh-metabolic-rateorgans,suchasheart,brainandliver.IF1istheprimaryendogenousinhibitoroftheF1Fo-ATPsynthasereversal.Therefore,itregulatesmetabolicadaptationtophysiologicalstressorsandpreventsATPdepletionunder ischaemia.Recentevidencesuggeststhattheinhibitoryprotein plays other key roles in mitochondrial function. Indeed, several human carcinomas arecharacterizedbyanincreasedlevelofIF1expression,whichprovidessurvivaladvantagetocancercellsby enhancing evasion of apoptosis, invasion and chemoresistance. Whereas, loss of IF1 activity isassociatedwithalterationinmitochondrialironassimilationandhemesynthesis,leadingtoanaemia.Here,weexaminedtheeffectof IF1onmitochondrial redoxbalanceandcristaeremodellingduringapoptosis,andfoundthattheanti-apoptoticproteinmaintainstheantioxidantcapacityofglutathione(GSH) and peroxiredoxin 3 (Prx3) systems through retention of mitochondrial ATP production.Alongside,IF1inhibitsthestress-inducedpeptidaseOMA1andpreventsthesubsequentcleavageofthepro-fusionproteinopticatrophy1(OPA1).IF1-mediatedpreservationofthemitochondrialantioxidantstatusandstabilizationofOPA1aretherebykeyeventsinimpedingmitochondrialderangementandcompletionofapoptosis.Thiswas further confirmed in IF1deficient zebrafishandmice,where theabsenceof functional IF1correlates with decreased levels of OPA1 in both brain and eye, causing neuronal loss and visualimpairment.WeproposethatIF1playsaroleinthecontrolofmitochondrialfunctionthroughthedual-regulationofmitochondrial bioenergetics and structure. By promoting cell growth and survival, IF1 not only is arelevantplayerincancer,butmightalsobeimportantlyinvolvedinthephysiologyofhighlyproliferativetissue,suchasthedevelopingneuralsystem.


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ElectrophysiologicalpropertiesofchannelformedbybovineFOF1ATPsynthaseinplanarlipidbilayerAndreaUrbani1,ChristophGerle2,ValentinaGiorgio1,3,VanessaChecchetto4,PaoloBernardi1,3andIldikòSzabò4

1Department of Biomedical Sciences,University of Padova, Padova (Italy); 2 Laboratory of ProteinCrystallography, Institute for Protein Research, Osaka University, Osaka (Japan); 3 CNR Institute ofNeuroscience(Italy);4DepartmentofBiology,UniversityofPadova,Padova(Italy)[email protected](PT)inmitochondriaistriggeredbyCa2+andspecificactivatorsandfinallyleadstoincreasedpermeabilitytoionsandsolutesoftheinnermitochondrialmembrane(IMM).InvitroPTisfollowedbymitochondrialswellingthatinturnpreventsATPsynthesis. Inthe1970stheideawasadvancedthatPTismediatedbyaCa2+-regulatedpore,namedthePermeabilityTransitionPore(PTP).PTPwasidentifiedasahigh-conductancechannelbypatch-clampingtheIMM,namedMitochondrialMegaChannel(MMC).DespitemanyyearsofstudyandthekeyroleofPTPopeningincelldeathandseveraldiseases, themolecularnatureof thePore remainsunclear. In2013Giorgioetal.providedevidencethatdimersofFOF1ATPsynthasepurifiedfromnativegelscanformthePTP/MMC[1]buttheexact mechanisms are still not clear. The present work was undertaken to further clarify theelectrophysiologicalpropertiesoftheATPsynthasebymeansofplanarlipidbilayer(PLB)singlechannelrecordings,exploitingaultra-purepreparationfrombovineheart[2].BovineFOF1ATPsynthasecomplexwasshown(i)tobeintactandinclusiveofallsubunits,bymeansofSDS-PAGEandmassspectrometry;and(ii)tobeactiveandhighlysensitivetooligomycin(>95%).FOF1ATPsynthasewasincorporatedinthePLBandthechannelactivitywasassessedinthepresenceofdifferentconcentrationsofCa2+andBz-423,asubstancethatisabletosensitizePTPtoCa2+.WereportevidenceofchannelactivitywiththeMMC-PTPkeyfeatures,includinghighconductance,activationbyCa2+,complexgatingkinetics,andinhibitionbyspecificPTPcompounds.[1]GiorgioVetal,PNAS110(2013)5887-9.[2]MaedaSetal,ActaCryst69(2013)1368–1370.


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MatrixCa2+isessentialforopeningofthemitochondrialpermeabilitytransitionporeLishuGuo,MichelaCarraro,ValentinaGiorgio,PaoloBernardiandValeriaPetronilli

Department of Biomedical Sciences, University of Padova and CNR Neuroscience Institute, Padova(Italy).lishu.guo@studenti.unipd.itIsolatedmitochondriacanundergoadramaticincreaseofpermeabilitytoionsandsolutesknownasthe permeability transition. The transition ismediated by the permeability transition pore (PTP), acyclosporineA(CsA)sensitivechannel,andismosteasilyobservedafterCa2+uptakeinthepresenceofawidevarietyof “inducingagents”.Recentlyweproposed thatdimersofF-ATPsynthase form thepermeability transition pore [1] and that Ca2+ binding to subunit β of F-ATP synthase induces aconformationalchangepotentiallyleadingtoPTPopening[2].However,althoughCa2+isessentialforPTPopening, ithasbeenreportedthataCsA-sensitivePTP inductioncanbeseenintheabsenceofaddedCa2+(orinthepresenceofEGTA)afterthetreatmentwiththeSHreagentphenylarsineoxide(PhAsO) [3] or with the arginine reagent p-hydroxyphenylglyoxal (OH-PGO) [4]. We investigatedwhethertheseeffectsdependonmatrixCa2+retainedduringtheisolationofmitochondria.InthisstudyweshowthatCa2+depletionwiththeionophoreA23187ormitochondrial loadingwiththechelatorBAPTApreventsPTPinductionbyPhAsOandOH-PGO.TogetherwiththefindingthatCa2+isanabsoluterequirementforopeningofthemegachannelingel-purified F-ATP synthase preparations incorporated in lipid bilayers [1], these results support theconclusionthatmatrixCa2+isaunique"permissive"factoressentialforPTPopening.[1]GiorgioVetal,PNAS110(2013)5887-5892.[2]GiorgioVetal,EMBORep(2017)inpress.[3]LenartowiczEetal,JBioenergBiomembr23(1991)679-688.[4]LinderMDetal,JBiolChem277(2002)937-942.


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The cytochrome b H291L mutation strongly impairs ubiquinol oxidation andprotontranslocationatthebacterialbc1QOsiteMarcoMalferrari1, Francesco Francia2, Pascal Lanciano3, Stefan Steimle3, FevziDaldal3andGiovanniVenturoli2

1DepartmentofChemistryGiacomoCiamician,UniversityofBologna(Italy);2DepartmentofPharmacyandBiotechnologies,UniversityofBologna(Italy);3DepartmentofBiology,UniversityofPennsylvania,Philadelphia(USA)[email protected] localizationofmetal ionbinding siteswas shown tobeavaluableapproach to identifyprotonpathways in redoxmembrane proteins. To this aim, the coordination of the Zn2+ binding site waselucidatedbyEXAFSspectroscopyintheavian,bovineandbacterialcytbc1complex[1].Specifically,putativeligandresidueshavebeenidentifiedforthepseudo-octahedralsiteofRhodobactercapsulatusandsuggestedtopartecipateintheprotonreleasewhichfollowsQH2oxidationattheQocatalyticsite.To test thisproposal, all the coordinating residuespickedupbyEXAFShavebeenmutated innon-protonatable residues. In a first screening, theH291Lmutantwas totally unable of photosyntheticgrowth.AnalysisperformedonnativemembranesshowedthatelectrontransferfromQotothehighpotentialFeS-cytcchain,aswellasreductionofcytbH,werestronglyinhibitedintheH291Lmutant.Moreover,protonreleaseassociatedwithQH2oxidationattheQositewasalsodramaticallyimpaired.Basedonthesefindings,ontheputativeroleofHis291inligandingZn2+,andonitssolventexposedandhighlyconservedposition,weproposethatHis291ofcytbiscriticalforprotonreleaseassociatedtoQH2oxidationattheQositeofcytbc1[2].[1]GiachiniLetal,BiophysJ88(2005)2038–2046.[2]FranciaFetal,BiochimBiophysActa1857(2016)1796-1806.


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Input of hydrogenases in H2 cycling and proton motive force generation inEscherichiacoliduringfermentationKarenTrchounian1,2andArmenTrchounian1,2

1 Scientific-Research Institute of Biology, Faculty of Biology, Yerevan State University, Yerevan(Armenia);2DepartmentofBiochemistry,Microbiology,andBiotechnology,FacultyofBiology,YerevanStateUniversity,Yerevan(Armenia).k.trchounian@ysu.amEscherichiacolihasthecapacitytoencodefourmembranebound[Ni-Fe]hydrogenases(Hyd)catalyzingthe redox reaction of 2H++2e-àH2. All hydrogenases are reversible: depending on pH and carbonsourcetheycanworkinH2uptakeorproducingmode.IthasbeenproposedthattheactivityofHydenzymesandtheirworkingdirectionalsodependonH2cyclingthroughthemembrane.HydenzymesformH2cycleduringglucoseorglycerolfermentationatpHof5.5-7.5.Moreover,itwasshownthatdeletionofthreeoftheHydenzymesdisturbsH2cycling.Probably,HydenzymesbyformingH2cycleandredoxchaintotheprotonF0F1ATPaseareworkingtowardsneutralizingtheendacids,equilibratingthecytoplasmaticpHandinvolvedinprotonmotiveforcegeneration.ItwasshownthatatpH7.5duringglycerolfermentationinhypFmutant(allHydenzymesareabsent)protonmotiveforcegeneratedishigher(122mV)comparedtowildtype(99mV).IncontrastatpH5.5inhypFmutantitwaslowerthaninwildtypeby~20mV.Inaddition,Hyd-1andHyd-2activityhasbeenshowntodependontheactivityofF0F1ATPaseatextremepHs(pH5.5andpH7.5)duringglucoseorglycerolfermentation.TakentogetheritmightbeconcludedthatbesidesF0F1ATPaseHydenzymesalsoare involved inprotonmotive forcegenerationduring fermentation.H2 cyclingasnewmembrane-associatedcyclecontributestowardsregulationofcytoplasmaticpHbyproducingoruptakingH2.Itissuggested that Hyd enzymes might be one of the main H+ sensing systems in the cell duringfermentativeconditions.


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AnewmitochondrialpotassiumchannelinvolvedincardioprotectionVanessaChecchetto1,AngelaPaggio2,DiegoDeStefani2,RosarioRizzuto2andIldikòSzabò1

1DepartmentofBiology,UniversityofPadova,Padova(Italy);2DepartmentofBiomedicalSciences,UniversityofPadova,Padova(Italy).vanessa.checchetto@unipd.itMitochondrialionchannelsareofgreatimportancetoensuretheproperfunctionofthisbioenergeticorganelleandtoregulatecellfate.However,inmanycasesourknowledgeconcerningtheirmolecularidentityandregulationisstilllimited.Recently,wecharacterizedanovelproteincomplexlocatedintheinner mitochondrial membrane that displays all known pharmacological and electrophysiologicalproperties of the long-sought mitochondrial KATP (mito KATP) channel. An important role has beenassignedtomitoKATP,especiallyintheheart,inprotectionfromI/Rinjury,pre-conditioningandpost-conditioningbutitscomposition,properties,regulationandfunctionconstitutethemostcontroversialtopicinthefieldofmitochondrialchannels.

SessionII:Specializedtransportersandporesstructure&function

23

VDAC3cysteineoxidativestateandchannelactivity:invitroandincellulostudies

SimonaReina1,RosariaSaletti2,MariaGaetanaGiovannaPittalà3,SerenaSineri1,FedericaZinghirino1andVitoDePinto3

1 Department of Biological, Geological and Environmental Sciences, Section of Molecular Biology,UniversityofCatania,Catania(Italy);2DepartmentofChemicalSciences,UniversityofCatania,Catania(Italy);3DepartmentofBiomedicalandBiotechnologicalSciences,UniversityofCatania,Catania(Italy).

[email protected]

Voltage-DependentAnionselectiveChannels (VDAC)arepore-formingproteins located in theoutermitochondrialmembrane.Theirrolesrangefromtheformationofahydrophilicconduitthroughthemembranethankstoitsbeta-barrelstructure,tolessunderstoodfunctionsthatmakethemkeyactorsin the cross-talkbetween themitochondrialmetabolismand thewhole cell. In this regard, despiteconserved sequences, similar structures, and the same gene organization, VDAC3 is the leastcharacterizedamongthethreeisoformsthatexistinmammals.Sincetherecentdiscoveryofitspeculiarchannelactivity[1],attentionhasbeenfocusedonunravelingthereasonsofthissurprisingbehavior.OneofthemostplausiblehypothesisseemstoresideinthegreaternumberofVDAC3cysteineresiduescomparedtoVDAC1.Basedonbioinformaticsprediction,allofthesixVDAC3cysteineswouldindeedbeexposedtowardsthe intermembranespace[2],makingthemapreferredtargetforoxidationbyReactiveOxygenSpecies.Accordingly,massspectrometryanalysisrevealedthateachcysteineresidueexistsinasetofdifferentoxidationstateswithinthesamemolecule[2,3].Moreover,studiesbasedonthe selective deletion of VDAC3 cysteines followed by in vitro conductance experiments andcomplementationassaysinΔporin1yeast,highlightedtheirroleinthefunctionoftheprotein[3].Alltogether,thesedataprovideevidenceforadirectinvolvementofcysteinesinVDAC3activity,raisingthepossibilitythatthisisoformcouldnotbefunctioningasasimplechannelwithinthecell.

[1]ChecchettoVetal,CellPhysiolBiochem34(2014)842-853.[2]ReinaSetal,Oncotarget7(2016)2249-2268.[3]SalettiRetal,BiochimBiophysActa1859(2017)301-311.


24

Structure/function relationships of human LAT1 amino acids transporter andscreeningofactive-sitetargetedinhibitorsMariafrancesca Scalise1, Lara Napolitano1, Michele Galluccio1, Ivano Eberini2,PanayiotisKoutentis3,AngeloCarotti4andCesareIndiveri1

1 Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and MolecularBiotechnology, University of Calabria, Arcavacata di Rende (Italy); 2 Dipartimento di ScienzeFarmacologiche e Biomolecolari, Università degli Studi di Milano, Milano (Italy); 3 Department ofChemistry,University of Cyprus,Nicosia (Cyprus); 4Dipartimento Farmaco-Chimico,Università degliStudiAldoMoro,Bari(Italy)[email protected](SLC7A5)isaplasmamembranetransporterforneutralandbranchedaminoacids;itbelongstotheHeterodimericAminoacidTransportergroup(HATs)andformsaheterodimerwiththeglycoproteinCD98(SLC3A2).LAT1mediatesanantiportreactionwitharandomsimultaneousmechanism.ThegreatinterestonLAT1islinkedtoitsover-expressioninmanycancersthatare“Glutamineaddicted”,needingalotofGlnfortheirpeculiarenergymetabolism.Inthisscenario,LAT1givesrisetoaGln/Leucycletogether with another transporter, ASCT2 feeding cancer cells. Then, LAT1 is considered as apharmacologicaltarget.ThedesignofgoodinhibitorsforhLAT1ischallengedbythelackof3DX-raystructure.Onlyhomologymodels,builtonthebasisoftheAdiCfromE.coli,areavailable.Recently,thebacterialover-expressionof recombinanthLAT1and its reconstitution inproteoliposomeshasbeenachieved.Key residues for substratebindingand translocationhavebeen identifiedbya combinedapproachofsite-directedmutagenesis,proteoliposometransportassayandbioinformatics:F252,S342,C335andC407arethemostcriticalresidues.Then,thepresenceofCysresiduesinthebindingsitehasbeen exploited to design inhibitors with higher potency and stability with respect to those so fardescribed.Ascreeningofanumberofdithiazoles,whichreactwiththiolgroups,hasbeenperformed;amongtestedcompounds,eighthavebeenidentifiedasbestinhibitorswithIC50inthemicromolarrange.PredictionbybioinformaticssuggeststhatinhibitionisduetomixeddisulfideformationwithatleastoneCysresidueoftheprotein.ThespecificC407Amutant,indeed,showeddecreasedreactivitytowardsdithiazoles indicatingthatthisresidue is involved inbinding. Interestingly, themostpotentinhibitorswerealsoabletoimpairviabilityofcancercellsexpressinghighlevelsofLAT1.Thesestudiesopennewimportantperspectivesforcancertherapy.


25

Regulation of the Mitochondrial Carnitine/Acylcarnitine Carrier by S-NitrosylationLaraConsole2,AnnamariaTonazzi1,NicolaGiangregorio1andCesareIndiveri1,2

1 CNR Institute of Biomembranes and Bioenergetics, Bari (Italy); 2 Department DiBEST (Biologia,Ecologia,ScienzedellaTerra)UnitofBiochemistryandMolecularBiotechnology,UniversityofCalabria,ArcavacatadiRende(Italy)[email protected] (NO)modulatesprotein functionsbypost-translationalmodifications.Oneof themostdiffusemechanism consists in S-nitrosylation of Cys residues of target proteins. Themitochondrialcarnitine/acylcarnitinecarrier (CAC)possessessixCys,someofwhicharemorereactivetowardsSHreagents. This transporter plays a central role in themitochondrial β-oxidation of fatty acids. ThepossibleregulationofCACfunctionbyS-nitrosylationwasstudiedbytreatingthetransporterwiththeNO-releasing compound, nitrosoglutathione, and testing the effect on transport activity inproteoliposomesreconstitutedwiththenativeortherecombinantratCAC.NOinhibitedtransportwithanIC50of74or71μM,respectively.TheparalleloccurrenceofS-nitrosylationwasrevealedbyanantiCys-NO antibody by WB analysis. To obtain information of NO effect under more physiologicalcondition,similarexperimentswereperformedwithintactmitochondria.Alsointhiscase,inhibitionofcarnitinetransportparalleltoS-nitrosylationwerefound.Interestingly,afterincubationoftheproteinwith DTE, both inhibition and S-nitrosylation were abolished, indicating that themodification wasreversibleandthatthereactionmechanismwasbasedonCysmodification.Thiswasthenestablishedby site-directedmutagenesis. Substitution of a specific Cys residue (C136) with Val, abolished theinhibitoryeffectofPTM.While,amultiplemutantcontainingonlyC136outofthesixCysresidues,behavedasthewildtype.Therefore,C136wasidentifiedasthetargetofNO.ThisresidueisconservedintheCACmembersofhigheranimalsandplantsbutnotininsectsorunicellulareukaryotes.InhibitionofCACoccursatNOconcentrationspresentincellsduringischemicconditions,thussuggestingthatnitrosylation may play an important role in controlling oxidation of fatty acids during altered cellmetabolismthuslimitingproductionofROS.


26

The second VDAC isoform in S. cerevisiae, an unknown protein: structural-functionalcharacterizationMariaCarmelaDiRosa1,AndreaMagrì1,SimonaReina1,CarloGuardiani2,MatteoCeccarelli3,DesirèeNicotra1andVitoDePinto4

1 Department of Biological, Geological and Environmental Sciences, Section of Molecular Biology,UniversityofCatania,Catania(Italy);2SchoolofEngineering,UniversityofWarwick,Coventry(UK);3Department of Physics, University of Cagliari, Cagliari (Italy); 4 Department of Biomedical andBiotechnologicalSciences,UniversityofCatania,Catania(Italy)[email protected] (Voltage Dependent Anion Channel) is the main channel of outer mitochondrial membrane(OMM) and mediates the exchanges of metabolites and ions between cytosol and inner ofmitochondria[1].Thisproteinishighlyconservedintheevolutionandwasstudiedinmanydifferentspecies. InyeastS.cerevisiaetherearetwogenesencodingtwoVDACisoforms:por1,encodingthemain isoform (yVDAC1), homologue to humanVDAC1, andmost abundant on theOMM,waswellcharacterized.Thesecondgenepor2encodesthesecondisoform,aputativeporinunknowmolecularandfunctional.S.cerevisiaedeletedofyVDAC1(∆por1strain)isunabletogrowthonnotfermentablecarbonsource,asglycerol,attherestrictivetemperatureof37°C.ThissuggeststheexistenceofanothermechanismthatensuresthemetabolitesfluxthroughtheOMMandapossiblefunctionforyVDAC2.TheoverexpressionofyVDAC2in∆por1straincomplementsyVDAC1absence[2].Overexpressionisnotaphysiologicalcompensativemechanism,since in∆por1strainthepresenceofpor2genedoesnotsupport the yeast growth, but an external stimulus has to be supplied to raise yVDAC2 levels andcomplement the growth impairment [3]. In this work we have purified and characterized therecombinant yVDAC2byelectrophysiologicalmethods. Furthermore, in silico computational studieshaveconfirmedourresults,highlightingmanystructuraldifferenceswithrespecttoVDAC1,thatcouldinfluencetheyVDAC2ownfunctionality.TheconclusionisthatyVDAC2hassimilar,butnotidentical,featureswithyVDAC1.[1]DePintoVetal,BiochimBiophysActa987(1989)1-7.[2]Blachly-DysonEetal,MolCellBiol17(1997)5727-5738.[3]MagrìAetal,BiochimBiophysActa1857(2016)789-798.


27

Structuralandfunctionalstudiesonmultidrugresistanceprotein6(MRP6):newinsightsAngelaOstuni,RocchinaMiglionico,FlaviaCuviello,MariaFrancescaArmentano,Maria Antonietta Castiglione Morelli, Monica Carmosino, Magnus Monnè andFaustinoBisaccia

DepartmentofSciences,UniversityofBasilicata,Potenza(Italy).angela.ostuni@unibas.itMRP6proteiniscodifiedbytheABCC6genebelongingtotheATP-bindingcassette(ABC)transporterssuperfamilythatuseenergyreleasedfromATPhydrolysistotransportsubstratesacrossmembranes[1]. Mutations of ABCC6 gene cause, through an unknown pathophysiological mechanism,pseudoxanthoma elasticum, a genetic disorder characterized by ectopic calcification of connectivefibers.ABCC6ismainlyexpressedinliverandkidneyandconferslowlevelresistancetosomeanticancerdrugsbutitsnaturalsubstrateremainundefined.TheMRP6structureconsistsoftwotransmembranedomains,twonucleotidebindingdomains[2,3]andanadditionalN-terminaltransmembranedomain(TMD0)connectedtocoreoftheproteinthroughtheloopL0.TMD0hasfiveTMsandaNout–Cinorientation[4];L0isinvolvedinthebasolateralmembranelocalizationoftheentireprotein[5].StableABCC6knockdownHepG2cellsshoweddysregulationofpro-andanti-mineralizationgenes[6],atypicalfeatureof replicativesenescenceandreductivestate [7].All these findingscertainlyallowus to re-evaluatetheroleofMRP6bothinphysiologicalandpathologicalconditions.[1]VasiliouVetal,HumanGenomics,3(2009)281–290.[2]OstuniAetal,ProtPeptLett17(2010)1553–8.[3]OstuniAetal,JBioenergBiomembr43(2011)465-71.[4]CuvielloFetal,FEBSLett589(2015)3921-3928.[5]MiglionicoRetal,JBioenergBiomembr48(2016)259–67.[6]MiglionicoRetal,CellMolBiolLett19(2014)517-26.[7]MiglionicoRetal,CellMolBiolLett22(2017)7.


28

DirectpharmacologicaltargetingofamitochondrialionchannelselectivelykillstumorcellsinvivoLuigi Leanza1, AndreaMattarei2, Katrin Anne Becker3,Michele Azzolini4,5, LuciaBiasutto4,5, Roberta Peruzzo1, Livio Trentin6, Mario Zoratti4,5, Erich Gulbins3,CristinaParadisi2andIldikòSzabò1

1Department of Biology,University of Padova, Padova (Italy); 2Department of Chemical Sciences,UniversityofPadova,Padova(Italy);3DepartmentofMolecularBiology,UniversityofDuisburg-Essen,Essen(Germany);4CNRInstituteofNeurosciences,Padova(Italy);5DepartmentofBiomedicalSciences,UniversityofPadova,Padova(Italy);6DepartmentofMedicine,UniversityofPadova,Padova(Italy)[email protected] are important oncological targets due to their crucial role in apoptosis. Our workidentifies a novel therapeutic tool that simultaneously exploits both the high expression of thepotassiumchannelKv1.3 in themitochondriaofvarious typesof cancercellsand thecharacteristicalteredredoxstateofmalignantcells,therebyleadingtotheselectiveeliminationofpathologicalcellsbytwomitochondria-targetedKv1.3 inhibitors. Indeed,theinhibitionofmitochondrialKv1.3bytwonoveldrugsaltersmitochondrialfunctionandleadstoROS-mediatedcelldeath.Theseinhibitorskilled98%ofexvivoprimarychronicB-lymphocytic leukemia tumorcellswhilesparinghealthyBcells. Inorthotopic mouse models of melanoma and pancreatic ductal adenocarcinoma, the compoundsreducedtumorsize,viaROS-mediatedselectiveapoptosisofcancercells,bymorethan90%and60%,respectively,withoutcausingsignificantsideeffectsofhealthytissues,likeimmune-depression,cardiactoxicityorhistologicalalteration.Thesefindingsthusoffertheperspectiveofamajoradvanceinthepharmacologicaltreatmentofsomehigh-impact,poor-prognosiscancers.

SessionIII:Specializedtransportersandpores,physiologicalandpathologicalroles

29

VDACinviabilityandenergeticcouplingstatusoftheinduciblePC12cellmodelofHuntington'sdiseaseDariaGrobys,AndonisKarachitos,KlaudiaKulczyńska,AdrianSobusiakandHannaKmita

LaboratoryofBioenergetics,InstituteofMolecularBiologyandBiotechnology,FacultyofBiology,AdamMickiewiczUniversityinPoznan,Poznan(Poland)[email protected](HD)isanautosomal-dominantandfatalneurodegenerativedisordercharacterizedbyaselectivelossofneurons.ItresultsfromCAGtrinucleotiderepeatexpansioninexon1ofHTTgeneencodinghuntingtin(Htt).Asthetrinucleotidecodesforglutamine,itsrepeatnumberhigherthan35resultsinanabnormallylongpolyglutaminetractinHttNterminusthatgivesrisetoitsmutantform(mHtt). It isnowobvious thatmitochondriaplayavital role inHDpathogenesisbut theunderlyingmechanismisstillelusive.Moreover,thefunctionalrelationshipsofHttandmHtttomitochondriaarestilluncertain.Thereforewedecidedtoinvestigatetheroleofthevoltage-dependentanionchannel(VDAC) inHttand/ormHtteffectonmitochondria.VDACtransports inorganic ionsandmetabolitesacrosstheoutermitochondrialmembraneandisregardedasadynamicregulator,orevengovernor,ofmitochondrialfunctions.TostudyVDACputativecontribution,weappliedPC12modelofHD,whichisbasedonPC12celllinesderivedfromapheochromocytomaoftheratadrenalmedullaandconsistsof PC-12HD-Q23andPC-12HD-Q74 cellswith inducible (bydoxycycline) andmonitoreddue toGFPlabeling, expression of Htt and mHtt, respectively. The obtained results including analysis ofreconstitutedVDACpropertiesaswellasthemodelcellviabilityandtheirenergeticcouplingstatusindicatethatVDACmayconstituteanimportantelementofcytotoxiceffecttriggeredbymHtt.Acknowledgments:thePC12celllineswereobtainedfromDavidRubinszteinandAndreasWyttenbach,UK; the studieswere supportedby the grant:NCN2011/01/B/NZ3/00359,GDWB-06/2015and theKNOWRNAResearchCentreinPoznanNo.01/KNOW2/2014.


30

Involvement of miRNAs in the acquired BRAF-I resistance of metastatic

melanomacells

SimonaBarbato1,AlessandraBaracca1,GiancarloSolaini1andMullerFabbri2

1DepartmentofBiomedicalandNeuromotorSciences,LaboratoryofBiochemistryandMitochondrialPathophysiology,University of Bologna, Bologna (Italy); 2Departments of Pediatrics andMolecularMicrobiology&Immunology,NorrisComprehensiveCancerCenter,KeckSchoolofMedicine,Universityof Southern California, Children’s Center for Cancer and Blood Diseases and The Saban ResearchInstitute,Children’sHospitalLosAngeles,LosAngeles(USA)[email protected] isahighlyaggressivecancer,withamedianoverall survival forpatientswithstageIVmelanomarangingfrom8to18months.ThemostcommonmutationBRAFV600E,thatinduceaconstitutiveactivationoftheMAPK/ERKpathway,canbeinhibitedbysmall-moleculekinaseinhibitors,among which Vemurafenib was approved in 2011 as themajor breakthrough in the treatment ofunresectableormetastaticmelanoma.Unfortunately,responsestoBRAFinhibitorsareshortlived,withevidenceofdiseaserelapsewithin6-8monthsafterthebeginningoftherapy,duetothedevelopmentofacquireddrugresistance,alongwithmitochondrialbioenergeticrearrangementofresistantcells[2].Intriguingly,severaloncogenicsignalingpathwayssupporttumorigenesisbymodulatingmicro-RNAs(miRNAs) [3] and data in the literature have strongly implicated aberrant miRNA expression inanticancerdrugresistanceandsensitivity.Nevertheless,onlylimiteddataexistontheimplicationofmiRNAsinmalignantmelanomasandlittleisstillknownaboutthefactorsunderlyingtheresistancetoBRAFinhibitionandtheirroleintheemergenceofmetabolicallyprivilegedcellphenotypes.Onthebasisofourpreliminaryresults,wehereinproposeamiRNAs-mediatedmechanismdrivingtheacquiredresistancetoBRAF-ItherapyandsetthestageforfurtherelucidationontheroleofmiRNAsasmetabolicswitchersinhumanmetastaticmelanomacells.[1]WellbrockCandHurlstoneA,BiochemPharmacol80(2010)561-567.[2]BaenkFetal,MolOncol10(2016)73-84.[3]BarbatoSetal,IntRevCellMolBiol(2017),inpress.


31

Novelpsoralen-derivativeswithincreasedsolubilityincancertreatmentRobertaPeruzzo.1,MicheleAzzolini2,3,MatteoRomio4,KatrinABecker5,AndreaMattarei4, Mario Zoratti2,3, Erich Gulbins5, Cristina Paradisi4, Luigi Leanza1 andIldikòSzabò1,2

1DepartmentofBiology,UniversityofPadova,Padova(Italy);2CNRInstituteofNeurosciences,Padova(Italy); 3DepartmentofBiomedical Sciences,Universityof Padova,Padova (Italy); 4DepartmentofChemicalSciences,UniversityofPadova,Padova(Italy);5DepartmentofMolecularBiology,UniversityofDuisburg-Essen,Essen(Germany).roberta.peruzzo@studenti.unipd.itIonchannelsareemergingasnewoncologicaltargets.Indeed,severalionchannelsshowadifferentexpressionpatterninnormalandcancercells.ThepotassiumchannelKv1.3hasamultiplesub-cellularlocalization, including both in the plasma membrane and in the inner mitochondrial membrane.Pharmacologicalinhibitionofthemitochondrialchannel(mtKv1.3),butnotoftheplasmamembranechannel, by membrane permeant blockers, Psora-4, PAP-1 and clofazimine, triggered apoptosis indifferent cancer cells. Cell death occurred even in the absence of Bax and Bak, by inducingmitochondrialmembranedepolarization,productionofmitochondrialROSandreleaseofcytochromec.DownregulationbysiRNAofKv1.3preventedalltheseeffects,indicatingspecificity.Sincemembranepermeant Kv1.3 inhibitors are characterized by poor water solubility, in order to increase theirbioavailability as well as their solubility, we have recently synthesized a few more soluble PAP-1derivatives.Thenewderivativeshavebeenfoundtoselectivelykillcancercellsandeveninvivoinamousemelanomapreclinicalmodel,withoutinducinganysideeffect.


32

ProteinKinaseAactivationpromotescancercellresistancetoglucosestarvationandAnoikisRoberta Palorini1,2*, Giuseppina Votta1*, Humberto De Vitto1, FrancescaRicciardiello1,KarstenHiller2andFerdinandoChiaradonna1

1 Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan (Italy); 2LuxembourgCentreforSystemsBiomedicine,Esch-sur-Alzette(Luxembourg).*[email protected] cells often rely on glycolysis to obtain energy and support anabolic growth. Several studiesshowedthatglycolyticcellsaresusceptibletocelldeathwhensubjectedtolowglucoseavailabilityortolackofglucose.However,somecancercells,includingglycolyticones,canefficientlyacquirehighertolerance to glucose depletion, leading to their survival and aggressiveness. Although increasedresistance to glucose starvation has been shown to be a consequence of signaling pathways andcompensatorymetabolicroutesactivation,thefullrepertoireoftheunderlyingmolecularalterationsremainelusive.Usingomicsandcomputationalanalyses,wefoundthatcAMP-PKAaxisactivationisfundamentalforcancercellsresistancetoglucosestarvationandanoikis.Notably,hereweshowthatsuchaPKA-dependentsurvivalismediatedbyparallelactivationofautophagyandglutamineutilizationthatinconcertconcurtoattenuatetheERstressandtosustaincellanabolism.Indeed,theinhibitionofPKA-mediatedautophagyorglutaminemetabolismincreasedthelevelofcelldeath,suggestingthattheinductionofautophagyandmetabolicrewiringbyPKAisimportantforcancercellularsurvivalunderglucose starvation. Importantly, both processes, togetherwith an increasedmitochondrial activity,activelyparticipatetocancercellsurvivalmediatedbysuspension-activatedPKAaswell.InadditionweidentifyalsoaPKA/Srcmechanismcapabletoprotectcancercellsfromanoikis.OurresultsrevealforthefirsttimetheroleoftheversatilePKAincancercellssurvivalunderchronicglucosestarvationandanoikisandmaybeanovelpotentialtargetforcancertreatment.

SessionIV:Bioenergeticsmetabolismandmitochondrialdysfunction

33

LonP1 differently modulates mitochondrial function and bioenergetics ofprimaryversusmetastaticcoloncancercellsLaraGibellini1,LorenaLosi2,SaraDeBiasi2,MilenaNasi3,DomenicoLoTartaro3,SimonePatergnani4,GianlucaCarnevale3,LucaRoncucci5,AndreaCossarizza1andMarcelloPinti2

1DepartmentofMedicalandSurgical Sciences forChildrenandAdults,UniversityofModenaandReggioEmilia,Modena(Italy);2DepartmentofLifeSciences,UniversityofModenaandReggioEmilia,Modena(Italy);3DepartmentofSurgery,Medicine,DentistryandMorphologicalSciences,UniversityofModenaandReggioEmilia,Modena(Italy);4DepartmentofMorphology,SurgeryandExperimentalMedicine,SectionofPathology,OncologyandExperimentalBiologyandLTTACenter,UniversityofFerrara,Ferrara(Italy);5DepartmentofDiagnostic,ClinicalMedicineandPublicHealth,UniversityofModenaandReggioEmilia,Modena(Italy)[email protected](mt)Lonprotease(LonP1)isamulti-functionenzymelocatedinthemtmatrix,abletobehave as a critical regulator in several human cancer, including colorectal cancer (CRC). ThemechanismsatthebasisofLonP1contributiontocolorectalcarcinogenesisarenotfullyunderstood.PreviousdataindicatedthatLonP1iscloselyrelatedtosurvivalinpatientswithCRC,andthatsilencingLonP1incoloncancercelllinesresultsinseveremtimpairmentandapoptosis.Here,weinvestigatedthe role of LonP1 in mt functions in colon primary tumour cells and in metastasis. First, LonP1expressionwasquantifiedbywesternblotbothinformalin-fixedparaffin-embeddedCRCtissuesandfresh frozen aberrant crypt foci (ACF), adenoma (Ad) and CRC. Second, the effects of LonP1overexpressiononmtstructure,functionsandbioenergetics,andoncellmetabolismwereanalysedinfourcoloncell lines.Exvivo, LonP1wasalmostabsent innormalmucosa,gradually increased fromsamplesofACFtoAd,andwasmostabundantinsamplesofCRC.Invitro,LonP1overexpressionleadstoasignificantreductionofoxygenconsumptionrate,andthisoccurredinSW620metastaticcoloncancercellsratherthaninSW480primarycoloncancercells.OverexpressionofLonP1inSW620cellsdeterminesdepolarizedmitochondria,reducedlevelsofmtsuperoxideandalteredmtultrastructure.LonP1overexpressioninSW480cellsprimarilyaffectstheexpressionofproteinsinvolvedinglycolysis,including lactate dehydrogenase and glucose transporter-1. To investigate whether metabolismreshaping by LonP1 reflects modification in cell aggressiveness, the levels of proteins involved inepithelial-mesenchymal transition were quantified in SW480 and SW620 overexpressing LonP1.Overexpressionof LonP1 inSW480cells leads to significantly lower levelsofE-cadherinandhigherlevelsofβ-ctn.Conversely,overexpressionofLonP1inSW620cellswasassociatedwithlowerlevelsofβ-ctn.


34

Theresponseofthemitochondrialqualitycontrolsystemtogradedtraumaticbraininjury

GiacomoLazzarino1,AngelaM.Amorini1,ValentinaDiPietro2,AntonioBelli2andBarbaraTavazzi1

1 Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Rome (Italy); 2Neuroscience andOphthalmology ResearchGroup, Institute of Inflammation and Ageing, School ofClinicalandExperimentalMedicine,CollegeofMedicalandDentalSciences,UniversityofBirmingham,Birmingham(UK).g.lazzarino@libero.itMitochondrialdynamicsareregulatedbyacomplexsystemofproteinsrepresentingthemitochondrialquality control (MQC). MQC balances antagonistic forces of fusion and fission determiningmitochondrialandcellfates.Inchronicandacuteneurodegenerations,suchastraumaticbraininjury(TBI), the leading cause of death below 45 years of age in Western countries, dysfunctionalmitochondria are thought to contribute to the pathophysiologicalmechanisms of cell damage.WepreviouslyfoundthatmildTBI(mTBI)causesatransientmitochondrialmalfunctioningaffectingtheirphosphorylating capacity, a transitory imbalance in tissue antioxidants and a temporary glucosedysmetabolism.Differently,sTBIproducedpermanentmitochondrialmalfunctioningwithdecreasedphosphorylatingcapacityandprofoundATPdepletion,permanentdecrease inGSH[58],permanentglucosedysmetabolismwithmarkedhyperglycolysisandexcitotoxicitycausedbyincreaseinglutamateandexcitatoryaminoacids,aswellasadecreaseofN-acetylaspartate.Usingbrainsamplesfromthesecohortsofanimals(controls,mTBIandsTBIrats),wehereevaluatedthemaingeneandproteinexpressioninvolvedintheMQCsystem.At6,24,48and120hoursaftermTBI or sTBI, gene and protein expressions of fusion and fission were measured in brain tissuehomogenates.Results showed that genes and proteins inducing fusion or fission were upregulated anddownregulated, respectively, inmTBI, but downregulated and upregulated, respectively, in sTBI. Inparticular,OPA1,regulatinginnermembranedynamics,cristaeremodelling,oxidativephosphorylation,waspost-translationallycleavedgeneratingmostlylongorshortOPA1inmTBIandsTBI,respectively.Corroboratedbydataoncitratesynthase,theseresultsconfirmthetransitory(mTBI)orpermanent(sTBI)mitochondrialdysfunction,enhancingMQCimportancetomaintaincellfunctionsandindicatinginOPA1anattractivepotentialtherapeutictargetforTBI.


35

Novel pathogenicmechanisms from investigationof humanHydrogen SulfidemetabolismFrancesca Malagrinò1,2, Henrique G. Colaço3, Karim Zuhra1,2, Paulo E. Santo4,5,ElenaForte2,AndréGutierres4,MarziaArese2,TiagoM.Bandeiras4,5,PaoloSarti2,PaulaLeandro6,JoséA.Brito4,JoãoB.Vicente4andAlessandroGiuffrè1

1CNRInstituteofMolecularBiologyandPathology,Rome(Italy);2DepartmentofBiochemicalSciences,SapienzaUniversityofRome(Italy);3InstitutoGulbenkiandaCiência,Oeiras(Portugal);4InstitutodeTecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras (Portugal); 5InstitutodeBiologiaExperimentaleTecnológica,Oeiras(Portugal);6ResearchInstituteforMedicinesand Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon(Portugal)[email protected](H2S),alongwithnitricoxide(NO)andcarbonmonoxide(CO),belongstoasmallgroupofgaseoussignallingmolecules,collectivelytermed‘gasotransmitters’.H2ShasbeenrecognizedtoplayaregulatoryroleincellbioenergeticsactingasaJanus-facedmolecule.Thegasisindeedabletoinhibitcomplex IV (COX) in themitochondrial respiratory chain at higher levels, but also to stimulateATPsynthesisatlowerconcentrationsactingasarespiratorysubstrate[1].H2Ssynthesisandcatabolismareaccomplished,partlyinthemitochondrion,byenzymesthatarecurrentlyinvestigatedbyourgroupincollaborationwithJoãoVicente(ITQB-UNL,Portugal).H2Sisinvolvedinimportantpatho-physiologicalprocesses; several pathologies are indeed associatedwith alterations of H2Smetabolism, includingoncologic,neurodegenerativeandmetabolicdiseases.Amongthese,classicalhomocystinuria,anotyetfully understood metabolic disease, results from mutations in the gene encoding a key enzymecontrollingthehomocysteineandH2Slevelsinhumans,thepyridoxal5’-phosphate(PLP)-dependentand heme-containing cystathionine β-synthase (CBS). CBS is positively regulated by S-adenosyl-L-methionine (AdoMet), but fully inhibited by CO orNO• [2-3]. Herewe report that the p.P49L CBSvariant,asrecombinantlyproducedinE.coliandpurified,displaysbothanimpairedH2S-generatingactivityrescuedbyPLPsupplementationalongtheproteinpurificationand,surprisingly,amarkedlyincreasedaffinityoftheferroushemeforCO,likelyleadingtoenzymeinhibitionatphysiologicalCOconcentrations[4].WesuggestthattheenhancedpropensitytoCOinhibitiondocumentedhereforthep.P49LCBSvariantcouldrepresentanovelpathogenicmechanisminclassicalhomocystinuria.[1]VicenteJBetal,BiochimBiophysActa1857(2016)1127-38.[2]VicenteJBetal,JBiolChem289(2014)8579-87.[3]VicenteJBetal,J.BiolChem291(2016)572-581.[4]VicenteJBetal,OxidMedCellLongev(2017)8940321.


36

AlisporivirrescuesmitochondrialrespiratorydysfunctionsinDuchennemusculardystrophymodelsMarco Schiavone1*, Alessandra Zulian1*, Sara Menazza1, Francesca Sardone1,ValeriaPetronilli1,FrancescoArgenton2,LucianoMerlini3,PatriziaSabatelli4andPaoloBernardi1*

1DepartmentofBiomedicalSciencesandCNRNeuroscience Institute,UniversityofPadova,Padova(Italy);2DepartmentofBiology,UniversityofPadova,Padova(Italy);3DepartmentofBiomedicalandNeuromotorSciences,UniversityofBologna,Bologna(Italy);4CNRInstituteofMolecularGeneticsandLaboratoryofMusculoskeletalCellBiology,IstitutoOrtopedicoRizzoli-IRCCS,Bologna(Italy).*[email protected];[email protected] dystrophy (DMD) is a devastatingmuscle disease of known ethiology withouteffective,orgenerallyapplicabletherapy.Mitochondriaareaffectedbythediseaseinanimalmodels(hamster,chickenandmouse)butwhethermitochondrialdysfunctionispartofthepathogenesis inpatientshasnotbeenaddressedbefore.WeshowthatprimaryculturesobtainedfrommusclebiopsiesofDMDpatients(i)displayadramaticdecreaseofrespiratoryreservecapacityand(ii)arepronetomitochondrialdysfunctionduetoopeningofthepermeabilitytransitionpore(PTP).TreatmentwiththecyclophilininhibitorAlisporivir-acyclosporinAderivativethatdesensitizesthePTPbutdoesnotinhibitcalcineurin- largelyrestoredthemaximalrespiratorycapacitywithoutaffectingbasaloxygenconsumptionincellfrompatients,thuseffectivelyreinstatinganormalrespiratoryreserve.TreatmentwithAlisporivir,butnotwithcyclosporinA,ledtoastrikingrecoveryofmusclestructureandrespiratoryfunction matching improved survival of sapje zebrafish, a severe model of DMD where theultrastructuralmuscledefectsareclosetothoseofDMDpatients.AlisporivirhasanexcellentsafetyprofileandcouldbeusedfortreatmentofDMD.


37

The role of mitochondria in the maintenance of endothelial function afterexposuretoredoxinjuryGinevra Nannelli1, Valentina Giorgio2, Sandra Donnini1, Daria Mochly-Rosen3,PaoloBernardi2andMarinaZiche1

1DepartmentofLifeSciences,UniversityofSiena,Siena(Italy)2DepartmentofBiomedicalSciences,University of Padova, Padova (Italy); 3 Department of Chemical and Systems Biology, StanfordUniversity,Stanford(USA)[email protected]

Eventhoughendothelialcells(ECs)appeartomeetmostoftheirenergyneedsanaerobically,theyhaveanextensivemitochondrialnetworkandconsumeoxygen.Theirmitochondriaarecoupledandpossessa considerable respiratory reserve,which is important in the response to oxidative stress, but themechanisticaspectsofthecrosstalkbetweenmetabolismandendothelialfunctionhaveonlystartedto become appreciated, with numerous questions remaining to be addressed [1]. Mitochondrialaldehydedehydrogenase(ALDH2)isresponsibleforthemetabolismofacetaldehydeandothertoxiclipidaldehydes.Ourdatadocumentedthatbetaamyloid(Aβ)peptidesinduceaprematuresenescencephenotypeproducingalterationsinendothelialfunctionbytargetingALDH2[2].Theaimofthisstudyis to investigate the role of Aβ1-40 and ALDH2 silencing on HUVECs mitochondrial function. Ourpreliminary results suggest that Aβ1-40 and ALDH2 silencing reduce maximal respiration andmitochondrialreservecapacity,whichisexpectedtoleadtodecreasedabilitytorespondtosecondaryenergetic stressors. We suggest that preserving ALDH2 activity may help preserve mitochondrialreservecapacityinstressconditions,andprovideanovelstrategyforthetreatmentofangiogenicormetabolicdiseases.

[1]PotenteMandCarmelietP,AnnuRevPhysiol79(2017)43–66.[2]SolitoRetal,JCellSci26(2013)1952–1961.

Acknowledgment:workwassupportedbyPrin-Bando2015Prot.20152HKF3Z

SessionV:ReactiveOxygenSpeciesandmitochondria

38

ReactiveoxygenspeciesdecreaseinhumancellsadaptedtohypoxiaGiuliaGorini,GianlucaSgarbi,AnnaCostanzini,SimonaBarbato,GiancarloSolaini,AlessandraBaracca.

DepartmentofBiomedicalandNeuromotor Sciences, LaboratoryofBiochemistryandMitochondrialPathophysiology,UniversityofBologna,Bologna(Italy).alessandra.baracca@unibo.itHypoxiainducesseverechangesincellsbioenergetics,dramaticallyimpairingmitochondrialrespirationand ATP production. Indeed, we have previously demonstrated how the oxphos complexes aredifferentlymodulatedwhenhypoxiccellsareculturedineitherthepresenceorabsenceofglucose[1].Whentheoxygentensiondecreases,thehypoxia-inducibletranscriptionfactorHIF-1αisstabilizedandaccumulates,inducingtheactivationofhundredsofgenesinvolvedintheadaptationtohypoxia[2].Interestingly,HIF-1αisalsostabilizedbynon-hypoxicstimuli,andROSarehypothesizedtobepartofsuchmechanism[3].Nevertheless,animportantissuestillcontroversialiswhetherROSlevelincreasesordecreasesinhypoxia,sinceconflictingresultswerereported.Inarecentstudy[4]weaddressedhowmoderate hypoxia (1%O2) affectedROS content in primary human fibroblasts grown in glucoseorglucose-freemedium:theoxygenlimitationcausedasteepdecreaseofROS,supportedinprolongedexposurebymitophagyincellsgrowninthepresenceofglucoseandmainlybytheenhancementofantioxidant enzymes in cells cultured in glucose-freemedium. These results will be discussed andcomparedwiththoseobtainedanalyzingatransformedcellmodel.

[1]BaraccaAetal,IntJBiochemCellBiol45(2013)1356-1365.[2]SemenzaGL,BiochemJ405(2007)1-9.[3]HamanakaRBandChandelNS,CurrOpinCellBiol21(2009)894-899.[4]SgarbiGetal,IntJBiochemCellBiol88(2017)133-144.


39

Misfolded SOD1 mutant alters VDAC1-Hexokinase 1 complexes promotingmitochondrialdysfunctioninanAmyotrophicLateralSclerosismodel

MariannaFloraTomasello1, FrancescaGuarino2,AndreaMagrì3, SimonaReina3,LoredanaLeggio2andAngelaMessina3

1 CNR Institute of Biostructures and Bioimaging, Catania (Italy); 2 Dept. Biological, Geological andEnvironmental Sciences, University of Catania, Catania (Italy); 3 Dept. Biological, Geological andEnvironmentalSciences,UniversityofCatania,Catania(Italy).

[email protected]

TheVoltage-DependentAnionChannel(VDAC)isoform1representsthemainporinofMitochondrialOuter Membrane (MOM) and allows metabolic exchanges between cytosol and mitochondria. Inphysiologicalconditions,VDAC1interactswiththeglycolyticenzymeHexokinase1(HK1),lettingittogainadirectaccesstothesynthetizedATP.Furthermore,thecontactbetweenVDAC1-HK1stimulatescellsurvivalbyhamperingVDAC1interactionwiththepro-apoptoticproteinBax.IntheneurodegenerativediseaseAmyotrophicLateralSclerosis(ALS),severalmisfoldedformsoftheantioxidantenzymeSuperoxideDismutase1(SOD1)werefoundboundtoVDAC1,asituationbelievedtocompromisethemitochondrialmetabolism[1].However,themitochondrialaccumulationofSOD1mutants occurs exclusively in the spinal cord, a tissue characterized by lower amounts of HK1 incomparisontothebrainortheliver;thissuggeststhatinmotorneuronsVDAC1couldbemorepronetointeractwithSOD1mutantsinsteadofinteractingwithHK1.Here,bymeansofinvitroandincelluloapproaches,weprovedtheinteractionbetweentheALS-relatedSOD1mutantG93AandVDAC1anddemonstratedthatSOD1G93AisincompetitionwithHK1forthesameVDAC1bindingsite(s).Moreover,wefoundthatSOD1G93Aisableto influencetheHK1sub-cellulardistributioninALScellmodelandthatitsoverexpressionrescuestheNSC34cellsfromSOD1G93Atoxicitybyincreasingcellviabilityandmitochondrialmembranepotential[2].Overall,ourresultsindicateaclearinvolvementofHK1inmitochondrialdysfunctionsinSOD1-mediatedALScasesandsuggestaprotectiveroleofHK1inALSpathology.[1]IsraelsonAetal,Neuron67(2010)575-587.[2]MagrìAetal,SciRep6(2016)34802.


40

Hexokinases1peptide impairsVDAC1-SOD1G93Amutanttoxic interaction inAmyotrophicLateralSclerosis:implicationsfortheneurodegenerativedisease’streatment

AndreaMagrì1,RamonaBelfiore2,StefanoContiNibali1,MariaCarmelaDiRosa1,LoredanaLeggio1,VitoDePinto2andAngelaMessina1

1Dept.Biological,GeologicalandEnvironmentalSciences,UniversityofCatania,Catania(Italy);2Dept.BiomedicalandBiotechnologicalSciences,UniversityofCatania,Catania(Italy).andrea.magri@unict.itMitochondrialdysfunctionintheneuromusculardisorderAmyotrophicLateralSclerosis(ALS)isanearlykeyeventofmotorneurondegenerationandcorrelateswiththeaccumulationofmisfoldedformsoftheantioxidantenzymeSuperoxideDismutase1(SOD1)ontothecytosolicsurfaceofmitochondria.TheVoltage-Dependence Anion Channel (VDAC) isoform 1 is the main porin of Mitochondrial OuterMembrane(MOM)andactsasadockingsiteforseveralSOD1mutants,aswellasforothermisfoldedproteins inmanyneurodegenerativediseases. Inaphysiological condition,VDAC1allowsmetabolicexchanges between cytosol and mitochondria and interacts with Hexokinase 1 (HK1) repressingapoptosis.However,theinteractionofSOD1G93AmutantwithVDAC1affectschannelconductanceandaltersHK1bindingtoVDAC1,indicatingacompetitionbetweenSOD1G93AandHK1bindingwithVDAC1.Starting from this information, we designed and tested a short peptide based on HK1 N-terminaldomainsequence(NHK1).NHK1wasabletospecificallybindVDAC1andmodulateitschannelactivityatthePlanarLipidBilayer,inasimilarmannertothatofthewholeHK1.Furthermore,theadditionofNHK1peptidetothepurifiedVDAC1ormitochondria,stronglypreventsSOD1G93Adeposition,andimprovesbothmitochondrialfunctionandcellviabilityintheALS-cellularmodel[1].Overall,ourresultsindicatethatNHK1peptideisapromisingtherapeutictoolforALStreatment.

[1]MagrìAetal,SciRep6(2016)34802.

Acknowledgments:FondazioneUmbertoVeronesi(Milano, Italy)forapost-doctoralfellowshiptoA.Magrì.


41

Mitochondrial reactive oxygen species sensitize T-ALL cells to apoptosis byengagingtheOMA1-OPA1axisGloria Scattolin1,Micol Silic-Benussi2, Ilaria Cavallari2, SoniaMinuzz12, SamuelaFrancescato3,PaoladelBianco2,GiuseppeBasso3,StefanoIndraccolo2,DonnaM.D’Agostino4andVincenzoCiminale1,2

1 Department of Surgery, Oncology, and Gastroenterology, University of Padova, Padova (Italy); 2IstitutoOncologicoVeneto-IRRCS,Padova(Italy);3DepartmentofWomanandChildHealth,Haemato-OncologyDivision,UniversityofPadova,Padova(Italy);4DepartmentofBiomedicalSciences,UniversityofPadova,Padova(Italy)[email protected]%ofpatientswithpediatricacuteT-lymphoblasticleukemia(T-ALL)arerefractorytocurrent glucocorticoid-based therapies. In the present study, we investigated the possibility toselectivelysensitizeT-ALLcellstoapoptosisbyincreasingtheirlevelsofmitochondrialreactiveoxygenspecies (mtROS). To this end, we employed NS1619, a synthetic benzimidazolone derivative thatincreasesmtROS production by opening the large conductance Ca2+-activated K+ (BK) channel anddehydroepiandrosterone (DHEA), which blunts ROS scavenging through inhibition of the pentosephosphatepathway.NS1619andDHEAincreasedmtROSandinduceddeathofT-ALLcelllines,patient-derivedxenografts(PDX)andprimarycellsfromT-ALLpatients(includingcasesofrefractoryT-ALL),butdid not induce death of normal human thymocytes or PBMC. The importance of depowering ROS-scavengingpathways to increase theefficacyofROS-producing treatmentswasunderscoredby thefindingthatNS1619andDHEAactivatedNRF2,themasterregulatorofantioxidantpathways.NS1619andDHEA induced cleavageofOPA1, amitochondrial protein that controls cristae remodeling andcytochrome cmobilization. OPA1 cleavage and cell deathwere inhibited by the ROS scavenger N-acetylcysteine and by siRNA-mediated knock-down of the mitochondrial protease OMA1, whosetargetsincludeOPA1.Importantly,NS1619andDHEAsensitizedT-ALLcellstodeathinducedbyTRAILor by the glucocorticoid dexamethasone, both of which are known to inducemitochondrial outermembranepermeabilization(MOMP)throughtheactivationofBid(TRAIL)orBim(Dexamethasone).Takentogether,ourfindingsprovidethefirstevidenceforaroleoftheOMA1-OPA1axisinsensitizingT-ALL cells to apoptosis, and suggest that effective targeting of refractory T-ALL requires amultidimensionalpharmacologicalapproachthatcombinesincreasedmtROSproduction,inhibitionofscavengingpathwaysandMOMP.


42

Dynamicphotosyntheticmembrane

AlexanderV.Ruban

DepartmentofCellandMolecularBiology,QueenMaryUniversityofLondon,London(UK)

[email protected]

Thephotosyntheticmembraneisthemostprotein-richbiologicalmembrane.Itperformsanumberofenergy transformation functions that include photon absorption, energy transfer between thephotosyntheticlightharvestingantennacomplexes,excitationenergytrappingbythereactioncentercomplexes, chargeseparationandelectron transportbetweenanumberof redoxelectroncarriers,coupledtoprotontranslocationacrossthemembrane,reductionofNADPandsynthesisofATP.Thissequenceofcoherently-coupledeventsisfinelyregulated,adjustingtochangesinenvironmentalandmetabolicfactors.Therefore,thestudyoftheseadaptivedynamicsofthephotosyntheticmembraneandthefactorsthatgovernthemfallincreasinglyinafocusofmodernphotosynthesisresearch.ThelightharvestingantennaofphotosystemII(LHCII)ofhigherplantscarriesupto70%ofallproteininthephotosynthetic membrane. It therefore inevitably governs membrane structure, dynamics andfunctions.Currently,agreatdealofinformationexistsontheorganizationoftheantennasofhigherplants around photosystems and attempts are being made to interpret the significance of thesestructuresforlightharvesting.However,verylittleissaidandactuallyknownaboutthepropertiesandprocessesinvolvedintheshort-termdynamicsoflightharvestingantennaunderlyingtheadaptationstotheenvironment.Here,IwillpresentaviewontheroleofthecompositionandfeaturesoftheLHCIIantenna in enabling the dynamics of the photosyntheticmembrane thatmakes its light harvestingfunctionflexibleevenonaminute’stimescale.

SessionVI:NewandnotablethemesinBioenergeticsandBiomembranes

43

Light-Drivenenergyproductionforcell-freemetabolicsystemsKyleAndrewMinor1,2,CarloDavidMontemagno1,2

1IngenuityLab,Alberta(Canada);2DepartmentofChemicalandMaterialsEngineering,UniversityofAlberta,Alberta(Canada)[email protected] and adjusting the energy needs at the nanoscale as enzymatic cofactors are the mostsignificantandcostprohibitivelimitationsofCell-freemetabolicsystems(CFMS).Ideally,adevicemustbecapableofregulatingcofactorbalancewithoutimpactingcarbonflux,onlyrequireinexpensiveandabundantinputsandbecompatiblewithanyCFMS.AnexampleofsuchadeviceisanartificialorganellewhichATPaseandBacteriorhodopsinareco-reconstitutedintoamembraneforATPregeneration[1,2].Hereweintroduceanengineeredbiotic/abioticorganellecapableofrecyclingNADH,merelyrequiringlight andwater [3]. This device pairs twomembrane protein complexes: Photosystem II (PSII) andNADH:Ubiquinoneoxidoreductase (CMI).Throughcontrolledvectorialassembly, these twoproteinsfunctioncollectivelytoreduceNAD+usingwaterassubstrateandgeneratingoxygenasthesoleby-product. In thiswork,weshowthe rateofNADHproduction isproportional toquantumfluxwhilecouplingefficienciesnearunity.Applying this technology tocell-freemetabolic systems (CFMS)willpermit control over NADH balance and eliminate constraints on the design of new biosyntheticpathways.[1]LuoTJMetal,NatureMeterials4(2005)220-224.[2]WendellDetal,NanoLett10(2010)3231-3236.[3]MinorKAetal(2016)underreview.


44

Flavin-basedelectronbifurcationinanaerobicBacteriaandArchaeaWolfgangBuckel

FachbereichBiologie,Philipps-Universität,35032Marburg,[email protected] complex III (cytochrome bc1) of the mitochondrial respiratory chain, quinone-based electronbifurcation isthewell-establishedheartoftheQ-cycle. In2008wediscoveredflavin-basedelectronbifurcationinstrictanaerobicbacteriaandarchaea,whichamplifiesthereducingpowerofanelectrontothelevelofferredoxinatthecostoftheotherelectronfromthesamedonorpair,suchasNADHorhydrogen [1,2]. In the anaerobic bacteriumAcidaminococcus fermentans the bifurcating system iscomposedoftwosolubleproteins,theelectrontransferringflavoprotein(EtfAB)andthebutyryl-CoAdehydrogenase (Bcd). EtfAB and Bcd together catalyze the bifurcation of 2 NADH (E0' = –320mV)exergonicallytocrotonyl-CoAyieldingbutyryl-CoA(E0'=–10mV)andendergonicallyto2ferredoxinsaffording 2 reduced ferredoxins (E' = –500 mV) [3, 4]. The reduced ferredoxin is mainly used forhydrogenproductionandgenerationofelectrochemicalNa+gradientsmediatedbymembrane-boundferredoxin-NAD reductase (Rnf) [5]. Acetogenic bacteria or methanogenic archaea bifurcate theelectronsofH2toferredoxinforthereductionofCO2,wherebyNADorheterodisulfideactaspositiveelectron acceptors [2]. The crystal structures of EtfAB fromA. fermentans [3] and the (EtfAB-Bcd)4complexfromClostridiumdifficile[submitted]gaveinsightsintothemechanismofelectronbifurcation.[1]HerrmannGetal,JBacteriol190(2008)784-791.[2]BuckelWetal,BiochimBiophysActa,1827(2013)94-113.[3]ChowdhuryNPetal,JBiolChem,289(2014)5145-5157.[4]ChowdhuryNPetal,FEBSJ,282(2015)3149-3160.[5]ChowdhuryNPetal,JBiolChem291(2016)11993-12002.


45

ExtracellularvesiclesareindependentmetabolicunitswithasparaginaseactivityNunzio Iraci1,10,EdoardoGaude2,TommasoLeonardi1,3,AnaS.H.Costa2,ChiaraCossetti1, Luca Peruzzotti-Jametti1, Joshua D. Bernstock1,4, Harpreet K. Saini3,Maurizio Gelati5,6, Angelo Luigi Vescovi6,7, Carlos Bastos8, Nuno Faria8, Luigi G.Occhipinti9,AntonJ.Enright3,ChristianFrezza2andStefanoPluchino1

1Wellcome Trust-Medical Research Council StemCell Institute, CliffordAllbutt Building, CambridgeBiosciencesCampus,DepartmentofClinicalNeurosciences-DivisionofStemCellNeurobiology,andNIHRBiomedicalResearchCentre,UniversityofCambridge,Cambridge(UK);2MRCCancerUnit,Universityof Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge (UK); 3EuropeanMolecularBiologyLaboratory,EuropeanBioinformaticsInstitute(EMBL-EBI),WellcomeTrustGenome Campus, Hinxton, Cambridge (UK); 4 Stroke Branch, National Institute of NeurologicalDisorders and Stroke, National Institutes of Health (NINDS/NIH), Bethesda (USA); 5 Stem CellsLaboratory,CellFactoryandBiobank,AziendaOspedaliera 'SantaMaria',Terni (Italy);6 IRCCSCasaSollievodellaSofferenza,SanGiovanniRotondo(Italy);7DepartmentofBiotechnologyandBiosciences,UniversityofMilano-Bicocca,Milano(Italy);8MedicalResearchCouncil-ElsieWiddowsonLaboratory,FulbournRoad,Cambridge(UK);9DepartmentofEngineering,ElectricalEngineeringDivision,UniversityofCambridge,Cambridge (UK);10Presentaddress:DepartmentofBiomedicalandBiotechnologicalSciences(BIOMETEC),UniversityofCatania,Catania(Italy)[email protected] vesicles (EVs) are membrane particles involved in the exchange of a broad range ofbioactivemoleculesbetweencellsandthemicroenvironment.WhileithasbeenshownthatcellscantrafficmetabolicenzymesviaEVsmuchremainstobeelucidatedwithregardtotheirintrinsicmetabolicactivity.Accordingly,hereinweassessedtheabilityofneuralstem/progenitorcell(NSC)-derivedEVstoconsumeandproducemetabolites.BothourmetabolomicsandfunctionalanalysesrevealedthatEVsharbourL-asparaginaseactivitycatalysedbytheenzymeAsparaginase-likeprotein1(Asrgl1).Critically,weshowthatAsrgl1activityisselectiveforasparagineandisdevoidofglutaminaseactivity.WefoundthatmouseandhumanNSC-derivedEVstrafficASRGL1.OurresultsdemonstrateforthefirsttimethatNSCEVsfunctionasindependent,extracellularmetabolicunitsabletomodifytheconcentrationsofcriticalnutrients,withthepotentialtoaffectthephysiologyoftheirmicroenvironment.


46

MitochondriamonitoringinstudiesofsuccessfulanhydrobiosisAndonisKarachitos1,MilenaRoszkowska2,MartynaOsiecka1,ŁukaszKaczmarek2,HannaKmita1

1LaboratoryofBioenergetics,InstituteofMolecularBiologyandBiotechnologyand2DepartmentofAnimalTaxonomyandEcology,InstituteofEnvironmentalBiology,FacultyofBiology,AdamMickiewiczUniversityinPoznań,[email protected] tardigrade species are capable of anhydrobiosis, defined as a reversible state induced bydesiccation (the lack of liquid water). The phenomenon includes entering, permanent and leavingstages corresponding to the dehydration, tun and rehydration stages, respectively. Undoubtedly,anhydrobiosis canbe regardedasanorganized stateandas such it requires some formsofenergysupply.Itisclearthatfunctionalmitochondriaguaranteethepropertunformation,butinthelightofthe available data mitochondria appear to be underestimated in studies on cellular/molecularmechanismsofsuccessfulanhydrobiosis.Thereforerecoveryfromthedesiccatedstageisatpresentthe only available indicative feature of successful anhydrobiosis. In our study we try to explainmitochondriacontributiontotardigradesuccessfulanhydrobiosiswithemphasisontheirroleduringthetunstageabletorecovertotheactivestage.Thereforeweapplyfunctionalmitochondrialabelingin living individuals ofHypsibius dujardini (Doyère, 1840) andRamazzottius subanomalus (Biserov,1985),representingtardigradespeciesofdifferentcapabilitytoundergoanhydrobiosis.Thelabelingisobtained by the cell-permeant, cationic, lipophilic fluorophore tetramethylrhodaminemethyl ester(TMRM)whichistransportedintomitochondriainthepresenceoftheinnermembranepotential.Theresultinglabelingismonitoredunderthefluorescencemicroscope.Thepreliminaryresultsindicatethatthis approachmay clarify the role ofmitochondria in the tun stage andmay add to descriptionofmitochondriafunctioningduringdifferentstagesofsuccessfulanhydrobiosis.Acknowledgements: the work was supported by the National Science Centre of Poland (grant no.2016/21/B/NZ4/00131), and performed with the contribution of the Biodiversity and AstrobiologyResearchgroup(BARg)atAdamMickiewiczUniversityinPoznań.


47

Genotype variation at the MSTN locus is associated with skeletal musclemitochondrial abundance and fibre composition in untrained Thoroughbredhorses.

MaryFRooney1,2,EmmelineWHill1,LisaMKatz3andRichardKPorter2

1UCDSchoolofAgricultureandFoodScience,UniversityCollegeDublin,Belfield(Ireland);2SchoolofBiochemistryandImmunology,TrinityBiomedicalSciencesInstitute(TBSI),TrinityCollegeDublin,Dublin(Ireland);3UCDSchoolofVeterinaryMedicine,UniversityCollegeDublin,Belfield(Ireland).

[email protected]

Variationinthemyostatin(MSTN)gene,mostnotablyasinglenucleotidepolymorphism(SNP)inthefirstintron(g.66493737C>T)andanSINEinsertionpolymorphism(Ins227bp)inthepromoterregion,hasbeenreported tobeassociatedwith racedistance,bodycompositionandskeletalmuscle fibrecompositioninthehorse.TheaimofthepresentstudywastotestthehypothesisthatMSTNvariationinfluencesmitochondrialphenotypesinequineskeletalmuscle.InasetofN=143horses,thetwoMSTNpolymorphismswere in complete concordance.Mitochondrial abundanceand skeletalmuscle fibretypesweremeasuredinwholemusclebiopsiesfromthegluteusmediusskeletalmuscleinN=82(n=37CC/II, n=34 CT/IN, n=11 TT/NN) untrained Thoroughbred horses (21 ± 3 months). Mitochondrialabundancewassignificantly(P<0.01)lessinthepresenceoftheC-allele/insertionalleleandskeletalmusclefibretypeproportionsweresignificantly(P<0.01)differentamongthethreeMSTNgenotypes.CC/IIhorseshadalowerproportionoftypeIfibresalongwithahigherproportionoftypeIIXfibrescompared toCT/INandTT/NNanimals, indicating that variation inMSTN genotype correlateswithmitochondrialabundanceandfibrecompositioninuntrainedThoroughbredhorses[1].TypeIskeletalmusclefibresaremoreoxidativeandcontainmoremitochondriathantheglycolytictypeIIXskeletalmuscle fibres, therefore the fibre composition data described is in agreement with the observedmitochondrialabundancedifferences.ThedatadescribedinthisstudyisalsoconsistentwithpreviouspublishedreportsthatthepresenceoftheSINEinsertionalleleisassociatedwithfewertypeIfibresandmoretypeIIXfibres.

[1]RooneyMFetal,BiochimBiophysActa–Bioenergetics1857(2016)Supple31.


48

Zebrafish(Daniorerio)asamodeltostudythepathophysiologicalroleofthemitochondrialchaperoneTRAP1

Claudio Laquatra1, Ionica Masgras1, Marco Schiavone1, Alessandra Zulian1,GiovanniMinervini1, Silvio Tosatto1, Andrea Vettori2, Francesco Argenton2, andAndreaRasola1

1DepartmentofBiomedicalSciencesandCNRInstituteofNeurosciences,UniversityofPadova,Padova(Italy);2DepartmentofBiology,UniversityofPadova,Padova(Italy).claudiolaquatra@gmail.comExpressionoftheHsp90-familychaperoneTRAP1isrestrictedtomitochondriaandincreasedinmosttumortypes.WehavepreviouslyshownthatTRAP1elicitsthestabilizationofthetranscriptionalfactorsHIF1α,thusgeneratingapseudohypoxicstatethatsupportsneoplasticgrowth.Followingoncogenicactivation of the Ras/ERK pathway TRAP1 is phosphorylated by ERK1/2, leading to inhibition ofsuccinatedehydrogenase(SDH),thecomplexIIofrespiratorychain,andtotheensuingaccumulationoftheoncometabolitesuccinate,whicheventuallystabilizesHIF1α.However,littleisknownaboutthetimingandimportanceofTRAP1inductionduringtheprocessofneoplasticonsetandgrowth,aswellasontheroleofTRAP1inthephysiologyofnon-transformedcells.ToanswerthesequestionswedecidedtoexploittheZebrafishmodel,whosebioenergeticfeaturesarestillpoorlyinvestigated.WefoundthatTRAP1ishighlyexpressedduringtheearlystagesofZebrafishembryodevelopment,butitslevelsaredramaticallydecreasedat96hpf.Notably,anincreaseinTRAP1proteinlevelwasdetectedafterHIF1αstabilization,causingareductioninSDHactivity,andaninsilicoanalysisofZebrafishTRAP1promotor showed thepresenceofhypoxic responsiveelementsmotifs.Moreover,TRAP1ishighlyexpressedinaZebrafishmodelofpancreaticadenocarcinomainducedbyKRasG12DexpressioninPtf1apositivecellswhereitcausesastrongdecreaseofSDHactivity,whileitisabsentinnormalpancreas.Altogether,thesedatasuggesttheexistenceofapositivefeedbackloopbetweenHIF1αandTRAP1,inwhich HIF1α stabilization induces TRAP1 expression acting at the transcriptional level, and in turnTRAP1,throughSDHinhibition,leadstoHIF1αstabilization.Thisregulatorymechanismcouldplayanimportant role for theadaptationof cells to fluctuating levelofoxygenboth inembryogenesisandduringtheprocessofneoplastictransformation.


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AnalysisoftheroleofLetm1inthedevelopmentofzebrafishSarfarazhussainFarooqui1,3,StefanHajny1,EvaScheuringer1,FriederikeSchlumm2,KristinTessmar-Raible1andKarinNowikovsky3

1MaxF.PerutzLaboratories,UniversityofVienna,CampusViennaBiocenter,Vienna(Austria);2TheResearchInstituteofMolecularPathology,ViennaBiocenter(VBC),Vienna(Austria);3Dept.ofInternalMedicine1,AnnaSpiegelCenterofTranslationalResearch,MedicalUniversityVienna,Vienna(Austria).sarfaraz.farooqui@univie.ac.atMitochondriaareindispensableforthecellularenergyproductionmostlybyoxidativephosphorylation,whichisverycriticalforenergydependenttissueslikenervoussystem.Additionally,theyhavealsobeen associated with a role in maintaining ion homeostasis (by the coordinated function of iontransporters and ion release exchange systems). LETM1 is an essential mitochondrial membraneproteinregulatingmitochondrialvolumeandcationhomeostasis.AbsenceofLETM1isassociatedwithseizures in WolfHirschhorn syndrome [1]. To explore the mechanism correlating mitochondrialdysfunctionwith seizures andbetter understanding ifmitochondrial ion homeostasis is involved inactionpotentialandepilepsywehavegeneratedazebrafishgeneticmodelofLETM1deficiencyusingTALENbasedgenomeediting technique. Here,wereport the resultsof theongoingexperiments inprogresstocharacterizetherelationshipofLetm1deficiencywithepilepsybyanalyzingtheirneuronalphenotypesinfivedifferentways(a)analyzingthelocomotorbehaviorunderastressinducingassay(b) calcium imaging in neuronal population [2] (c) mitochondrial dynamics using mitofish [3] (d)analyzinggrowthduringdevelopment. Theprogress in theseexperimentswillbepresented in themeeting.[1]HartLetal,DisModelMech7(2014)535-545.[2]AhrensMBetal,NatMethods10(2013)413-420.[3]PlucinskaGetal,JNeurosci32(2012)16203-16212.


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InductionofautophagyandmitochondrialbiogenesisbythenaturalcompoundPterostilbene:mechanismsandbiomedicalpotentialMartina La Spina1,2,Michele Azzolini1,2,Marco Schiavone2,Mario Zoratti1,2 andLuciaBiasutto1,2

1CNRInstituteofNeuroscience,Padova(Italy);2DepartmentofBiomedicalSciences,UniversityofPadova,Padova(Italy)[email protected](Pt),aphenoliccomponentofblueberries,hasbeenproposedtobebeneficialinmanyhumanpathologiessuchasthemetabolicsyndrome,neurodegenerativediseasesandcancer.However,the molecular mechanisms underlying its effects have been poorly characterized. Several paperssuggest that Ptmay induce autophagy. This process ismainly regulatedby Transcription Factor EB(TFEB),whoseaction iscontrolledby inhibitoryphosphorylationbymTORC1,whichretains it inthecytosol, and dephosphorylation by Ca2+-dependent Calcineurin, which promotes its nucleartranslocationandthusitstranscriptionalactivity.We confirmed that Pt at physiologically relevant (low µM) levels promotes autophagy in vitro byinducingTFEBnuclearmigration,andtracedbacktheupstreamsignalingcascadetoROSgenerationbymitochondria. Indeed,pre-treatmentwithanantioxidantreducedtheTFEBnucleus/cytosolratio. Inagreement,weobservedinhibitionofmitochondrialrespiratorychaincomplexIbyPt.ROSseemtobeconnectedtoanincreaseofcytoplasmicCa2+whichactivatesCalcineurin.CyclosporinA,whichblocksCalcineurin,reducedPt-inducednucleartranslocationofTFEBaswell.BothROSandCa2+areinvolvedinAMPKactivation.AMPKinturninhibitsmTORC1,facilitatingTFEBmigration. This metabolic sensor is known to play an important role in promoting mitochondrialbiogenesisthroughPgc-1α.Accordingly,wefoundthatPtbothup-regulatesPgc-1αbyactingonAMPK,andincreasesthemitochondrialcontentofcells.TheseresultsledustotestthebiomedicalpotentialofPtinazebrafishmodelofCollagenVImyopathiescharacterizedbyadefectiveautophagyandaccumulationofdamagedmitochondria.Strikingly, thismolecule partially rescued both motor and structural abnormalities in these fish at sub-µMconcentrations. Preliminary experiments suggest that these effects are indeed associatedwith theactivationofautophagyandmitochondrialbiogenesis.


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Flavonoidtransportinplantmicrosomes:characterizationofquercetinuptake.

AntonioFilippi,ElisaPetrussaandEnricoBraidot

DepartmentofAgricultural,Food,AnimalandEnvironmentalSciences,UniversityofUdine,Udine(Italy)[email protected] flavonoids are one of themost studiedmembers of natural polyphenolic family due to theirbiochemical, physiological and pharmacological effects in both plants and animals. The biologicalactivities of plant flavonoids depend on concentration and pH and for this reason theirtransport/absorptionandstoragesystemsareunderstrictattention,althoughaclearscenarioisfartobe elucidated. Besides transport attributable only to energized transporter systems, recentobservationshypothesizethatflavonoidscouldpassthroughmembraneswithoutthesupportofATPhydrolysis,openinganewinterestingandunknownmechanismofmembranepermeation.Inthiswork,wedevelopedanewmethodologyfortheanalysisofinvivotransportofquercetin(QC)acrossplantbio-membranes.Tothispurpose,weusedmicrosomalvesiclesobtainedfromPisumsativumetiolatedstemsasamodel.WetestedthevariationoffluorescencedependentonpH,thetransportmodulationusingoxidative\reducing agents in addition to protein crosslinker and the effective entranceofQCwithinthemicrosomestroughmetaholicextractions.Thisworkhasallowedustoproposeaneweasy-to-useandfastassay,aimingatmonitoringflavonoidpassivetransportacrossbio-membranes.


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LISTOFPARTICIPANTS

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AgrimiGennaro [email protected]

AvetisyanArmine [email protected]

BaraccaAlessandra [email protected]

BarbatoSimona [email protected]

BarileMaria [email protected]

BernardiPaolo [email protected]

BuckelWolfgang [email protected]

BuschKarin [email protected]

CapitanioNazareno [email protected]

ChecchettoVanessa [email protected]

ChiaradonnaFerdinando [email protected]

CoccoTiziana [email protected]

ConsoleLara [email protected]

ContiNibaliStefano [email protected]

DePintoVito [email protected]

DiRosaMariaCarmela [email protected]

FaccendaDanilo [email protected]

FarooquiSarfarazhussain [email protected]

FilippiAntonio [email protected]

ForteElena [email protected]

FranciaFrancesco [email protected]

FrezzaChristian [email protected]

GalberChiara [email protected]

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GibelliniLara [email protected]

GiuffrèAlessandro [email protected]

GoriniGiulia [email protected]

GrobysDaria [email protected]

GuarinoFrancesca [email protected]

GuoLishu [email protected]

IndiveriCesare [email protected]

IraciNunzio [email protected]

KarachitosAndonis [email protected]

KmitaHanna [email protected]

LaspinaMartina [email protected]

LaquatraClaudio [email protected]

LazzarinoGiacomo [email protected]

LeanzaLuigi [email protected]

LeggioLoredana [email protected]

LippeGiovanna [email protected].

MagrìAndrea [email protected]

MalagrinòFrancesca [email protected]

MalferrariMarco [email protected]

MancusoFrancesca [email protected]

MartinelliFabio [email protected]

MessinaAngela [email protected]

MinorKyle [email protected]

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NannelliGinevra [email protected]

NicotraDesirè [email protected]

NowikovskyKarin [email protected]

OstuniAngela [email protected]

PacelliConsiglia [email protected]

PereiraClara [email protected]

PeruzzoRoberta [email protected]

PiccoliClaudia [email protected]

PintiMarcello [email protected]

PittalàMariaGaetanaG. [email protected]

PorterRichard [email protected]

RapaportDoron [email protected]

ReinaSimona [email protected]

RooneyMary [email protected]

RubanAlexander [email protected]

ScaliseMariafrancesca [email protected]

ScattolinGloria [email protected]

SchiavoneMarco [email protected]

SineriSerena [email protected]

SolainiGiancarlo [email protected]

TomaselloFlora [email protected]

TrchounianKaren [email protected]

TretterLazlo [email protected]

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TrevisanElena [email protected]

UrbaniAndrea [email protected]

VivarelliSilvia [email protected]

WalkerJohn [email protected]

ZinghirinoFederica [email protected]

ZuhraKarim [email protected]

ZulianAlessandra [email protected]

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