Deciphering the molecular basis of ferroportin resistance to hepcidin: Structure/function analysis of rare SLC40A1 missense mutations found in suspected hemochromatosis type 4 patients

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Deciphering the molecular basis of ferroportin resistance to hepcidin: Structure/function analysis of rare

SLC40A1 missense mutations found in suspected hemochromatosis type 4 patients

M. Le Tertre, C. Ka, J. Guellec, I. Gourlaouen, C. Férec, I. Callebaut, G. Le Gac

To cite this version:

M. Le Tertre, C. Ka, J. Guellec, I. Gourlaouen, C. Férec, et al.. Deciphering the molecular basis of

ferroportin resistance to hepcidin: Structure/function analysis of rare SLC40A1 missense mutations

found in suspected hemochromatosis type 4 patients. Transfusion Clinique et Biologique, Elsevier,

2017, 24 (4), �10.1016/j.tracli.2017.07.002�. �hal-01679064�

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TransfusionCliniqueetBiologique24(2017)462–467

Research perspectives

Deciphering the molecular basis of ferroportin resistance to hepcidin:

Structure/function analysis of rare SLC40A1 missense mutations found in suspected hemochromatosis type 4 patients

Mécanismes de résistance de la ferroportine à l’hepcidine : analyses structure/fonction de mutations faux-sens rares du gène SLC40A1 identifiées chez des patients présentant une surcharge en fer

typique de l’hémochromatose de type 4

M. Le Tertre

, C. Ka

, J. Guellec

, I. Gourlaouen

, C. Férec

, I. Callebaut

, G. Le Gac

aInsermUMR1078,facultédemédecineetdessciencesdelasanté,universitéBretagneLoire–universitédeBretagneOccidentale,IBSAM,IBRBS,22,rue Camille-Desmoulins,29200Brest,France

bLaboratoryofExcellenceGR-Ex,institutImagine,24,boulevardduMontparnasse,75015,Paris,France

cLaboratoiredegénétiquemoléculaireethistocompatibilité,hôpitalMorvan,CHRUdeBrest,2,avenueFoch,29200Brest,France

dÉtablissementfran¸caisdusang,Bretagne,sitedeBrest,46rueFélix-Le-Dantec,29200Brest,France

eAssociationGaetan-Saleun,29,rueFélix-Le-Dantec,29200Brest,France

fMuséumd’histoirenaturelle,IRDUMR206,case115,IMPMC,Sorbonneuniversités–UMRCNRS7590,UPMCuniversitéParis06,4,placeJussieu,75252 Pariscedex05,France

Availableonline18August2017

Abstract

Geneticmedicineappliedtothestudyofhemochromatosishasidentifiedthesystemicloopcontrollingironhomeostasis,centeredonhepcidin- ferroportininteraction.Currentchallengesaretodissectthemolecularpathwaysunderlyingliverhepcidinsynthesisinresponsetocirculatory iron,HFE,TFR2,HJV,TMPRSS6andBMP6functions,andtodefinethemajorstructuralelementsofhepcidin-ferroportininteraction.Webuilt afirst3Dmodelofhumanferroportinstructure,usingthecrystalstructureofEmrD,abacterialdrugeffluxtransporteroftheMajorFacilitator Superfamily,astemplate.Themodelenabledstudyofdisease-associatedmutations,andguidedmutagenesisexperimentstodeterminetheroleof conservedresiduesinproteinstabilityandirontransport.Resultsrevealednovelaminoacidsthatarecriticalfortheironexportfunctionandthe hepcidin-mediatedinhibitionmechanism:forexample,tryptophan42,localizedintheextracellularendoftheferroportinporeandinvolvedinboth biologicalfunctions.Here,weproposeastrategythatisnotlimitedtostructureanalysis,butintegratesinformationfromdifferentsources,including humandisease-associatedmutationsandfunctionalinvitroassays.ThefirstmajorhypothesisofthisPhDthesisisthatferroportinresistanceto hepcidinreliesondifferentmolecularmechanismsthatarecriticalforferroportinendocytosis,andincludeatleastthreefundamentalsteps:(i) hepcidinbindingtoferroportin,(ii)structuralreorganizationoftheN-andC-terferroportinlobes,and(iii)ferroportinubiquitination.

©2017ElsevierMassonSAS.Allrightsreserved.

Keywords: Ironmetabolism;Hemochromatosis;Ferroportin;Hepcidin;Gain-of-functionmutations Résumé

L’étudedesformesraresd’hémochromatoseacontribuéàunemeilleureconnaissancedesmécanismescellulairesetsystémiquesquiparticipent aumaintiendel’homéostasieduferetquidépendentdel’axehepcidine-ferroportine.Desquestionsfondamentalesmajeuresrestentposéessurles voiesd’activationdelasynthèsed’hepcidineenfonctiondelaquantitédeferplasmatiqueetdel’actionàlasurfacedeshépatocytesdesprotéines

∗Correspondingauthor.

E-mailaddress:marlene.letertre@etudiant.univ-brest.fr(M.LeTertre).

http://dx.doi.org/10.1016/j.tracli.2017.07.002

1246-7820/©2017ElsevierMassonSAS.Allrightsreserved.

M.LeTertreetal./TransfusionCliniqueetBiologique24(2017)462–467 463 HFE,TFR2,HJV,TMPRSS6etBMP6,ainsiquesurlesbasesstructuralesdel’interactionhepcidine-ferroportine.Nousavonsconstruitunpremier modèletridimensionneldelaferroportinehumaineàpartirdelastructurecristallographiéed’EmrD,unexportateurdedroguesbactérienqui appartientàlafamilledestransporteurssecondairesMFS(«MajorFacilitatorSuperfamily»).Cettemodélisationapermis,enlienavecl’étude fonctionnelledemutationsfaux-sensidentifiéeschezdespatientsprésentantdesphénotypestypiquesd’hémochromatosedetype4,d’identifier desacidesaminésquiontunefonctioncritiquedanslastabilitédelaferroportine,lafonctiond’exportdufer,oularégulationparl’hepcidine.

Letryptophaneenposition42s’est,parexemple,révéléimportantàlafoispourlapriseenchargeduferetl’endocytosedelaferroportineàla suitedelafixationdel’hepcidine.Nousproposonsiciunestratégiequicombineanalysesstructurales,corrélationsgénotype/phénotypeettests fonctionnelsinvitro.Cetravaildethèseviseàrévélerlesdifférentsmécanismesmoléculairesquisous-tendentleprocessusdedégradationdela ferroportine.Ceprocessussemblecomprendretroisétapesessentielles:(i)fixationdel’hepcidineàlaferroportine,(ii)réorganisationstructurale deslobesNetC-terminauxdelaferroportine,et(iii)ubiquitinationetinternalisationdutransporteur.

©2017ElsevierMassonSAS.Tousdroitsr´eserv´es.

Motsclés:Métabolismedufer;Hémochromatose;Ferroportine;Hepcidine;Mutations«gain-de-fonction»

1. Introduction

Ironisessentialtonormalcellbiology.Asacomponentof heme,itisparticularlyimportantforerythropoiesisandoxygen transport.However,ironexcessand“free”reactiveironistoxic.

Bodyironlossisinsufficient,andintertwinedmechanismshave evolvedtomaintainintracellularandsystemicironhomeostasis.

Ferroportin (FPN1,also referred toas SLC40A1, SLC11A1, MTP1andIREG1)istheonlyknownironexporterinmammals andisconsideredakeycoordinatorofironbalancebetweenthe twocompartments[1].

Ferroportin is expressed in all types of cell that handle majorironflow,includingiron-recyclingmacrophages,absorp- tiveenterocytesandstoringhepatocytes(Fig.1).Ferroportinis predominantlyregulatedbytheliver-derivedpeptidehepcidin, whichbindsferroportinonthecellsurface,inducinginternal- izationanddegradation[2].Hepcidin-ferroportininteractionis critical inbothnormal ironhomeostasis andincommoniron metabolismpathologies,includingnotonlyinheriteddisorders andironoverloadbutalsonon-geneticdiseasesandanemia[3].

1.1. Regulationofferroportinactivity

Ferroportin expression is regulated transcriptionally, post- transcriptionally,andpost-translationally.Atthecellularlevel, ferroportinsynthesis is regulated via the iron-responsive ele- ment/ironregulatoryproteinssystem.Thissystemorchestrates thepost-transcriptionalregulationofseveralotherimportantiron metabolismgenes[4].Attheleveloftheorganism,ferroportinis subjecttopost-translationaldownregulationviatheliver-derived peptidehepcidin[2,5].Themolecularmechanismofhepcidin- mediatedferroportindownregulationisnotfullyunderstood,but includesatleastthreefundamentalsteps:

• hepcidin bindingtoferroportin, astep that involvesamino acidresiduep.Cys326[6];

• ferroportinubiquitination, astepthat is thoughtto involve severallysineresiduesbetweenpositions229and269[7];

• ferroportintraffickingtothemultivesicularbodyanddegra- dationinthelateendosome/lysosomecompartment[8].

1.2. DichotomouspatternsofhumanSLC40A1mutation Inhumans,mostferroportinmutationsaffectplasmamem- branelocationand/orironexportability.Theseloss-of-function mutationsexplaintheclassicalreportsofisolatedserumferritin elevation,relativeplasmairondeficiencyandpreferentialiron retentionincellsofthereticuloendothelialsystem.Thishisto- logicalpicture iscommonlyreferredtoas ferroportindisease [9].

In contrast, some SLC40A1 mutations do not impair the abilityofferroportintoexportironbutresultinpartialtocom- pleteresistancetohepcidin.Patientswiththesegain-of-function mutations(hemochromatosistype4)usually displayhistolog- ical andclinical presentationssimilar to thoseof the typical HFE-relatedhemochromatosis.Moreespecially,duetogreater flowofironthroughiron-recyclingmacrophagesandincreased ironabsorption,transferrinsaturationisexpectedtobemarkedly elevatedinthesepatients[10,11].

1.3. Ferroportinstructure

Thestructuralorganizationofhumanferroportininthelipid bilayeriscontroversial.Wallaceandcollaboratorsreportedthe first modelof the3D structureof humanferroportin, with12 predicted transmembrane helices built de novo (i.e., without homology).Theysubsequentlyfittedthemodeltoexperimental data,usingEscherichiacoliglycerol-3-phosphate(GlpT)trans- porter as template [12]. This template belongs to the Major Facilitator Superfamily (MFS),whichis the largest groupof secondaryactivemembrane transporters,transporting arange ofdiversesubstratesacrossmembranes.

BonaccorsidiPatiandcollaboratorsdescribedafairlysimilar 3D model of ferroportin, based on threading/ab initio mod- eling [13]. Thisisnot surprising,since,independently of the algorithms used, the Italian group predicted an inward-open conformationoftheferroportinirontransporter,usingGlpTas template.TheyusedadifferentMFSmember,E.coliL-fucose protonsymporter(FucP),tobuildanoutward-openconforma- tionofferroportin.Theswitchbetweenthetwoconformations isbelievedtodriveirontransportacrosstheplasmamembrane.

Fig.1.Schematicrepresentationofironmetabolism.IronispartlyimportedinduodenalenterocytesbyDMT1(DivalentMetalTransporter1),storedinhepatocytes andrecycledfromsenescentredbloodcells(RBC)inmacrophages.Ferroportin(FPN1)mediatesironexportintothebloodstreamfromiron-absorptiveenterocytes, iron-recyclingmacrophagesandiron-storinghepatocytes.Theironreleasedintotheplasmaistransportedbytransferrinmainlytothebonemarrowforerythropoiesis.

Cellularironexportisregulatedbyhepcidin,whichdirectlybindsferroportinandleadstoitsinternalizationanddegradation.

Webuiltathird3Dmodelofferroportin,usingatruecom- parativemodelingapproach,withsensitivemethodsforremote homology detectionbased on profile–profile alignments, and foralignmentrefinement(specifichydropathyprofiles,deduced fromhydrophobicclusteranalysis),andvalidatingtheuseofthe crystalstructureofoneMFSantiporter,E.colimultidrugefflux proteinD(EmrD),astemplate[14].ThestructureofEmrDwas determined inanoccluded state.It ismorecompactthanthe crystalstructureofGlpT,andthe12transmembranehelicesare arrangedaroundacentralpore.

Acomparisonofthe3Dmodelsofallpossiblestatesaccom- panyingirontransportisessentialforunderstandingthestructure andbiologyofhumanferroportin. Inadditiontotheoutward- andinward-facingstates,MFStransportersarethoughttoforma transitionaloccludedstate,inwhichthesubstrateissequestered frombothsidesofthemembrane.Therecentreportofthecrys- talstructuresof aputativebacterial homologueof ferroportin (BbFPN) andthe description of intra-domain conformational rearrangementsduringirontransportopensupnewavenuesfor predictingtheatomicdetailsoftheorganizationofferroportin transmembranehelicesandelucidatingthedetailedmechanisms ofironegressandhepcidin-mediateddownregulation[15].

2. Objectives

Fundamentalquestionsconcerningferroportinstructureand biologyremaintobeanswered.Basedonstructuralandmuta- tionalanalyses,weaimto:

• getinsightintotheinteractionofhepcidinwithferroportin;

• characterizesequenceinvariantsthatmayplayakeyrolein conformationalchangesfollowinghepcidinbinding;

• improveinterpretationof raremissensemutationsfoundin suspectedhemochromatosispatients.

Ourresearchprojectisorganizedaroundthreequestions.

2.1. Howcanthestudyofmissensemutationsidentifiedin hemochromatosispatientshelptorevealthemolecular mechanismsunderlyingphenotypicvariabilityandtoshed lightonproteinfunction?

As mentioned above, ferroportin gene mutations fall into 2 functional categories, responsible for different phenotypes (hemochromatosis type 4 vs. ferroportin disease). Most are unable to export iron (loss-of-function mutants; ferroportin disease), while others retain full capability but are insensi- tivetodown-regulationbyhepcidin(gain-of-functionmutants;

hemochromatosistype4).Mostferroportinmutantsareprivate, and most alteramino acids. Thishinders bothdiagnosis and therapeuticdecision-making,butstructure-functionstudiesmay shedlightonferroportinfunctionanditsrelationshipwithhep- cidin.Thisisexemplifiedbytheresultsoftwopreviousstudies:

thefirststudieddisease-causingmutationsandrevealedaclear dichotomybetweengain-of-functionmutations,whichclustered in a regionexposed to the extracellular milieu, accessibleto hepcidin,andloss-of-functionmutations,locatedatthecytoplas- micinterface,whereintracellularironisdeliveredtoferroportin forexport[14];thesecondfunctionallycharacterizedmissense mutationsofunknownsignificanceandidentifiednovelcritical aminoacids[11].

M.LeTertreetal./TransfusionCliniqueetBiologique24(2017)462–467 465

Fig.2.Ferroportindown-regulationmechanisms.A.Gain-of-functionferroportinmutationsidentifiedinpatientsandfunctionallycharacterized.Substitutionresidues generatingpartialresistanceofferroportintohepcidinarelabeledingreen,andthosegeneratingstrongresistanceinred.B.Modelofhepcidin-mediateddown- regulationofferroportin.HepcidinbindstotheferroportinC-terlobe,preventingitsreturntotheinwardconformation.Intracellularloopaccessisimproved,enabling ubiquitinationbyubiquitinligases,triggeringinternalizationanddegradationoftheironexporter(adaptedfromTaniguchietal.,2015[15]).

2.2. Howtoimproveourknowledgeofthehepcidindocking process?

Acriticalpointintheelucidationofthestructureandbiol- ogyof ferroportinwasreached withthe recentpublication of theoutward-andinwardfacingstructuresofaputativebacterial homologueof ferroportin(BbFPN).Taniguchi andcollabora- torssuppliedveryimportantcluesforidentifyingaminoacids involvedintheiron-bindingsiteandfordecipheringinteraction networksbetweentransmembranehelicesintheoutward-and inward-facingextremestructures.Theyfurtherrevealedthepiv- otalroleofacentralcavitythathoststheiron-bindingsiteinthe intra-andextra-cellularopenstatesofBbFPNandislikelyto betargetedbyhepcidinintheoutward-facingconformationof humanferroportin[15].Althoughveryimportant,thesedataare notthelastword,butwillserveasthestartingpointtopredict atomicdetailsoftheorganizationofferroportintransmembrane helicesandtoachieve accuratemodeling ofthe humanferro- portin3Dstructure.

Itnoteworthy thatmissensemutationsleading tothe high- est degrees of hepcidin resistance are located in the C-ter lobe of ferroportin [6,11,16–18], in the vicinity of Cys326, whichis known tobe essential to hepcidin/ferroportin inter- action,while severalmissense mutationslocated inthe N-ter lobe have proved to induce partial resistance to hepcidin (Fig.2A)[6,16,19,20].Results,however,areconflicting,prob- ablybecausedifferentconcentrationsofhepcidinanddifferent experimentalprocedureswereusedbythevariousgroups.Fur- thermore,manymissensemutationsidentifiedinpatientswith atypicalhemochromatosisphenotypehavenotbeenevaluated

functionally.Homogenousproceduresareneeded,tobetterdis- cern gain-of-functionmutations andconfirm the existence of afunctional dichotomybetweenthe N- andC-terferroportin lobes.

2.3. Howtoidentifyferroportindomainsthatareimportant forhepcidin-inducedendocytosis?

Ingeneral,ligand-inducedendocytosisofplasmamembrane transportersistriggeredbyaconformationalchangefollowed byphosphorylationand/or ubiquitinationoftheir cytoplasmic segments[21].Qiaoetal.demonstratedthattheubiquitination oflysinesbetweenresidues229and269inthethirdintracellu- larloopofferroportinisthemajorsignalforincorporationinto themultivesicularbodyanddegradationinthe lateendosome compartment.Theyfurthershowedthatthehumanferroportin mutationp.Lys240Glu(K240E) causedresistance tohepcidin invitrobyinterferingwithferroportinubiquitination[7].The specificdetailsofferroportin’scellularinternalizationprocess, including which ubiquitin-binding adaptor proteins may be involved,remaintobedetermined.Inaddition,nothingisyet known aboutthe conformationalchanges that resultfrom the interactionofhepcidinwithferroportin.

ThefactthatseveralmissensemutationsintheN-terlobeare associatedwithpartialresistancetohepcidinwithoutaffecting theprimarydockingstep[6]suggeststhatlocalchangesinthe firstsixhelicesplayanimportantrole(Fig.2B).Here,weplan tofunctionally characterize10 missensemutations locatedin transmembranehelices1,2,4,5 and6.Thiscouldhelpiden- tifyinter-domaininteractionsandresiduesthatarecriticalfor

hepcidin-inducedendocytosis,inastepthatdependsonhepcidin binding and comes before ubiquitination of lysines between residues229and269.

3. Experimentalprocedures 3.1. Geneticstudies

Inrecentyears,wecontributedtoreportingahandfulofnew mutationsanddiseasephenotypesinpatientswithironoverload.

WewillcapitalizeonourexpertisetoinvestigatenewSLC40A1 missensemutations inpatients withwell-definedphenotypes.

Thiswillbefacilitatedbyanationalmedicalresearchprogram (PHRC)todeterminetheprevalenceandseverityofferroportin- associated ironoverload.Eighteen missensemutations,found in44Frenchpatients,havealreadybeenreportedandfunction- allycharacterized[11].Fivemissensemutationsarestillunder investigation,andwecanconfidentlypredictthatafewothers willbeidentifiedinthecomingtwoyears.

3.2. Functionalinvitrotesting

To testthe degree of resistanceof the ferroportin variants tohepcidin, we will useWestern blot analysis,flow cytome- try andmeasurements of radiolabeled ⁵⁵Fe.We will use the HEK293T cells as a cellular model to transiently or stably (tetracyclin-inducible system) overexpress ferroportin, while humanhepcidinwillbeproducedinstablytransfectedHep3B cells. This will enable us toperform co-culture experiments and improve our ability to reveal subtle differences between mutationsthatcauseresistancetohepcidin.AC-terbiotinylated hepcidinsyntheticpeptidewillbeusedinpull-downandWest- ernblottingexperimentstotesttheinteractionbetweenhepcidin andferroportinmutants.¹²⁵I-Hepcidinuptakeexperimentsand global analysis of lysine ubiquitinationwill be performed to investigateaminoacidsthatdonotdirectlyinteractwithhepcidin butcouldbeinvolvedinferroportinendocytosis.

3.3. Structuralstudies

Wewillseektoprovidearefinedmodelofhumanferroportin despitearelativelylowlevelofsequenceidentity(24%)with thebacterialtemplate.Thiswillbecompletedbyamolecular- dockingapproachtostudytheinteractionbetweenhepcidinand ferroportin.Thistheoreticalanalysis,combinedwithexperimen- taltesting,willbenefitfromourpastexperienceinmembrane proteinmodeling(e.g.,CFTR)[22,23].

4. Milestones

• M1-Accuratestructurepredictionofhumanferroportin;

• M2-Betterdefinitionof thehepcidin-bindingsiteonferro- portin;

• M3-Identificationofthefunctionalnetwork(keyresiduesand interactions) that orchestrate hepcidin-induced ferroportin endocytosis;

• M4-Betterunderstandingofthemolecularandphysiological basesofhemochromatosis,bycharacterizingnewmutations andreportingwell-definedphenotypes.

Disclosureofinterest

Theauthorsdeclarethattheyhavenocompetinginterest.

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