Tuesday,February10,2015463a
AMPHhashigherpotencythandopamine.Thesedatasupportthehypothesis
thatmonoaminetransportersubstrate-inducedcurrentcoupleselectricallyto
L-typeCa
2t
channelsbutnottohigh-voltage-activatedCaV2.2channels.Sup-
port:NIHR01DA033930
2328-PosBoardB465
AKineticAssessmentofLigandBindingtoMonoamine-Transporters
PeterS.Hasenhuetl,MichaelFreissmuth,HaraldH.Sitte,WalterSandtner.
Dep.forPharmacology,MedicalUniversityofVienna,Vienna,Austria.
Inthisstudywepresentanovelelectrophysiologicalapproachtomeasurethe
bindingkineticsofligandsthatbindtotheSerotoninTransporter(SERT)and
theDopamineTransporter(DAT).Themethodsexploredallowforthemea-
surementofonandoff-ratesofdrugswithinawideaffinityrange.Herewe
determinedtherespectiveratesforcocainebindingtoSERTandDATand
weshowthatthederivedkineticparameterscanverywellpredictcocaineaf-
finityinequilibrium.WealsoexploredMethyphenidatbinding(licensedas
Ritalin)toSERTandDAT.ThisdrugisknowntobindpotentlytoDATand
onlyweaklytoSERT.Ourkineticassessmentrevealedthatthedifferencein
theobservedaffinitycanbesolelyattributedtodifferencesintherespective
on-ratesofMethyphenidat,whereastherespectiveoff-ratesweresimiliar.
Ourfindingthereforechallengestheprevalentviewthatdifferencesinpotency
originatefromdifferingdissociationrates.Additionallyourapproachmaypro-
videguidanceintherationaldesignofnewdrugsthatselectivelytargetSERT
orDAT.
2329-PosBoardB466
ProtonTransportMechanismoftheE.coliCopperTransportEfflux
Pump
ArmanFathizadeh,FatemehKhalili-Araghi.
UniversityofIllinoisatChicago,Chicago,IL,USA.
GramNegativeBacteriasuchasE.Coliuseatripartitecomplexsystemtoexpel
toxicchemicals,suchasantibiotics,andionsoutofthecell.InE.Coli,
CusCBA,whichisacomplexofaninnermembranetransporter(CusA),acon-
nectingfusionprotein(CusB),andanoutermembranechannel(CusC),utilizes
theprotonmotiveforcetotransportcopperandsilverionsoutofthecell.Three
chargedresiduesinthetransmembranedomainhavebeensuggestedtoplaya
crucialroleintheprotontransportacrossthemembrane.Thecrystalstructures
ofthecomplexofCusAandCusBhaverecentlybeenresolvedinthepresence
andabsenceofthecopperions,providingthefirstatomicresolutionimagesof
theassemblyofthetransporterandthefusionproteinsinthetripartitefamily.
UsingthecrystalstructureoftheApo-CusBA,wehavestudiedtheproton
transportmechanisminaseriesofunbiasedmoleculardynamicssimulations
correspondingtodifferentprotonationschemesofthesethreeresidues.The
simulationshaverevealedtwoseparatewaterpermeationpathwaysinthetrans-
membranedomainthatcoincidewiththethreekeyresiduesinvolvedinthepro-
tontransportprocess.Thepresence,stability,andthenumberofwater
moleculesinthetwocanalsshowastrongcorrelationwithprotonationstate
ofthethreekeyresidues.Forinstance,protonationofAsp405leadstoentrance
ofsignificantlyhighernumberofwatermoleculesintotheprotein,anddepro-
tonationofLys984leadstoareductioninthenumberofwatermolecules.
Moreover,protonationofAsp405intheapostateresultedinconformational
changesinthetransmembraneregion(TM8)andtheperipelasmiccleftof
thepump,initiatingatransitiontowardtheCu-boundconformationofthe
protein.
2330-PosBoardB467
TheRoleofHistidineResiduesintheSpecificityoftheHumanZincTrans-
porterhZIP4
RobertDempski,SagarAntala,ElizabethBafaro.
WorcesterPolytechnicInstitute,Worcester,MA,USA.
ZIPtransporters,namedafterthezincregulated(Zrt)andironregulated(Irt)
transportproteins,areessentialforzincandirontranslocationacrosscellular
membranes.Theseproteinsfunctiontoincreasethecytosolicconcentration
oftransitionmetals.Whilebothzincandironareessentialmicronutrients
whicharerequiredforthestructureand/orfunctionofhundredsofcellularpro-
teins,themolecularmechanismofZIPtransportersisnotwellunderstood.
Complicatingmechanisticstudiesistheobservationthattheconcentrationof
freezincandironisnanotopicomolar.Ourinteresthasbeentoelucidate
themechanismofzinctransportmediatedbythehuman(h)ZIP4.hZIP4is
locatedattheprimarylocationofzincuptakeinhumansandhasbeendirectly
implicatedinmultiplediseasestatesincludingAcrodermatitisenteropathica
andpancreaticcancer.Howeverthemechanismoftransportisnotknown.
Wehavepreviouslyshownthatzinc,nickelandcoppercanbetransported
byhZIP4,followingheterologousexpressioninX.laevisoocytes,wherethere
aretwobindingaffinities(inthenMandmMrangeofbiometal).Currently,our
researchinterestshavebeentotargetresiduesoffunctionalimportance.Here,
wewilldescribesomeofourrecentefforts.
GeneticandEpigeneticRegulatorySystems
2331-PosBoardB468
HybridMicrornaControlofColonCancerStemCellAsymmetricDivision
XilingShen.
ElectricalEngineering,BiomedicalEngineering,CornellUniversity,Ithaca,
NY,USA.
Coloncancerstemcells(CCSC)undergobothsymmetricandasymmetricdi-
vision,whichbalancedifferentiationversusself-renewalinthetumorcellpop-
ulation.ThisdecisionisdeterminedbythemicroRNAmiR-34a,whosespatial
segregationgeneratesabimodalNotchresponsethatdeterminescellfateout-
comes.Thisbimodalresponseiscausedbykineticmutualsequestrationbe-
tweenmiR-34aandNotchmRNA.
However,threequestionshaveremainedsincewereportedtheabovefindings
(Buetal,CellStemCell,2013).First,doesmiR-34ageneratebimodalre-
sponsesfromalltargetgenes?Second,whatistherelationshipbetweenmiR-
34aandthecanonicalcellfatedeterminantproteinNumb,whichalsotargets
Notchtoregulatecellfatesymmetry?Andthird,dothesemechanismsexist
innormalstemcells?
(1)AsystematicstudydemonstratesthatthekineticsofmicroRNAregulation
istarget-specific.Quantitativesingle-cellanalysisrevealedthatmiR-34agen-
eratesbimodalresponsesfromasmallsubsetofgenesthatareinvolvedincell
fatedetermination,butregulatingthemajorityofgenes(e.g.,metabolicand
growthgenes)inanon-bimodalmanner.
(2)WereportthatmiR-34aandNumbdirectlyinteracttoformanincoherent
feedforwardloop.ThismicroRNA-proteinhybridcircuitrysynergisticallyen-
hancesNotchandcellfateasymmetrybyordersofmagnitudeandexhibits
adaptivebehaviortooffsetinterferencefromothermiR-34atargetgenes,hence
bufferingasymmetriccellfateoutcomesfromfluctuationsinmiR-34alevels.
(3)Weshowthatthehybridcontrolmechanismisactiveintheintestinalstem
cellniche.Usinginnovativeabdominalwindowand3-D-printedintestinal
insert,chronicinvivomultiphotonimagingfurtherrevealedthereal-time
spatiotemporaldynamicsofthehybridcircuitryinlivemice.Thiscontrolis
graduallysubvertedinlate-stagecancerstemcells,contributingtotheirprolif-
erationandmalignancy.
2332-PosBoardB469
NucleocytoplasmicShuttlingofaGata-FamilyTranscriptionFactor
FunctionsasaDevelopmentTimer
HuaqingCai
1
,MarikoKatoh-Kurasawa
2
,TetsuyaMuramoto
3
,
BalajiSanthanam
2
,YuLong
1
,LeiLi
4
,MasahiroUeda
3
,PabloA.Iglesias
5
,
GadShaulsky
2
,PeterN.Devreotes
1
.
1
CellBiology,JohnsHopkinsUniversity,Baltimore,MD,USA,
2
Baylor
CollegeofMedicine,Houston,TX,USA,
3
OsakaUniversity,Osaka,Japan,
4
UniversityofVirginia,Charlottesville,VA,USA,
5
JohnsHopkins
University,Baltimore,MD,USA.
Biologicaloscillationsareuniversalinnature,fascinating,andcriticalatmany
levelsofcellularorganization.InthesocialamoebaeDictyosteliumdiscoi-
deum,starvation-triggeredmulticellulardevelopmentisorchestratedbyperi-
odicextracellularcAMP(3
0
-5
0
-cyclicadenosinemonophosphate)waves,
whichprovidebothchemotacticcuesanddevelopmentalsignals.Repeatedoc-
cupancyoftheG-proteincoupledcAMPreceptors(cARs)promotesoptimal
developmentwhereascontinuousstimulationsuppressestheprogram.While
recognizednearly40yearsago,theunderlyingmechanismforthisintriguing
stimulus-responsepatternhasnotbeenelucidated.Inthisstudy,wereport
thataGATAfamilytranscriptionfactor,GtaC,whichisessentialfordevelop-
mentalprogression,exhibitsrapidnucleocytoplasmicshuttlinginresponseto
cAMPwaves.Thisbehaviorrequirescoordinatedactionofanintrinsicnuclear
localizationsignal(NLS)andreversiblecAR-mediatedphosphorylation.Dis-
ruptingGtaCshuttlingbyaddinganexogenousNLSormutatingtheresidues
involvedinphospho-cyclingleadstoprecociousdevelopment.Intriguingly,
whilecAMPisrequiredtoactivatetheexpressionofdevelopmentalgenes,it
alsodrivesGtaCintothecytosol.Asaresult,eachpeakofthecAMPoscillation
generatesatransientburstofGtaC-dependenttranscription,andthedeclineof
cAMPallowsGtaCtoreturntothenucleusandresensitizesthesystem.We
demonstratethatthisdesign,likean‘‘edgetrigger’’logicalcircuit,filtersout
highfrequencysignalsandcountsthoseadmitted,therebyenablingcellsto
modulategeneexpressionaccordingtothedynamicpatternoftheexternal
stimuli.
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