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RESEARCHOpenAccessIntracellularcomplexesoftheearly-onsettorsiondystonia-associatedAAA+ATPaseTorsinAHuiLi1,Hui-ChuanWu1,ZhonghuaLiu1,3,LuciaFZacchi2,JeffreyLBrodsky2andMichalZolkiewski1*AbstractAsingleGAGcodondeletioninthegeneencodingtorsinAislinkedtomostcasesofearly-onsettorsiondystonia.
TorsinAisanER-localizedmembrane-associatedATPasefromtheAAA+superfamilywithanunknownbiologicalfunction.
WeinvestigatedtheformationofoligomericcomplexesoftorsinAinculturedmammaliancellsandfoundthatwildtypetorsinAassociatesintoacomplexwithamolecularweightconsistentwiththatofahomohexamer.
Interestingly,thedystonia-linkedvarianttorsinAΔEdisplayedareducedpropensitytoformtheoligomerscomparedtothewildtypeprotein.
WealsodiscoveredthatthedeletionoftheN-terminalmembrane-associatingregionoftorsinAabolishedoligomerformation.
Ourresultsdemonstratethatthedystonia-linkedmutationinthetorsinAgeneproducesaproteinvariantthatisdeficientinmaintainingitsoligomericstateandsuggestthatERmembraneassociationisrequiredtostabilizethetorsinAcomplex.
Keywords:Early-onsetdystonia,TorsinA;AAA+ATPase;ProteinassociationBackgroundEarly-onsettorsiondystonia(EOTD)isthemostcom-monandsevereformofprimarydystonia,aneurologicaldisorderthatmanifestsasuncontrollablemovementsandabnormalbodypostures.
MostcasesofEOTDareassoci-atedwithadeletionofasingleGAGcodonintheDYT1gene.
Asaresult,asingleglutamicacidresidueisabsentintheEEpairlocatedintheC-terminalregionoftorsinA(Ozeliusetal.
1997).
TorsinAisaputativememberoftheAAA+superfamilyofATPasesassociatedwithdifferentactivities(Neuwaldetal.
1999).
ThetorsinAmRNAiswidelyexpressedinvarioushumantissues,includingthecentralnervoussystem,butthebiologicalroleoftorsinAisnotcompletelyclear(reviewedin(Tanabeetal.
2009;ZolkiewskiandWu2011)).
AAA+ATPasesareenergy-driven"molecularmachines",whichremodeltheconformationofmacromoleculesanddisassemblemacromolecularcomplexes(HansonandWhiteheart2005).
ProteinsfromtheAAA+familyformring-shapedhexamericcomplexes,whichenclosetheirsubstratemole-cules.
HexamerformationisessentialfortheactivityofAAA+ATPases(Barnettetal.
2000).
NumeroustorsinApartnershavebeenidentifiedandtheassociationwithsomeoftheseiscompromisedwhenthemutantgeneproductisexpressed(Naismithetal.
2009;ZolkiewskiandWu2011).
However,theidentityofatorsinAsubstratethatiscriticalforitscellularactivityisunknownand,morefundamentally,whethertorsinAevenformsahex-amerincellshasnotbeenfullyestablished.
Moreover,itisunclearhowtheglutamatedeletionaffectsthesebiochem-icalpropertiesandinturn,whichdefect(s)associatedwiththemutantproteinarelinkedtoEOTD.
ThetorsinAsequencecontainsanN-terminalER-targetingsignalpeptidethatiscleaveduponimportintotheERlumen,producingthemature36-kDaformoftheprotein(Liuetal.
2003).
Thesignalsequenceisfollowedbya20-residue-longhydrophobicsegmentthatisresponsibleformembraneassociation(Liuetal.
2003)andERretention(VanderHeydenetal.
2011).
TheAAA+moduleoftorsinAislocateddownstreamofthemembrane-bindingdomainandcontainsanon-canonicalATP-bindingWalker-Amotif(Nagyetal.
2009;ZolkiewskiandWu2011)andsixcysteinesthatareabsentfromotherAAA+ATPases(Zhuetal.
2008;ZolkiewskiandWu2011).
Thesiteofthedystonia-linkedglutamatedeletion(E302/E303)islocatedwithintheC-terminalAAA+subdomain,whichsupportsoligo-merizationofotherAAA+ATPases(Barnettetal.
2000).
*Correspondence:michalz@ksu.
edu1DepartmentofBiochemistryandMolecularBiophysics,KansasStateUniversity,Manhattan,KS66506,USAFulllistofauthorinformationisavailableattheendofthearticleaSpringerOpenJournal2014Lietal.
;licenseeSpringer.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/4.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
Lietal.
SpringerPlus2014,3:743http://www.
springerplus.
com/content/3/1/743StudieswithpurifiedrecombinanttorsinArevealedeitheramonomericprotein(Kustedjoetal.
2003;Zhuetal.
2008)oraspectrumofhigh-molecularweightparticles(Zhaoetal.
2013).
Incontrast,torsinAassembliesrangingfrommonomersanddimerstohexamersweredetectedinlysatesfrommammaliancells(Kustedjoetal.
2000;GordonandGonzalez-Alegre2008;VanderHeydenetal.
2009;Jungwirthetal.
2010).
Howtheseassembliesareaf-fectedbythedisease-causingmutationorthehydrophobicmembraneanchorhasnotyetbeenestablished.
Tothisend,weinvestigatedthesizeofhumantorsinAcomplexesafterisolationfromculturedmammaliancells.
WefoundthatthemainoligomericspeciesisconsistentwiththeformationoftorsinAhexamers,butthisstruc-turebecomeslessstablewhenthedystonia-linkedproteinvariantisexpressed.
Wealsofoundthatthemembrane-boundhydrophobicsegmentstabilizesthetorsinAoligo-mer.
ThesedataaddfundamentalnewinsightstoourunderstandingoftorsinAstructureandsuggestwhythelossofasingleaminoacidcanexhibitprofoundcellulareffects.
ResultsanddiscussionTodeterminewhetherhumantorsinAandthedystonia-linkedtorsinAΔEvariantoligomerizeinthecell,weexpressedeachproteinintwocelllines,HEK293andCHOcells.
Afterpreparationofcelllysatesindodecyl-maltoside,BN-PAGEandimmunoblottingwithananti-torsinAantibodywasusedtoobservethedistributionofthetorsinA-containingspecies(Figure1).
BothstablytransfectedcelllinesproducedcomparableamountsoftorsinAandtorsinAΔE(Figure1A,B,lowerpanels).
InadditiontosomemonomerictorsinAandtorsinAΔE(shownbythebandsbelow66kDa),BN-PAGEdetectedasinglemajorimmunoreactivespeciesmigratingclosetothe200-kDacomplexofβ-amylase,butslowerthanthe242-kDaproteinstandard(Figure1A,B,upperpanels).
ThemigrationofthetorsinAoligomerinBN-PAGEisconsistentwiththatofahomohexamer(predictedmo-lecularweight216kDa)andisconsistentwiththeforma-tionofaspeciesofsimilarsizeinBN-PAGEusinglysatespreparedfromU2OScells(VanderHeydenetal.
2009).
Itcannotbeexcluded,however,thatthedetectedspeciescorrespondstoahetero-oligomercontainingtorsinAandothercomponents,suchasthetorsinAbindingpartnersLAP1andLULL1(GoodchildandDauer2005;Zhaoetal.
2013;Sosaetal.
2014).
WealsofoundthatthedeletionofGlu302intorsinAapparentlydestabilizestheoligomericspecies(Figure1A,B,upperpanels).
Thisresultsuggeststhatthedystonia-linkedtorsinAvariantmaybedefectiveineitherself-associationorinteractionswithotherpro-teins.
Indeed,theefficiencyoftorsinAΔEinteractionwithLAP1andLULL1iscompromisedrelativetothewildtypeprotein(Naismithetal.
2009;Zhaoetal.
2013).
Incontrast,thedystonia-linkedtorsinAΔEvariantshowsanenhancedbindingaffinityfornesprin(Neryetal.
2008).
Thus,theapparentlossofthedetectedoligomericspeciesinthetorsinAΔEproducingcells(Figure1)suggeststhattheobservedtorsinAcomplexdoesnotincludenesprin.
Nevertheless,thedatapresentedinFigure1indicatethattheEOTD-associatedmutationhasaprofoundeffectonoligomerand/orcomplexformation,andweproposethatthisdefectmightimpacttheprotein'sfunctionanddiseasepresentation.
Asnotedabove,previousstudiesonpurifiedfull-lengthtorsinAfailedtodetectoligomericspecies,whichcouldhavebeentheresultofdetergent-induceddestabilizationofintersubunitcontacts(Kustedjoetal.
2003).
Otherex-perimentsusingapurifiedproteindetectedhighermo-lecularweightspeciesthatwerenotfurtherresolved(Zhaoetal.
2013).
Toobtainenrichedsolubleproteinintheabsenceofadetergent,weproducedatruncatedtor-sinAvariantlackingthehydrophobicmembrane-bindingregion,torsinAΔ40(Liuetal.
2003).
TorsinAΔ40wasexpressedinS2cellsandpurifiedfromtheculturemedia(seeMaterialsandMethods).
Interestingly,torsinAΔ40ΔEwaspoorlysecretedinS2culture(Liuetal.
2003),whichisconsistentwiththeapparentmislocalizationofthisdys-toniavariantfromtheERlumentothenuclearenvelope(GoodchildandDauer2004;Naismithetal.
2004).
ThecirculardichroismspectrumoftorsinAΔ40(Figure2A)wassimilartothatofthefull-lengthtorsinApurifiedwithdetergent(Kustedjoetal.
2003),whichindicatesthatade-letionofthehydrophobicsegmentdoesnotinhibitfoldingoftorsinA.
TorsinAΔ40wasstrictlymonomeric(~30kDa,Figure2B),regardlessofwhethersizeexclusionchroma-tographywasrunintheabsenceofnucleotidesorinthepresenceofATPorADP,whichinothercasesstabilizeAAA+hexamers(Akoevetal.
2004).
Tocorroboratethesedata,wenextinvestigatedtheoligomericstateoftorsinAΔ40inmammaliancelllysates(Figure2C).
Incontrasttothefull-lengthprotein(WT),thetorsinAΔ40andtorsinAΔ40ΔEvariantsagainfailedtoformoligomericspeciesinBN-PAGE.
Thisresultisinac-cordancewiththepropertiesofpurifiedtorsinAΔ40(Figure2B)andindicatesthatthe20residue-longN-terminalhydrophobicsegmentisessentialtostabilizetorsinAcomplexes.
Twomechanismscanbeproposedtoaccountforthisresult.
First,thehydrophobicseg-mentmaydirectlyparticipateineitherself-associationorhetero-associationwithanotherprotein'smembrane-embeddeddomain.
Second,themembraneassociationoftorsinAanditsretentionintheERlumenmayincreasethelikelihoodofformingthehomo-orheterooligomers.
Recently,hetero-hexamersoftorsinAandLAP1werereconstitutedwithpurifiedproteins(Sosaetal.
2014).
SinceLAP1isatransmembraneprotein,itsinteractionwithtorsinAinthecellmightbeefficientonlyiftorsinALietal.
SpringerPlus2014,3:743Page2of5http://www.
springerplus.
com/content/3/1/743isalsotargetedtotheERmembranebyitsN-terminalhydrophobicsegment.
Futureeffortswillbedirectedtoaddressthishypothesis.
ConclusionsInsummary,wefoundthattorsinAformsadiscretehigh-molecularweightcomplexinmammaliancells.
However,thecomplexisdestabilizedbythedystonia-linkedmutation,butisstabilizedbythemembranean-chor.
EstablishingalinkbetweendefectsintorsinAΔEoligomerizationandEOTDwillbeanimportantfocusoffutureresearchefforts.
MethodsPlasmids,antibodies,andreagentsDNAconstructscontainingthehumantorsinAsequenceinpcDNA3vectorweredescribedbefore(Liuetal.
2003)andanti-torsinantibodieswereobtainedasdescribed(Zacchietal.
2014).
Sweetpotatoβ-amylasewasfromSigma.
NativeelectrophoresisproteinstandardswerefromInvitrogen/LifeTechnologies.
CellcultureHEK293cellsweremaintainedinDMEM(Invitrogen)supplementedwith10%fetalbovineserum(BioWhittaker)Figure1BN-PAGEanalysisoftorsinAcomplexes.
Full-lengthhumantorsinA(WT)orthedystonia-linkedtorsinAΔEprotein(ΔE)wasexpressedinHEK293(A)andCHO(B)cells.
ProductionofthetorsinAvariantswasconfirmedbySDS-PAGEfollowedbyimmunoblottingwithanti-torsinAantibodies(lowerpanels)usinguntransfectedcellsasacontrol(C).
ThecelllysateswereseparatedonBN-PAGEfollowedbyimmunoblotting(upperpanels).
ForBN-PAGE,themigrationpositionsofthenative-electrophoresisstandardsareindicated.
Themigrationpositionofβ-amylase(200kDa)isindicatedwithanarrow.
ProteinmigrationinBN-PAGEcanreflectotherbiophysicalproperties,besidesthemolecularweight,sothemolecularweightdeterminationisonlyapproximate.
Thefigureshowsarepresentativeresultfromtwoindependentexperiments.
Lietal.
SpringerPlus2014,3:743Page3of5http://www.
springerplus.
com/content/3/1/743at37°Cinthepresenceof5%CO2.
CHO-K1cells(ATCC)weremaintainedinF-12Kmedium(Invitrogen)sup-plementedwith10%fetalbovineserum.
Cellsweretrans-fectedwithpcDNA3expressionvectorscontainingthetorsinAvariantsusingFuGene6transfectionreagent(Roche)accordingtothemanufacturer'sinstructions.
Stablytransfectedcellswereselectedinthepresenceof1mg/mLG418(Invitrogen).
Blue-nativePAGEBN-PAGEisanativegelelectrophoresistechnique,wheretheCoomassieBrilliantBluedyebindstomembranepro-teincomplexesandprovidestheelectricchargefortheelectrophoreticseparation.
BN-PAGEwascarriedoutaspreviouslydescribed(Wittigetal.
2006;VanderHeydenetal.
2009;Jungwirthetal.
2010).
Briefly,~90%confluentcellswerecollectedandsolubilizedinalysisbuffer(50mMimidazolepH7.
0,50mMNaCl,2mM6-aminohexanoicacid,4mMMgCl2,2mMEDTA,2mMATP,1mMPMSF,and0.
25%dodecylmaltoside)for15minin4°C,andcentrifugedtwicefor15minat15,000rpminIECMicromaxbenchtopcentrifuge.
Thesupernatantsupplementedwith0.
0625%CoomassieblueG-250and5%glycerolwasloadedontoa9%polyacryl-amidegel.
Followingelectrophoresis,separatedproteinsweretransferredontoPVDFmembraneandsubjectedtoimmunoblottingusinganti-torsinAantibodies,followedbyhorseradishperoxidase-conjugatedanti-rabbitIgGan-tibodies(SouthernBiotechnology).
SignaldetectionwasperformedwithWestPicochemiluminescencekit(Pierce).
ProteinpurificationTheS2celllinestablytransfectedwithatorsinAΔ40ex-pressionplasmidcontainingtheBiPsignalsequencefollowedbyanN-terminalHis-tagwasproducedaspre-viouslydescribed(Liuetal.
2003).
Cellsweregrownat23°CinS2medium(Invitrogen)supplementedwith0.
5%DMSOwhenthedensityreached107cells/mlandproteinexpressionwasinducedafter24hwith0.
7mMCuSO4.
Thecellswereseparatedfromthecultureme-diumbycentrifugation6dayspostinduction.
Theme-diumwasfilteredthrougha0.
45μmmembraneandloadedontoa2.
5-mlChelatingSepharosecolumn(GEHealthcare).
ProteinswereelutedwithanimidazolestepFigure2OligomerizationoftheN-terminallytruncatedtorsinAvariants.
(A)Far-UVcirculardichroismspectraofpurifiedtorsinAΔ40(1mg/ml,solidline)andthedialysisbuffer(dottedline)areshown.
(B)Gel-filtrationanalysisoftorsinAΔ40intheabsenceofnucleotidesorinthepresenceof2mMATPorADPisshown.
Theelutiontimesofmolecularweightstandards(kDa)areindicated.
(C)BN-PAGE(upperpanel)andSDS-PAGE(lowerpanel)analysiswasfollowedbyimmunoblottingwithanti-torsinAantibodiesoflysatesfromHEK293andCHOcellsexpressingeitherfull-lengthtorsinA(WT),torsinAΔ40(Δ40),torsinAΔ40ΔE(Δ40ΔE)oruntransfectedcells(C).
ForBN-PAGE,themigrationpositionsofthenative-electrophoresisstandardsareindicated.
Themigrationpositionofβ-amylase(200kDa)isindicatedwithanarrow.
ProteinmigrationinBN-PAGEcanreflectotherbiophysicalproperties,besidesthemolecularweight,sothemolecularweightdeterminationisonlyapproximate.
Thefigureshowsarepresentativeresultfromtwoindependentexperiments.
Lietal.
SpringerPlus2014,3:743Page4of5http://www.
springerplus.
com/content/3/1/743concentrationgradient.
FractionscontainingtorsinAΔ40(elutedat10–50mMimidazole)werepooled,concen-tratedonCentriplusYM-10(Millipore),anddialyzedin50mMTris–HClpH7.
5,100mMNaCl,20mMMgCl2,and10%glycerol.
CirculardichroismspectroscopyCDspectraweremeasuredwithaJascoJ-720spectrometerusinga0.
01-cmcylindricalcuvetteatroomtemperature.
GelfiltrationchromatographyGelfiltrationanalysiswasperformedatroomtem-peraturewithaShimadzuHPLC.
TorsinAΔ40samples(20μl,~0.
2mg/ml)wereanalyzedat0.
04ml/minonaSuperdex200PC3.
2/30column(GEHealthcare)equil-ibratedin50mMTris/HClpH7.
5,0.
2MKCl,20mMMgCl2withoutnucleotidesorwith2mMATPorADP.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsThisstudywasconceivedanddesignedbyMZandJLB.
TheexperimentswereperformedbyHL,H-CW,ZL,andLFZ.
ThemanuscriptwaswrittenbyMZandJLB.
Allauthorsreadandapprovedthefinalversionofthemanuscript.
AcknowledgementsThisresearchwassupportedbyagrant(toJLBandMZ)andapostdoctoralfellowship(toLFZ)fromtheDystoniaMedicalResearchFoundationandbygrantGM75061fromtheNationalInstitutesofHealthtoJLB.
Thisiscontribution15-105-JfromtheKansasAgriculturalExperimentStation.
PublicationofthisarticlewasfundedinpartbytheKansasStateUniversityOpenAccessPublishingFund.
Authordetails1DepartmentofBiochemistryandMolecularBiophysics,KansasStateUniversity,Manhattan,KS66506,USA.
2DepartmentofBiologicalSciences,UniversityofPittsburgh,Pittsburgh,PA15260,USA.
3Presentaddress:DepartmentofEmbryology,CarnegieInstitution,Baltimore,MD21218,USA.
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