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Mutant Cu/Zn superoxide dismutase deposition and dismutation kinetics in cell culture
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Permanent Link: http://ufdc.ufl.edu/IR00001403/00001
 Material Information
Title: Mutant Cu/Zn superoxide dismutase deposition and dismutation kinetics in cell culture
Physical Description: Archival
Creator: HHMI/UF Creativity in the Arts & Sciences ( Conference )
Workman, Aron S.
Brown, Hilda
Borchelt, David R.
Publication Date: 2008
 Notes
Acquisition: Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Aron Workman.
General Note: Support and PI by David R. Borchelt. Radiometric assay by Hilda Brown. All other figures and text by Aron S. Workman.
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Source Institution: University of Florida Institutional Repository
Holding Location: University of Florida
Rights Management: All rights reserved by the submitter.
System ID: IR00001403:00001

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Figure2.35S cysteinepulse chasedetergentextractions. 24hafterHEKcell transfection,sampleswereradiolabeledfor1handharvestedimmediately (pulse)oraftergrowing24hin cold medium(chase).Aggregatesat49h post transfectionarecomposedofproteinsthatwerepredominantlysoluble 24hbeforeforbothD101NandD101G,butnotWT. Figure1.Sequentialtimeddetergentextractions. D101NandD101GdepositionlevelsintransfectedHEKcellsasassayedbydetergent/ultracentrifugation extraction(detailedinmaterialsandmethods).D101Gaggregatesappearpr omptlyandabundantlywhereasD101Nisconsiderablyslowertonucleate. WTdoes notformdetergent insolubleaggregatesatanytime. EverytoxicSOD1mutantassayedthusfarformsdistinctbutrelatively variabledetergent insolubleaggregateswhereaswildtype( WT)andother innocuousSOD1sremainpersistentlysoluble(1,Fig.1).Relativetoamutantstandard,mutantaggregationpotentialinHEKcell cultureiscorrelatewithashorterdiseasedurationinpatientcohorts(1).Spinalcordextractsofsymptomatictransgenicmiceshowrapid accumulationofaggregateSOD1overthecourseofdisease(2),and depositionoccursnear selectivelyintheventralhorn(3).PathogenicSOD1speciesaberrantlyoligomerize,andaportionofthesemultimersformcytoplasmicmassaggregates. Aggregatedepositionsareprimarilydetectedbiochemicallyand histologically,thedataofwhichhasledtoseveralimplicateobservations:. AmyotrophicLateralSclerosis(ALS)isacommonneuromusculardiseasewithouteffectivetreatment characterizedbytheselectivedegenerationofmotorneuronsresultingin paralyticdeath.InafamilialsubsetofALS,mutantCu/Znsuperoxide dismutase(SOD1 )enzymesinitiateandsustainthisdisorderdose dependently.SOD1isahighlyconserved153 residue barrelhomodimer thatservesasthepredominantintracellularscavengerofneurotoxic superoxideanionradical( O2 ).Determiningthegain of function mechanismofSOD1toxicityfortherapeuticinterventioninALShasbeen thesubjectofcontinuingresearchforoverfifteenyears. Mutant Cu/Zn superoxide dismutase deposition and dismutationkineticsin cell cultureDepartment of Neuroscience and the McKnight Brain Institute University of Florida, Gainesville, FL 32610 AronWorkman*,HildaBrown,andDavidR.Borchelt MutantSOD1dossiers:D101NandD101GHistorically,mutantcomparisonshavebeenhelpfulfordelineatingtoxic function.Forexamples:theverifiedmetal free(consequently,inactiveor scavenging free)doubleCu bindinghistidineknockoutH46R/H48Qis stillparalyticandaggregate prone,asisamutantmouseSOD1witha glycinelesionhomologicaltohumanG85R.Thisstudyinvestigates mutantisoformsatasparticacidresidue101.D101Nisarelatively commonALSmutant,butwithuniqueWT likeproperties:itsmetallation status,H/Dexchange,DSCprofile,UVandEPRspectroscopydatamatch thatofWT(4).D101Ndoesnotaggregateat24hinHEKcellculture, whereasD101Gdoessorobustlyseeminglyassoonasproteinis synthesized.PatientscarryingD101NandD101Gareclinicallyidentical withaverageagesofonsetof41.010and48.09.1yearsandsurvival timesof2.40.9and2.50.4years,respectively(N=14and3,1). 1. PrudencioM etal. 2009HumMolGenet. 18:3217. 2. KarchCM etal .2009PNAS 106:7774. 3. WangJ etal .2009PNAS 106:1392. 4. RodriguezJA etal .2005PNAS 102:10516. 5. BeauchampCandFridovichI.1971AnalBiochem 44:276.REFERENCES Figure 3. Native nitro blue tetrazoliumsuperoxide scavenging gel assay. D101N, D101G, and WT among other mutant SOD1s in 3T3s. D101G loses dismutase activity, whereas D101N appears WTlike except with decreased mouse heterodimerformation. Extracellular Mn SOD (SOD3) is noted, as are homodimericendogenous mouse SOD1 (<), mouse/human SOD1 heterodimer(<<), and human SOD1 homodimer (<<<) on the basis of electrophoreticmobility. MATERIALSANDMETHODSCellcultureandtransfections. SOD1cDNAscodedinthemammalianexpressionvectorpEF BOSwerepreparedby doubleCsCl/EtBrdensitygradientandconfirmedbyautomatedsequencingandagaroseelectrophoresis.4 gcDNA transfectantwaspreparedwithLipofectamine2000(Invitrogen)andaddedtoconfluent60mmdishesofhuman embryonickidneycells(HEKcells,lineHEK293FT)ormouseembryonicfibroblasts(3T3s,lineNIH3T3).Cellswere passagedinhigh glucoseDMEMwith10%serum(horseforHEKcells,newborncalffor3T3s),supplementedwithL glutamine.Freshmediawasadded4haftertransfection,andcellpelletswereharvestedto 80 Cforstorageafterrinsing threetimesinphosphate bufferedsaline(PBS). SOD1aggregateextractionandimmunoblotting.Cellpelletswerethawedoniceandresuspendedin100 L1xTEN(10 mMTrispH7.4,1mMEDTApH8.0,100mMNaCl).100 L1xTENwith1%NonidetP40(NP 40)and1%protease inhibitorcocktail(PI,Roche)wasthenaddedforafinalconcentrationof0.5%NP 40.Thismixturewasthensonicated andcentrifugedfor5minat>100,000x g inaBeckmanAirfuge.Thesolubleportionwassavedas S1 andthepellet resuspendedin200 Lofrinsebuffer:1xTENwith0.5%NP 40and1%PI.Thepelletwassonicatedandcentrifuged again,thistimediscardingthesolublefraction.Thepelletwasfinallyresuspendedandresonicatedin1xTENwith0.5% NP 40,0.25%SDS,0.5%deoxycholate,and1%PIandsavedas P2 .ProteinconcentrationswereassayedbytheBCA method(BSAstandard).5 gS1or20 gP2werebroughtupto20 Lin1xTENwithlaemmlibuffer(5% mercaptoethanol).Sampleswereboiledat95 Cfor6minandloadedon18%tris glycineacrylamidegels.Gelswere transferredtonitrocellulosemembranefor2hat400mAandthenblockedfor15 40minsin5%lowfatdrymilkinPBS T (PBSwith0.1%Tween 20).Primaryanti m/hSOD1antibodywasadded1:5000inmilkfromPBS Tfor1 16hfollowedby 3xrinseinPBS Taloneandsecondarygoatanti rabbitHRPat1:2500inmilkfromPBS Tfor1h.Blotswererinsedagain andvisualizedandpixel quantifiedwithECLchemiluminescenceonaFujifilmLAS 3000.35S cysteinepulse chaseanalysis. 24haftertransfection,HEKcellswerepulseradiolabeledwith35S cysteineforone hour.Somesampleswereharvestedimmediately,othersampleswerechasedwithcoldmediafor24hbeforeharvesting fordetergentextractionandimmunoprecipitationwithanti m/hSOD1,followedbySDS PAGEandautoradiography. Superoxideanionscavengingactivityassay. <100 gtotalcelllysatepreparedfrom10xpelletvolumesonicated0.1% NP 40in1xTNwasrunon8%or4 20%tri glycineacrylamidegelsin1xTGwith20%methanolat4 Cand100constant Vfor4 6h,withoutdenaturantorreductantadded.Thegelwasretrievedandsoakedin50mMpotassiumbicarbonate buffercontaining65 g/mLriboflavinand280 g/mLnitrobluetetrazolium,pH7.6(5).Afterincubating2 40m,the solutionwasaspiratedand0.1%TEMEDin50mMpotassiumbicarbonatebufferwasaddedtothegel.Thegelwas immediatelyexposedtowhitelightfromabrightboxan dimagedonaAgfaDuoscan,withcontrastincreased.