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Mutant Cu/Zn superoxide dismutase deposition and dismutation kinetics in cell culture 2ed
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Permanent Link: http://ufdc.ufl.edu/IR00001406/00001
 Material Information
Title: Mutant Cu/Zn superoxide dismutase deposition and dismutation kinetics in cell culture 2ed
Physical Description: Conference Proceedings
Creator: Workman, Aron S.
Slunt-Brown, Hilda
Borchelt, David R.
Conference: HHMI/UF Creativity in the Arts & Sciences
Publication Date: 2010
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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 Slunt-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: IR00001406:00001

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Mutant Cu/Zn superoxide dismutase deposition and dismutation kinetics in cell cultureDepartment of Neuroscience and the McKnight Brain Institute University of Florida, Gainesville, FL 32610 AronS.Workman*,HildaSlunt-Brown,andDavidR.Borchelt •EverytoxicSOD1mutantpublishedthusfarformsdistinctbutrelatively variabledetergent insolubleaggregateswhereaswildtype( WT )andother innocuousSOD1sremainpersistentlysoluble(1andreferencestherein).•Relativetoamutantstandard,mutantaggregationpotentialinHEKcell cultureiscorrelatewithashorterdiseasedurationinpatientcohorts(1).•Spinalcordextractsofsymptomatictransgenicmiceshowrapid accumulationofaggregateSOD1overthecourseofdisease(2),and depositionoccursnear selectivelyintheventralhorn(3).PathogenicSOD1speciesaberrantlyoligomerize,andaportionofthesemultimersformmito/cytoplasmicmassaggregates. Aggregatedepositionsareprimarilydetectedbiochemicallyand histologically,thedataofwhichhasledtoseveralimplicateobservations:. AmyotrophicLateralSclerosis(ALS)isacommonneuromusculardiseasewithouteffectivetreatment characterizedbytheselectivedegenerationofmotorneuronsresultingin paralyticdeath.InafamilialsubsetofALS,mutantCu/Znsuperoxide dismutase( SOD1 )enzymesinitiateandsustainthisdisorderdose dependently.SOD1isa153 residue barrelhomodimerthatservesasthe predominantcytosolicscavengerofneurotoxicsuperoxideanionradical ( O2 ).Determiningthegain of functionmechanismofSOD1toxicityfor therapeuticinterventioninALShasbeenthesubjectofcontinuing researchforoverfifteenyears. 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 METHODSCellcultureandtransfections. SOD1cDNAscodedinthemammalianexpressionvectorpEF BOSwere preparedbydoubleCsCl/EtBrdensitygradientandconfirmedbyautomatedsequencingandagarose electrophoresis.4 gcDNAtransfectantwaspreparedwithLipofectamine2000(Invitrogen)andaddedto confluent60mmdishesofhumanembryonickidneycells (HEKcells,lineHEK293FT).Cellsweremaintainedin high glucoseDMEMwith10%horseserumsupplementedwithL glutamine.Freshmediawasadded4hafter transfection,andcellpelletswereharvestedto 80 Cforstorageafterrinsingthreetimesinphosphate buffered saline(PBS). SOD1aggregateextractionandimmunoblotting .Cellpelletswerethawedoniceand resuspendedin100 L1xTEN(10mMTrispH7.4,1mMEDTApH8.0,100mMNaCl).100 L1xTENwith1% NonidetP40(NP 40)and1%proteaseinhibitorcocktail(PI,Roche)wasthenaddedforafinalconcentrationof 0.5%NP 40.Thismixturewasthensonicatedandcentrifugedfor5minat>100,000x g inaBeckmanAirfuge. Thesolubleportionwassavedas S1 andthepelletresuspendedin200 Lofrinsebuffer:1xTENwith0.5%NP 40and1%PI.Thepelletwassonicatedandcentrifugedagain,thistimediscardingthesolublefraction.The pelletwasfinallyresuspendedandresonicatedin1xTENwith0.5%NP 40,0.25%SDS,0.5%deoxycholate,and 1%PIandsavedas P2 .ProteinconcentrationswereassayedbytheBCAmethod(BSAstandard).5 gS1or20 gP2werebroughtupto20 Lin1xTENwithlaemmlibuffer(5% mercaptoethanol).Sampleswereboiledat 96 Cfor6minandloadedon18%tris glycineacrylamidegels.Gelsweretransferredtonitrocellulose membranefor2hat400mAandthenblockedfor15 240minsin5%lowfatdrymilkinPBS T(PBSwith0.1% Tween 20).Primaryanti m/hSOD1antibodywasadded1:5000inmilkfromPBS Tfor1 16hfollowedby3x rinseinPBS Taloneandsecondarygoatanti rabbitHRPat1:2500inmilkfromPBS Tfor1h.Blotswererinsed againandvisualizedandpixel quantifiedwithECLchemiluminescenceonaFujifilmLAS 3000. Semi native analysis. EquivalentproteinfromS1s,P2s,orraw1xTNfreezethaw(F/T)extractswereloadedwith~20% glycerolandminimaldyeon4 20%1xTGgelswithoutSDSandrunat100constantvoltsat4 Cfor4 8hours. Gelswereretrieved.Somegelsweretreatedwith2%SDS2% ME1xTGandmicrowavedforpulsesof20 secondseach,2minutestotalforin gelreduction.Nativegelsweretransferredtonitrocelluloseand immunolabeledasdescribedabove. CONCLUSIONS BothD101NandD101Gformsignificantdetergent insolubleaggregatesin HEKcellculture,althoughD101Nnucleationisdelayed.D101N&G carriershavejust2.5yearsaveragetimefromdiagnosistodeath. D101N&Ghavesimilarratesofdepositionandasimilarthresholdbut dissimilarnucleation,indicatingmetricsthatmaybemorecorrelativewith clinicaldata.Thisisnotthecaseforallvariablemutantsatoneposition. Moretimecourseandpulsechaseexperimentsareunderwaytotestthis hypothesis. TheD101Glesionresultsinalossofdismutaseactivity(andintraoxidized protein),whereastheD101Nmutantiselectrophoreticallyand enzymaticallyWT like.Notably,theapparenttrimersandpentamersare resistantto5% MEandappearinboththeS1andP2fractions,asdo E133V’s ME resistantoxidizeddisulfides.E133Vretainsnoaggregate intrareducedproteineveninextendedcellculture.TheE133V::YFPfusion proteiniscomparabletoWT::YFPinthatneitheraggregateinadherentcells eveninextendedcellculture. SOD1sextractedinthepresenceofNP 40andEDTAhavediminished activityandtightenedbandsrelativetoTNextracts(datanotshown).Thus S1assaysareanunderrepresentationofactualactivity.However,itdoes notappearthedegreetowhichdetergentsorchelatorseffectSOD1proteins varysignificantlyinterspecifically.AlthoughitisunlikelyinsolubleSOD1s areactive,thedetergentextractdesign(rinsesandSDS/deoxycholate)isnot suitableforenzymaticanalysis. ThisworksupportedinpartbyanHHMI/UFScienceforLifeawardtoASW. RelativeaggregationTime of harvest (hours post-transfection) 24 6 12 18 24 30 36 42 48A4VD101N Figure4.QuantifiedmutantSOD1aggregation. Deposition levelsintransfectedHEKcellsasassayedby detergent/ultracentrifugationextraction(detailedinmaterials andmethods).Immunolabeledmutantproteinatthe apparentlyreducedmonomericpositionwaspixel quantified,with24hA4Vsetto1.D101Gaggregatesappear promptlyandabundantlywhereasD101Nisconsiderably slowertonucleate.Wildtype,familialmutantE133V,andtwo ofthreeartificial*mutantsdonotformdetergent insoluble aggregatesatanytime.N=2 4. Figure5.D101Naggregatenucleationisacomplexreaction involvingoligomericintermediatesorotherSOD1 reactive adducts. Apparenttrimersandapparentpentamers(each withtwoisoforms,between40 60kDaandat80kDa respectively,basedsolelyonpost stainedmolecularweight markers)arenoted.Preemptive24happarentpentamerN=2, allelseN=3.D101Nexpressionstabilizesbetween12and18 hours.Thereisvirtuallynoexpressionat6hourspost transfection,andthe6hourlaneshownisrepresentativeof high exposurebackgroundincontrolP2s.