Title Author(s) The role of lipoylation-related genes in energy metabolism in Drosophila Dan, Jeonghwa Citation Issue Date URL 2014-03-25 http://hdl.handle.net/10748/6909 DOI Rights Type Textversion Thesis or Dissertation author http://www.tmu.ac.jp/ 首都大学東京 機関リポジトリ 修 士 学 位 論 文 題名 Theroleoflipoylation-relatedgenesinenergy metabolisminDrosOρh〃7a シ ョ ウ ジ ョ ウ バ エ の エ ネ ル ギ ー 代 謝 に お』 け る リ ポ イ ル 化 関 連 遺 伝 子 の 役 割(英 文) 指導教授 相垣 敏郎 教授 平 成26年1月10日 提 出 首都大学東京 理工学研究科 大学 院 生命科学専攻 学修番号12881328 氏 名 段 晶華 Theroleoflipoylation-relatedgenesinenergymetabolisminDrosoρhila シ 子 ョ ウ ジ の 役 割(英 ョ ウ バ エ の エ ネ ル ギ ー 代 謝 に お け る リ ポ イ ル 化 関 連 遺 伝 文) 細 胞 遺 伝 学 研 究 室 ABSTRACT Lipoicacid(LA),alsocalledthiocticacidworksasananti-oxidantlikevitaminC, vitaminEandcoenzymeQIotoprotectourbodyfromtheharmoffreeradicals. Unlikewatersolublevitamins,LAcanworkinfattytissuesbecauseitissolublein bothwaterandfat.LAisacofactorofseveralenzymesincludingpyru.vate dehydrogenase(PDH)and2-oxoglutaratedehydrogenase(OGDH),whichare involvedinthemaJ'orenergyproducingpathways,glycolysisandTCAcycle.The pathwayoflipoylationofPDHandOGDHhasbeenwellcharacterizedinE.coli.It hasbeenshownthatthelipoylationprocessinvolvesthreegenes;lipoicacid synthetase(lipA),lipoate-proteinligase(lpIA),andlipoyltransferase(lip.B). Eukaryotesgenomescontainorthologsoftheseenzymes,buttheirfUnctionsinLA synthesisandincorporationintoproteinsremainedelusive. Inthisthesis,tounderstandthegeneticbasesoflipoylationineukaryotes,Iused DrosophilaasamodelorganismandinvestigatedtherolesofLas,L,plA,LiT2,the orthologsofbacteriallipA,LρIA,andlipB,respectively.IusedtheGAL4-UASsystem tooverexpressorRNA-mediatedgeneknockdowninDrosoρhila,andanalyzedtheir phenotypesincludingviability,lifespan,metabolomes.Ialsoanalyzedthree 2 段 晶 華 transposoninsertionlinesinwhich-P-elementsareinsertednearbyeachofthethree loci. IfoundthatubiquitousknockdownorthetransposoninsertioninLas,LiT2showed defectinlipoylationofPDHandOGDHandtheirviabilitywasseverelyimpaired. TheseresultsindicatedthatLas,-L,plAandLipT2areessentialfbrviabilityandare requiredfbrthelipoylationpathwayofPDHandOGDHinDrosoρhila.Thus,the lipoylationpathwayislikelytobedifferentfromthatinE.coli. 3 要 旨 水 溶 性 で 脂 溶 性 で も あ る リボ 酸 は チ オ ク ト酸 と 呼 ば れ て い て 体 内 抗 酸 化 剤 機 能 を 有 す る 。 ビ タ ミ ンC、 ビ タ ミ ンEと コ エ ン ザ イ ムQ10と と も に 抗 酸 化 ネ ッ トワ ー ク を 形 成 し て フ リー ラ ジ カ ル 危 害 を 防 御 す る。 こ れ ま で の 研 究 か ら、 エ ネ ル ギ ー 代 謝 に か か わ る 酵 素 の 障 害 は 発 達 障 害 や 精 神 疾 患 の 発 症 と密 接 に 関 連 し て い る こ と が 示 唆 さ れ て い る 。 最 近 、 タ ン パ ク質 の 翻 訳 後 修 飾 の ひ とつ で あ る リボ 酸 修 飾 に 関 わ る 遺 伝 子 の 異 常 と発 達 障 害 の 関 連 を 示 唆 す る 研 究 成 果 が 報 告 され た 。解 糖 系 の 酵 素 で あ る ピ ル ビ ン 酸 脱 水 素 酵 素 や ク エ ン 酸 回 路 の 酵 素 で あ る2一 オ ク ソグル タル 酸 脱 水 素 酵 素 が リ ポ イ ル 化 修 飾 を うけ る 。大 腸 菌 で は こ れ ら の 酵 素 の リポ イ ル 化 に つ い て は よ く研 究 され 、 リ ポ イ ル 化 に 関 わ る3つ の 遺 伝 子 が 同 定 され て い る。 しか し、真 核 生 物 に お い て リボ酸 修 飾 に 関 わ る遺 伝 子 の詳 細 な研 究 は行 われ て い な い。 シ ョ ウ ジ ョ ウバ エ と ヒ トは7割 近 く の 遺 伝 子 が 共 通 し て お り、 中 で も代 謝 、 神 経 機 能 、個 体 の 成 長 や 寿 命 な ど、 生 物 の 基 本 的 な 機 能 に 関 わ る遺 伝 子 は 保 存 性 が 高 い こ と が わ か っ て い る 。 本 研 究 で は 、 シ ョ ウ ジ ョ ウバ エ を用 い て 大 腸 菌 で 同 定 され て い る リ ボ 酸 修 飾 に 関 わ る 酵 素 の 遺 伝 子 の シ ョ ウ ジ ョ ウバ エ ホ モ ロ ジ ー に 注 目 し 、そ れ らの リ ポ イ ル 化 に お け る 機 能 、お よ び 生 体 内 の機 能 を 明 らか に す る こ と を 目的 と した 。 具 体 的 に は リポ イ ル トラ ン ス フ ェ ラ ー ぜ(liρB、 シ ョ ウ ジ ョ ウ バ エ で はLiioT2)、 リボ 酸 シ ン セ タ ー ゼ(1i .,oA、 シ ョ ウ ジ ョ ウバ エ で はLas)、 リボ 酸 塩 タ ン パ ク 質 リガ ー ゼ(lplA、 シ ョ ウ ジ ョ ウバ エ で はLplA)の3つ の 遺 伝 子 に つ い て 機 能 破 壊 、機 能 抑 制 変 異 や 過 剰 発 現 トラ ン ス ジ ェ ニ ッ ク 個 体 を 作 製 し そ れ を 解 析 し た 。 Lasの 過 剰 発 現 とLi .,oT2変 異 体 及 び ノ ッ ク ダ ウ ン シ ョ ウ ジ ョ ウバ エ は ピル ビ ン 酸 脱 水 素 酵 素 と2一 オ ク ソ グ ル タ ル 酸 脱 水 素 酵 素 の リポ イ ル 化 が 減 少 し 、酵 素 の 活 性 が 低 下 し た 。 メ タ ボ ロ ー ム 解 析 の 結 果 、解 糖 系 の ピル ビ ン酸 、 ク エ ン 酸 回 路 の2一 グ ル タ ル 酸 が 蓄 積 され て い る こ と が 分 か っ た 。ATPの 生 合 成 量 、NADH+/NAD比 オ クソ が低 下 し た こ と か ら 、エ ネ ル ギ ー 代 謝 系 に 影 響 を 及 ぼ して い る こ とが 示 唆 され た 。加 齢 に 伴 う体 重 の 減 少 と ト リグ リセ リ ド減 少 が 加 速 され た こ と か ら 、脂 質 異 化 に よ る エ ネ ル ギ ー 産 生 で 補 わ れ て も の と推 測 され る 。ま た 、LasとLplAの ウバ エ は リー サ ル に な っ て し ま い 、LipT2ノ ノ ッ クダ ウ ンシ ョウジ ョ ッ ク ダ ウ ン と三 つ の リポ イ ル 化 関 連 遺 伝 子 の 過 剰 発 現 シ ョ ウ ジ ョ ウ バ エ も 寿 命 が 短 く な っ た 結 果 と合 わ せ て み た ら 三 っ の 遺 伝 子 は シ ョ ウ ジ ョ ウバ エ に は な く て は な ら な い 大 事 な 役 割 を し て い て,発 現 量 が 少 な 4 く て も 多 く て も代 謝 に は 副 影 響 を 与 え る の が 分 か っ た 。シ ョ ウ ジ ョ ウ バ エ で の ピ ル ビ ン 酸 脱 水 素 酵 素 と2一 オ ク ソ グ ル タ ル 酸 脱 水 素 酵 素 リポ イ ル 化 プ ロ セ ス は 大 腸 菌 と は 異 な っ て い る と考 え られ る 。 5 MASTER'STHESIS Theroleoflipoylation-relatedgenesinenergy metabolisminDrosophila JeonghwaDan CellularGeneticsLaboratory DepartmentofBiologicalSciences TokyoMetropolitanUniversity 6 CONrENrs 8 ABSTRACT 0 1 N O n C U D O R T N I 3 1 MATERIALSANDMETHODS 7 1 S T L U S E R 0 2 N ㎜ D 2 2 ACKNOWLEDGEMENTS 3 2 聡 ㎜ R 5 2 FIGURELEGENDS 8 2 ㎜ F 7 ABSTRACT Lipoicacid(LA),alsocalledthiocticacidworksasananti-oxidantlikevitaminC, vitaminEandcoenzymeQIotoprotectourbodyfromtheharmoffreeradicals. Unlikewatersolublevitamins,LAcanworkinfattytissuesbecauseitissolublein bothwaterandfat.LAisacofactorofseveralenzymesincludingpyruvate dehydrogenase(PDH)and2-oxoglutaratedehydrogenase(OGDH),whichare involvedinthemajorenergyproducingpathways,glycolysisandTCAcycle.The pathwayoflipoylationofPDHandOGDHhasbeenwellcharacterizedinE.coli.It hasbeenshownthatthelipoylationprocessinvolvesthreegenes;lipoicacid synthetase(lipL・4),lipoate-proteinligase(ILPl∠1),andlipoyltransferase(lip、8). Eukaryotesgenomescontainorthologsoftheseenzymes,buttheirfUnctionsinLA synthesisandincorporationintoproteinsremainedelusive. Inthisthesis,tounderstandthegeneticbasesoflipoylationineukaryotes,Iused Drosophilaasamodelorganismandinvestigatedtherolesof五as,∠{ρ theorthologsofl)acterialli]A,L,pIA,andli.8,respectively.Iusedthe(}AL4一 systemtooverexpressorRNA-mediatedgeneknockdowninDrosophila,and analyzedtheirphenotypesincludingviability,lifespan,metabolomes.Ialsoanalyzed threetransposoninsertionlinesinwhich-P-elementsareinsertednearbyeachofthe threeloci. Ifbundthatubiquitousknockdownorthetransposoninsertionin五as,LiT2showed defectinlipoylationofPDHandOGDHandtheirviabilitywasseverelyimpaired. Theseresultsindicatedthat五as,一 ∠1フ ゐ4and五ipT2areessentialfbrviabilityandare 8 乙4,andLipT2, 乙IAS requiredfbrthelipoylationpathwayofPDHandOGDHinDrosoρhila.Thus,the lipoylationpathwayislikelytobedifferentfromthatin-E.coli. 9 INrRODUCTION Lipoicacid(LA)isanorganosulfurcompoundderivedfromoctanoicacid(1).Itisa fattyacidthatactsasananti-oxidantlikevitaminC,vitaminEandcoenzymeQlo. LAcanworkinbothwaterandfat,forminganti-oxidationnetworktogetherwith otheranti-oxidantstoprotectourbodyfromharmoffreeradical.LAispresentin almostallfbods,andatslightlyhigherconcentrationinkidney,heart,liver,spinach, broccoli,andyeastextract(2). EndogenouslysynthesizedLAisuniversallyrequiredfbraerobicmetabolism,works asacofactorofpyruvatedehydrogenasecomplex(PDH)and2-oxoglutarate dehydrogenase(OGDH),involvedinthemajorenergyproducingpathways, glycolysisandTCAcycle(Figurel)(3,4).Inthecell,verylittleLAexistsasafree acidandmostofthemarecovalentlyattachedtotheE2subunitoftheseenzyme complexesthroughaminobondtoaspecificlysineresidue(5).Thispost-translational modificationisessentialfortransferringreactionintermediatesamongactivesitesof theenzymecomplexinbothcasesLAincoq)orationintoproteinpathwayin prokaryoteswasalreadyuncovered(3). ThepathwayoflipoylationofPDHandOGDHhasbeenwellcharacterizedinE. coli(Figure2)(6).Thepathwayinvolvesthreelipoylation-relatedgenes;lipoicacid synthetase(liA),lipoate-proteinligase(lplA),lipoyltransferase(li.8).Previous studiesindicatethatE.co1'hasatleasttwopathwaysfbrattatchingLAtolipoyl domains(LDs)(7).Precursortolipoicacid,octanoicacid,ismadeviafattyacid biosynthesisintheformofoctanoyl-acylcarrierprotein.Thelipoyltransferase(lipB) cantransfereitheralipoyloroctanoylgroupfromaacylcarrierprotein(ACP)to lipoyl-acceptingdomains.ThereactionisfbllowedbyadditionofsulfUrbylipAto producelipoylappendage.Lipoate-proteinligase(ろ ρ乙4)catalyesthetwosteps. Followingthisreaction,lipoicacidsynthaseconvertstheoctanoylateddomainsof targetproteinsintolipoylmoiety(8). Basedontherecentreport,lilpレ4-deficient-E.colibecomesauxotrophicforLA,which canbetakenupandmetabolizedviathesalvagepathway.Incontrast,eukaryotesare 10 strictlydependentonthedenovosynthesisofthelipoylgroupwithinmitochondria. ThishasbeenshownintheSaccharomアcescerevisiaelipsmutant(五asinDrosophilの, whichisunabletoutilizeLAsuppliedinthegrowthmedium.Itwasalsofbundthat earlyembryoniclethalityofLasknockoutmicecannotbeovercomeoramelioratedby fc)edingofLAtopregnantheterozygousmice(7). DefectofPDHcausesmetabolicacidosisaccumulatingpynlvateandlactate,and completedeficiencyofPDHresultsindevelopmentaldefectsofthenervoussystem, muscularspasticityandearlydeath(9).Inzebrafish,amutationinthePDH-E2 subunitcausesneurologicaldysfUnctionandembryoniclethality(10).Diminished OGDHfUnctionalsocausesmetabolicdeficiencyresultinginneuronaldysfUnction. Inaddition,enzymeactivityofOGDHismarkedlydeclinedinsome neurodegenerativediseases,suchasParkinson'sdisease,Wernick-korsakoffsyndrome andAlzheimer'sdisease,suggestingthatimpairmentsofglucosemetabolismcould triggerneuronaldeathinthesediseases(ll).Amutationinlipoicacidsynthasegene (五as)causedanovelmitochondrialdisease(12).Patientshaveseveralsymptomsthat oralautomatismsofleftarmandlegwhichlastedforapproximatelyoneminute.They alsoobservedincludingrecurrentapneas,reducedconsciousness,worsenedhypotonia andlactateacidosiswhichisassociatedwithahighleveloflactateinthebloodof newbom.Thesesymptomsmaybecausedbydeficiencyof五asandconcludethat defectsinanyofthesestepsmightresultinasimilarbiochemicalandclinical phenotype(13).Reductionof-Lassignificantlydecreaseslipoylationlevelsofboth PDHandOGDH.Thus,thesymptomsofLasdeficiencyislikelytocausethe disru.ptionofthesemitochondrialenzymes.Althougheukaryotesappearedtocontain orthologsoftheseenzymes,themechanismofLAsynthesisandincorporationinto proteinsremainedelusiveineukaryotes. HereIusedDrosophilatoinvestigatetherolesofgenesinvolvedinLAsynthesis andcovalentattatchmentoflipoatetoPDHandOGDHthroughmanipulatingthree lipoylation-relatedgenes,Las,Lp∠!4,and-Z}ipT2,thecounterpartsofbacteriallipA, lplA,andlipB,respectively.TorevealtherolesoftheDrosoρhilahomologsoflipA lplAandlipB,IusedRNAi-mediatedgene㎞ockdown(KD)andoverexpression(OE) 11 ofeachgeneinDrosophilausingtheGAL4一 乙fASsystem.Thesystemhastwoparts: theGAL4gene,encodingtheyeasttranscriptionactivatorproteinGAL4,andtheUAS (UpstreamActivationSequence),anenhancertowhichGAL4specificallybindsto activategenetranscription.Ialsoanalyzedthreetransposoninsertionlinesinwhich -P-elementsareinsertednearbyeachofthethreeloci. Icharacterizedthephenotypesincludingviability,lifespan,metabolomes.Ifbund thatthechangeinexpressionlevelofeachlipoylation-relatedgenesresultedina shortendlifespananddecreasedlipoylationlevelinPDHandOGDH.Theactivityof PDHwasalsodecreased,suggestingthatthreelipoylation-relatedgenesparticipate thelipoylationofPDHandtheycan'treplaceeachothertocompletethismechanism. Inaddition,thelipoylation-relatedgenesOEmodelsshowsdecreasedNAD+and NADHratioandATPproduction.Ialsodemonstratedthatlowerbodyweightand triglyceride(TGA)levelsinLasandL,plAOEorKDflies.Theseresultsprovided evidencethatthreelipoylation-relatedgenesplayanimportantroleinenergy metabolisminDrosoρhila. 12 MATERIALSandMETHODS FIystrainsandmaintenance Thestandardlaboratorystocksyw,w川8usedascontrolstraintotransposoninsertion linesandthefliesgeneratedbycrossingw11180rしIAS-GFPtoGA-L41ineswereused ascontrolstraintoUASlines.actin-GAL4,elav-GAL4, .pρ1-GAL4,UAS-GFP, -P{EP}LasG6544,SUPor-P?CG428371KGO94467,wereobtainedfromthe BloomingtonStockCenter.-PBac♂WH}CG9804/05071wasobtainedfromthe ExelixisCollectionatHarvardMedicalSchoolandしIAS-Las(CG5231?-IR(5231R-4?, UAS-LipT2(℃G9804)-IR(9804R-3?,UAS-LρIA(CG8446)-1Rで8446R-2りwerefromFly StockofNationalInstituteofGenetics.uAS-Las(CG5231ノ,uAS-LipT2ピCG9804ノ, UAS-∠{ρ 乙41℃G8446)weregeneratedbyourselftousefbrexperimentsasa overexpressionstrain.Allstockswerebackcrossedtow1118strainatleastfbrsix generationsbefbreusingfbrexperiments.Strainsusedinthisstudyaresummarized withdetailinformation(Tablel).Allflieswererearedonastandard glucose-yeast-agarmediumcontainingpropionicacidandn-butylp-hydroxybenzoate asmoldinhibitors.Fliesweremaintainedat250Cthroughoutdevelopment.Flieswere trasfc)rredtofreshvialseverythreedays QuantitativeRT・PCR TotalR[NAwasextractedfromadultfliesusingTRIzolreagent(lnvitrogen)andpoly A(+)RNAwasreversetranscribedbySuperScriptIIItranscriptase(Invitrogen). QuantitativeRT-PCRwasperformedbyusingsYBRPremixExTaq(TaKaRa)and Chromo4Four-ColorReal-TimeSystem(Bio-Rad).Valueswerenormalizedagainst thelevelofrp-49mRNA.PrimersusedinthisRT-PCRarelistedinbelow. rρ一49primer Forward:AAGATCGTGAAGAAGCGCAC Reverse:TGTGCACCAGGAACTTCTTG Lasprimer Forward:AAGATCGTGAAGAAGCGCAC 13 Reverse:TGTGCACCAGGAACTTCTTG LipT2primer Forward:AAGATCGTGAAGAAGCGCAC Reverse:TGTGCACCAGGAACTTCTTG LplAprimer Forward:AAGATCGTGAAGAAGCGCAC Reverse:TGTGCACCAGGAACTTCTTG Western止)lotting Adultflieswerehomogenizedinl×SDS-PAGEsamplebuffer.Thesampleswere heatedfbr5minat950C.ProteinsinthesupernatantwereseparatedonalO% SDS-PAGEgelandtransferredtoaHi-bondPmembrane(GEHealthcare,Waukesha, WI).Afterblockingwithasolutioncontaining5%BSA,themembranewas incubatedwithrabbitanti-a-Lipoicacidantibody(1:4000dilution,Calbiochem,city), rabbitanti-phospho-AMPKantibody(1:4000dilution,CellsignalingTechnology, Danvers,MA),fbrovernightat40C,rinsedwithTBST(TBSwithO.1%Tween20), treatedwiththeblockingsolutionfbr5min,andthenincubatedwith HRP-cor巾gatedanti-rabbitIgG(GEHealthcare).Signalsweredetectedusing ECL-plusreagents(GEHealthcare). LOngevityteSt Newlyeclosedflieswerekeptinaglassvialcontainingstandardglucose-yeast medium,transferredtofreshmediaevery2-3day,andthenumberofdeadflieswas countedatthetimeoftransfer.Atleastl20fliespereachgenotypewereusedfbrthe lOngeVityteSt. Climbingassay Climbingactivityassaywasperformedasdescribedpreviously(14).Twentyadult maleflieswereplacedinalongvial(2cmindiameter;20cminlength),andbumped downtothebottom.Pictu.resweretakenat8safterthebumping,andusedtomeasure 14 theheighteachindividualclimbedup.Foreachsample,3trialswerecarriedoutto determinetheaverageclimbingactivity. Triglyceridemeasurement Tenadultflieswereweighedandhomogenizedin100ulofO.1%Tween20(Wako), heatedfbr5minat700Candstoredat-80。C.Thesampleswerethawedonice,and centrifUgedfbrlOminatl3,000rpmatroomtemperature.Theamountoftriglycerides wasdeterminedbyusingFreeglycerolreagent,Triglyceridereagent(Sigma)to determinethetriglyceride.Concentrationofsolubleproteinwasmeasuredbyusing Bio-Radproteinassayreagent.Assayswereperfbrmedatleastintriplicate. MeasurementofPDHandOGDHactivity Mitochondriawereisolatedfromadultfliesasdescribed(15,16).Thirtyflieswere homogenizedinlmlchilledmitochondrialisolationmedium(MIM:250mM sucrose,10mMTrispH7.4,0.15mMMgCl2).Thesampleswerecentrifugedtwice f()r5minatl,000gat40Ctoremovedebris.Thesupematantwasthenspunfbr5 minatl3,000gat40C.ThepelletwaswashedwithlmlofMIMandresuspendedin 50μlofMIM.MeasurementofPDHactivitywascarriedoutaccordingto Hinman,smethod(17).Briefly,50μgofmitochondrialsuspensionwasmixedwith anassaybuffer(2.5mMNAD(Sigma-Aldrich,St.Louis,MO),0.lmMcoenzyme A,0.2mMthiaminpyrophosphate,0.3mMdithiothreitol,lmMMgCl2,lmg/ml BSA,0.05Mphosphatebuffer,pH7.8,0.6mMIodonitroterazoliumchloride (Sigma-Aldrich),0.lmg/mldihydrolipoicaciddehydrogenase).Thereactionwas initiatedbyadditionof5mMpyruvateandscannedat500nmfbr5min.OGDH activitywasmeasuredusingtheHumphriesandSzweda'smethod.Briefly(18),50 μgofmitochondriasuspensionwasplacedtoanassaymixture(5mMMgCl2,0.5mM EDTA,35mMphosphatebufferpH7.4,200μMthiaminepyrophosphate,0.5mM NAD,130μMCoenzymeA(Sigma-Aldrich),2.5μMrotenone(Sigma-Aldrich)). Thereactionwasstartedbyadding2mM2-oxoglutarateandtheabsorbanceat340 nmwasmonitoredfbr2mintomeasureNADHgeneration.Allmeasurementswere 15 performedinatleasttriplicate. Measurementofglucosemetabolites Tenadultflieswerehomogenizedineitheracetonitrile/water(3:1)onice. Homogenateinacetonitrile/waterwascentrifUged(fbrlOminatl20,00×g)to removedebris.Homogenateinwaterwasmixedwith3volumeofacetonitrilebefbre centrifUgation.ThesupernatantwasdriedupusingmiVacSampleConcentrator (Genevac)anddissolvedin20μlofdistilledwater.Thesampleswereanalyzedby liquidchromatographyquadropoletime-of-flightmassspectrometrysystemAcquity uPLcandxevoQTofMs(waters,Milfbrd,MA).chromatographicseparationwas performedat45。cusinganAcQuITYuPLcHssT3column(1.oxloomm,1.8 μm;waters)oranAcQuITYuPLcBEHcl8column(1.ox50mm,1.7μm; Waters):thecolumnswereequilibratedwithlOmMDibutylammoniumacetate (pH4.95)andcompoundswereelutedwithagradientofmethanolconcentration.The MSsystemwasequippedwithadualelectrosprayionization(ESI)probeand operatedinthenegativeionmodewiththesourcetemperatureatl200CThemass scanningrangewasm/zsO-1000.Massmeasurementwascalibratedbylockmasses toobtainhighmassaccuracyofanalysis.Leucineenkephalin([M-H]一=554.2620) wasusedasalockmass.Obtainedionintensityofeachcompoundwasnormalized againstionintensityofL-Phenylalanine-13C9,15N. Statisticalanalysis Statisticalanalysesforsurvivalswereperformedusingalog-ranktest.Forallother experiments,meanscomparisonswereanalyzedusingaStudent'st-test. 16 Results Lipoylation・relatedgenesareimportantforviabilityin・DrosOphita Tricarboxylicacidcycle(TCA)cycleandglycolysisarethemostimportant carbohydratemetabolismpathwaysfbrallaerobicorganisms.Thelipoylgroupandits associatedsubunitE2serveasthecoreofthisgiganticfactory(9).LiT2(lipBin -E.coli)and五as(勿 レ4in-E.co1')isrequiredfbrlipoylationoftwomitochondrialtarget proteins;E2subunitsofPDHandOGDH(9).TheL,plA(LρIAinE.coli)isalso requiredfbranothermechanismthattheorganismcanutilizefreeLAfromthe mediumandalsouseoctanoicacidasasubstrate,albeitwithreducedefficiency(9). Thismechanismisalreadyuncoverdin-E.coliandIfbundthatthesethreegenesare alsoconsevedinDrosophilamelanogaster. Inordertoinvestigateallthethreelipolation-relatedgenes加vivofUnctionof Drosophila,Iconstru.ctedeachgene'soverexprssiontransgenicfliesandobtained somemutantand㎞ockdown(KD)fliesfromstockcenter.Quantitativereal-time PCRanalysisshowedthatmRNAexpressionlevelshavechanged(Figure4A&B).I analyzedthephenotypesoftheseflies.FirstIcheckedlifc)spanoftheseflies(Figure 5A&B).FlieshomozygousforLasKOmutantandLasKDaswellasLρIAKDflies werelethal.LiT2KD(drivedbyactinGA五4)shortendlifespandramaticall》i.In addition,五asOE,.LρIAOE(drivedbyactinα4五4)alsoshortendlifespan.Theresults suggestedthatalloftheselipoylation-relatedgenesareimportanttoDrosoρhilaalive andincreasedexpressionlevelofthesegenesarenotbenefitfbrDrosophila's metabolism. Contri-butionoflipoylationandmetabolicdisruption Lipoylation-relatedgenesarerequiredfbrthelipoylmodificationoftargetproteins, E2subunitsofPDH(CG5261,PDH-E2)andOGDH(CG5214,0GDH-E2). TherefbreIcomparedthelevelsoflipoylatedproteinsbetweencontrolfliesandKO orKDfliesfbreachgenebyimmunoblottinganalysisusinganti-LAantibodythat recognizeslipoicacidcovalentlyboundtoproteins.Predictedmolecularweightsof 17 PDH-E2andOGDH-E2wereapproximately54kand50k,respectively.Twodistinct immunoreactivebandsweredetectedinwild-typefliesandamountsofthesebands wereclearlyreducedinLasOE(drivedbyactinGAL4)andLiLpT2mutantflies (Figu.re6A&B&C&D).TheresultssuggestedthatPDH-E2andOGDH-E2arethe targetproteinsofLasandLipT2.Thesegenesplayanimportantroleandcontributeto thelipoylationPDH-E2andOGDH-E2inDrosophila.TheLasOEfliesshowsa reducedleveloflipoylationinbothPDH-E2andOGDH-E2,suggestingthatincreased expressionlevelofLasalsoimpairsthenomalmetabolism.Imeasuredtheenzymatic activitiesofPDHandOGDHinmitochondrialfractionisolatedfromwholebodyof adultf[ies.PDHactivitiewasincreasedinallmodelfliescomparedtocontrolf[ies (Figu.re5D).IndicatingthatlipoylationisessentialfbrthefUnctionoftheseenzymes inDrosophila.Inglycolysis,PDHconvertspyruvatetoacetyl-CoAandinTCAcycle OGDHconvert2-oxoglutaratetosuccinyl-CoA.ThusIexpectthatthesemodelf[ies mayhavedefectsinglucosemetabolism;glycolysisandTCAcycle.Impairmentof metabolicpathwaysoftenresultsinintermediarymetaboliteimbalances.Therefore,I quantifiedthecontentsofmetabolitesrelatedtoglycolysisandTCAcyclebyliquid chromatographyquadropoletime-of-flightmassspectrometry(LC-MS).InLasOE (drivenbyactin-GAL4)Ifbundthatthereareaccumulationofpyru.vateand 2-oxoglutaratewhicharesubstratesfbrPDHandOGDH(Figure7A).Itprovidedin vivoevidencethatPDHandOGDHaredefectiveinLasOEflies.Thiscouldbethe reasonwhyPDHandOGDHactivitiesweredecreasedorwhyTCAcycleslowing downinLasOEflies. Theenergysynthesis GlucosemetabolismthroughglycolysisandTCAcyclereducesNADtoNADH finallyresultinginATPgeneration.Toestimatethemetabolicactivitiesofglycolysis andTCAcycle,ImeasuredtheNADHANADratioandATPcontentincontroland modelflies(Figure7B&C).LasOEandLplAOE(drivedbyactin-GAL4)shows lowerlevelinbothNADH∠NADratioandATPcontentcomparedtocontrolflies, suggestingthatLasOEandLplAOEflieshavemetabolicdeficiencywithinefficient 18 energysynthesis. Metabolicanalysis Inmetabolicprocesses,ATPasanenergysourceconvertsitbackintoitsprecursors. WhentherearenoenoughATPtomaintainthemetabolism,theywilluse魚tbodyasa secondenergysourcetokeepthemetabolicbalance.Ioverexpressedor㎞ock downedeachlipoylation-relatedgenesinDrosophila'sfatbodyusingppl-(}A五4asa driver,andfbundthatLα51Rf[iesshowsslowerdevelopmentspeedthancontrolf[ies (datanotshown).TheaveragebodyweightofthelO-day-oldflieswassignificantly reducedfromthatof1-day-oldonesin五asIRand々 フIAIRf[ies,whiletherewasno significantdifferenceinbodyweightofwild-typefliesduringthesameperiod(Figu.re 8A&C).InDrosophila,thefatbodyistheprimarytissuefbrenergystorageofneutral fat,suchasdiacylglyceridesandtriglycerides(TGA)(Figure8B&D).ThentheTGA levelinLα50EincreasedingeneralwhentheywerebomedbutafterlOdaysLα50E and五asIRbothincreasedinTGAlevels,whichisdifferentwithcontrolfliesthat showsincreasedlevelinTGAafterlOdays.TGAlevelsinLρIAOEaswellasllρ 肩 IRweresignificantlylessthanthatofsame-agedcontrol.Theseresultsimplythatfat bodycatabolismwaspromotedin五asandZ(ρ 乙40EorKDf[iesbecauseofthe dysfUnctionofglycolysisandTCAcycle. Lipoylation・relatedgenesinthe1)rosophitaneuroussystem Todeterminethephysiologicalphenotypeoflipoylation-relatedgenes,Imeasured theirlocomotoractivitybyclimbingassay.Lipoylation-relatedgeneswere overexpressedinneuronsusingelav-GAL4asadriver.AlloftheseOE,KDand mutantfliesweredefectiveinclimbingabilitywhencomparedwithcontrolflies (Figure9). 19 Disscusion Lipoylationisanessentialpost-translationalmodificationfbrPDH&OGDHtowork inglycolysisandTCAcyle,themajorenergymetabolismpathways.Mostofstudies onlipoylationpathwayhasbeencarriedoutinprokaryotes,andhaveshownthatthe lypolationprocessinvolvesthreegenes;lipoylsynthaseLas,lipoyltransfc)rase五ip7『2, lipoateproteinligaseLpIA.InthisstudyIusedDrosophilaasamulticellularmodelto investigatetheroleoflipoylationineukaryotes.Thoughnaturalpopulationand laboratoryselectedDrosoρhilawithlipoylation-relatedgenescontroledhasbeenwell studied.Itookgeneticapproachtoidentifymoleculesinvolvedinthemechanism underlyinglipoylation. Ifbundthatthreelipoylation-relatedgenesalsoplayimportantrolesinDrosophila. Thelipoylation-relatedgenesmutantsandRNAi㎞ockdownaswellas overexpressionDrosoρhiladramaticallyshortenedlifespaninLasOEand五iT2KD flies.五asand∠(ρ 乙4KDfliesbecomelethalwhentheyareinearlypupastage.It's meanthattheselipoylation-relatedgenesarereallyimportantinDrosoρhila metabolism.Unexpectedresultfromshortenedlifespanin五asOEsuggestthat reducedorincreasedmRNAleveloflipoylation-relatedgenewasnotbenefitfbr Dros()phila.Westemblottingwithlipoicacidantibodyshoweddecreasedamountof lipoylatedPDHandOGDHin五asOEandLipT2mutantf[iesandPDHenzyme acitivitywasreducedinalllines.Itissuggestedthatthreelipoylation-relatedgenes areimportantfbrtheviabilityinDrosophila.五asandLiLpT2arerelatedtoenergy metabolismthroughlipoylationofPDHorOGDH.Unexpectedly,overexpressed lipoylation-relatedgenesarenotcontributetoDrosophila'smetabolism.Onthe contrary,theyleadmetabolicdeficiencyinDrosophila.Excessed五as〃zの theProteinCatabolismSystemsothedecreasedquantityofPDHleadsdecreased leveloflipoylatedPDHin五asOE.Anothercasetherearemaybeinduceproteins competitiontogainLaswithPDHwhentheLasoverexpreesed.Ithasbeenunknown whetherLplAparticipatethelipoylationineukaryotes.Itmaybehasalightertaskin thispathwayorLplAmaybeworksinanotherpathwayinsteadworksinlipoylation 20 めepromote mechanism.Accordingtotheresultsofwestemblotting,manipulationof lipoylation-relatedgenesexpressionlevelhadslightlystrongerwithPDHcomparedto OGDH.Theroleofthreelipoylation-relatedgenesmayhavedifferentmechanism dependingonthesubstrate. Lipoylation-relatedgenesalsohadanimpactonmetabolitesinglucosemetabolism. In五asOEthereareaccumulationsofpyru.vateand2-oxoglutarateandreductionsof othermetabolites.Therefbre,thesemodel且iesmayhavedefectsinglucose metabolism;glycolysisandTCAcycle.ThendecreasedATPcontentandNADH+ NADratioin五asOEsupportedthatPDHandOGDHaredefectiveinthese Drosophila,andtherecducedPDHactivitiesandTCAcycleappearedslowingdown in五asOEflies.EffectsonthebodyweightandTGAlevelsimpliedthatfatbody catabolismwaspromotedinLasandLplAOEorKDf[iesbecauseofthedysfUnction ofglycolysisandTCAcycle.Thethreelipoylation-relatedgenesareimportantfbr energymetabolism.Atleasttwolipoylation-relatedgenes五asandLiLpT2are participatethelipoylationpathwayandtheycannotbereplacedeachotherto completeit. ClimbingassayusedallDrosoρhilalines(drivenbyθ1α γ一GA五4)showdeficiencyin climbingability.InDrosophilalipoylation-relatedgenesmaycontributetothe neuronalfUnctionssuchasGCS(glycinecleavagesystem)inhumans. Accordingtothepreviousstudy,LipT2mutantshowednochangeinthiermetabolic conditionwhentheyhaveenoughfreelipoicacidinthierfbod,meansL,plAin Drosophilamaynotuse丘eelipoicacidtocompletethelipoylation.Inconclusion therearesomedifferencesbetweeneukaryotesandprokaryotesinlipoylation mechanism.IthinkDrosophiladidn'thavetwoindependentpathways(oneisfrom fattyacidsynthesis,anotheroneisusefreeacidfromfood)tocompletelipoylation.In thisstageIcan'tdecidethatwhichgenegoesfirstinthismechanismbutmaybethe threegenesworkinthesamelineinthismechanism.LpIAinDrosophilamaynot fUnctionaslipoateligaseproteinlike.E.coli,butitisanimportantgenefbrviabilityin Drosophila. 21 Acknowledgement AlwaysappreciatemyProf.ToshiroAigakiforhisadvicesandsupportsinmylifeof studyingaboard.Alwaysencouragemeandgivemesuchvaluablechancetostudyin hisworldandthought.IalsogainedmanyexperiencesinthistwoyearsthatIcouldn't learnfrombooks.It'savividmemoriesfbrme.Iamalsoconverymyappreciateto ourcellulargeneticslabmemberswhoalwaysgavemeahandwhenIwasintrouble. 22 References 1.TortF,Ferrer-cort6sx,Thi6M,Navarro-sastreA,MatalongaL,Quintana E,BujanN,AriasA,Garcia-VilloriaJ,AcquavivaC,Vianey-SabanC,Artuch R,Garcia-CazorlaA,BrionesP,RibesA(2013)Mutationsinthelipoyltransferase LIPTlgenecauseafataldiseaseassociatedwithaspecificlipoylationdefectof the2-ketoaciddehydrogenasecomplexes.HumMolGenet.2013,1-9. 2.LesterPacker,EricH,Witt,andHansJurgenTritschler(1995)Alpha-LipoicAcdi asaBiologicalAntioxidant.FreeRadicalBiology&Medicinel9,227-250. 3.JordanSW&CronanJE,Jr.(1997)Biosynthesisoflipoicacidand posttranslationalmodificationwithlipoicacidinEscherichiacoliMethods Enzymol279,176-183. 4.ReedLJ&HackertML(1990)Structure-functionrelationshipsin dihydrolipoamideacyltransferasesJBiolChem265,8971-8974. 5.FujiwaraK,Okamura-IkedaK,&MotokawaY(1996)Lipoylationof acyltransferasecomponentsofalpha-ketoaciddehydrogenasecomplexesJBiol Chem271,12932-12936. 6.SquireJ.Booker(2004)UnravelingthePathwayofLipoicAcidBiosynthesis. Chemistry&Biologyll,10-12. 7.MelissaS.Schonauer,AlexanderJ.Kastaniotis,V.A.SamuliKursu,J.Kalervo HiltunenandCarolL.Dieckmann(2009)LipoicAcidSynthesisandAttachment inYeastMitochondria.THEJOURNALOFBIOLOGICALCHEMISTRY284, 23234-23242 8.FatemahA.HermesandJohnE.Cronan(2013)TheroleoftheSaccharomyces cerevisiaelipoateproteinligasehomologue,Lip3,inlipoicacidsynthesis.Yeast 30,415-427 9.RobinsonBH(2006)LacticacidemiaandmitochondrialdiseaseMolGenetMetab 89,3-13. 23 10.BrownGK,OteroLJ,LeGrisM,&BrownRM(1994)Pyruvatedehydrogenase deficiencyJMedGenet31,875-879. ll.TretterL&Adam-ViziV(2005)Alpha-ketoglutaratedehydrogenase:atargetand generatorofoxidativestressPhilosTransRSocLondBBiolSci360,2335-2345. 12.JohannesA.Mayr,FranzA.Zimmermann,ChristineFauth,ChristaBergheim, DavidMeierhoferDorisRadmayr,JohannesZschocke,JohannesKoch,and WolfgangSperl(2011)LipoicAcidSynthetaseDeficiencyCausesNeonatal-Onset Epilepsy,DefectiveMitochondrialEnergyMetabolism,andGlycineElevation. TheAmericanJoumalofHumanGenetics89,792-797. 13.MayrJA,ZimmermannFA,FauthC,BergheimC,MeierhoferD,RadmayrD, ZschockeJ,KochJ,&SperlW(2011)Lipoicacidsynthetasedeficiencycauses neonatal-onsetepilepsy,defectivemitochondrialenergymetabolism,andglycine elevationAmJHumGenet89,792-797. 14.TsudaM,OotakaR,OhkuraC,KishitaY,SeongKH,MatsuoT,&AigakiT (2010)LossofTrx-2enhancesoxidativestress-dependentphenotypesin DrosophilaFEBSLett584,3398-3401. 15.SchwarzeSR,WeindruchR,&AikenJM(1998)Oxidativestressandaging reduceCOXIRNAandcytochromeoxidaseactivityinDrosophilaFreeRadic BiolMed25,740-747. 16.LoisM.Hinmant:andJohnP.Blass(1981)AnNADH-linked SpectrophotometricAssayforPyruvateDehydrogenaseComplexinCru.de TissueHomogenate.Receivedfbrpublication256,6583-6586. 17.HinmanLM&BlassJP(1981)AnNADH-linkedspectrophotometricassayfbr pyruvatedehydrogenasecomplexincrudetissuehomogenatesJBiolChem256, 6583-6586. 18.HumphriesKM&SzwedaLI(1998)Selectiveinactivationofalpha-ketoglutarate dehydrogenaseandpyruvatedehydrogenase:reactionoflipoicacidwith 4-hydroxy-2-nonenalBiochemistry37,15835-15841. 24 FIGURELEGENDS Tablel.Strainsusedinthisstudy Allstockswerebackcrossedtowlll8strainatleastfbrsixgenerationsbefbreusing f()rexperiments. Figurel.Schematicrepresentationofcentralpathwaysofenergymetabolism Glycolysisdegradesglucoseandproducespyruvate,whichisconvertedinto acetyl-CoAbypyru.vatedehydrogenasecomplex(PDH)and2-oxoglutaratetransfered tosuccinyl-CoAby2-oxoglutaratedehydrogenase(OGDH)intheTCAcycle.Lipoic acidworksasacoenzymeofthesetwoproteins. Figure2.Pathwayforlipoylincoporationin・E.co〃 Thepathwayinvolvesthreelipoylation-relatedgenes;lipoicacidsynthetase(lipA), lipoate.proteinligase@ム4),lipoyltransferase(lipB).-E.colimaintainatleasttwo pathwaysfbrattatchingthelipoatetolipoyldomains(LDs).Precursortolipoic acid,octanoicacid,ismadeviafattyacidbiosynthesisintheformofoctanoyl-acyl carrierprotein.Thelipoyltransferase(lipB,LipT2inDrosoρhilのcantransfereithera lipoyloroctanoylgroupfromacylcarrierprotein(ACP)tolipoyl-acceptingdomains. ThereactionisfbllowedbyadditionofsulfUrbylipoylsynthesis(伽 ン4,Lasin Drosoρhila,)toproducelipoylappendage.Lipoate-proteinligase(1ρIA,々 フ!Ain Drosophilのcatalyesthetwosteps.Followingthisreaction,lipoicacidsynthase convertstheoctanoylateddomainsoftargetproteinsintolipoylmoiet》 Figure3.TheG4L4'U4Ssystem GAL4一 乙IASsystemisalwaysusedasabiochemicalmethodtostudygene expressionandfunctioninorganismssuchasDros()phila. 25 乙 Figure4.QuantitativeRT'PcRanalysis QuantitativeRT-PcRanalysesofeachmodelflies.ThemRNAexpressionlevels werechanged.Unexpectly,P-elementinsertionLasmutantfliesshowsincreaselevel ofmRNAexpressionthanWT.Daterepresentmean±SEMofatleastthree experiments(Stu.dent'sttest;[*]p<0.05[**]p<0.Ol[***]p<0.001). Figure5.ShortenedLifespaninmodellines Survivalcurvesofmodelfliesandcontrolfliesmaleandfemale.Thelongevityof mutantsandKDaswellasOEflieswasdramaticallyreducedcomparedtothatof control. Figure6.Reductionoflipoylatedproteinsandtheiractivity Detectionoflipoylatedproteinsinadultflies(male)usingrabbitanti-lipoicacid antibody(A).AllRNAiandoverexpressionlineswerecrossedtoact-GAL4,a ubiquitousdriver.EnzymaticactivitiesofPDHcomplex(B)weremeasuredusing mitochondrialfraction.LipoylatedPDHenzymesweremeasuredwithsoftware (C&D).Daterepresentmean±SEMofatleastthreeexperiments(Student'sttest;[*] p<0.05[**]p<0.Ol[***]p<0.001) Figure7.Metabolicdisruptioninglucosemetabolism RelativeamountsofmetabolitesinTCAcycleandglycolysis(A).TheNADH/NAD ratio(B),ATPcontent(C)weresignificantlydecreasedinLasOEflies.Allrelative valueswerecalculatedagainstthedataofcontrolflies.Daterepresentmean±SEMof atleastthreeexperiments.(Student'sttest;[*]p<0.05[**]p<0.Ol[***]p<0.001) Figure8.ComparisonofthebodyweightandamountsofTGA AIIRNAiandoverexpressionlineswerecrossedtopρ1-GAL4,whichasadriverin fatbody.(A).LasOEandKDflieslosebodyweightwithage.Triglycerides(TGA) contentsweremeasuredatdaylanddaylO(B)it'sdecreased.(C)LplAOEandKD fliesalsoshowlooseweightwithage.LplAOEandKDfliesshowsdecreased 26 Triglycerides(TGA)contentswhentheyareindaylOthandayl(D)Datarepresent mean±SEMofatleastthreeexperiments.(Student'sttest;[*]p<0.05[**] p<0.Ol[***]p<0.001) Figure9.Clim-bingactivity AllRNAiandoverexpressionlineswerecrossedtoelav-GAL4,whichisexpressed inneuron.(AandB)Locomotoractivity.Climbingactivitiesofthemodellinesflies andcontrolflies.Theactivitywasdeterminedatday3aftereclosion.Theactivitiesof alllinelowerthanthatofcontrolinclimbingassay.(Stu.dent'sttest;[*]p<0.05[**] p<0.Ol[***]p<0.001) 27 Tablel.Strainsusedinthisstudy straln Genotype Ref Lα51R y[*]w【*】;加5(5231R-4)/TM6,Sb,Tb N【G Lψ721R y[*]w[*];P{w[+mW.hs]=GawB}CGlO74[NP5461] NIG 9 /TM6,P{w[一]=UAS-lacZ.UW23-1}UW23-1 1μ41R y[*]w[*];P{w[+mW.hs]=GawB}CG8446[NP6343] N【G /CyO,P{w[一]=UAS-lacZ.UWl4}UWl4 Lα50E y[1]w[*];UAS一 五α3(CG5231)/CyO Thisstudy Lψ720E y[1]w[*];UAS一 五ψ72(CG9804)/CyO Thisstudy 1μ40E y[1]w[*];UAS一 ムρ班(CG8446)/TM3,Sb[1]Ser[1] Thisstudy Lα5Mutant y[1]w[*];P{w[+mC]=EP}五 α3[G6544]/TM3,Sb[1] Bloomington Ser[1] Lψ72Mutant y[1]w[*];Mi{y[+mDint2]=MIC}CG9804[MIO5013] Havardmedical school 4μ4Mutant y[1];P{y[+mDint2]w[BR.EBR]=SUPor-P}CG4283 7[KGO9446]CG44243[KGO9446]/CyO;ry[506] Bl・ ・mingt・n Figurel.Schematicrepresentation ofcentralpathwaysofenergy metabolism 「 Glucose Glycolysis 自 臆● Acetyl・CoA Dehydrogenase Complex Citte。GDH 2■oxoglutarate dehydrogenase ate TCA CYCLE Fumrate Suc lsocirate Figure2.PathwayforlipoylincoporationinE.coti 触 ㍉● ● 禦>>>NlsH li3± ●HSvqll● ●(Las) Fattyac■dbiosynthesis Frommedium O e (LplA)lplA CAMP ウ 0 AMP 0 IplA(LplA) @9 。H竺L l3± lipoyltransferase lipoicacidsynthetase lipoate-proteinligase 30 Figure3。TheGAL4-UASsystem Tissuespecific romotor UAS GAL4 1■ipoylation ene 8 畢 ー 少 匠 1■ipoylation UAS ene Figure4.QuantitativeRT'PCRanalysisofallmodelstrains A 6 5 4 3 2 1 ■ ①︾2 ︽ 差 ∈ ①ξ 6■ ①匡 ★★ ★ ★ 「を ノ 幽 ズ認 一 一 ボげ ざ( 撃 認 〆 0 一t ★★★ 幽_ B T ﹄⋮ 2 1 1 1 β ∩ U 一 ①>2 ︽ 差 β ∩U 鴻 2 OU ∩U ∈ ①ξ 6■ ①匡 0 WT Las+1一 LplA-1一 Figure5.Lifespan A 00 1 MALE 80 承 ■6≧ ︾﹂5の 'ミ:● ● ●●●●●●Act1+ 6 0 ・・・…Act>GFP 40 20 Act>Las 0 Act>LplA 0 4060 20 80 Act>LipT21R Days B FEMALE 100 一〇≧ ヒ 5の 80 ●●●●●●Act/+ 60 ・・… ・Act>GFP 40 ⊂一■-gAct>Las 20 Act>LplA 0 0 20 4060 80 Days 33 100 Figure6.Reductionoflipoylatedproteinsandtheiractivity A 障・ PDHE2、 鞭獄少 OGDHE2ノ 試試ア Actin B 皿 ¶凸 8 戸 0 ** 4 り 2 ロ { U ■ ①︾①■エ(= ℃g 6∼ oΩコ * * 鶴髄 甥 寧 o ∪ PDcActMty(nmol/min/mg》 、 LipoylatedOGDHlevel 9-●-NNww8L oいoいoいoいo ooooooooo ooooooooo 。 駕 、 轟 駕 、.匁 曳 秘 %め 亭 %《 堵** ≒ 日一 気 礁 曳、 一 £ 呈 自8P謁 Figure7.Metabolicdisruptioninglucosemetabolism A ︻﹂ 亀 乙 ら乙 く﹂ -占 -占 ︻J 0 ∩U の↑= ①乞 ﹂O り ① ︾ 場 6 ■① 匡 1 撮 勲欝 摩 エ エ ■Act1+ ■Act>Las Act>LplA B 8 旧 一歪 1.2 ★ 1 O︽ミ エO︽Z Φ︾旧 一﹄ Φ匡 0.8 ★ 0.6 0.4 0.2 0 一 一 一T Act1+ 一 一 一 Act>Las Act>LplA C 1.2 1 ★ 8。 0 あ 0 鴻 0 2 0 0 3 =2 =Oり ﹂﹂︽ ①≧ 紹 ■ 2﹄ T 1.4 Act>LplA Act>Las Act1+ Figure8.ComparisonofthebodyweightandamountsofTGA A 0.006 ^0.005 導 ∠0.004 ロ 'di O.0033 ■Day1 言O・002 0 diO.001 囲Day10 0 評 〆 〆 瀦 メ B 嚢嚢 言旧 ①ぢ ﹂轟 ∈ 、。。∈ ︾︽2 0.700 0.600 0.500 0.400 0.300 ■DAY1 0.200 ■DAY10 0.100 0.000 評 《 〆続 〆銘 〆誘 C 嚢** ( ◎◎︾ 主 りo旧 ①3 言 om 0.00500 0.00400 0.00300 0.00200 ■Day1 0.00100 ■Day10 0.00000 〆 〆ノ D **嚢 嚢嚢★ 「 0.8 ぬ εO・7 「 20.6 9 90.5 凶 ∈0.4 、 ooO.3 ■Day1 ■DaylO ∈ とo・2 繹o.1 0 PPL》GFP PPL》LplA 39 PPL>LplAIR Figure9.Climbingactivity A ★ ∩U 0 O︾ 5 0σ 0 00 5 ξ8。 §6。 婁4・ ξ2・ 0 WTLas+1-LplA-1一 〆 §∼100 誤 ノ ^120 ゐ ♂8 評 詞 評 ボ ノ 〆 ワ﹁ 馴 か 00 0 (3 ︾﹀彗 旧 ぢ 帽 O=旧 ρ∈ 旧 圏O 1 B
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