学 位 論 文

修

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 大学 院

理工学研究科 生命科学専攻

学修番号12881328

氏 名 段 晶華

Theroleoflipoylation‑relatedgenesinenergymetabolisminDrosoρhila

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)

細 胞 遺 伝 学 研 究 室 段 晶 華

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

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 オ ク ソ

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 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 ノ ッ ク ダ ウ ン シ ョ ウ ジ ョ ウバ エ は リー サ ル に な っ て し ま い 、LipT2ノ 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

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

CONrENrs ABSTRACT

MATERIALSANDMETHODS

NOnCUDORTNI STLUSER

ACKNOWLEDGEMENTS

N聡  

㎜㎜㎜

DRF 803701112

FIGURELEGENDS

23582222

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 乙4,andLipT2,

theorthologsofl)acterialli]A,L,pIA,andli.8,respectively.Iusedthe(}AL4一 乙IAS

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

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

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.

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.

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 めepromote

theProteinCatabolismSystemsothedecreasedquantityofPDHleadsdecreased

leveloflipoylatedPDHin五asOE.Anothercasetherearemaybeinduceproteins

competitiontogainLaswithPDHwhentheLasoverexpreesed.Ithasbeenunknown

whetherLplAparticipatethelipoylationineukaryotes.Itmaybehasalightertaskin

thispathwayorLplAmaybeworksinanotherpathwayinsteadworksinlipoylation

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.

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》 乙

Figure3.TheG4L4'U4Ssystem

GAL4一 乙IASsystemisalwaysusedasabiochemicalmethodtostudygene

expressionandfunctioninorganismssuchasDros()phila.

25

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

straln9 Genotype Ref

Lα51R y[*]w【*】;加5(5231R‑4)/TM6,Sb,Tb N【G

Lψ721R y[*]w[*];P{w[+mW.hs]=GawB}CGlO74[NP5461]

/TM6,P{w[一]=UAS‑lacZ.UW23‑1}UW23‑1

NIG

1μ41R y[*]w[*];P{w[+mW.hs]=GawB}CG8446[NP6343]

/CyO,P{w[一]=UAS‑lacZ.UWl4}UWl4

N【G

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]

Ser[1]

Bloomington

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 metabolism

ofcentralpathwaysofenergy

「

Glucose Glycolysis 自

臆 ●

ate

Fumrate

Suc

Acetyl・CoA

TCA CYCLE

Dehydrogenase Complex

Citte。GDH

2■oxoglutarate dehydrogenase

lsocirate

Figure2.PathwayforlipoylincoporationinE.coti

触 ㍉● 禦>>>NlsH

li3±●HSvqll●

Fattyac■dbiosynthesis

Frommedium

ウ

0

。H竺L

IplA(LplA)

(LplA)lplA

AMP@9

0

●

●(Las)

O

e

CAMP l3±

lipoyltransferase lipoicacidsynthetase lipoate‑proteinligase

Figure3。TheGAL4‑UASsystem

Tissuespecific

romotor GAL4 UAS 1■ipoylation ene

8

畢

ー

少 匠

1■ipoylation

UAS _ene

Figure4.QuantitativeRT'PCRanalysisofallmodelstrains

A

★ ★

★ ★ 「を

幽

一t

654321

■①︾2︽差∈①ξ6■①匡 0

ノ

認

ボげ〆

撃ざ( 認

B T

﹄⁝21ββ鴻2011∩U∩UOU∩U

一①>2︽差∈①ξ6■①匡

LplA‑1一 Las+1一

WT

Figure5.Lifespan

A

00806040201

承■6≧︾﹂5の 0

0 20

'ミ:● ●

4060 Days

80

MALE

●●●●●●Act1+

・・・…Act>GFP

Act>Las

Act>LplA

Act>LipT21R

B

一〇≧ヒ5の

100

80 60 40

20

0

0 20 4060

Days

80 100

FEMALE

●●●●●●Act/+

・・… ・Act>GFP

⊂一■‑gAct>Las

Act>LplA

33

Figure6.Reductionoflipoylatedproteinsandtheiractivity

A

PDHE2、

OGDHE2ノ

鞭獄少

障・

試試ア

Actin

B

皿 ¶凸8戸042{U   り ロ■①︾①■エ(=℃g6〜oΩコ

*

**

*

鶴髄 甥 寧

PDcActMty(nmol/min/mg》

9‑●‑NNww8L oいoいoいoいo ooooooooo ooooooooo

、

%め

≒ 日 一

︑ 轟 駕 ︑ . 匁

∪

LipoylatedOGDHlevel

。 ￡ 呈 自8P謁

駕

曳 秘

亭

気 一

%《

礁 曳 、

o

Figure7.Metabolicdisruptioninglucosemetabolism

A

︻﹂ら乙く﹂‑占︻J∩U亀乙‑占0

の↑=①乞﹂Oり①︾場6■①匡

1

撮 勲欝 摩

■Act1+

■Act>Las

Act>LplA

B

8旧一歪O︽ミエO︽ZΦ︾旧一﹄Φ匡

1.2 1 0.8 0.6 0.4 0.2

0 ^{一 一 一T  一 一 一}

★

★

Act1+ Act>Las Act>LplA

★

Act>LplA Act>Las

T

Act1+

1.4 1.2 1︒8あ鴻20000 0 C 3=2=Oり﹂﹂︽①≧紹■2﹄

Figure8.ComparisonofthebodyweightandamountsofTGA

A

0.006

^0.005

導

∠0.004 'diロ

O.0033

言O・002 0 diO.001

0

評 〆 〆 瀦メ

■Day1 囲Day10

B

嚢嚢

言旧①ぢ﹂轟∈︑︒︒∈︾︽2

0.700 0.600 0.500 0.400 0.300 0.200 0.100 0.000

評 《 〆続 〆銘 〆誘

■DAY1

■DAY10

C

嚢**

(◎◎︾主りo旧①3言om

0.00500 0.00400 0.00300 0.00200 0.00100 0.00000

〆 〆 ノ

■Day1

■Day10

D

0.8

ぬ

εO・7 20.6

9 90.5 凶

∈0.4

、 ooO.3

∈ とo・2

繹o.1 0

**嚢

「

嚢嚢★

「

■Day1

■DaylO

PPL》GFP PPL》LplA PPL>LplAIR

39

Figure9.Climbingactivity

A ★05050馴∩UO︾0σ0000ワ﹁1(3︾﹀彗旧ぢ帽O=旧ρ∈旧圏O 〆

誤 ノ ゐ ♂ 8

評詞

か

評ボ

ノ 〆

B

^120

§〜100

ξ8。

§6。

婁4・

ξ2・

0

WTLas+1‑LplA‑1一