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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
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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
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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
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Figure5.Lifespan
A
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1
MALE
80
承 ■6≧ ︾﹂5の
'ミ:● ●
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6
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⊂一■-gAct>Las
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20
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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
■
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、
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駕
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≒ 日一
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一
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呈
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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
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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
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Act>LplA
C
1.2
1
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8。
0
あ
0
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0
0
3 =2 =Oり ﹂﹂︽ ①≧ 紹 ■
2﹄
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1.4
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Act>Las
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Figure8.ComparisonofthebodyweightandamountsofTGA
A
0.006
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