分子醫(yī)學(xué) 代謝疾病

分子醫(yī)學(xué)課程“代謝疾病”

1.多功能代謝酶的生理學(xué)意義2.代謝紊亂與腫瘤發(fā)生發(fā)展多功能代謝酶：

糖代謝酶在基因轉(zhuǎn)錄調(diào)節(jié)

過程中的“兼職”功能以及

生理學(xué)意義

（模式分析系統(tǒng)：

S-期組蛋白基因表達的調(diào)控）EukaryotictranscriptionmachineriesarefarmorecomplicatedthantheprokaryoticonesFromBobRoeder,2003LaskerAwardCommentaryActivitiesofeukaryotic“sigmafactors”canbeaffectedbydiverseenvironmentalorintracellularcues:viralinfection,stress,signalsfromcytokines,hormones,smallmetabolitesormolecules,cellularredoxstatus/metabolicstates,etcHistoneexpressionisS-phase-specificand

inconcertwithDNAreplicationcore&linkerhistoneshistonegenetranscriptionfactorsand/orcofactorsH2A/H2B/H3/H4/H1NuclearProtein,Ataxia-Telangiectasialocus

S-phaseentryHistoneExpressionisS-phase-specificandinconcertwithDNAReplicationG1G1/SSS/G2G2MDNAsynthesis

HistoneexpressionH2Bβ-actinCouplingofHistoneExpressionandDNAReplicationintheS-phase1.

NewlysynthesizedDNAispackagedimmediatelyintonucleosomeswithhistones(chromatinassembly)2XH2A,H2B,H3&H42.IncompleteorimproperchromatinassemblymakesDNAmorevulnerabletomutagenicassaultsChromatinassemblylagstheDNAreplicationforkbyonlyoneOkazakifragment(~400ntinmammaliancells=~40mSpolymerizationtime)Watsonetal.,Mol.Biol.Cell

1. Histonegenesareorganized

intothenuclearsub-organelles

dubbed

CajalBodies(CBs)

2.

CycE/cdk2,itssubstrateNPAT,

andhistonetranscriptionfactors andcofactorsareallco-localized

intheCBsinmammaliancells

=2xH2A,H2B,H3&H4CBs=NPATfociHistone2B(H2B)TranscriptionTATABoxGTFs/PolIIOctamer(ATTTGCAT)ElementS-phase-inducibleH2BPromoterActivationUSA(PCs)OCA-Sp38/GAPDHp36/LDHOCA-S:Oct-1CoActivatorinS-phase,amulti-componentcomplexOct-1S-phase

signalingGTFs/PolII=GeneralTranscriptionFactors+RNAPolymeraseII(RNAPII)USA=Mediator+additionalPositiveCofactors

Octamer

bindingfactor1p65/HSP70Oct-1p36/LDHp38/GAPDHp20/nm23-H1p18/nm23-H2p36/UNGFNSFNS-IPp60/Sti1EarlierdefinedcomponentsoftheOCA-ScomplexOCA-Swasinitiallyidentifiedbytediousbiochemicalisolation/purificationComponentsofOCA-ScomplexProtein(kDa)IdentityEnzymeActivityRolesinOCA-Sp65Hsp70ATPase

ComplexAssembly?p38GAPDHGAPDH(NAD+)aTranscription,Oct-1-interacting

&redoxsensorp36LDHLDH(NADH)bTranscription,&redoxsensor(andredoxmodulator?)p36UNGUNG(DNArepair)

?p20/p18nm23H1/H2(d/r)NDP

KinasescTranscription?p60Sti1-dComplexAssembly?p10Unpublisheda.k.a.FNS-eNPAT-interacting(Y2H),&linkingcyclinE/cdk2?a,b,

knock-downabolishesH2Btranscriptionc,d,e, knock-downreducesH2BtranscriptionNetYield:1glucoseto2lactateand2ATPEleven-stepGlycolytic

Pathway**GAPDHLDHp38/GAPDHandp36/LDHareEssentialforH2BTranscriptioninLivingCellsp36/LDHKnock-downp38/GAPDHKnock-downOCA-SRoleinH2BTranscription

Oct-1Zheng,Roeder&Luo,Cell2003GTFs/RNAPIIH2BpromoterOctamerATTTGCATTATABoxPNPATCyclinE/cdk2OCA-Sp38/GAPDHp36/LDHChromatinImmuno-Precipitation(ChIP)assaysonsynchronizedcellsH2BPromoterOct-1p38/GAPDHNPATp60/Sti1Na?veIgGRG1SG2anti-OCA-Sp60/Sti1Sti1:Stressinduced1USA(PCs)Moonlightingrolesofp38/GAPDHandp36/LDHinH2Btranscriptionhavetwophysiologicalimplications:1)One-protein-multi-functionconcept2)AreNAD(H),thecoenzymesforenzymaticGAPDHandLDH,involvedinmodulatingmammalianH2Btranscription?

HumanH2BpromoterTATAboxGTFs+RNAPIIcomplexOct-1p38/GAPDHp36/LDHOCA-SATTTGCAT(Octamer)CyclinE/cdk2NPATDNAreplication,

S-phaseentry&progressionMetabolic/Redox

SignalingOtherhistonegenesCyclinE/cdk2andhistoneexpressionPO4p60/Sti1indirectdirectCell2003JBC2008CellCycle2009histone

expressioncoordinationH2BTranscriptioninaNuclearExtractDevoidofEndogenousNAD(H)andTitratedwithExogenousNAD+orNADHNAD+NADHinvitroH2BTranscriptionRepressedActivatedLowLevelRepressedRepressedOptimalRedoxWindow?H2BTranscriptioninaNuclearExtractDevoidofEndogenousNAD(H)andSupplementedwithInhibitoryNADH(0.4mM)andIncreasingNAD+AModelforRedox-modulatedH2BTranscriptionOct-1NAD+OCA-ScomplexS-phaseinducibleH2BpromoterActivationp36/LDHRedoxsensitivep38/GAPDHRedoxsensitiveNADHOptimal

WindowOctamersiteATTTGCATTATAMotifBasalTranscriptionMachineryinvivoH2BtranscriptionInvivoRedoxPerturbationbyNAD+p38/GAPDHOct-1OctamerTATAInitiatorGTFsH2BpromoterChIPp38/GAPDHOct-1OctamerTATAInitiatorGTFsH2BpromoterinvivoH2BtranscriptionChIPInvivoRedoxPerturbationbyNADHH2BTranscriptioninaNuclearExtractDevoidofEndogenousNAD(H)andSupplementedwithDifferentialNAD+/NADHratiosQ:Istherea

naturalfluctuation

ofthe

NAD+/NADHratiosinacellcycleaccountingfortheoscillatoryH2Btranscriptionlevels?OscillationoftheNAD+/NADHredox(ratios)inacellcycleRe-culturingoftheG1-phasecells

RG1G1/SSRG1SG2Centrifugally-elutriatedHeLacellsYeastMetabolicCycle(YMC)dO2

Tuetal.,Science,2005Chenetal.,Science,2007:certainyeastmutantstrains

canshifttheS-phaseleft-orright-wardandaccumulatespontaneousmutationsatanincreasedrateMammalianMetabolicCycle(MMC)?OptimalRedoxWindowS-phaseredoxwindow●reductiveenoughtoprotectgenome(DNAduringreplicationismorevulnerabletooxidativedamage)●sufficiently(butnotoverly)

oxidative,allowingbothoptimalhistonetranscriptionandgenomeprotectionNAD+NADHOptimalRedoxWindowS-phaseS-phaseG1-&G2-phases&NAD+perturbationNAD+-depletion (incl.DNAdamage/repair?)&NADHperturbationinvivo H2BTranscriptionOff+++++OffTheH2BTranscriptioninLivingMammalianCellsisS-phase-specific

and

ConfinedtoanOptimalS-phase

RedoxWindowCoordinatedHistoneExpressionItiscommonlythoughtthatthehistoneexpressioncoordinationisduetoS-phasefeedbacksignals=2xH2A,H2B,H3&H4CBs=NPATfociCoordinatedHistoneExpression

4hours24hoursRepressingH2BexpressioncancoordinatelybringabouttherepressionofexpressionofothercorehistonegeneswithoutS-phasefeedbacksADual-signalingModelforHistoneExpressionRegulation

Cajal

Bodies(CBs)(w/oS-phasefeedback)FNSHumanH2BpromoterTATAboxGTFs+RNAPIIcomplexOct-1p38/GAPDHp36/LDHOCA-SATTTGCAT(Octamer)CyclinE/cdk2FNS

NPATDNAreplication,

S-phaseentry&progressionMetabolic/Redox

SignalingOtherhistonegenesRXLRXLLFDCyclinE/cdk2andhistoneexpressionPO4FacilitatorofNPAT-mediatedSignalingp60/Sti1indirectdirectHomosapiensHistonepromotersTATAboxGTFs+RNAPIIcomplexHistoneGeneRegulatoryElement(s)HomosapiensCyclinE/cdk2FNS

NPATDNAreplication,

S-phaseentry&progressionRXLRXLLFDSignalingduringhistoneexpressionPO4USA(PCs)p300/CBPHATactivityTip60(HATactivity/complex)histonegene-specificTxn

(co)activatorsFX(D/E)(X)3(L/I)HistoneAcetylTransferaseFacilitatorofNPAT-mediatedSignaling(FNS)andNPATinteractinayeasttwo-hybridscreenandinvitroY2HGST-pulldownInputNPATGST-FNSGSTonlyNPAT+FNS-NPAT-FNSFNS:co-localizationwithNPATandaroleinhistoneexpressionControlRNAiFNSRNAiDAPIAnti-FNSAnti-NPATMergeFNSRNAiHistonemRNALevelsConservedrolesofdrosophilamelanogasterFNS[dmFNS]

andGAPDHinhistoneexpression

CtrlRNAi18nM37nMdmFNS

RNAiTubulindmFNS****0.00.20.40.60.81.01.2Ctrl18nM37nMRelativegeneexpressiondmH2BdmH4HLBGAPDHHLB/GAPDHMergeHLB:HistoneLocusBody

dmFNSTranslocationduringInter-phasedmFNSdmFNS/GAPDHMerge/DAPI

G1phase

G2phase

Sphase

Cytoplasm/nucleusoutermembraneNucleusCytoplasm/nucleusoutermembranedmFNSEye-specificRNAiControlRNAidmFNS

RNAidmFNShasanessentialroleforcelldivisionduringeyeorganogenesisHistonepromotersTATAboxGTFs+RNAPIIcomplexHistoneGeneRegulatoryElement(s)HomosapiensCyclinE/cdk2FNS

NPATDNAreplication,

S-phaseentry&progressionRXLRXLLFDWhereisthehistonegenespecificity?PO4USA(PCs)p300/CBPHATactivityTip60(HATactivity/complex)histonegene-specificTxn

(co)activatorsFX(D/E)(X)3(L/I)HistoneAcetylTransferaseHumanH2BpromoterTATAboxGTFs+RNAPIIcomplexOct-1p38/GAPDHp36/LDHOCA-SATTTGCAT(Octamer)CyclinE/cdk2FNS

NPATDNAreplication,

S-phaseentry&progressionStein&ZhaoLabsRXLRXLLFDCyclinE/cdk2andhistoneexpressionPO4p60/Sti1FNS-IP?OtherhistonegenetranscriptionregulatorsHATsHistone

GenePromotersTATAboxGTFs+RNAPIIcomplexHistoneGeneRegulatoryElement(s)CyclinE/cdk2FNS

NPATDNAreplication,

S-phaseentry&progressionRXLRXLLFDMulti-layerinteractionsandspecificityPO4USA(PCs)p300/CBPTip60complexhistonegene-specificTxn

(co)activatorsFX(D/E)(X)3(L/I)Homosapiens休息10分鐘1.多功能代謝酶的生理學(xué)意義2.代謝紊亂與腫瘤發(fā)生發(fā)展惡性腫瘤的多階段多基因發(fā)生發(fā)展過程

例1：大腸癌

Vogelgram，

ProcNatl

Acad

SciUSA.2008105(11):4283–4288.我們的優(yōu)勢：積累40余年的各種階段的大腸癌樣本以及癌旁組織樣本腫瘤研究所鄭樹（轉(zhuǎn)移進展期腺癌-腺癌-腺瘤-增生-正常-

）(75-90%)(80-95%)(50-75%)(45-55%)(7-10%)我們的優(yōu)勢：各種發(fā)展期樣本癌變的多階段多基因發(fā)生發(fā)展過程（Hruban-gram）

例2：胰腺癌（1）持續(xù)生長信號

（2）避開生長抑制

（3）抗拒細胞死亡

（4）永久復(fù)制模式

（5）促進血管生成（6）侵潤/轉(zhuǎn)移激活（7）代謝紊亂失控（8）逃脫免疫摧毀9）炎癥反應(yīng)（準特性）30余年探索總結(jié)出的腫瘤細胞9大特征糖代謝腫瘤細胞代謝紊亂失控最主要體現(xiàn)在:對葡萄糖的依賴2.異常糖酵解途徑（和分叉代謝途徑）由此產(chǎn)生的微環(huán)境會加劇其它數(shù)種特性的體現(xiàn)：增加腫瘤細胞抗死亡能力，侵潤/轉(zhuǎn)移能力和逃脫免疫監(jiān)控能力

高頻率突變只涉及很少幾個基因除了基因突變導(dǎo)致/獲得的腫瘤細胞生存生長優(yōu)勢，維持優(yōu)勢的助力是？（代謝紊亂—微環(huán)境—表觀遺傳改變）

代謝途徑的改變鮮有是由于相關(guān)基因突變而引起的報道糖代謝紊亂牽涉到包括細胞轉(zhuǎn)運，胞內(nèi)代謝，胞內(nèi)外乳酸化以及乳酸再利用的蛋白質(zhì)/酶腫瘤微環(huán)境對其發(fā)生發(fā)展的影響（例如乳酸性）WhyEnergy-inefficientGlycolysis?GlucoseG-6-PPEPPyruvate

Lactate糖酵解途徑(1glucoseproduces2ATP)線粒體氧化磷酸化途徑(1glucoseproduces36ATP)Cancercellsmostlyuseaerobicglycolysis(Warburgeffect,1927)獲取生長優(yōu)勢？2501501007550372520kDapH47710Pre-Vacserum15250150100755037252015Post-Vac3serum134567892250150100755037252015134CBBproteinstain2567891:HSP602:Prolyl4-hydroxylase3:Calumenin

isoformaprecursor4:RhoGDPdissociationinhibitoralpha5:Pyruvate

kinaseM26:Enolasealpha7:AnnexinA28:L-lactatedehydrogenasechainM9:Peptidylprolyl

isomeraseZheng

etal.2011Glucosemetabolismenzymesarepancreaticcancerassociatedantigens在尋找腫瘤特異性抗原的努力中不斷地發(fā)現(xiàn)糖代謝酶糖酵解途徑與分叉代謝途徑GlucoseG-6-PPEPPyruvate

Lactate(lacticacidosis)

糖酵解途徑戊糖途徑R-5-P+NADPH1.還原力2.介入所有還原合成3.細胞存話/

生長必需因子NADPH:Enolase(alpha)PK(M2)LDH(A)核苷酸DNA/RNA氨基酸蛋白質(zhì)脂肪酸++BiomassBuilding

lacticacidosis乳酸化NADPH1.還原力2.介入所有還原合成3.細胞存話/

生長必需因子NADPH:++BiomassBuilding（添磚加瓦）胞內(nèi)NADPH濃度（或NADPH/NADP+比例）臨界點1細胞生存臨界點2細胞生長臨界點3細胞旺盛生長？腫瘤細胞代謝紊亂失控最主要體現(xiàn)在:對葡萄糖的依賴2.異常糖酵解途徑（和分叉代謝途徑）由此產(chǎn)生的微環(huán)境會加劇其它數(shù)種特性的體現(xiàn)：增加腫瘤細胞抗死亡能力，侵潤/轉(zhuǎn)移能力和逃脫免疫監(jiān)控能力

TxnipexpressionistightlycorrelatedwithglucoselevelsMondoMondoMLXMLXCACGTG-----CACGTGCarbohydrateResponseElement(ChoRE)Txnip2XChoREsMondo

和MLX:葡萄糖敏感的轉(zhuǎn)錄因子復(fù)合體Anegativefeed-backloopforregulatingglucosehomeostasisatthecellularlevelGlucoseTxnip

ChoREs

TxnipGlutHexokinase-PG-6-P-PHexokinaseMLXMondoGlycolysisGlut=GlucosetransporterMechanism(s)bywhichglucose-uptake

isinhibitedbyTxnipiscurrentlynotfullyunderstoodG6PGADPMLXMondo3PGGlutGlucosepyruvateGlycolysisTxnipOXPHOSTCAATPGAPDHLDH

LactateDifferentiatedcellsLactateProliferativecellsCancercells(Warburgeffect)?LinkingTxnipexpressiontometaboliccues生長信號脂肪酸代謝氨基酸代謝核酸代謝能量狀態(tài)etcTxnipChoREsNuclei在許多種類的癌細胞里Txnip

的表達急劇下降GlucoseTxnip

ChoREs

TxnipGlutHexokinase-PG-6-P-PHexokinaseMLXMondoGlycolysisGlut=GlucosetransporterMechanism(s)bywhichglucose-uptake

isinhibitedbyTxnipiscurrentlynotfullyunderstood糖酵解途徑與分叉代謝途徑GlucoseG-6-PPEPPyruvate

Lactate(lacticacidosis)

糖酵解途徑戊糖途徑R-5-P+NADPH1.還原力2.介入所有還原合成3.細胞存話/

生長必需因子NADPH:Enolase(alpha)PK(M2)LDH(A)核苷酸DNA/RNA氨基酸蛋白質(zhì)脂肪酸++BiomassBuilding代謝途徑的改變鮮有是由于相應(yīng)基因突變而引起的報道DukesD(Livermetastasis)DukesB/C轉(zhuǎn)移性大腸癌差異性表達的基因包括代謝酶unpublished不同期大腸癌（包括肝轉(zhuǎn)移）細胞內(nèi)碳水化合物代謝酶和相應(yīng)代謝物的變化

戊糖途徑加強戊糖R-5-P回流到糖酵解途徑減弱unpublishedGlucoseG-6-PPEPPyruvate

Lactate(lacticacidosis)

糖酵解途徑R-5-P+NADPH1.還原力2.介入所有還原合成3.細胞存話/

生長必需因子NADPH:Enolase(alpha)PK(M2)LDH(A)++BiomassBuilding戊糖R-5-P經(jīng)過旁路回流到糖酵解途徑戊糖途徑糖酵解途徑與分叉代謝途徑糖酵解途徑與分叉代謝途徑GlucoseG-6-PPEPPyruvate

Lactate(lacticacidosis)

糖酵解途徑戊糖途徑R-5-P+NADPH1.還原力2.介入所有還原合成3.細胞存話/

生長必需因子NADPH:Enolase(alpha)PK(M2)LDH(A)核苷酸DNA/RNA氨基酸蛋白質(zhì)脂肪酸++BiomassBuilding糖酵解途徑與分叉代謝途徑GlucoseG-6-PPEPPyruvate

Lactate(lacticacidosis)

糖酵解途徑戊糖途徑R-5-P+NADPH1.還原力2.介入所有還原合成3.細胞存話/

生長必需因子NADPH:Enolase(alpha)PK(M2)LDH(A)核苷酸DNA/RNA氨基酸蛋白質(zhì)脂肪酸++BiomassBuildingRegulatoryprocessorpathwayPutativepancreaticcancertumorantigens**Pathwaysgeneticallyalteredinmostpancreaticcancers*KRASsignalingRhoGDPdissociationinhibitoraCellAdhesionAnnexinA2Galectin-3IntegrinsignalingLamininreceptor-likeproteinLAMRL5ApoptosisTranslationallycontrolledtumorprotein1(TCTP)CytoskeletonKeratin19Pathwaysnotfrequentlyalteredinpancreaticcancers*ProteinchaperoneHeatshockprotein60Heatshockprotein27UbiquitinpathwayOtubain1MetabolismPyruvatekinasetypeM2EnolaseaLDH-MPeptidylprolylisomerase(cyclophilinA)S-adenosylhomocysteinehydrolaseNucleosidediphosphatekinaseSolutecarrierfamily25,member24Putativepancreatictumorassociatedmetabolicenzymesarenotmutated*Jonesetal.Science2008**NotmutatedZheng

etal.unpublisheddataBaylinandJones,NatRevCancer,2011與代謝酶相關(guān)的腫瘤特異性抗原的表達受相應(yīng)基因的表觀遺傳學(xué)調(diào)控狹義的表觀遺傳概念：基因啟動子上的DNA甲基化狀態(tài)的改變，以及染色質(zhì)上組蛋白乙?；癄顟B(tài)的改變已完成和進行中的全基因組遺傳學(xué)及表觀遺傳學(xué)研究全基因組測序全基因組甲基化/miRNA研究人胰腺癌原代培養(yǎng)細胞20銖已完成（Vogelstein，Science

2009）未計劃人胰腺癌間質(zhì)組織N/A待進行人胰腺癌成纖維細胞N/A進行中人胰腺癌浸潤淋巴細胞N/A待進行小鼠胰腺癌KPC細胞進行中待進行小鼠胰腺癌KPCA-/-細胞（非轉(zhuǎn)移性）進行中待進行小鼠胰腺癌KPC細胞成瘤后的間質(zhì)N/A待進行小鼠胰腺癌KPCA-/-細胞（非轉(zhuǎn)移性）成瘤后的間質(zhì)N/A待進行小鼠胰腺癌panc02細胞肝轉(zhuǎn)移模型進行中未計劃人大腸癌已完成（Vogelstein，Science

2008）基因組甲基化已完成（Baylin）miRNA研究已完成（S.Zheng)人大腸腺瘤已完成（D.Zhou&S.Zheng,待發(fā)表)未計劃小鼠大腸癌CT26細胞肝轉(zhuǎn)移模型已完成；發(fā)現(xiàn)119

missense

mutations進行中除注明出處外，其余來源于L.

ZHENG和他的合作者糖酵解途徑與分叉代謝途徑GlucoseG-6-PPEPPyruvate

Lactate(lacticacidosis)

糖酵解途徑戊糖途徑R-5-P+NADPH1.還原力2.介入所有還原合成3.細胞存話/

生長必需因子NADPH:Enolase(alpha)PK(M2)LDH(A)核苷酸DNA/RNA氨基酸蛋白質(zhì)脂肪酸++BiomassBuilding酶抑制劑

shikonin

是Pyruavte

KinaseM2（PKM2;丙酮酸激酶）抑制劑JChen&XHuetal.2011Oncogene30:4297-4306.abc動物模型:同源大腸癌或胰腺癌細胞移植到正常非免疫缺陷小鼠

小鼠腫瘤肝轉(zhuǎn)移的半脾切除模型；Zhengetal.2011Saline105CT26ABCMurineModelofIsolatedHepaticColorectalMetastases4weekspostchallenge大腸癌肝轉(zhuǎn)移小鼠模型KrasG12DTrp53R172H

胰腺特異性插入的轉(zhuǎn)基因小鼠：研究胰腺腫瘤多階段發(fā)展的動物模型（KPC小鼠fromD.Tuveson）Recapitulatetheprogressionfromlow-gradePanINlesionstoinvasivepancreaticadenocarcinoma(Zhengetal.unpublisheddata)RegulatoryprocessorpathwayPutativepancreaticcancertumorantigens**Pathwaysgeneticallyalteredinmostpancreaticcancers*KRASsignalingRhoGDPdissociationinhibitoraCellAdhesionAnnexinA2Galectin-3IntegrinsignalingLamininreceptor-likeproteinLAMRL5ApoptosisTranslationallycontrolledtumorprotein1(TCTP)Cytoskeleto