分子医学 代谢疾病

分子医学课程“代谢疾病”

1.多功能代谢酶的生理学意义2.代谢紊乱与肿瘤发生发展多功能代谢酶：

糖代谢酶在基因转录调节

过程中的“兼职”功能以及

生理学意义

（模式分析系统：

S-期组蛋白基因表达的调控）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/Sti1NaïveIgGRG1SG2anti-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分钟1.多功能代谢酶的生理学意义2.代谢紊乱与肿瘤发生发展恶性肿瘤的多阶段多基因发生发展过程

例1：大肠癌

Vogelgram，

ProcNatl

Acad

SciUSA.2008105(11):4283–4288.我們的优势：积累40余年的各种阶段的大肠癌样本以及癌旁組织样本肿瘤研究所郑树（转移进展期腺癌-腺癌-腺瘤-增生-正常-

）(75-90%)(80-95%)(50-75%)(45-55%)(7-10%)我們的优势：各种发展期样本癌变的多阶段多基因发生发展过程（Hruban-gram）

例2：胰腺癌（1）持续生长信号

（2）避开生长抑制

（3）抗拒細胞死亡

（4）永久复制模式

（5）促进血管生成（6）侵润/转移激活（7）代謝紊乱失控（8）逃脫免疫摧毀9）炎症反应（准特性）30余年探索总结出的肿瘤细胞9大特征糖代谢肿瘤细胞代謝紊乱失控最主要体现在:对葡萄糖的依赖2.异常糖酵解途径（和分叉代謝途径）由此产生的微环境会加剧其它数种特性的体现：增加肿瘤细胞抗死亡能力，侵润/转移能力和逃脱免疫监控能力

高频率突变只涉及很少几個基因除了基因突变导致/获得的肿瘤细胞生存生长优势，维持优势的助力是？（代謝紊乱—微环境—表观遗传改变）

代谢途径的改变鲜有是由于相关基因突变而引起的报道糖代谢紊乱牵涉到包括细胞转运，胞内代谢，胞内外乳酸化以及乳酸再利用的蛋白质/酶肿瘤微环境对其发生发展的影响（例如乳酸性）WhyEnergy-inefficientGlycolysis?GlucoseG-6-PPEPPyruvate

Lactate糖酵解途径(1glucoseproduces2ATP)线粒体氧化磷酸化途径(1glucoseproduces36ATP)Cancercellsmostlyuseaerobicglycolysis(Warburgeffect,1927)获取生长优势？2501501007550372520kDapH47710Pre-Vacserum15250150100755037252015Post-Vac3serum134567892250150100755037252015134CBBproteinstain2567891:HSP602:Prolyl4-hydroxylase3:Calumenin

isoformaprecursor4:RhoGDPdissociationinhibitoralpha5:Pyruvate

kinaseM26:Enolasealpha7:AnnexinA28:L-lactatedehydrogenasechainM9:Peptidylprolyl

isomeraseZheng

etal.2011Glucosemetabolismenzymesarepancreaticcancerassociatedantigens在寻找肿瘤特异性抗原的努力中不断地发现糖代谢酶糖酵解途径与分叉代謝途径GlucoseG-6-PPEPPyruvate

Lactate(lacticacidosis)

糖酵解途径戊糖途径R-5-P+NADPH1.还原力2.介入所有还原合成3.细胞存话/

生长必需因子NADPH:Enolase(alpha)PK(M2)LDH(A)核苷酸DNA/RNA氨基酸蛋白质脂肪酸++BiomassBuilding

lacticacidosis乳酸化NADPH1.还原力2.介入所有还原合成3.细胞存话/

生长必需因子NADPH:++BiomassBuilding（添砖加瓦）胞内NADPH浓度（或NADPH/NADP+比例）临界点1细胞生存临界点2细胞生長临界点3细胞旺盛生長？肿瘤细胞代谢紊乱失控最主要体现在:对葡萄糖的依赖2.异常糖酵解途径（和分叉代謝途径）由此产生的微环境会加剧其它数种特性的体现：增加肿瘤细胞抗死亡能力，侵润/转移能力和逃脱免疫监控能力

TxnipexpressionistightlycorrelatedwithglucoselevelsMondoMondoMLXMLXCACGTG-----CACGTGCarbohydrateResponseElement(ChoRE)Txnip2XChoREsMondo

和MLX:葡萄糖敏感的转录因子复合体Anegativefeed-backloopforregulatingglucosehomeostasisatthecellularlevelGlucoseTxnip

ChoREs

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

isinhibitedbyTxnipiscurrentlynotfullyunderstoodG6PGADPMLXMondo3PGGlutGlucosepyruvateGlycolysisTxnipOXPHOSTCAATPGAPDHLDH

LactateDifferentiatedcellsLactateProliferativecellsCancercells(Warburgeffect)?LinkingTxnipexpressiontometaboliccues生长信号脂肪酸代谢氨基酸代谢核酸代谢能量状态etcTxnipChoREsNuclei在许多种类的癌细胞里Txnip

的表达急剧下降GlucoseTxnip

ChoREs

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

isinhibitedbyTxnipiscurrentlynotfullyunderstood糖酵解途径与分叉代謝途径GlucoseG-6-PPEPPyruvate

Lactate(lacticacidosis)

糖酵解途径戊糖途径R-5-P+NADPH1.还原力2.介入所有还原合成3.细胞存话/

生长必需因子NADPH:Enolase(alpha)PK(M2)LDH(A)核苷酸DNA/RNA氨基酸蛋白质脂肪酸++BiomassBuilding代谢途径的改变鲜有是由于相应基因突变而引起的报道DukesD(Livermetastasis)DukesB/C转移性大肠癌差异性表达的基因包括代谢酶unpublished不同期大肠癌（包括肝转移）細胞内碳水化合物代谢酶和相应代谢物的变化

戊糖途径加强戊糖R-5-P回流到糖酵解途径减弱unpublishedGlucoseG-6-PPEPPyruvate

Lactate(lacticacidosis)

糖酵解途径R-5-P+NADPH1.还原力2.介入所有还原合成3.细胞存话/

生长必需因子NADPH:Enolase(alpha)PK(M2)LDH(A)++BiomassBuilding戊糖R-5-P经过旁路回流到糖酵解途径戊糖途径糖酵解途径与分叉代謝途径糖酵解途径与分叉代謝途径GlucoseG-6-PPEPPyruvate

Lactate(lacticacidosis)

糖酵解途径戊糖途径R-5-P+NADPH1.还原力2.介入所有还原合成3.细胞存话/

生长必需因子NADPH:Enolase(alpha)PK(M2)LDH(A)核苷酸DNA/RNA氨基酸蛋白质脂肪酸++BiomassBuilding糖酵解途径与分叉代謝途径GlucoseG-6-PPEPPyruvate

Lactate(lacticacidosis)

糖酵解途径戊糖途径R-5-P+NADPH1.还原力2.介入所有还原合成3.细胞存话/

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

etal.unpublisheddataBaylinandJones,NatRevCancer,2011与代谢酶相关的肿瘤特异性抗原的表达受相应基因的表观遗传学调控狭义的表观遗传概念：基因启动子上的DNA甲基化状态的改变，以及染色质上组蛋白乙酰化状态的改变已完成和进行中的全基因组遗传学及表观遗传学研究全基因组测序全基因组甲基化/miRNA研究人胰腺癌原代培养细胞20铢已完成（Vogelstein，Science

2009）未计划人胰腺癌间质组织N/A待进行人胰腺癌成纤维细胞N/A进行中人胰腺癌浸润淋巴细胞N/A待进行小鼠胰腺癌KPC细胞进行中待进行小鼠胰腺癌KPCA-/-细胞（非转移性）进行中待进行小鼠胰腺癌KPC细胞成瘤后的间质N/A待进行小鼠胰腺癌KPCA-/-细胞（非转移性）成瘤后的间质N/A待进行小鼠胰腺癌panc02细胞肝转移模型进行中未计划人大肠癌已完成（Vogelstein，Science

2008）基因组甲基化已完成（Baylin）miRNA研究已完成（S.Zheng)人大肠腺瘤已完成（D.Zhou&S.Zheng,待发表)未计划小鼠大肠癌CT26细胞肝转移模型已完成；发现119

missense

mutations进行中除注明出处外，其余来源于L.

ZHENG和他的合作者糖酵解途径与分叉代谢途径GlucoseG-6-PPEPPyruvate

Lactate(lacticacidosis)

糖酵解途径戊糖途径R-5-P+NADPH1.还原力2.介入所有还原合成3.细胞存话/

生长必需因子NADPH:Enolase(alpha)PK(M2)LDH(A)核苷酸DNA/RNA氨基酸蛋白质脂肪酸++BiomassBuilding酶抑制剂

shikonin

是Pyruavte

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

小鼠肿瘤肝转移的半脾切除模型；Zhengetal.2011Saline105CT26ABCMurineModelofIsolatedHepaticColorectalMetastases4weekspostchallenge大肠癌肝转移小鼠模型KrasG12DTrp53R172H

胰腺特异性插入的转基因小鼠：研究胰腺肿瘤多阶段发展的动物模型（KPC小鼠fromD.Tuveson）Recapitulatetheprogressionfromlow-gradePanINlesionstoinvasivepancreaticadenocarcinoma(Zhengetal.unpublisheddata)RegulatoryprocessorpathwayPutativepancreaticcancertumorantigens**Pathwaysgeneticallyalteredinmostpancreaticcancers*KRASsignalingRhoGDPdissociationinhibitoraCellAdhesionAnnexinA2Galectin-3IntegrinsignalingLamininreceptor-likeproteinLAMRL5ApoptosisTranslationallycontrolledtumorprotein1(TCTP)Cytoskeleto