膽固醇代謝平衡調控

機制和合成膽固醇代謝平衡調控

機制和合成提綱膽固醇的生物學功能膽固醇相關疾病膽固醇的生物合成膽固醇在血液中的運輸家族型高膽固醇血癥NPC疾病膽固醇的降解與清除膽固醇代謝的負反饋調控機制—SCAP-SREBP途徑膽固醇代謝的負反饋調控機制—HMGCR降解途徑飲食膽固醇吸收的分子途徑提綱膽固醇的生物學功能Cholesterol（膽固醇）Cholesterol（膽固醇）一、膽固醇的生物學功能MembraneFluidityBileAcidsHormones、VitaminDSynapseHedgehogModificationSignalTransduction……一、膽固醇的生物學功能MembraneFluidity哺乳動物細胞模式圖哺乳動物細胞模式圖膜上的微結構域膜上的微結構域Cholesterolandcholesterolderivatives

雌激素雄激素Cholesterolandcholesterolde膽固醇代謝平衡調控機制和合成課件脂肪酸、糖等提供能量（ATP）膽固醇并不提供能量脂肪酸、糖等提供能量（ATP）二、膽固醇相關疾病二、膽固醇相關疾病CholesterolisthemajorcauseofatherosclerosisCholesterolisthemajorcause心腦血管疾病惡性腫瘤肺炎、流感感染性疾病意外死亡慢性阻塞性肺病肝病及肝硬化糖尿病腎病其它43.8%22.3%FromHeJ.etal.,NEnglJMed,(2005),353:1124-1134.中國死亡原因統計心腦血管疾病惡性腫瘤肺炎、流感感染性疾病意外死亡慢性阻塞性肺蒙娜麗莎患有高膽固醇？意大利帕勒莫大學的病理解剖學教授弗蘭克，研究認為蒙娜麗莎飲食不健康，患有高膽固醇癥。正是眼部的脂肪瘤，造成了蒙娜麗莎這副神秘莫測的表情。畫中的她（或者說達芬奇的這個模特）正在為自己體內過高的膽固醇而擔憂。蒙娜麗莎患有高膽固醇？意大利帕勒莫大學的病理解膽結石老年癡呆癥CholesterolandDiseasesNiemann-PickTypeCDisease糖尿病/肥胖癥膽結石老年癡呆癥CholesterolandDiseasFromIMSHealth,MIDAS

藥品銷售排行榜NameCompanyIndicationSalestoJune07($bn)LipitorPfizerHypercholesterolaemia（高膽固醇血癥）13.5NexiumAstraZenecaGastroesophagealreflux（胃食管返流）,gastriculcers（胃潰瘍）6.9SeretideGSKAsthma（哮喘）6.7PlavixBristol-MyersSquibbAtheroscleroticevents（動脈粥樣硬化癥）5.8AranespAmgenAnaemia（貧血癥）5.1FromIMSHealth,MIDAS藥品銷售排行榜N三、膽固醇的生物合成三、膽固醇的生物合成CholesterolisnotrequiredinthedietCholesterolisanessentialmoleculebutisnotrequiredinthedietbecauseallcellscansynthesizeitfromsimpleprecursorsCholesterolisnotrequiredinCholesterolismadefromacetyl-CoAinfourstagesallofitscarbonatomsareprovidedbyasingleprecursor–acetateCholesterolismadefromacetyStage1Threeacetateunitscondensetoformasixcarbonintermediate,mevalonateStage1ThreeacetateunitsconTwomoleculesofacetate-CoAcondenseformingacetoacetyl-CoA.Acetoacetyl-CoAcondenseswithacetyl-CoAtoyieldb-hydroxy-b-methylglutaryl-CoA(HMG-CoA)Twomoleculesofacetate-CoAcThefinalstepthereductionofHMG-CoAtomevalonate,catalyzedbyHMG-CoAreductase.ThefinalstepthereductionoStage2ConversionofmevalonateintoactivatedisopreneunitsIsoprenecontainingmoleculesareimportantintermediatesincholesterolbiosynthesisStage2ConversionofmevalonatStage3Polymerizationofsix5-carbonisopreneunitstoformthe30-carbonlinearstructureofsqualene.Stage3Polymerizationofsix5Cyclizationofsqualeneformsthefourringsofthesteroidnucleus.Subsequentmodificationsleadstothefinalproduct,cholesterol.Stage4Cyclizationofsqualeneforms膽固醇代謝平衡調控機制和合成課件MostofthecholesterolmadeintheliverisexportedMuchofcholesterolsynthesistakesplaceinthe

liverMostisexportedMostofthecholesterolmadeiCholesterolisexportedinthreeforms1.Bilesalts–amphipathiccholesterolderivativesthataidlipiddigestion2.Cholesterol–tobile3.Cholesterylesters–transportedandsecretedin lipoproteinparticlestoother tissuesthatusecholesterol orarestoredintheliverCholesterolisexportedinthr四、膽固醇在血液中的運輸四、膽固醇在血液中的運輸CholesterylesterformationFormedintheliverConvertingcholesteroltoamorehydrophobicformCholesterylesterformationForCholesteroltransport:theproblemCholesterolandcholesterylestersareessentiallyinsolubleinwaterThesemoleculesmustbemovedfromthetissueoforigintothetissuesinwhichtheyarestoredorareconsumedCholesteroltransport:theproCholesteroltransport:thesolutionCholesterolandcholesterylestersarecarriedinthebloodplasmafromonetissuetoanotherasplasmalipoproteinsCholesteroltransport:thesolCholesterolestersentercellsby

receptormediatedendocytosisCholesterolestersentercells五、家族型高膽固醇血癥LDLreceptor(LDLR)突變五、家族型高膽固醇血癥LDLreceptor(LDLR)家族性高膽固醇血癥(FamilialHypercholesterolemia,FH)雜合子患者血清總膽固醇較正常人高出1～2倍純合子患者血清總膽固醇較正常人高出6～8倍雜合子患者發生率為1/500純合子患者發生率為1/1,000,000雜合子患者男性30～40歲時，患CAD，23%患者在50歲以前死于CAD，>50%患者在60歲時明顯的CAD癥狀；純合子患者十幾歲時，有嚴重的心血管事件甚至死亡家族性高膽固醇血癥雜合子患者血清總膽固醇較正常人高出1～2倍LDLR突變----黃色瘤LDLR突變----黃色瘤FH患者LDLR突變----眼底脂質滲出正常人FH患者LDLR突變----眼底脂質滲出正常人六、

Niemann-PicktypeC(NPC)疾病溶酶體堆積型疾病（LysosomalStorageDisorders）膽固醇在溶酶體中堆積進行性神經細胞死亡、肝脾腫大、小腦共濟失調、癡呆、語言吞咽困難、青春期之前死亡1：120，000發病，攜帶者1：100NPC1（大的膜蛋白）或NPC2（小的可溶蛋白）基因突變臨床嘗試用環化糊精進行治療六、Niemann-PicktypeC(NPC)疾病正常細胞NPC1突變細胞正常細胞NPC1突變細胞膽固醇代謝平衡調控機制和合成課件膽固醇代謝平衡調控機制和合成課件七、膽固醇的降解與清除七、膽固醇的降解與清除DegradationofcholesterolTheringstructureofcholesterolcannotbemetabolizedtoCO2andH2OinhumansTheintactsterolringiseliminatedfromthebodyby:Conversiontobileacids,whichareexcretedinfecesSecretionofcholesterolintothebile,whichtransportsittotheintestineforeliminationDegradationofcholesterolTheSteroidhormonesareformedfromcholesterolAllsteroidhormonesarederivedformcholesterolInthecortexofadrenalglandstwoclassesofhormonesaresynthesized–mineralocorticoidsandglucocorticoidsInthemaleandfemalegonads–sexhormonesareproducedSexhormonesinclude–progesterone,androgensandestrogensSteroidhormonesareformedfr八、膽固醇代謝的負反饋調控機制（一）SCAP-SREBP途徑八、膽固醇代謝的負反饋調控機制（一）SCAP-SREBP途徑膽固醇代謝平衡調控機制和合成課件脂質代謝的關鍵蛋白質及其功能調控的臨床意義LDLReceptorsLDLHMGCRSREBPPathwayHMGCRinhibitor脂質代謝的關鍵蛋白質及其功能調控的臨床意義LDLLDLHMGInsigInsigSREBP—膜結合的轉錄因子WangX,BriggsMR,HuaX,YokoyamaC,GoldsteinJL,BrownMS.Nuclearproteinthatbindssterolregulatoryelementoflowdensitylipoproteinreceptorpromoter.II.Purificationandcharacterization.JBiolChem.1993Jul5;268(19):14497-504.YokoyamaC,WangX,BriggsMR,AdmonA,WuJ,HuaX,GoldsteinJL,BrownMS.SREBP-1,abasic-helix-loop-helix-leucinezipperproteinthatcontrolstranscriptionofthelowdensitylipoproteinreceptorgene.Cell.1993Oct8;75(1):187-97.WangX,SatoR,BrownMS,HuaX,GoldsteinJL.SREBP-1,amembrane-boundtranscriptionfactorreleasedbysterol-regulatedproteolysis.Cell.1994Apr8;77(1):53-62.SREBP—膜結合的轉錄因子WangX,BriggsM膽固醇代謝平衡調控機制和合成課件25-Hydroxycholesterol-irradiationMutantcellssurvive(25-RA)25-Hydroxycholesterol-irradiaAmphotericinB-irradiationCholesterolauxotrophs(M19)HumanfibroblasmgenomicDNAtoM19cellsHfT1M19(c)Growinlipid-deficientmediumGrowinlipid-deficientmediumHfT1M19(c)genomicDNAtoM19cellsHfT2M19(c)Growinlipid-deficientmediumHfT2M19(c)genomicDNAtoM19cellsHfT3M19(c)Inter-AluPCR,usethisprobetoscreengenomicPAClibrary,transfectPACcloneintoM19,sequencePACcloneandcomparewithEST.AmphotericinB-irradiationChS2PcDNAtoCHOcellsBrieflyincubatedwithLDL,AmphotericinBselection-irradiationLowfluorescentLDLuptake-irradiationSRD-12BS2PcDNAtoCHOcellsBrieflyiDeBose-BoydRA,BrownMS,LiWP,NohturfftA,GoldsteinJL,EspenshadePJ.Transport-dependentproteolysisofSREBP:relocationofsite-1proteasefromGolgitoERobviatestheneedforSREBPtransporttoGolgi.Cell.1999Dec23;99(7):703-12.NohturfftA,YabeD,GoldsteinJL,BrownMS,EspenshadePJ.RegulatedstepincholesterolfeedbacklocalizedtobuddingofSCAPfromERmembranes.Cell.2000Aug4;102(3):315-23.SterolRegulatedTransportofSCAPDeBose-BoydRA,BrownMS,LiWPurificationSchemeforSCAP-interactingProteinsYangetal.,Cell(2002)489-500PurificationSchemeforSCAP-iTwoInsigs:Insig-1andInsig-2A.SequenceAlignmentB.HydropathyPlotYabeetal.,PNAS(2002)12753-8TwoInsigs:Insig-1andInsig-InsigInsig九、膽固醇代謝的負反饋調控機制（二）HMGCR降解途徑九、膽固醇代謝的負反饋調控機制（二）HMGCR降解途徑FromHeJ.etal.,NEnglJMed,(2005),353:1124-1134.andIMSHealth,MIDAS

藥品銷售排行榜NameCompanyIndicationSalestoJune07($bn)LipitorPfizerHypercholesterolaemia（高膽固醇血癥）13.5NexiumAstraZenecaGastroesophagealreflux（胃食管返流）,gastriculcers（胃潰瘍）6.9SeretideGSKAsthma（哮喘）6.7PlavixBristol-MyersSquibbAtheroscleroticevents（動脈粥樣硬化癥）5.8AranespAmgenAnaemia（貧血癥）5.1FromHeJ.etal.,NEnglJMeHMG-CoAReductase(HMGCR):theRate-LimitingEnzymeinCholesterolBiosyntheticPathway(HMGCR)HMG-CoAReductase(HMGCR):theProposedModelforINSIG-mediatedRegulationof

HMGCoAReductaseandSCAPProposedModelforINSIG-mediaSterol-RegulatedDegradationofHMG-CoAReductaseisBlockedbyInhibitorsofthe26SProteasome

Sterol-RegulatedDegradationoWorkingHypothesisforSterol-RegulatedDegradationofHMG-CoAReductaseWorkingHypothesisforSterol-Sterol-StimulatedUbiquitinaionofHMGCRRequiresInsigSever,Song,Yabe,etal.,JBC,2003Sterol-StimulatedUbiquitinaioAminoAcidSequenceoftheHMG-CoAReductase

MembraneDomainAminoAcidSequenceoftheHMGLysines89and248areRequiredfortheSterol-RegulatedUbiquitinationandDegradationofHMGCR

Lysines89and248areRequireStrategyforPurifyingtheE3ofHMG-CoAReductaseStrategyforPurifyingtheE3E3ActivityCo-immunoprecipitateswithInsig-1inSterol-TreatedCellsE3ActivityCo-immunoprecipitaIdentificationofgp78andVCPasInsig-1-associatingproteinsIdentificationofgp78andVCP膽固醇代謝平衡調控機制和合成課件MolecularPathwayforSterol-regulatedDegradationofHMGCRSongetal.,MolCell,2005Whatareotherproteinsinvolvedinthispathway?MolecularPathwayforSterol-rIdentificationofUfd1asagp78interactingproteinIP:Endogenousgp78IP:Endogenousgp78Transfect&CoIPIP:Flag-Ufd1ABCIdentificationofUfd1asagpUfd1isrequiredfortheubiquitinationofendogenousHMGCRUfd1isrequiredfortheubiquUfd1enhancestheE3activityofgp78invitroUfd1enhancestheE3activityDualrolesofUfd1:EnhancingE3activityandPromotingdegradationCaoetal.,CellMetab,2007DualrolesofUfd1:Enhancing十、飲食膽固醇吸收的分子途徑十、飲食膽固醇吸收的分子途徑(HMGCR)DenovoCholesterolSynthesisisaEnergy-ConsumingProcess18

Acetyl-CoACholesterol27

NADPH11

O218

ATP(HMGCR)DenovoCholesterolSynCholesterolBiosynthesisandAbsorptioninHuman300-500mgDietaryAbsorptionBiosynthesis600-900mg~1000mgBiliaryre-absorptionCholesterolBiosynthesisandADietaryCholesterolAbsorptionbyIntestinalEnterocytesCholesterolEsterHydrolysisCholesterolCholesterolCholesterolEsterChylomicronIntestinallumenLymphACAT2EREnterocyteTightJunctionEzetimibe2001CholesterolabsorptioninhibitorEzetimibeNPC1L12004IdentificationofNPC1L1DietaryCholesterolAbsorptionHumanNiemannPickC1

Like1(NPC1L1)HumanNiemannPickC1Like1(NPC1L1mediatesthere-absorptionofcholesterolfrombileApicalHepatocytesBasolateralApicalNPC1L1mediatesthere-absorptNPC1L1recyclesbetweenPMandERCrespondingtocholesterollevelABNPC1L1recyclesbetweenPMandOverexpressionofNPC1L1increasesfreecholesteroluptakeOverexpressionofNPC1L1increKnockingdownofNPC1L1proteininL02cellsattenuatestheuptakeoffreecholesterolKnockingdownofNPC1L1proteiStructuresofdifferentsterolsSterolAbsorption:<5%plantsterols>50%cholesterolStructuresofdifferentsterolSterol-specificityforNPC1L1-mediatedinternalizationABSterol-specificityforNPC1L1-Hypothesis:NPC1L1mediatescholesterolupta