胆固醇代谢平衡调控机制和合成课件.ppt

1、胆固醇代谢平衡调控胆固醇代谢平衡调控机制和合成机制和合成提纲提纲1.胆固醇的生物学功能2.胆固醇相关疾病3.胆固醇的生物合成4.胆固醇在血液中的运输5.家族型高胆固醇血症6.NPC疾病7.胆固醇的降解与清除8.胆固醇代谢的负反馈调控机制SCAP-SREBP途径9.胆固醇代谢的负反馈调控机制HMGCR降解途径10. 饮食胆固醇吸收的分子途径Cholesterol（胆固醇）（胆固醇）一、胆固醇的生物学功能一、胆固醇的生物学功能u Membrane Fluidityu Bile Acidsu Hormones、Vitamin Du Synapseu Hedgehog Modificationu Si

2、gnal Transductionu 哺乳动物细胞模式图哺乳动物细胞模式图膜上的微结构域膜上的微结构域Cholesterol and cholesterol derivatives 雌激素雌激素雄激素雄激素l脂肪酸、糖等提供能量（脂肪酸、糖等提供能量（ATP）l胆固醇并不提供能量胆固醇并不提供能量二、胆固醇相关疾病二、胆固醇相关疾病Cholesterol is the major cause of atherosclerosis心脑血管疾病心脑血管疾病恶性肿瘤恶性肿瘤肺炎、流感肺炎、流感感染性疾病感染性疾病意外死亡意外死亡慢性阻塞性肺病慢性阻塞性肺病肝病及肝硬化肝病及肝硬化糖尿病糖尿病肾病肾病

3、其它其它43.8%22.3%From He J. et al., N Engl J Med, (2005), 353: 1124-1134. 中国死亡原因统计中国死亡原因统计蒙娜丽莎患有高胆固醇？蒙娜丽莎患有高胆固醇？ 意大利帕勒莫大学的病理解意大利帕勒莫大学的病理解剖学教授弗兰克，研究认为剖学教授弗兰克，研究认为蒙娜蒙娜丽莎饮食不健康，患有高胆固醇丽莎饮食不健康，患有高胆固醇症症。 正是正是眼部的脂肪瘤，造成了眼部的脂肪瘤，造成了蒙娜丽莎这副神秘莫测的表情蒙娜丽莎这副神秘莫测的表情。画中的她（或者说达芬奇的这个画中的她（或者说达芬奇的这个模特）模特）正在为自己体内过高的胆正在为自己体内过高的

4、胆固醇而担忧固醇而担忧。胆结石胆结石老年痴呆症老年痴呆症Cholesterol and DiseasesNiemann-Pick Type C Disease糖尿病糖尿病/肥胖症肥胖症From IMS Health, MIDAS 药品销售排行榜药品销售排行榜NameCompanyIndicationSales to June 07($bn)LipitorPfizerHypercholesterolaemia（高胆固醇血症）（高胆固醇血症）13.5NexiumAstraZenecaGastroesophageal reflux（胃食管返流）, gastric ulcers（胃溃疡）6.9Sere

5、tideGSKAsthma（哮喘）6.7PlavixBristol-Myers SquibbAtherosclerotic events（动脉粥样硬化症）5.8AranespAmgenAnaemia（贫血症）5.1三、胆固醇的生物合成三、胆固醇的生物合成Cholesterol is not required in the diet Cholesterol is an essential molecule but is not required in the diet because all cells can synthesize it from simple precursorsCholes

6、terol is made from acetyl-CoA in four stages all of its carbon atoms are provided by a single precursor acetateStage 1 Three acetate units condense to form a six carbon intermediate, mevalonate Two molecules of acetate-CoA condense forming acetoacetyl-CoA. Acetoacetyl-CoA condenses with acetyl-CoA t

7、o yield b-hydroxy-b-methylglutaryl-CoA (HMG-CoA) The final step the reduction of HMG-CoA to mevalonate, catalyzed by HMG-CoA reductase.Stage 2 Conversion of mevalonate into activated isoprene units Isoprene containing molecules are important intermediates in cholesterol biosynthesisStage 3 Polymeriz

8、ation of six 5-carbon isoprene units to form the 30-carbon linear structure of squalene. Cyclization of squalene forms the four rings of the steroid nucleus. Subsequent modifications leads to the final product, cholesterol.Stage 4Most of the cholesterol made in the liver is exported Much of choleste

9、rol synthesis takes place in the liver Most is exportedCholesterol is exported in three forms1. Bile salts amphipathic cholesterol derivatives that aid lipid digestion2. Cholesterol to bile3. Cholesteryl esters transported and secreted in lipoprotein particles to other tissues that use cholesterol o

10、r are stored in the liver四、胆固醇在血液中的运输四、胆固醇在血液中的运输Cholesteryl ester formationFormed in the liver Converting cholesterol to a more hydrophobic formCholesterol transport: the problem Cholesterol and cholesteryl esters are essentially insoluble in water These molecules must be moved from the tissue of o

11、rigin to the tissues in which they are stored or are consumedCholesterol transport: the solution Cholesterol and cholesteryl esters are carried in the blood plasma from one tissue to another as plasma lipoproteinsCholesterol esters enter cells by receptor mediated endocytosis五、家族型高胆固醇血症五、家族型高胆固醇血症LD

12、L receptor (LDLR) 突变突变杂合子患者血清总胆固醇较正常人高出杂合子患者血清总胆固醇较正常人高出1 12 2倍倍纯合子患者血清总胆固醇较正常人高出纯合子患者血清总胆固醇较正常人高出6 68 8倍倍杂合子患者发生率为杂合子患者发生率为1/5001/500纯合子患者发生率为纯合子患者发生率为1/1,000,0001/1,000,000杂合子患者男性杂合子患者男性30304040岁时，患岁时，患CADCAD， 23% 23% 患者在患者在5050岁以前岁以前死于死于CADCAD，50% 50% 患者在患者在6060岁时明显的岁时明显的CADCAD症状；症状；纯合子患者十几岁时，有严重

13、的心血管事件甚至死亡纯合子患者十几岁时，有严重的心血管事件甚至死亡LDLR突变突变-黄色瘤黄色瘤LDLR突变突变-眼底脂质渗出眼底脂质渗出六、六、 Niemann-Pick type C(NPC) 疾病疾病u溶酶体堆积型疾病（溶酶体堆积型疾病（Lysosomal Storage DisordersLysosomal Storage Disorders）u胆固醇在溶酶体中堆积胆固醇在溶酶体中堆积u进行性神经细胞死亡、肝脾肿大、小脑共济失调、痴呆、进行性神经细胞死亡、肝脾肿大、小脑共济失调、痴呆、语言吞咽困难、青春期之前死亡语言吞咽困难、青春期之前死亡u1 1：120120，000000发病，携带

14、者发病，携带者1 1：100100uNPC1NPC1（大的膜蛋白）或（大的膜蛋白）或NPC2NPC2（小的可溶蛋白）基因突变（小的可溶蛋白）基因突变u临床尝试用临床尝试用环化糊精环化糊精进行治疗进行治疗正常细胞正常细胞NPC1突变细胞突变细胞七、胆固醇的降解与清除七、胆固醇的降解与清除Degradation of cholesterolThe ring structure of cholesterol cannot be metabolized to CO2 and H2O in humansThe intact sterol ring is eliminated from the body

15、by:1.Conversion to bile acids, which are excreted in feces2.Secretion of cholesterol into the bile, which transports it to the intestine for eliminationSteroid hormones are formed from cholesterol All steroid hormones are derived form cholesterol In the cortex of adrenal glands two classes of hormon

16、es are synthesized mineralocorticoids and glucocorticoids In the male and female gonads sex hormones are produced Sex hormones include progesterone, androgens and estrogens八、胆固醇代谢的负反馈调控机制八、胆固醇代谢的负反馈调控机制（一）（一）SCAP-SREBP途径途径脂质代谢的关键蛋白质及其功能调控的临床意义脂质代谢的关键蛋白质及其功能调控的临床意义LDLReceptorsLDLHMGCRSREBP PathwayHMG

17、CR inhibitorInsigSREBP膜结合的转录因子膜结合的转录因子Wang X, Briggs MR, Hua X, Yokoyama C, Goldstein JL, Brown MS. Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization. J Biol Chem. 1993 Jul 5;268(19):14497-504.Yokoyama C, Wang X,

18、Briggs MR, Admon A, Wu J, Hua X, Goldstein JL, Brown MS. SREBP-1, a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene. Cell. 1993 Oct 8;75(1):187-97.Wang X, Sato R, Brown MS, Hua X, Goldstein JL. SREBP-1, a membrane-bound transcrip

19、tion factor released by sterol-regulated proteolysis. Cell. 1994 Apr 8;77(1):53-62. 25-Hydroxycholesterol -irradiation Mutant cells survive (25-RA)Amphotericin B -irradiation Cholesterol auxotrophs (M19)Human fibroblasm genomic DNA to M19 cellsHfT1M19(c)Grow in lipid-deficient mediumGrow in lipid-de

20、ficient mediumHfT1M19(c) genomic DNA to M19 cellsHfT2M19(c)Grow in lipid-deficient mediumHfT2M19(c) genomic DNA to M19 cellsHfT3M19(c)Inter-Alu PCR, use this probe to screen genomic PAC library, transfect PAC clone into M19, sequence PAC clone and compare with EST.S2P cDNA to CHO cellsBriefly incuba

21、ted with LDL,Amphotericin B selection -irradiation Low fluorescent LDL uptake -irradiation SRD-12B DeBose-Boyd RA, Brown MS, Li WP, Nohturfft A, Goldstein JL, Espenshade PJ. Transport-dependent proteolysis of SREBP: relocation of site-1 protease from Golgi to ER obviates the need for SREBP transport

22、 to Golgi. Cell. 1999 Dec 23;99(7):703-12.Nohturfft A, Yabe D, Goldstein JL, Brown MS, Espenshade PJ. Regulated step in cholesterol feedback localized to budding of SCAP from ER membranes. Cell. 2000 Aug 4;102(3):315-23.Sterol Regulated Transport of SCAPPurification Scheme for SCAP-interacting Prote

23、insYang et al., Cell (2002) 489-500Two Insigs: Insig-1 and Insig-2A.Sequence AlignmentB.Hydropathy PlotYabe et al., PNAS (2002) 12753-8Insig九、胆固醇代谢的负反馈调控机制九、胆固醇代谢的负反馈调控机制（二）（二）HMGCR降解途径降解途径From He J. et al., N Engl J Med, (2005), 353: 1124-1134. and IMS Health, MIDAS 药品销售排行榜药品销售排行榜NameCompanyIndicat

24、ionSales to June 07($bn)LipitorPfizerHypercholesterolaemia（高胆固（高胆固醇血症）醇血症）13.5NexiumAstraZenecaGastroesophageal reflux（胃食管返流）, gastric ulcers（胃溃疡）6.9SeretideGSKAsthma（哮喘）6.7PlavixBristol-Myers SquibbAtherosclerotic events（动脉粥样硬化症）5.8AranespAmgenAnaemia（贫血症）5.1HMG-CoA Reductase (HMGCR): the Rate-Limi

25、ting Enzyme in Cholesterol Biosynthetic Pathway(HMGCR)Proposed Model for INSIG-mediated Regulation ofHMG CoA Reductase and SCAPSterol-Regulated Degradation of HMG-CoA Reductase is Blocked by Inhibitors of the 26S ProteasomeWorking Hypothesis for Sterol-Regulated Degradation of HMG-CoA ReductaseStero

26、l-Stimulated Ubiquitinaion of HMGCR Requires InsigSever, Song, Yabe, et al., JBC, 2003Amino Acid Sequence of the HMG-CoA Reductase Membrane DomainLysines 89 and 248 are Required for the Sterol-Regulated Ubiquitination and Degradation of HMGCRStrategy for Purifying the E3 of HMG-CoA ReductaseE3 Activ

27、ity Co-immunoprecipitates with Insig-1 in Sterol-Treated CellsIdentification of gp78 and VCP as Insig-1-associating proteinsMolecular Pathway for Sterol-regulated Degradation of HMGCRSong et al., Mol Cell, 2005What are other proteins involved in this pathway?Identification of Ufd1 as a gp78 interact

28、ing proteinIP: Endogenous gp78IP: Endogenous gp78Transfect & CoIPIP: Flag-Ufd1ABCUfd1 is required for the ubiquitination of endogenous HMGCRUfd1 enhances the E3 activity of gp78 in vitroDual roles of Ufd1: Enhancing E3 activity and Promoting degradation Cao et al., Cell Metab, 2007十、饮食胆固醇吸收的分子途径十、饮食

29、胆固醇吸收的分子途径(HMGCR)De novo Cholesterol Synthesis is a Energy-Consuming Process18 Acetyl-CoACholesterol27 NADPH11 O218 ATPCholesterol Biosynthesis and Absorption in Human300-500 mgDietary AbsorptionBiosynthesis 600-900 mg1000 mgBiliary re-absorptionDietary Cholesterol Absorption by Intestinal Enterocyt

30、esCholesterol EsterHydrolysisCholesterol Cholesterol CholesterolEster ChylomicronIntestinal lumenLymphACAT2EREnterocyteTight JunctionEzetimibe2001 Cholesterol absorption inhibitor EzetimibeNPC1L12004 Identification of NPC1L1Human Niemann Pick C1 Like 1 (NPC1L1)NPC1L1 mediates the re-absorption of ch

31、olesterol from bileApicalHepatocytesBasolateralApicalNPC1L1 recycles between PM and ERC responding to cholesterol levelABOverexpression of NPC1L1 increases free cholesterol uptakeKnocking down of NPC1L1 protein in L02 cells attenuates the uptake of free cholesterolStructures of different sterols Ste

32、rol Absorption:50% cholesterolSterol-specificity for NPC1L1-mediated internalization ABHypothesis: NPC1L1 mediates cholesterol uptake through vesicle endocytosisNPC1L1CholesterolCytoskeleton is Essential for Vesicular TransportFrom Cai et al., 2006 Dev CellDisruption of microfilaments ablates the en

33、docytosis of NPC1L1-EGFP and cellular uptake of cholesterol Cholesterol ReplenishmentCholesterol ReplenishmentIdentification of NPC1L1-Associating ProteinsClathrin/AP-2 Mediates Vesicular EndocytosisFrom Conner et al., 2003 NatureKnockdown of CHC and 2 attenuates the internalization of NPC1L1 and ch

34、olesterolCholesterol Absorption Inhibitor (Ezetimibe)Ezetimibe (EZ)CtrEZStatinEZ+StatinEzetimibe inhibits the association of NPC1L1 and clathrinEzetimibe Blocks Both NPC1L1 Endocytosis and Cholesterol UptakeA working model for the NPC1L1-mediated cholesterol uptake筛选潜在的胆固醇吸收抑制剂筛选潜在的胆固醇吸收抑制剂0 min60 min120 minDMSOC6NPC1L1CholesterolNPC1L1CholesterolCompound 6 Inhibits NPC1L1 Endocytosis and Cholesterol UptakeThank you!