课件：胆固醇代谢科大英才班.ppt

胆固醇代谢平衡调控,中国科学院上海生命科学研究院 生物化学与细胞生物学研究所,宋保亮 研究员,提纲,胆固醇的生物学功能 胆固醇相关疾病 胆固醇的生物合成 胆固醇在血液中的运输 家族型高胆固醇血症 NPC疾病 胆固醇的降解与清除 胆固醇代谢的负反馈调控机制SCAP-SREBP途径 胆固醇代谢的负反馈调控机制HMGCR降解途径 饮食胆固醇吸收的分子途径,Cholesterol（胆固醇）,一、胆固醇的生物学功能,Membrane Fluidity Bile Acids Hormones、Vitamin D Synapse Hedgehog Modification Signal Transduction ,哺乳动物细胞模式图,膜上的微结构域,Cholesterol and cholesterol derivatives,雌激素,雄激素,脂肪酸、糖等提供能量（ATP） 胆固醇并不提供能量,二、胆固醇相关疾病,Cholesterol is the major cause of atherosclerosis,From He J. et al., N Engl J Med, (2005), 353: 1124-1134.,中国死亡原因统计,蒙娜丽莎患有高胆固醇？,意大利帕勒莫大学的病理解剖学教授弗兰克，研究认为蒙娜丽莎饮食不健康，患有高胆固醇症。 正是眼部的脂肪瘤，造成了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模特）正在为自己体内过高的胆固醇而担忧。,胆结石,老年痴呆症,Cholesterol and Diseases,Niemann-Pick Type C Disease,糖尿病/肥胖症,From IMS Health, MIDAS,药品销售排行榜,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 precursors,Cholesterol is made from acetyl-CoA in four stages,all of its carbon atoms are provided by a single precursor acetate,Stage 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 to 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 biosynthesis,Stage 3,Polymerization of six 5-carbon isoprene units to form the 30-carbon linear structure of squalene.,Stage 4,Cyclization of squalene forms the four rings of the steroid nucleus. Subsequent modifications leads to the final product, cholesterol.,Most of the cholesterol made in the liver is exported,Much of cholesterol synthesis takes place in the liver Most is exported,Cholesterol is exported in three forms,1. Bile salts amphipathic cholesterol derivatives that aid lipid digestion 2. Cholesterol to bile 3. Cholesteryl esters transported and secreted in lipoprotein particles to other tissues that use cholesterol or are stored in the liver,四、胆固醇在血液中的运输,Cholesteryl ester formation,Formed in the liver Converting cholesterol to a more hydrophobic form,Cholesterol transport: the problem,Cholesterol and cholesteryl esters are essentially insoluble in water These molecules must be moved from the tissue of origin to the tissues in which they are stored or are consumed,Cholesterol transport: the solution,Cholesterol and cholesteryl esters are carried in the blood plasma from one tissue to another as plasma lipoproteins,Cholesterol esters enter cells by receptor mediated endocytosis,五、家族型高胆固醇血症,LDL receptor (LDLR) 突变,家族性高胆固醇血症 (Familial Hypercholesterolemia, FH),杂合子患者血清总胆固醇较正常人高出12倍 纯合子患者血清总胆固醇较正常人高出68倍,杂合子患者发生率为1/500 纯合子患者发生率为1/1,000,000,杂合子患者男性3040岁时，患CAD， 23% 患者在50岁以前死于CAD，50% 患者在60岁时明显的CAD症状； 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-黄色瘤,FH患者,LDLR突变-眼底脂质渗出,正常人,六、 Niemann-Pick type C(NPC) 疾病,溶酶体堆积型疾病（Lysosomal Storage Disorders） 胆固醇在溶酶体中堆积 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 1：120，000发病，携带者1：100 NPC1（大的膜蛋白）或NPC2（小的可溶蛋白）基因突变 临床尝试用环化糊精进行治疗,正常细胞,NPC1突变细胞,七、胆固醇的降解与清除,Degradation of cholesterol,The ring structure of cholesterol cannot be metabolized to CO2 and H2O in humans The intact sterol ring is eliminated from the body by: Conversion to bile acids, which are excreted in feces Secretion of cholesterol into the bile, which transports it to the intestine for elimination,Steroid hormones are formed from cholesterol,All steroid hormones are derived form cholesterol In the cortex of adrenal glands two classes of hormones 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,（一）SCAP-SREBP途径,脂质代谢的关键蛋白质及其功能调控的临床意义,Insig,SREBP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, 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 transcription factor released by sterol-regulated proteolysis. Cell. 1994 Apr 8;77(1):53-62.,25-Hydroxycholesterol,-irradiation,Mutant cells survive (25-RA),2019/4/19,51,可编辑,Amphotericin B,-irradiation,Cholesterol auxotrophs (M19),S2P cDNA to CHO cells,Briefly incubated 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 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 SCAP,Purification Scheme for SCAP-interacting Proteins,Yang et al., Cell (2002) 489-500,Two Insigs: Insig-1 and Insig-2,A.Sequence Alignment,B.Hydropathy Plot,Yabe et al., PNAS (2002) 12753-8,Insig,九、胆固醇代谢的负反馈调控机制,（二）HMGCR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,HMG-CoA Reductase (HMGCR): the Rate-Limiting Enzyme in Cholesterol Biosynthetic Pathway,Proposed Model for INSIG-mediated Regulation of HMG CoA Reductase and SCAP,Sterol-Regulated Degradation of HMG-CoA Reductase is Blocked by Inhibitors of the 26S Proteasome,Working Hypothesis for Sterol-Regulated Degradation of HMG-CoA Reductase,Sterol-Stimulated Ubiquitinaion of HMGCR Requires Insig,Sever, Song, Yabe, et al., JBC, 2003,Amino Acid Sequence of the HMG-CoA Reductase Membrane Domain,Lysines 89 and 248 are Required for the Sterol-Regulated Ubiquitination and Degradation of HMGCR,Strategy for Purifying the E3 of HMG-CoA Reductase,E3 Activity Co-immunoprecipitates with Insig-1 in Sterol-Treated Cells,Identification of gp78 and VCP as Insig-1-associating proteins,Molecular Pathway for Sterol-regulated Degradation of HMGCR,Song et al., Mol Cell, 2005,What are other proteins involved in this pathway?,Identification of Ufd1 as a gp78 interacting protein,IP: Endogenous gp78,IP: Endogenous gp78,Transfect & CoIP IP: Flag-Ufd1,A,B,C,Ufd1 is required for the ubiquitination of endogenous HMGCR,Ufd1 enhances the E3 activity of gp78 in vitro,Dual 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,De novo Cholesterol Synthesis is a Energy-Consuming Process,18 Acetyl-CoA,Cholesterol,27 NADPH 11 O2 18 ATP,Cholesterol Biosynthesis and Absorption in Human,300-500 mg,Dietary Absorption,Biosynthesis,600-900 mg,1000 mg,Biliary re-absorption,Dietary Cholesterol Absorption by Intestinal Enterocytes,Cholesterol Ester,Hydrolysis,Cholesterol,Cholesterol,Cholesterol Ester,Chylomicron,Intestinal lumen,Lymph,ACAT2,ER,Enterocyte,Tight Junction,NPC1L1,2004 Identification of NPC1L1,Human Niemann Pick C1 Like 1 (NPC1L1),NPC1L1 mediates the re-absorption of cholesterol from bile,NPC1L1 recycles between PM and ERC responding to cholesterol level,A,B,Overexpression of NPC1L1 increases free cholesterol uptake,Knocking down of NPC1L1 protein in L02 cells attenuates the uptake of free cholesterol,Structures of different sterols,Sterol Absorption:,50% cholesterol,Sterol-specificity for NPC1L1-mediated internalization,A,B,Hypothesis: NPC1L1 mediates cholesterol uptake through vesicle endocytosis,NPC1L1,Cholesterol,Cytoskeleton is Essential