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研究生: 張薰勻
Chang, Hsur-Yun
論文名稱: AMP蛋白激酶的活化對金屬硫蛋白基因表現之影響
Effect of AMP-activated Protein Kinase Activation on Metallothionein Gene Expression
指導教授: 林立元
Lin, Lih-Yuan
口試委員:
學位類別: 碩士
Master
系所名稱: 生命科學暨醫學院 - 分子與細胞生物研究所
Institute of Molecular and Cellular Biology
論文出版年: 2009
畢業學年度: 97
語文別: 中文
論文頁數: 47
中文關鍵詞: 金屬硫蛋白能量缺失
外文關鍵詞: AMPK, MT, energy-deprivation
相關次數: 點閱:3下載:0
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  • 金屬硫蛋白 (Metallothionein, MT)在生理上重要的功能為調節細胞內金屬的恆定性和對抗氧化壓力,而近幾年很多研究指出MT可能參與能量代謝的生理活動,但是仍沒有明確的機制。在研究中我們使用能量代謝抑制劑NaNB3B和2-deoxyglucose (2DG)處理HEK293細胞使之處於能量缺乏的情況,發現MT基因的表現受到抑制,且NaNB3B和2DG因阻斷細胞內ATP的生成而促進能量監督者AMPK的活性。由於AMPK調控細胞中能量的平衡,包括參與代謝的蛋白質或基因,於是我們測試NaNB3B是否透過活化AMPK而抑制MT基因的表現,我們利用Dominant-negative AMPK (DN-AMPK)與AMPK siRNA抑制AMPK的活性,發現當AMPK的活性被減低後,MT基因表現被NaNB3B抑制的情形減緩,故推測NaNB3B透過活化AMPK來調控MT基因的表現。當細胞遭受能量低迷的時候,活化的AMPK會藉由磷酸化受質來調控下游的代謝途徑與基因的表現,以維持細胞中的能量平衡,於是我們測試了與代謝有關的c-Myc和AMPK的受質Forkhead box O3 (FOXO3)是否參與在NaNB3B抑制MT基因表現的途徑中,結果顯示c-Myc和FOXO3均可能會影響MT基因表現,但不參與在NaNB3B活化AMPK而抑制MT基因表現的途徑。


    中文摘要 I Abstract II 目次 III 圖次 IV 緒論 1 材料與方法 8 1.化學藥品及製備 8 2.細胞株與培養條件 8 3.細胞質RNA萃取 (Extraction Cytoplasmic RNA) 9 4.甲醛變性瓊脂凝膠電泳 (Formaldehyde/denaturating agarose gel electrophoresis) 9 5.反轉錄 (reverse transcription) 10 6.即時定量聚合酶連鎖反應 (Quantitative Real-time PCR) 11 7.蛋白質定量 11 8.西方墨點法 (Western Blot) 12 9.轉殖作用 (Transfection) 14 10.報導基因檢定 (luciferase assay) 16 結果 17 1. NaN3抑制HEK293細胞的MT基因表現、且活化細胞中的AMPK 17 2. 2DG抑制HEK293細胞的MT基因表現、且活化細胞中的AMPK 17 3. NaN3透過活化AMPK來調控MT基因的表現 18 4.FOXO3不參與NaN3活化AMPK進而調控MT基因的途徑 21 5.c-Myc不參與NaN3活化AMPK進而調控MT基因的途徑 22 討論 25 參考文獻 33

    Andersen RD, Taplitz SJ, Wong S, Bristol G, Larkin B, Herschman HR (1987) Metal-dependent binding of a factor in vivo to the metal-responsive elements of the metallothionein 1 gene promoter. Mol Cell Biol 7(10): 3574-3581

    Andrews GK (2000) Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem Pharmacol 59(1): 95-104

    Barthel A, Schmoll D, Kruger KD, Roth RA, Joost HG (2002) Regulation of the forkhead transcription factor FKHR (FOXO1a) by glucose starvation and AICAR, an activator of AMP-activated protein kinase. Endocrinology 143(8): 3183-3186

    Beattie JH, Wood AM, Newman AM, Bremner I, Choo KH, Michalska AE, Duncan JS, Trayhurn P (1998) Obesity and hyperleptinemia in metallothionein (-I and -II) null mice. Proc Natl Acad Sci U S A 95(1): 358-363

    Benassi B, Fanciulli M, Fiorentino F, Porrello A, Chiorino G, Loda M, Zupi G, Biroccio A (2006) c-Myc phosphorylation is required for cellular response to oxidative stress. Mol Cell 21(4): 509-519

    Bhat R, Weaver JA, Wagner C, Bodwell JE, Bresnick E (1996) ATP depletion affects the phosphorylation state, ligand binding, and nuclear transport of the 4 S polycyclic aromatic hydrocarbon-binding protein in rat hepatoma cells. J Biol Chem 271(51): 32551-32556

    Brugnera E, Georgiev O, Radtke F, Heuchel R, Baker E, Sutherland GR, Schaffner W (1994) Cloning, chromosomal mapping and characterization of the human metal-regulatory transcription factor MTF-1. Nucleic Acids Res 22(15): 3167-3173

    Carling D (2004) The AMP-activated protein kinase cascade--a unifying system for energy control. Trends Biochem Sci 29(1): 18-24

    Dalton T, Palmiter RD, Andrews GK (1994) Transcriptional induction of the mouse metallothionein-I gene in hydrogen peroxide-treated Hepa cells involves a composite major late transcription factor/antioxidant response element and metal response promoter elements. Nucleic Acids Res 22(23): 5016-5023

    Dang CV (1999) c-Myc target genes involved in cell growth, apoptosis, and metabolism. Mol Cell Biol 19(1): 1-11

    Datta J, Majumder S, Kutay H, Motiwala T, Frankel W, Costa R, Cha HC, MacDougald OA, Jacob ST, Ghoshal K (2007) Metallothionein expression is suppressed in primary human hepatocellular carcinomas and is mediated through inactivation of CCAAT/enhancer binding protein alpha by phosphatidylinositol 3-kinase signaling cascade. Cancer Res 67(6): 2736-2746

    Davies SP, Helps NR, Cohen PT, Hardie DG (1995) 5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C alpha and native bovine protein phosphatase-2AC. FEBS Lett 377(3): 421-425

    Ghoul M, Pommepuy M, Moillo-Batt A, Cormier M (1989) Effect of carbonyl cyanide m-chlorophenylhydrazone on Escherichia coli halotolerance. Appl Environ Microbiol 55(4): 1040-1043

    Giannakou ME, Goss M, Junger MA, Hafen E, Leevers SJ, Partridge L (2004) Long-lived Drosophila with overexpressed dFOXO in adult fat body. Science 305(5682): 361

    Greer EL, Oskoui PR, Banko MR, Maniar JM, Gygi MP, Gygi SP, Brunet A (2007) The energy sensor AMP-activated protein kinase directly regulates the mammalian FOXO3 transcription factor. J Biol Chem 282(41): 30107-30119

    Hardie DG, Scott JW, Pan DA, Hudson ER (2003) Management of cellular energy by the AMP-activated protein kinase system. FEBS Lett 546(1): 113-120

    Hawley SA, Selbert MA, Goldstein EG, Edelman AM, Carling D, Hardie DG (1995) 5'-AMP activates the AMP-activated protein kinase cascade, and Ca2+/calmodulin activates the calmodulin-dependent protein kinase I cascade, via three independent mechanisms. J Biol Chem 270(45): 27186-27191

    Hemminki A, Markie D, Tomlinson I, Avizienyte E, Roth S, Loukola A, Bignell G, Warren W, Aminoff M, Hoglund P, Jarvinen H, Kristo P, Pelin K, Ridanpaa M, Salovaara R, Toro T, Bodmer W, Olschwang S, Olsen AS, Stratton MR, de la Chapelle A, Aaltonen LA (1998) A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature 391(6663): 184-187

    Heuchel R, Radtke F, Georgiev O, Stark G, Aguet M, Schaffner W (1994) The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression. EMBO J 13(12): 2870-2875

    Huang MJ, Cheng YC, Liu CR, Lin S, Liu HE (2006) A small-molecule c-Myc inhibitor, 10058-F4, induces cell-cycle arrest, apoptosis, and myeloid differentiation of human acute myeloid leukemia. Exp Hematol 34(11): 1480-1489

    Hurley RL, Anderson KA, Franzone JM, Kemp BE, Means AR, Witters LA (2005) The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases. J Biol Chem 280(32): 29060-29066

    Hwangbo DS, Gershman B, Tu MP, Palmer M, Tatar M (2004) Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body. Nature 429(6991): 562-566

    Jelluma N, Yang X, Stokoe D, Evan GI, Dansen TB, Haas-Kogan DA (2006) Glucose withdrawal induces oxidative stress followed by apoptosis in glioblastoma cells but not in normal human astrocytes. Mol Cancer Res 4(5): 319-330

    Jiang LJ, Maret W, Vallee BL (1998) The ATP-metallothionein complex. Proc Natl Acad Sci U S A 95(16): 9146-9149

    Johnson LN, Noble ME, Owen DJ (1996) Active and inactive protein kinases: structural basis for regulation. Cell 85(2): 149-158

    Kagi JH, Himmelhoch SR, Whanger PD, Bethune JL, Vallee BL (1974) Equine hepatic and renal metallothioneins. Purification, molecular weight, amino acid composition, and metal content. J Biol Chem 249(11): 3537-3542

    Kagi JH, Valee BL. (1960) Metallothionein: a cadmium- and zinc-containing protein from equine renal cortex. J Biol Chem, Vol. 235, pp. 3460-3465.

    Kahn BB, Alquier T, Carling D, Hardie DG (2005) AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1(1): 15-25

    LaRochelle O, Gagne V, Charron J, Soh JW, Seguin C (2001) Phosphorylation is involved in the activation of metal-regulatory transcription factor 1 in response to metal ions. J Biol Chem 276(45): 41879-41888

    Li Z, Van Calcar S, Qu C, Cavenee WK, Zhang MQ, Ren B (2003) A global transcriptional regulatory role for c-Myc in Burkitt's lymphoma cells. Proc Natl Acad Sci U S A 100(14): 8164-8169

    McGee SL, Hargreaves M (2008) AMPK and transcriptional regulation. Front Biosci 13: 3022-3033

    McGee SL, van Denderen BJ, Howlett KF, Mollica J, Schertzer JD, Kemp BE, Hargreaves M (2008) AMP-activated protein kinase regulates GLUT4 transcription by phosphorylating histone deacetylase 5. Diabetes 57(4): 860-867

    Miles AT, Hawksworth GM, Beattie JH, Rodilla V (2000) Induction, regulation, degradation, and biological significance of mammalian metallothioneins. Crit Rev Biochem Mol Biol 35(1): 35-70

    Murphy BJ, Andrews GK, Bittel D, Discher DJ, McCue J, Green CJ, Yanovsky M, Giaccia A, Sutherland RM, Laderoute KR, Webster KA (1999) Activation of metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1. Cancer Res 59(6): 1315-1322

    Nilsson JA, Cleveland JL (2003) Myc pathways provoking cell suicide and cancer. Oncogene 22(56): 9007-9021

    Partanen JI, Nieminen AI, Makela TP, Klefstrom J (2007) Suppression of oncogenic properties of c-Myc by LKB1-controlled epithelial organization. Proc Natl Acad Sci U S A 104(37): 14694-14699

    Plisov SY, Nichiporenko MG, Shkapenko AL, Kumarev VP, Baranova LV, Merkulova TI (1994) The immediate vicinity of mouse metallothionein-I gene contains two sites conferring glucocorticoid inducibility to the heterologous promoter. FEBS Lett 352(3): 339-341

    Powell SR (2000) The antioxidant properties of zinc. J Nutr 130(5S Suppl): 1447S-1454S

    Quaife CJ, Findley SD, Erickson JC, Froelick GJ, Kelly EJ, Zambrowicz BP, Palmiter RD (1994) Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia. Biochemistry 33(23): 7250-7259

    Rutter GA, Da Silva Xavier G, Leclerc I (2003) Roles of 5'-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochem J 375(Pt 1): 1-16

    Sampson VB, Rong NH, Han J, Yang Q, Aris V, Soteropoulos P, Petrelli NJ, Dunn SP, Krueger LJ (2007) MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells. Cancer Res 67(20): 9762-9770

    Sanders MJ, Grondin PO, Hegarty BD, Snowden MA, Carling D (2007) Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochem J 403(1): 139-148

    Searle PF, Stuart GW, Palmiter RD (1985) Building a metal-responsive promoter with synthetic regulatory elements. Mol Cell Biol 5(6): 1480-1489

    Shaw RJ, Bardeesy N, Manning BD, Lopez L, Kosmatka M, DePinho RA, Cantley LC (2004) The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6(1): 91-99

    Stein SC, Woods A, Jones NA, Davison MD, Carling D (2000) The regulation of AMP-activated protein kinase by phosphorylation. Biochem J 345 Pt 3: 437-443

    Thirumoorthy N, Manisenthil Kumar KT, Shyam Sundar A, Panayappan L, Chatterjee M (2007) Metallothionein: an overview. World J Gastroenterol 13(7): 993-996

    Thors B, Halldorsson H, Thorgeirsson G (2004) Thrombin and histamine stimulate endothelial nitric-oxide synthase phosphorylation at Ser1177 via an AMPK mediated pathway independent of PI3K-Akt. FEBS Lett 573(1-3): 175-180

    Vervoorts J, Luscher-Firzlaff J, Luscher B (2006) The ins and outs of MYC regulation by posttranslational mechanisms. J Biol Chem 281(46): 34725-34729

    Waelput W, Verhee A, Broekaert D, Eyckerman S, Vandekerckhove J, Beattie JH, Tavernier J (2000) Identification and expression analysis of leptin-regulated immediate early response and late target genes. Biochem J 348 Pt 1: 55-61

    Wang SH, Chang CY, Chen CF, Tam MF, Shih YH, Lin LY (1997) Cloning of porcine neuron growth inhibitory factor (metallothionein III) cDNA and expression of the gene in Saccharomyces cerevisiae. Gene 203(2): 189-197

    Westin G, Schaffner W (1988) A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I gene. EMBO J 7(12): 3763-3770

    Winder WW, Hardie DG (1999) AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes. Am J Physiol 277(1 Pt 1): E1-10

    Witters LA, Kemp BE (1992) Insulin activation of acetyl-CoA carboxylase accompanied by inhibition of the 5'-AMP-activated protein kinase. J Biol Chem 267(5): 2864-2867

    Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D, Schlattner U, Wallimann T, Carlson M, Carling D (2003) LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 13(22): 2004-2008

    Xie M, Zhang D, Dyck JR, Li Y, Zhang H, Morishima M, Mann DL, Taffet GE, Baldini A, Khoury DS, Schneider MD (2006) A pivotal role for endogenous TGF-beta-activated kinase-1 in the LKB1/AMP-activated protein kinase energy-sensor pathway. Proc Natl Acad Sci U S A 103(46): 17378-17383

    Zou MH, Hou XY, Shi CM, Nagata D, Walsh K, Cohen RA (2002) Modulation by peroxynitrite of Akt- and AMP-activated kinase-dependent Ser1179 phosphorylation of endothelial nitric oxide synthase. J Biol Chem 277(36): 32552-32557

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