臺灣博碩士論文加值系統

一氧化氮 (Nitric Oxide) 是一個與細胞訊息傳遞有關的氣體分子，由 L-arginine 經一氧化氮合成酶 (NOS) 催化而生成。當細胞面臨刺激時，誘發型一氧化氮合成酶 (iNOS) 會大量表現進而製造高濃度的一氧化氮，此高濃度的 NO 對細胞通常具有毒性。許多研究藉由抑制 iNOS 基因的轉錄或調控 iNOS 的後轉譯修飾，來降低 NO 對細胞的傷害。本論文主要探討酪氨酸磷酸化對 iNOS 蛋白質的穩定度、二聚體的形成以及蛋白質之間的交互作用中所扮演的角色。 我們發現 tyrosine kinase inhibitors 能有效的抑制脂多糖 (LPS) 和干擾素 (IFNγ) 所產生的 NO 含量、 iNOS 蛋白質表現量與降低 iNOS 蛋白質的穩定度，並且降低 iNOS 二聚體/單聚體比例。經由免疫沉澱的實驗發現在 MES-13 細胞內， iNOS 能與 caveolin 1 (cav 1) 和 heat shock protein 90 (hsp90) 產生交互作用；且此交互作用在加入 Genistein 、 PP1 或去除 iNOS 蛋白上酪氨酸的磷酸根，並不會受到影響。因此，在 MES-13 細胞內， tyrosine kinase inhibitors 可加速 iNOS 蛋白質的降解的機轉可能是透過減少其二聚體的形成有關，而並非依靠與 cav 1 之間的交互作用。

Nitric oxide (NO), a messenger gas generated from L-arginine, is a product catalyzed by nitric oxide synthase (NOS). Inducible NOS (iNOS) produces high concentrations of NO when cells are challenged with endotoxins or cytokines. NO synthesis by iNOS was mostly regulated at transcriptional level and also by the post-translational modification of iNOS. It has also been reported recently that iNOS activity was mediated by tyrosine phosphorylation. In this present study, roles of tyrosine phosphorylation in mediating iNOS protein stability, dimer formation and protein-protein interactions were investigated in glomerular mesangial cell line, MES-13 cells. Tyrosine kinase inhibitors (either genistein or PP1) effectively inhibited lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-induced NO production and iNOS protein expression in MES-13 cells. In addition, both genistein and PP1 perturbed LPS/ IFNγ-induced iNOS protein stability in MES-13 cells. The dimer to monomer ratio of iNOS protein in MES-13 cells was attenuated with the addition of either genistein or PP1. Association of iNOS protein with caveolin 1 (cav 1) and heat shock protein 90 (hsp90) in MES-13 cells was confirmed by immunoprecipitation experiments. Neither genistein nor PP1 interrupted the interaction of iNOS protein with caveolin 1 in MES-13 cells. Furthermore, dephosphorylation of iNOS by LAR tyrosine phosphatase (LAR) did not affect its interaction with caveolin 1 in vitro. Our data indicated that tyrosine kinase inhibitors (either genistein or PP1) accelerated iNOS protein degradation through reducing dimer to monomer ratio, but not its interaction with caveolin 1 in MES-13 cells.

誌謝…………………………………………………………………3
中文摘要……………………………………………………………5
英文摘要……………………………………………………………7
縮寫表………………………………………………………………9
序論…………………………………………………………………12
實驗材料……………………………………………………………22
實驗方法……………………………………………………………24
研究動機……………………………………………………………29
實驗結果……………………………………………………………30
討論…………………………………………………………………34
圖表…………………………………………………………………38
附錄…………………………………………………………………55
文獻參考……………………………………………………………62

http://www.doh.gov.tw/CHT2006/index_populace.aspx

Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262, 5592-5595.

Aktan F (2004) iNOS-mediated nitric oxide production and its regulation. Life Sci 75, 639-653.

Andrew PJ, Mayer B (1999) Enzymatic function of nitric oxide synthases. Cardiovasc Res 43, 521-531.

Bogdan C, Thuring H, Dlaska M, Rollinghoff M, Weiss G (1997) Mechanism of suppression of macrophage nitric oxide release by IL-13: influence of the macrophage population. J Immunol 159, 4506-4513.

Cattell V (2002) Nitric oxide and glomerulonephritis. Kidney Int 61, 816-821.

Chen Y, Panda K, Stuehr DJ (2002) Control of nitric oxide synthase dimer assembly by a heme-NO-dependent mechanism. Biochemistry 41, 4618-4625.

Cho HJ, Xie QW, Calaycay J, Mumford RA, Swiderek KM, Lee TD, Nathan C (1992) Calmodulin is a subunit of nitric oxide synthase from macrophages. J Exp Med 176, 599-604.

Dawn B, Bolli R (2002) Role of nitric oxide in myocardial preconditioning. Ann N Y Acad Sci 962, 18-41.

Delgado M (2003) Inhibition of interferon (IFN) gamma-induced Jak-STAT1 activation in microglia by vasoactive intestinal peptide: inhibitory effect on CD40, IFN-induced protein-10, and inducible nitric-oxide synthase expression. J Biol Chem 278, 27620-27629.

Eissa NT, Haggerty CM, Palmer CD, Patton W, Moss J (2001) Identification of residues critical for enzymatic activity in the domain encoded by exons 8 and 9 of the human inducible nitric oxide synthase. Am J Respir Cell Mol Biol 24, 616-620.

Eissa NT, Strauss AJ, Haggerty CM, Choo EK, Chu SC, Moss J (1996) Alternative splicing of human inducible nitric-oxide synthase mRNA. tissue-specific regulation and induction by cytokines. J Biol Chem 271, 27184-27187.

Felley-Bosco E, Bender FC, Courjault-Gautier F, Bron C, Quest AF (2000) Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells. Proc Natl Acad Sci U S A 97, 14334-14339.

Furchgott RF, Zawadzki JV (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 373-376.

Ghosh DK, Rashid MB, Crane B, Taskar V, Mast M, Misukonis MA, Weinberg JB, Eissa NT (2001) Characterization of key residues in the subdomain encoded by exons 8 and 9 of human inducible nitric oxide synthase: a critical role for Asp-280 in substrate binding and subunit interactions. Proc Natl Acad Sci U S A 98, 10392-10397.

Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA (1996) Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J Biol Chem 271, 695-701.

Hausel P, Latado H, Courjault-Gautier F, Felley-Bosco E (2006) Src-mediated phosphorylation regulates subcellular distribution and activity of human inducible nitric oxide synthase. Oncogene 25, 198-206.

Ignarro LJ, Byrns RE, Buga GM, Wood KS (1987) Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ Res 61, 866-879.

Kagami S, Kondo S (2004) Beta1-integrins and glomerular injury. J Med Invest 51, 1-13.

Kim HS, Loughran PA, Billiar TR (2008) Carbon monoxide decreases the level of iNOS protein and active dimer in IL-1beta-stimulated hepatocytes. Nitric Oxide 18, 256-265.

Kleinert H, Pautz A, Linker K, Schwarz PM (2004) Regulation of the expression of inducible nitric oxide synthase. Eur J Pharmacol 500, 255-266.

Kleinert H, Schwarz PM, Forstermann U (2003) Regulation of the expression of inducible nitric oxide synthase. Biol Chem 384, 1343-1364.

Kolodziejski PJ, Musial A, Koo JS, Eissa NT (2002) Ubiquitination of inducible nitric oxide synthase is required for its degradation. Proc Natl Acad Sci U S A 99, 12315-12320.

Kone BC, Kuncewicz T, Zhang W, Yu ZY (2003) Protein interactions with nitric oxide synthases: controlling the right time, the right place, and the right amount of nitric oxide. Am J Physiol Renal Physiol 285, F178-190.

Kox M, Wijetunge S, Pickkers P, Hughes AD (2007) Inhibition of Src family tyrosine kinases prevents lipopolysaccharide-induced hyporeactivity in isolated rat tail arteries. Vascul Pharmacol 46, 195-200.

Larsen RI (1966) Air pollution from motor vehicles. Ann N Y Acad Sci 136, 277-301.

Lin YS, Hsieh M, Lee YJ, Liu KL, Lin TH (2008) AH23848 accelerates inducible nitric oxide synthase degradation through attenuation of cAMP signaling in glomerular mesangial cells. Nitric Oxide 18, 93-104.

Liu T, Huang Y, Likhotvorik RI, Keshvara L, Hoyt DG (2008) Protein Never in Mitosis Gene A Interacting-1 (PIN1) regulates degradation of inducible nitric oxide synthase in endothelial cells. Am J Physiol Cell Physiol 295, C819-827.

Lo HW, Hsu SC, Ali-Seyed M, Gunduz M, Xia W, Wei Y, Bartholomeusz G, Shih JY, Hung MC (2005) Nuclear interaction of EGFR and STAT3 in the activation of the iNOS/NO pathway. Cancer Cell 7, 575-589.

Lowenstein CJ, Padalko E (2004) iNOS (NOS2) at a glance. J Cell Sci 117, 2865-2867.

Miller MJ, Sandoval M (1999) Nitric Oxide. III. A molecular prelude to intestinal inflammation. Am J Physiol 276, G795-799.

Mitani T, Terashima M, Yoshimura H, Nariai Y, Tanigawa Y (2005) TGF-beta1 enhances degradation of IFN-gamma-induced iNOS protein via proteasomes in RAW 264.7 cells. Nitric Oxide 13, 78-87.

Musial A, Eissa NT (2001) Inducible nitric-oxide synthase is regulated by the proteasome degradation pathway. J Biol Chem 276, 24268-24273.

Nathan C, Xie QW (1994) Regulation of biosynthesis of nitric oxide. J Biol Chem 269, 13725-13728.

Palmer RM, Ferrige AG, Moncada S (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524-526.

Pan J, Burgher KL, Szczepanik AM, Ringheim GE (1996) Tyrosine phosphorylation of inducible nitric oxide synthase: implications for potential post-translational regulation. Biochem J 314 ( Pt 3), 889-894.

Park JH, Na HJ, et al. (2002) Nitric oxide (NO) pretreatment increases cytokine-induced NO production in cultured rat hepatocytes by suppressing GTP cyclohydrolase I feedback inhibitory protein level and promoting inducible NO synthase dimerization. J Biol Chem 277, 47073-47079.

Parsons SJ, Parsons JT (2004) Src family kinases, key regulators of signal transduction. Oncogene 23, 7906-7909.

Rao KM (2000) Molecular mechanisms regulating iNOS expression in various cell types. J Toxicol Environ Health B Crit Rev 3, 27-58.

Saha RN, Pahan K (2006) Regulation of inducible nitric oxide synthase gene in glial cells. Antioxid Redox Signal 8, 929-947.

Sakai K, Suzuki H, et al. (2006) Phosphoinositide 3-kinase in nitric oxide synthesis in macrophage: critical dimerization of inducible nitric-oxide synthase. J Biol Chem 281, 17736-17742.

Shah OJ, Kimball SR, Jefferson LS (2002) The Src-family tyrosine kinase inhibitor PP1 interferes with the activation of ribosomal protein S6 kinases. Biochem J 366, 57-62.

Takaki H, Minoda Y, Koga K, Takaesu G, Yoshimura A, Kobayashi T (2006) TGF-beta1 suppresses IFN-gamma-induced NO production in macrophages by suppressing STAT1 activation and accelerating iNOS protein degradation. Genes Cells 11, 871-882.

Thirunavukkarasu M, Juhasz B, Zhan L, Menon VP, Tosaki A, Otani H, Maulik N (2007) VEGFR1 (Flt-1+/-) gene knockout leads to the disruption of VEGF-mediated signaling through the nitric oxide/heme oxygenase pathway in ischemic preconditioned myocardium. Free Radic Biol Med 42, 1487-1495.

Walker G, Pfeilschifter J, Otten U, Kunz D (2001) Proteolytic cleavage of inducible nitric oxide synthase (iNOS) by calpain I. Biochim Biophys Acta 1568, 216-224.

Ye X, Liu SF (2002) Lipopolysaccharide down-regulates Sp1 binding activity by promoting Sp1 protein dephosphorylation and degradation. J Biol Chem 277, 31863-31870.

Ying WZ, Sanders PW (2003) Accelerated ubiquitination and proteasome degradation of a genetic variant of inducible nitric oxide synthase. Biochem J 376, 789-794.

Yoshida M, Xia Y (2003) Heat shock protein 90 as an endogenous protein enhancer of inducible nitric-oxide synthase. J Biol Chem 278, 36953-36958.

Zhang W, Kuncewicz T, Yu ZY, Zou L, Xu X, Kone BC (2003) Protein-protein interactions involving inducible nitric oxide synthase. Acta Physiol Scand 179, 137-142.

Zhang Y, Brovkovych V, Brovkovych S, Tan F, Lee BS, Sharma T, Skidgel RA (2007) Dynamic receptor-dependent activation of inducible nitric-oxide synthase by ERK-mediated phosphorylation of Ser745. J Biol Chem 282, 32453-32461.

Ziolo MT, Kohr MJ, Wang H (2008) Nitric oxide signaling and the regulation of myocardial function. J Mol Cell Cardiol 45, 625-632.