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研究生:呂俊宏
研究生(外文):Jyun-Hong Lyu
論文名稱:Nap1酵素性質與啟動子作用分析
論文名稱(外文):Enzymatic Character and Promoter Assay of Nap1 gene
指導教授:簡靜香簡靜香引用關係
指導教授(外文):Chin-Hsiang Chien
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:44
中文關鍵詞:抑癌基因
外文關鍵詞:tumor suppressorNap1
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NAP1目前是潛在的抑癌因子,然而對於其酵素功能及基因調控作用鮮少被研究。過去我們實驗室的研究顯示人類NAP1具有低活性的nitrilase特性,我們進一步了解其酵素性質,測定出人類NAP1具有ω-amidase活性,可以作用於受質α-ketoglutarmate以及succinamate,另外觀察到ω-amidase活性受到glycylglycine競爭性抑制,以及iodoacetamide不可逆抑制作用。以luciferase assay 觀察NAP1基因調控作用,在Huh7細胞顯示有高轉錄活性,預測人類及其他物種NAP1 promoter轉錄因子結合位置,顯示在許多物種保存有Sp1轉錄因子,進一步以mithramycin A作用於Huh7細胞,觀察到NAP1基因表現降低,以及轉錄能力降低,顯示Sp1轉錄因子活化NAP1基因,扮演調控NAP1基因的重要角色。了解經由Sp1活化NAP1基因作用,以TGF-β以及histone deacetylase抑制劑trichostatin A (TSA)作用於Huh7細胞,非預期結果顯示TGF-β無法使NAP1基因表現增加,而TSA反而抑制NAP1基因表現,目前為止,經由Sp1何種訊息傳遞路徑誘導NAP1基因表現還不清楚。
NAP1 is known as a putative tumor suppressor. Few studies have described the enzyme function and gene regulation until recent three years. Previous studies demonstrated that human Nap1 showed low nitrilase activity. In this study, human Nap1 with ω-amidase activity was determined. Human Nap1 catalyzed α-ketoglutarmate and succinamate to corresponding hydroxamate under acyl-transfer reaction. The ω-amidase activity can be inhibited competitively by glycylglycine and irreversibly inhibited by iodoacetamide. To understand transcriptional regulation of NAP1 promoter, we applied a NAP1 promoter clone with luciferase tag. Luciferase assay results show high basal NAP1 promoter activity in Huh7 cell. Sequence analysis of the promoter region among many species exhibited several putative transcription factor binding sites for Sp1. Treatment of Huh7 cell with mithramycin A resulted to reduce the NAP1 gene expression and transcriptional level. It indicates that Sp1 activate NAP1 gene. To investigate the Sp1-mediated NAP1 activation, we treated Huh7 cell with TGF-β and trichostatin A (TSA), a histone deacetylase inhibitor. Unexpected results showed no obvious increase of NAP1 gene expression by TGF-β and showed inhibitory effect by TSA. The signaling pathway of Sp1-mediated NAP1 gene expression is still unclear.
論文電子檔著作權授權書 i
論文審定同意書 ii
誌謝 iii
中文摘要 iv
Abstract v
目錄 vi
縮寫對照表 viii
一、 緒論 1
Nap1研究起源 1
Nitrilase superfamily 1
NitFhit 4
Nit1 4
Nap1 5
Nap1酵素性質及結構研究 6
Nitrile代謝及其偵測方法 6
Amidase反應偵測方法 7
ω-amidase 8
Sp1 10
研究目的與策略 11
二、 材料與方法 12
A. 材料 12
1. 細胞株及細胞培養液 12
2. 菌株及細菌培養液 13
3. 實驗使用酵素 15
4. 試劑套件(kit) 15
5. 實驗用質體 16
6. 實驗用藥品及溶液 16
7. 其他藥品 20
8. 儀器設備 20
9. 軟體 22
10. 其他 22
B. 方法 23
1. 細胞培養技術 23
2. 細菌培養技術 23
3. 質體抽取技術 24
4. 限制酶檢定 24
5. Nap1蛋白質純化 24
6. RNA抽取技術 26
7. 反轉錄聚合酶連鎖反應RT-PCR 27
8. 膠體電泳Agarose electrophoresis 28
9. 變性膠體電泳 SDS-PAGE 29
10. 預測Nap1 promoter轉錄因子結合位置 29
三、 結果 30
A. 人類Nap1酵素性質研究 30
1. 純化人類Nap1蛋白質 30
2. 人類Nap1 ω-amidase活性性質 30
3. 人類Nap1 ω-amidase活性抑制作用 30
四、 討論 32
純化Nap1蛋白質重點 32
五、 結論 33
六、 參考文獻 34
1. Hsieh, Y.C., et al., Influenza pandemics: past, present and future. J Formos Med Assoc, 2006. 105(1): p. 1-6.
2. 陳瑜絢, 利用神經胺酸酶之高保存性胺基酸序列選殖streptomyces albus 之基因, in 生物化學研究所. 1984, 國立陽明大學. p. -.
3. 黃裕智, 鏈黴菌神經胺酸 基因之選殖及表現 英文論文名稱: Cloning and expression of the neuraminidase gene from Streptomyces albus, in 生物化學研究所. 1989, 國立陽明大學 p. 82.
4. 黃麗櫻, 研究Clostridium Perfringens大型神經胺�@酸�D對OVCAR-3細胞的影響, in 生物化學研究所. 2000, 國立陽明大學. p. 64.
5. 簡麗敏, Clostridium perfringens 細胞內大型sialidase的結構與功能之研究, in 生物化學研究所. 2000, 國立陽明大學. p. 76.
6. 鄭雅文, 與Clostridium perfringens大型神經胺酸甘脢交互作用及與凝血相關蛋白之研究, in 生物化學研究所. 2001, 國立陽明大學. p. -.
7. 林俊宏, 人類nit2與fhit基因之選殖、定序與結構功能之研究, in 生物化學研究所. 2001, 國立陽明大學. p. 72.
8. Pace, H. and C. Brenner, The nitrilase superfamily: classification, structure and function. Genome Biol, 2001. 2(1): p. reviews0001.1 - reviews0001.9.
9. Brenner, C., Catalysis in the nitrilase superfamily. Curr Opin Struct Biol, 2002. 12(6): p. 775-82.
10. Veitia, R.A., Rosetta Stone proteins: "chance and necessity"? Genome Biol, 2002. 3(2): p. INTERACTIONS1001.
11. Pace, H.C., et al., Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers. Curr Biol, 2000. 10(15): p. 907-17.
12. Nakai, T., et al., Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases. Structure, 2000. 8(7): p. 729-37.
13. Novo, C., et al., Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity. Biochem J, 2002. 365(Pt 3): p. 731-8.
14. Hung, C.L., et al., Crystal structure of Helicobacter pylori formamidase AmiF reveals a cysteine-glutamate-lysine catalytic triad. J Biol Chem, 2007. 282(16): p. 12220-9.
15. Kimani, S.W., et al., Structure of an aliphatic amidase from Geobacillus pallidus RAPc8. Acta Crystallogr D Biol Crystallogr, 2007. 63(Pt 10): p. 1048-58.
16. Barglow, K.T., et al., Functional Proteomic and Structural Insights into Molecular Recognition in the Nitrilase Family Enzymes. Biochemistry, 2008. 47(51): p. 13514-13523.
17. Lin, C.H., et al., Growth inhibitory effect of the human NIT2 gene and its allelic imbalance in cancers. Febs J, 2007. 274(11): p. 2946-56.
18. Semba, S., et al., Biological functions of mammalian Nit1, the counterpart of the invertebrate NitFhit Rosetta stone protein, a possible tumor suppressor. J Biol Chem, 2006. 281(38): p. 28244-53.
19. Pekarsky, Y., et al., Nitrilase and Fhit homologs are encoded as fusion proteins in Drosophila melanogaster and Caenorhabditis elegans. Proc Natl Acad Sci U S A, 1998. 95(15): p. 8744-9.
20. Sun, J., et al., Nit1 and Fhit tumor suppressor activities are additive. J Cell Biochem, 2009.
21. Zhang, H., et al., Mammalian nitrilase 1 homologue Nit1 is a negative regulator in T cells. Int Immunol, 2009.
22. Ishii, H., et al., Potential cancer therapy with the fragile histidine triad gene: review of the preclinical studies. JAMA, 2001. 286(19): p. 2441-9.
23. 林俊宏, 人類nit基因功能之研究, in 生化暨分子生物研究所. 2006, 國立陽明大學. p. -.
24. 陳文彬, 內生性Nap1的調控與大量表現Nap1對HeLa細胞的影響, in 生化暨分子生物研究所. 2006, 國立陽明大學. p. -.
25. Banerjee, A., R. Sharma, and U.C. Banerjee, The nitrile-degrading enzymes: current status and future prospects. Appl Microbiol Biotechnol, 2002. 60(1-2): p. 33-44.
26. Martinkova, L., V. Vejvoda, and V. Kren, Selection and screening for enzymes of nitrile metabolism. J Biotechnol, 2008. 133(3): p. 318-26.
27. Foerstner, K.U., et al., A nitrile hydratase in the eukaryote Monosiga brevicollis. PLoS ONE, 2008. 3(12): p. e3976.
28. Bugg, T., Introduction to enzyme and coenzyme chemistry. 2nd ed. 2004, Oxford, UK ; Malden, MA, USA: Blackwell Pub. x, 292 p.
29. Fournand, D., F. Bigey, and A. Arnaud, Acyl transfer activity of an amidase from Rhodococcus sp. strain R312: formation of a wide range of hydroxamic acids. Appl Environ Microbiol, 1998. 64(8): p. 2844-52.
30. Bisswanger, H., Enzyme kinetics : principles and methods. 2nd, rev. and updated ed. 2008, Weinheim: Wiley-VCH. xviii, 301 p.
31. Meister, A., Preparation of enzymatic reactions of the keto analogues of asparagine and glutamine. J Biol Chem, 1953. 200(2): p. 571-89.
32. Cooper, A.J.L., et al., alpha-Keto acid omega-amidase from rat liver, in Methods in Enzymology. 1985, Academic Press. p. 350-358.
33. Krasnikov, B.F., et al., Assay and purification of omega-amidase/Nit2, a ubiquitously expressed putative tumor suppressor, that catalyzes the deamidation of the alpha-keto acid analogues of glutamine and asparagine. Anal Biochem, 2009.
34. Meister, A., et al., Hydrolysis and transfer reactions catalyzed by omega-amidase preparations. J Biol Chem, 1955. 215(1): p. 441-60.
35. Cooper, A.J., Asparagine transaminase from rat liver. J Biol Chem, 1977. 252(6): p. 2032-8.
36. Cooper, A.J., S.P. Raps, and A. Meister, Fluorometric determination of alpha-ketosuccinamic acid in rat tissues. Anal Biochem, 1987. 167(2): p. 312-20.
37. Hersh, L.B., Rat liver omega-amidase. Purification and properties. Biochemistry, 1971. 10(15): p. 2884-91.
38. Calderon, J., E. Morett, and J. Mora, Omega-amidase pathway in the degradation of glutamine in Neurospora crassa. J Bacteriol, 1985. 161(2): p. 807-9.
39. Cooper, A.J., et al., UBIQUITOUSLY EXPRESSED TOUSLY EXPRESSED ω-AMIDASE IS A POTENTIAL NEUROPROTECTANT IDENTICAL TO TUMOR SUPPRESSOR NIT2. J Neurochem, 2009. 108(s1): p. 182.
40. Vergara, F., T.E. Duffy, and F. Plum, Alpha-ketoglutaramate, a neurotoxic agent in hepatic coma. Trans Assoc Am Physicians, 1973. 86: p. 255-63.
41. Cooper, A.J., The role of glutamine transaminase K (GTK) in sulfur and alpha-keto acid metabolism in the brain, and in the possible bioactivation of neurotoxicants. Neurochem Int, 2004. 44(8): p. 557-77.
42. Tang, B., et al., The methionine salvage pathway compound 4-methylthio-2-oxobutanate causes apoptosis independent of down-regulation of ornithine decarboxylase. Biochem Pharmacol, 2006. 72(7): p. 806-15.
43. Wierstra, I., Sp1: emerging roles--beyond constitutive activation of TATA-less housekeeping genes. Biochem Biophys Res Commun, 2008. 372(1): p. 1-13.
44. Tan, N.Y. and L.M. Khachigian, Sp1 phosphorylation and its regulation of gene transcription. Mol Cell Biol, 2009. 29(10): p. 2483-8.
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