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研究生:陳玫孝
研究生(外文):Mei-Hsiao Chen
論文名稱:利用以活性探針為基礎的蛋白質體學方法偵測神經細胞分化過程中蛋白質磷酸水解酶的變化
論文名稱(外文):Profiling Protein Phosphatases Activities of Neuronal Differentiated Cells using Mechanism-Based Activity Probes
指導教授:林敬哲林敬哲引用關係
指導教授(外文):Jing-Jer Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:58
中文關鍵詞:蛋白質體學蛋白質磷酸水解酶神經細胞分化
外文關鍵詞:proteomicsactivity probesLCL-2neuronal differentiationprotein phosphatasesquinone methide intermediate
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  • 被引用被引用:1
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隨著人類基因體計畫的完成,蛋白質體學,proteomics的時代來臨,鑑別基因產物的功能為首要之務。基因序列傳統蛋白質體學,二維膠體電泳和質譜儀分析在於偵測蛋白質的表現含量。受膠體解析度的影響,表現量少的蛋白,是測不到的。且靜態的表現量不等於細胞內蛋白的動態活性改變。針對這樣的缺點,我們設計合成activity probes,以活性為基礎的探針來標示蛋白,非以蛋白質的富含量為考量。活性探針為酵素的催化受質,進行水解時會啟動補抓的機制,致使活性探針以共價鍵結標示於酵素蛋白質上。我們設計並合成一活性探針,LCL-2來偵測蛋白質磷酸水解酶的活性。利用純化的重組蛋白PTP1B當作一個模版,我們可以看到LCL-2的標示反應在30℃時,幾乎在四分鐘內就全部完成了,此外,其可測得的最少量PTP1B大約是10 ng。因為磷酸水解酶抑制劑Na3VO4可以抑制反應的發生,ATPase inhibitor, AMP-PNP卻不能。因此,我們的實驗結果顯示LCL-2對蛋白質磷酸水解酶的標示反應是具專一性的。接著,以N18 neuroblastoma cell line and PC12 pheochromcytoma cell line作為研究神經細胞分化的實驗模版,將probes應用於測定神經分化過程的蛋白質磷酸水解酶。我們在N18 and PC12分化系統鑑定到幾個LCL-2-enriched和分化有關的蛋白質。然而鑑定到的蛋白質多屬豐富性蛋白。可能的原因是探針活化後的quinone methide intermediate造成的非專一性結合。針對此現象,我們也設計了semi-solid phase probe,期望減少與非專一性蛋白結合的機會而可以在複雜的蛋白質群體中分離到PTPs。
As the completion of human genome project, biological research is entering a new era in which experimental focus shift to identify the function of gene products. Conventional proteomic analysis combining two-dimensional gel electrophoresis and mass spectrometry detects protein expression level of a proteome. However steady state protein level cannot represent the dynamic change of cells. To overcome this apparent drawback of proteomic analysis, we designed and synthesized activity probes to label their target proteins based on the activity rather than abundance. Activity probes are substrates of enzymes that are capable of turning on the tapping mechanism during the action of enzymatic hydrolysis, leading to covalent modification of the enzyme. We have designed and synthesized an activity probe, LCL-2, that could detect the phosphatase activity within a proteome. Using purified recombinant PTP1B as a model, we showed that the kinetics and sensitivity of LCL-2 toward phosphatases. The labeling is almost complete within 4 minutes at 30℃. We could detect PTP1B with as few as 10 ng of proteins. We also showed that the labeling is specific for phosphatases as the labeling reaction was inhibited by phosphatase inhibitor Na3VO4, but not by ATPase inhibitor, AMP-PNP. LCL-2 was applied to detect protein phosphatases during neuronal differentiation using N18 neuroblastoma cell line and PC12 pheochromocytoma cell line as the model system for our analysis. Several LCL-2 and differentiation dependent protein bands in both N18 and PC12 differentiation systems were detected. However, most of the identified proteins were abundant protein. This may resulted from the nonspecific binding of quinone methide intermediate. To overcome this problem, we also designed a second generation probe, the semi-solid phase probe. This probe should eliminate the problem of LCL-2 and enable the profiling of PTPs in a proteome.
1. Adam, G.C., Cravatt, B.F., and Sorensen, E.J. (2001). Profiling the specific reactivity of the proteome with non-directed activity-based probes. Chem. Biol. 8, 81-95.
2. Alonso, A., Sasin, J., Bottini, N., Friedberg, I., Friedberg, I., Osterman,A., Godzik,A., Hunter,T., Dixon,J., and Mustelin,T. (2004). Protein tyrosine phosphatases in the human genome. Cell 117, 699-711.
3. Amano, T., Richelson, E., and Nirenberg, M. (1972). Neurotransmitter synthesis by neuroblastoma clones (neuroblast differentiation-cell culture-choline acetyltransferase-acetylcholinesterase-tyrosine hydroxylase-axons-dendrites). Proc. Natl. Acad. Sci. U. S. A 69, 258-263.
4. Banker, G. and Goslin, K. (1988). Developments in neuronal cell culture. Nature 336, 185-186.
5. Bogyo, M., Verhelst, S., Bellingard-Dubouchaud, V., Toba, S., and Greenbaum, D. (2000). Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs. Chem. Biol. 7, 27-38.
6. Campbell, D.A. and Szardenings, A.K. (2003). Functional profiling of the proteome with affinity labels. Curr. Opin. Chem. Biol. 7, 296-303.
7. Cox, R.P., Gilbert, P., Jr., and Griffin, M.J. (1967). Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase. Biochem. J. 105, 155-161.
8. Denu, J.M., Stuckey, J.A., Saper, M.A., and Dixon, J.E. (1996). Form and function in protein dephosphorylation. Cell 87, 361-364.
9. Desai, C.J., Gindhart, J.G., Jr., Goldstein, L.S., and Zinn, K. (1996). Receptor tyrosine phosphatases are required for motor axon guidance in the Drosophila embryo. Cell 84, 599-609.
10. Desai, C.J., Sun, Q., and Zinn, K. (1997). Tyrosine phosphorylation and axon guidance: of mice and flies. Curr. Opin. Neurobiol. 7, 70-74.
11. Fenn, J.B., Mann, M., Meng, C.K., Wong, S.F., and Whitehouse, C.M. (1989). Electrospray ionization for mass spectrometry of large biomolecules. Science 246, 64-71.
12. Figeys, D. (2003). Proteomics in 2002: a year of technical development and wide-ranging applications. Anal. Chem. 75, 2891-2905.
13. Gharahdaghi, F., Weinberg, C.R., Meagher, D.A., Imai, B.S., and Mische, S.M. (1999). Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 20, 601-605.
14. Gordon, J.A. (1991). Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol. 201, 477-482.
15. Greenbaum, D., Medzihradszky, K.F., Burlingame, A., and Bogyo, M. (2000). Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem. Biol. 7, 569-581.
16. Greenbaum, D.C., Baruch, A., Grainger, M., Bozdech, Z., Medzihradszky, K.F., Engel, J., DeRisi, J., Holder, A.A., and Bogyo, M. (2002). A role for the protease falcipain 1 in host cell invasion by the human malaria parasite. Science 298, 2002-2006.
17. Greene, L.A., Farenelli, S.E., Cunningham, M.E., and Park, D.S. (1998). Culture and experimental use of the PC12 rat pheochromocytoma cell line. In Culturing Nerve Cells, 2nd Edition, G.Banker and K.Goslin, ed. (MIT Press, Cambridge, MA: pp. 161-187.
18. Greene, L.A. and Tischler, A.S. (1976). Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. U. S. A 73, 2424-2428.
19. Guan, K.L. and Dixon, J.E. (1991). Evidence for protein-tyrosine-phosphatase catalysis proceeding via a cysteine-phosphate intermediate. J. Biol. Chem. 266, 17026-17030.
20. Gygi, S.P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H., and Aebersold, R. (1999). Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994-999.
21. Han, D.K., Eng, J., Zhou, H., and Aebersold, R. (2001). Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat. Biotechnol. 19, 946-951.
22. Hunter, T. (2000). Signaling--2000 and beyond. Cell 100, 113-127.
23. Karas, M. and Hillenkamp, F. (1988). Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 60, 2299-2301.
24. Kidd, D., Liu, Y., and Cravatt, B.F. (2001). Profiling serine hydrolase activities in complex proteomes. Biochemistry 40, 4005-4015.
25. Kozarich, J.W. (2003). Activity-based proteomics: enzyme chemistry redux. Curr. Opin. Chem. Biol. 7, 78-83.
26. Krueger, N.X., Van Vactor, D., Wan, H.I., Gelbart, W.M., Goodman, C.S., and Saito, H. (1996). The transmembrane tyrosine phosphatase DLAR controls motor axon guidance in Drosophila. Cell 84, 611-622.
27. Li, L. and Dixon, J.E. (2000). Form, function, and regulation of protein tyrosine phosphatases and their involvement in human diseases. Semin. Immunol. 12, 75-84.
28. Liu, Y., Patricelli, M.P., and Cravatt, B.F. (1999). Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. U. S. A 96, 14694-14699.
29. Lo, L.C., Pang, T.L., Kuo, C.H., Chiang, Y.L., Wang, H.Y., and Lin, J.J. (2002). Design and synthesis of class-selective activity probes for protein tyrosine phosphatases. J. Proteome. Res. 1, 35-40.
30. Longo, F.M., Martignetti, J.A., Le Beau, J.M., Zhang,J.S., Barnes,J.P., and Brosius,J. (1993). Leukocyte common antigen-related receptor-linked tyrosine phosphatase. Regulation of mRNA expression. J. Biol. Chem. 268, 26503-26511.
31. Mann, M., Hojrup, P., and Roepstorff, P. (1993). Use of mass spectrometric molecular weight information to identify proteins in sequence databases. Biol. Mass Spectrom. 22, 338-345.
32. Mullen, R.J., Buck, C.R., and Smith, A.M. (1992). NeuN, a neuronal specific nuclear protein in vertebrates. Development 116, 201-211.
33. Myers, J.K. and Widlanski, T.S. (1993). Mechanism-based inactivation of prostatic acid phosphatase. Science 262, 1451-1453.
34. Rogers, M.V., Buensuceso, C., Montague, F., and Mahadevan, L. (1994). Vanadate stimulates differentiation and neurite outgrowth in rat pheochromocytoma PC12 cells and neurite extension in human neuroblastoma SH-SY5Y cells. Neuroscience 60, 479-494.
35. Speers, A.E. and Cravatt, B.F. (2004a). Chemical strategies for activity-based proteomics. Chembiochem. 5, 41-47.
36. Speers, A.E. and Cravatt, B.F. (2004b). Profiling enzyme activities in vivo using click chemistry methods. Chem. Biol. 11, 535-546.
37. Stoker, A.W. (2001). Receptor tyrosine phosphatases in axon growth and guidance. Curr. Opin. Neurobiol. 11, 95-102.
38. Tisi, M.A., Xie, Y., Yeo, T.T., and Longo, F.M. (2000). Downregulation of LAR tyrosine phosphatase prevents apoptosis and augments NGF-induced neurite outgrowth. J. Neurobiol. 42, 477-486.
39. Tonks, N.K. and Neel, B.G. (1996). From form to function: signaling by protein tyrosine phosphatases. Cell 87, 365-368.
40. Tonks, N.K. and Neel, B.G. (2001). Combinatorial control of the specificity of protein tyrosine phosphatases. Curr. Opin. Cell Biol. 13, 182-195.
41. Tsuneizumi, K., Kume, T., Watanabe, T., Gebbink, M.F., Thomas, M.L., and Oishi, M. (1994). Induction of specific protein tyrosine phosphatase transcripts during differentiation of mouse embryonal carcinoma (F9) cells. FEBS Lett. 347, 9-12.
42. Wright, J.H., Drueckes, P., Bartoe, J., Zhao, Z., Shen, S.H., and Krebs, E.G. (1997). A role for the SHP-2 tyrosine phosphatase in nerve growth-induced PC12 cell differentiation. Mol. Biol. Cell 8, 1575-1585.
43. Yan, J.X., Wait, R., Berkelman, T., Harry, R.A., Westbrook, J.A., Wheeler, C.H., and Dunn, M.J. (2000). A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21, 3666-3672.
44. Zhang, Z.Y. (2003). Chemical and mechanistic approaches to the study of protein tyrosine phosphatases. Acc. Chem. Res. 36, 385-392.
45. Zolnierowicz, S. and Bollen, M. (2000). Protein phosphorylation and protein phosphatases. De Panne, Belgium, September 19-24, 1999. EMBO J. 19, 483-488.
46. 江盈霖.(2003) 以催化機制為基礎的活性探針純化蛋白質磷酸水解酶 國立陽明大學生物藥學研究所碩士論文
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