臺灣博碩士論文加值系統

PRMT1最早是藉由與哺乳類TIS21早期立即蛋白交互作用而被選殖出來，為哺乳類動物細胞中最主要的精胺酸甲基轉移酶，其提供了大於90%以上的精胺酸甲基化活性。為了了解PRMT1的酵素作用能力，我們以GST融合的PRMT1針對hnRNP A2此甲基化受質，在不同條件下進行甲基化反應，並以SDS-PAGE及fluorography分析。我們發現，二價陽離子的存在會影響PRMT1酵素催化能力，其中又以鈣離子有最顯著的影響，而單價陽離子則影響較小。
藉由與TIS21/BTG1 基因產物或是interferon receptor的結合，PRMT1的活性會受到調控，而此兩類蛋白質被認為在調控細胞身生長上扮演重要角色，因此我們在RAT1此大鼠纖維母細胞中篩選出過度表現PRMT1的穩定群落，並選取其中一群落65-3，透過計算生長曲線以及BrdU嵌合分析其生長特性，發現其與RAT1細胞在生長上並未有顯著差別。同時，我們針對其細胞粹出物進行活體外甲基化實驗，以測試過度表現PRMT1的酵素活性，結果顯示群落65-3與RAT1細胞中PRMT1的酵素活性並未有明顯差異。

PRMT1 (protein-arginine N-methyltransferase 1) is the predominant arginine methyltransferase in mammalian cells that contributes > 90% of protein-arginine methylated activity. PRMT1 was first cloned because of its ability to interact with mammalian TIS21 immediate-early protein. To understand the enzymatic activity of PRMT1, we used hnRNP A2 as a substrate for GST-PRMT1. We showed that bivalent cations affected the methyltransferase activity of PRMT1. That display distinct methylated degrees in the presences of different cations and concentrations and we found that Ca2+ has conspicuous influence. Relative, univalent cation has slightly effect.
PRMT1 activity appears to be positively regulated through its association with two proteins, the TIS21/BTG1 gene product and the interferon receptor, each of which plays an important role in cell growth regulation. We characterized the growth property of RAT1 cells overexpressing HA-PRMT1 by growth curve counting and BrdU incorporation analysis, and demonstrated that there is no difference between RAT1 and RAT1 overexpressing HA-PRMT1 cells in growth patterns. In addition, in vitro methylation of Trx-hnRNP A2, using cell lysates from RAT1 or RAT1 overexpressing HA-PRMT1, showed that there was no apparent difference in methyltransferases activity.

目 錄 ………………………………………………………………………………Ⅰ
縮 寫 表 ………………………………………………………………………………Ⅱ
圖次目錄 ………………………………………………………………………………Ⅳ
中文摘要 ………………………………………………………………………………1
英文摘要 ………………………………………………………………………………2
緒 論 ………………………………………………………………………………3
研究目標 ………………………………………………………………………………10
實驗材料 ………………………………………………………………………………11
實驗方法 ………………………………………………………………………………19
實驗結果 ………………………………………………………………………………33
討 論 ………………………………………………………………………………43
參考文獻 ………………………………………………………………………………49
圖 ………………………………………………………………………………56
附 圖 ………………………………………………………………………………75

1.Clarke, S. (1993). Protein methylation. Current Opinion in Cell Biology, 5: 977-983.
2.Shapiro, M. J., Chakrabarti, I., and Koshland, D. E., Jr. (1995). Contributions made by individual methylation sites of the Escherichia coli aspartate receptor to chemotactic behavior. Proceedings of the National Academ of Sciences of the United States of America, 92: 1053-1056.
3.Alon, U., Surette, M. G., Barkai, N., and Leibler, S. (1999). Robustness in bacterial chemotaxis. Nature, 397: 168-171.
4.Turowski, P., Fernandez, A., Favre, B., Lamb, N. J., and Hemmings, B. A. (1995). Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression. Journal of Cell Biology, 129: 397-410.
5.McFadden, P. N., Clarke, S. (1982). Methylation at D-aspartyl residues in erythrocytes: possible step in the repair of aged membrane proteins. Proceedings of the National Academ of Sciences of the United States of America, 79: 2460-2464.
6.Ashby, M. N. (1998). CaaX converting enzymes. Current Opinion in Lipidology, 9: 99-102.
7.Zhang, F. L., and Casey, P. J. (1996). Protein prenylation: molecular mechanisms and functional consequences. AnnualReview of Biochemistry, 65: 241-269.
8.Okamoto, Y., and Yount RG. (1985). Identification of an active site peptide of skeletal myosin after photoaffinity labeling with N-(4-azido-2-nitrophenyl)-2-
aminoethyl diphosphate. Proceedings of the National Academy of Sciences of the United States of America, 82: 1575-1679.
9.Swanson, R. V., and Glazer, A. N. (1990). Phycobiliprotein methylation. Effect of the gamma-N-methylasparagine residue on energy transfer in phycocyanin and the phycobilisome. Journal of Molecular Biology, 214: 787-796.
10.Solomon, L. R., and Rubenstein, P. A. (1987). Studies on the role of actin's N tau-methylhistidine using oligodeoxynucleotide-directed site-specific mutagenesis. Journal of Biological Chemistry, 262: 11382-11388.
11.Ghosh, S. K., Paik, W. K., and Kim, S. (1988). Purification and molecular
identification of two protein methylases I from calf brain. Myelin basic protein- and histone-specific enzyme. Journal of Biological Chemistry, 263: 19024-19033.
12.Najbauer, J., Johnson, B. A., Young, A. L., and Asward, D. W. (1993). Peptides with sequences similar to glycine arginine rich motifs in proteins interacting with RNA are efficiently recognized by methyltransferases modifying arginine in numerous proteins. Journal of Biological Chemistry, 268: 10501-10509.
13.Rajpurohit, R., Lee, S. O., Park, W. K., and Kim, S. (1994a). Enzymatic methylation of recombinant heterogeneous nuclear RNP protein A1. Dual substrate specificity for S-adenosylmethionine: histone-arginine N-methyltransferase. Journal of Biological Chemistry, 269: 1075-1082.
14.Liu, Q., and Dreyfuss, G. (1995). In vivo and in vitro arginine methylation of RNA-binding proteins. Molecular and Cellular Biology, 15: 2800-2808.
15.Gary, J. D., and Clarke, S. (1998). RNA and protein interaction modulated by protein arginine methylation. Progress in Nucleic Acid Research and Molecular Biology, 61: 65-131.
16.Lin, W. J., Gary, J. D., Yang, M. C., Clarke, S., and Herschman, H.R. (1996). The mammalian immediate-early TIS21 protein and the Leukemia-associated BTG1 protein interact with a protein-arginine N- methyltransferase. Journal of Biological Chemistry, 271: 15034- 15044.
17.Tang, J., Frsnkel, A., Cook, R. J., Kim, S., Paik, W. K., Williams, K. R., Clarke, S., and Herschman, H. R. (2000). PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells. Journal of Biological Chemistry, 275: 7723-7730.
18.Varnum, B. C., Reddy. S. T., Koski, R. A., and Herschman, H. R. (1994). Synthesis, degradation, and subcellular localization of proteins encoded by the primary response genes TIS7/PC4 and TIS21/PC3. Journal of Cellular Physiology, 158: 205-213.
19.Fletcher, B. S., Lim, R. W., Vamum, B. C., Kujubu, D. A., Koski, R. A., and Herschman, H. R. (1991). Structure and expression of TIS21, a primary resonse gene induced by growth factors and tumor promoters. The Journal of Biological Chemistry, 266: 14511-14518.
20.Montagnoli, A., Guardavaccaro, D., Starace, G., and Tirond, F. (1996). Overexpression of the nerve growth factor-inducible pC3 immediate-early gene is associated with growth inhibition. Cell Growth and Differentiation, 7: 1327-1336.
21.Dreyfuss, G., Matunis, M. J., Pinol-Roma, S., and Burd, C. G. (1993). hnRNP proteins and the biogenesis of mRNA. Annual Review of Cell Biology, 62: 289-321.
22.Soulard, M., Della, V. V., Siomi, M. C., Pinol-Roma, S., Codogno, P., Bauvy, C., Bellini, M., Lacroix, J. C., Monod, G., and Dreyfuss, G. (1993). hnRNP G:sequence and characterization of a glycosylated RNA-binding protein. Nucleic Acids Research, 21: 4210-4217.
23.Cobianchi, F., Calvio, C., Stoppini, M., Buvoli, M., and Riva, S. (1993). Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity. Nucleic Acids Research, 21: 949-955.
24.Boffa, L. C., Karn, J., Vidali, G., and Allfrey, V. G. (1997). Distribution of NG, NG,-dimrthylarginine in nuclear protein fractions. Biochemical and Biophysical Research Communications, 74: 969-976.
25.Rajpurohit, R., Paik, W. K., and Kim, S. (1992). Enzymatic methylation of heterogeneous nuclear ribonucleoprotein in isolated liver nuclei. Biochimica et Biophysica Acta, 1122: 183-188.
26.Kim, S. Merrill, B. M., Rajpurohit, R., Kumar, A., Stone, K. L., Papov, V. V., Schneiders, J. M., Szer, W., Wilson, S. H., Paik, W. K., and Williams, K. R. (1997). Identification of N(G)-methylarginine residues in human heterogeneous RNP protein A1: Phe/Gly-Gly-Gly-Arg-Gly-Gly-Gly/Phe is a preferred recognition motif. Biochemistry, 36: 5185-5192.
27.Rajpurohit, R., Paik, W. K., and Kim, S. (1994). Effect of enzymic methylation of heterogeneous ribonucleoprotein particle A1 on its nucleic-acid binding and controlled proteolysis. Biochemical Journal, 304: 903-909.
28.Siomi, H., and Dreyfuss, G. (1995). A nuclear localization domain in the hnRNP A1 protein. Journal of Cell Biology, 129: 551-560.
29.Michael, W. M., Choi, M., and Deryfuss, G. (1995). A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell, 83: 415-422.
30.Deryfuss, G., Matunis, M. J., Pinol-Roma, S., and Burd, C. G. (1993). hnRNP protein and the biogenesis of mRNA. Anuual Review of Biochemistry, 62: 289-321.
31.Nichols, R. C., Wang, X. W., Tang, J., Hamilton, B. J., High, F. A., Herschman, H. R., and Rigby, F. C. (2000). The RGG domain in hnRNP A2 affects subcellular localization. Experimental Cell Research, 256: 522-532.
32.Tockman, M. S., Erozan, Y. S., Gupta, P., Piantadosi, S., Mulshine, J. L., and Ruckdeschel, J. C. (1994). The early detection of second primary lung cancers by sputum immunosaining. Chest, 106: 385-390.
33.Abramovich, C., Yakobson, B., Chebath, J., and Revel, M. (1997). A protein- arginine methyltransferase binds to the intrcaytoplasmic domain of the IFNAR1 chain in the type I interferon receptor. EMBO Journal, 16: 260-266.
34.Mowen, K. A., Tang, J., Zhu, W., Schurter, B. T., Shuai, K., Herschman, H. R., and David, M. (2001). Arginine methylation of STAT1 modulates IFNα/β-induced transcription. Cell, 104: 731-741.
35.Tang, J., Kao, P. N., and Herschman, H. R. (2000). Protein-arginine methyltransferase 1, the predominant protein-arginine methyltransferase in cells, interacts with and is regulated by interleukin enhancer-binding factor 3. Journal of Biological Chemistry, 275: 19866-19876.
36.Paik, W. K., and Kim, S. (1967). Enzymatic methylation of protein fractions from
calf thymus nuclei. Biochemical and Biophysical Research Communications, 29: 14-20.
37.Rawal N., Rajpurohit, R., Paik, W. K., and Kim, S. (1994). Purification and characterization of S-adenosylmethionine-protein-arginine N-methyltransferase from rat liver. Biochemical Journal, 300: 483-489.
38.Tang, J., Gary, J. D., Clarke, S., and Herschman, H. R. (1998). PRMT 3, a type I protein arginine N-methyltransferase that differs from PRMT1 in its oligomerization, subcellular localization, substrate specificity, and regulation. Journal of Biological Chemistry, 273(27): 16935-16945.
39.Najbauer, J., Johnson, B. A., and Aswad, D. W. (1993). Analysis of protein methylation in cultured cells. Archives of Biochemistry and Biophysics, 293(1): 85-92.
40.Siegel, F. L., and Wright, L. S. (1985). Calmodulin-stimulated protein methylation in rat liver cytosol. Archives of Biochemistry and Biophysics, 237(2): 347-353.
41.Huzoor, A. (1992). Role of protein methylation in agonist-induced signal transduction in human platelets. SAAS Bulletin, Biochemistry and Biotechnology, 5: 7-12.