Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/16670
標題: 新抑癌素蛋白的苯丙胺酸78殘基突變影響生色團釋放的再探討
Further Study of the effect of Phe78 Mutation on Chromophore Release From Neocarzinostatin Chromoprotein Complex
作者: 盧志明
Lu, Zhi-Ming
關鍵字: neocarzinostatin;新抑癌素;aponeocarzinostatin;Apo-NCS;NCS;mutation;殘基突變;新抑癌素蛋白
出版社: 化學系所
引用: 1. Ishida, N., Miyazaki, K., Rikimaru, M., “Neocarzinostatin, an antibiotic of high molecular weight.” J. Antibiotics. 1965, 18, 8-76 2. Koide, Y., Ishii, F., Hasuda, K., Koyama, Y., Edo, Katamine, S., Kitame, F., & Ishida, N. “Isolation of a non-protein component from neocarzinostatin (NCS) and their biological activities.” J. Antibiotics. 1980, 33 (3), 342-346. 3. Napier, M. A., Holmquist, B., Strydom, D. J., & Goldberg, I. H. “Neocarzinostatin chromophore: purification of the major active form and characterization of its spectral and biological properties.” Biochemistry. 1981, 20, 5602-5608. 4. Edo, K., Mizugaki, M., Koide, Y., Seto, H., Furihata, K., Oake, N. & Ishida, N. “The struture of neocarzinostatin chromophore possessing a novel bicyclo-[7,3,0]dodecadiyne system.” Tetrahedron Letters. 1985, 26, 331-334. 5. Kappen, L. S., Napier, M. A., Goldberg, I. H., “Roles of chromophore and apo-protein in neocarzinostatin action.” Proc. Natl. Acad. Sci. 1980, 77, 1970-1974. 6. 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H., “Neocarzinostatin chromophore binds to deoxyribonucleic acid by intercalation.” Biochemistry, 1981, 14, 4007-4014. 26. Hensens, O. D., Dewey R. S., Liesch, J. M., Napier, M. A., Reamer R. A., Smith J. L., Schönberg, G. A., Goldberg I. H., “Neocarzinostatin chromophore: presence of a highly strained ether ring and its reaction with mercaptan and sodium borohydride.” Biochem. Biophys. Res. Commun. 1983, 113, 538-547 27. Napier, M. A., Holmquist, B., Strydom, D. J., & Goldberg, I. H. “Neocarzinostatin chromophore: purification of the major active form and characterization of its spectral and biological properties.” Biochemistry. 1981, 20, 5602-5608. 28. Albers-Schonberg, G., Dewey, R. S., Hensens O. D., Liesch, J. M., Napier, M. A., Goldberg, I. H., “Neocarzinostatin: chemical characterization and partial structure of the non-protein chromophore.” Biochem. Biophys. Res. Coummun. 1980, 95, 1351-1356. 29. Schönberg, G. A., Dewey, R. S., Hensens, O.D., Liesch, J. M., Napier, M. 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Tanaka, T., Hirama, M., Fujita, K.-I., Imajo, S., and Ishiguro, M., “Solution structure of the antitumor antibiotic neocarzinostatin, a chromophore-protein complex” (1993) J. Chem. Soc., Chem. Commun., 1993, 15: 1205-1207. 35. Goldberg, I. H., “Mechanism of neocarzinostatin action: Role of DNA microstructure in determination of chemistry of bistranded oxidative damage.” Acc. Chem. Res. 1991, 2, 191-198. 36. Andrew G. Myers, Michael E. Kort, and Marlys Hammond, “A Comparison of DNA Cleavage by Neocarzinostatin Chromophore and Its Aglycon: Evaluating the Role of the Carbohydrate Residue” J. Am. Chem. Soc. 1997, 119(13), 2965-2972 37. Myers, A. G., “Proposed structure of the neocarzinostatin chromophore-methyl thioglycolate adduct; a mechanism for the nucleophilic activation of neocarzinostatin.” Tetrahedron Letters. 1987, 28 (39), 4493-4496. 38. Lee, S. H., Goldberg, I. H., “Role of Epoxide in Neocarzinostatin Chromophore Stability and Action”, Mol Pharmacol, 1988, 33, 396-401. 39. 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C., Jiang, Z.-W., Goldberg, I. H., “Neocarzinostatin-Mediated DNA Damage in a Model AGT˙ACT Site: Mechanistic Studies of Thiol-Sensitive Partitioning of C4' DNA Damage Products” Biochemistry 1992 31, 1917-1927. 48. Kikuchi, M., Shoji, M., Ishida, N., “Pre-neocarzinostatin, a specific antagonist of neocarzinostatin.” J. Antibiot. 1974, 27(10), 766-74 49. Maeda, H., Kuromizu, K., “Spontaneous deamidation of a protein antibiotic, neocarzinostatin, at weakly acidic pH. Conversion to a homologous inactive preneocarzinostatin due to change of asparagine 83 to aspartic acid 83 accompanied by conformational and biological alterations.” J. Biochem. 1977, 81(1), 25-35 50. Meienhofer, J., Maeda, H., Glaser, C. B., Czombos, J., & Kuromizu, K. “Primary structure of neocarzinostatin, an antitumor protein.” Science. 1972, 178, 875-876. 51. Maeda, H., Glaser, C. B., Czombos, J., & Meienhofer, J. “Structure of the antitumor protein neocarzinostatin.” Arch. Biochem. Biophys. 1974, 164, 369-378. 52. 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摘要: 
新抑癌素(Neocarzinostatin)是1960年代於日本發現的一種抗腫瘤抗生素藥物。新抑癌素主要能分成兩個部份;一是具有高度細胞毒性、不穩定的小分子藥物新抑癌素生色團。二是負責保護生色團,具有高度穩定性、大分子量的蛋白質載體新抑癌素蛋白。在溶液中,新抑癌素蛋白以非共價鍵的形式與生色團緊密結合(Kd=0.1 nM),能夠保護生色團不被環境所分解。但當進行細胞毒殺的時候,新抑癌素蛋白必須釋放生色團,讓生色團抵達並嵌入細胞DNA鏈的小凹槽(minor groove),被硫醇激活後奪取DNA磷糖骨架的氫,造成細胞DNA鏈斷裂,藉此抑制細胞的生長甚至殺死細胞。令科學家感到好奇的是,新抑癌素究竟是利用哪種機制,調控生色團在適當的時間與地點釋放?蛋白與生色團結合的作用力,以及蛋白釋放生色團時的作用機制,至今仍有許多值得我們深入研究與探討的問題。
由文獻與實驗室學長姐的實驗結果分析,除了新抑癌素蛋白疏水性穴口內部對生色團的作用力之外,我們已知位於穴口外部的78號苯丙胺酸殘基對生色團的釋放扮演著關鍵性的角色。藉由含有新抑癌素蛋白基因序列的質體載體,我們以聚合酶鏈鎖反應為基礎的技術,我們突變對應78號殘基的密碼子,表現並純化得到78號苯丙胺酸殘基被置換的新抑癌素突變蛋白。在得到一系列置換為不同殘基的新抑癌素突變蛋白後。我們利用圓二色光譜測量,與核磁共振分析氮十五標記蛋白等技術,證實新抑癌素突變蛋白的二級、三級和疏水性穴口的構形幾乎沒有改變,排除穴口構形改變所可能造成的影響。我們藉由當生色團由蛋白中被釋放出來,可被溶液中的硫醇激活環化生成產物一,導致螢光強度明顯的變強,利用螢光強度變化的特性,我們偵測並計算新抑癌素突變蛋白釋放生色團的速率。藉由釋放速率的改變,我們推測新抑癌素78號位置的苯丙胺酸與生色團之間的作用力。
由實驗結果得知,帶有芳香環結構的殘基支鏈似乎能夠穩定蛋白與生色團之間的結合。非極性的脂肪族殘基隨著支鏈長度的增加,生色團的釋放速率也會有變快的傾向,而78號殘基支鏈形狀對生色團作用力也有影響。帶有正電荷的殘基突變能於不影響新抑癌素蛋白整體的結構的情況下嚴重的影響新抑癌素蛋白與生色團的結合。新抑癌素蛋白是已知的良好藥物載體,如何調控藥物的釋放是臨床上重要的課題,我們的研究結果應該能在這方面提供有用的資訊。

Neocarzinostatin is a potent antitumer antibiotic chromoprotein, consisting of a labile cytotoxic chromophore that is non-covalently associated with a protective carrier protein, aponeocarzinostatin. The release of chromophore from the chromoprotein has been demonstrated to be a key step in DNA cleavage on the course of its cytotoxic activity. We previously reported that F78 residue on the opening of the binding cleft of aponeocarzinostatin plays an important role in gating the chromophore release. To further investigate the releasing mechanism, here we continue our mutation study at the position of F78 residue. Various mutants bearing different side-chains in place of F78 in aponeocarzinostatin have been made. The results obtained from circular dichroism and nuclear magnetic resonance spectroscopic analyses indicate that the native protein conformation is conserved in these mutants. After chromophore reconstitution, kinetic studies of chromophore release were performed on each mutant by monitoring the time course of fluorescence. Comparison of release rate among neocarzinostatin mutants suggests that mutants bearing aromatic side-chains at position 78 are able to sustain similar release rate as that of wild-type. While in circumstances aliphatic residues are in place of F78, the release rate increases corresponding approximately to the length of aliphatic side-chains. Meanwhile, shape and hydrophobicity of the aliphatic side-chain also appear to influence the release rate. In addition, mutants bearing basic amino acid residues in place of F78 lead release rate to increase significantly, even though theses mutants maintain the same architecture as the one wild-type has. The above results may provide potentially useful information for improving the drug delivery system in neocarzinostatin.
URI: http://hdl.handle.net/11455/16670
其他識別: U0005-1508200814370300
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