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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 詹迺立(Nei-Li Chan) | |
dc.contributor.author | Min-Hao Wu | en |
dc.contributor.author | 吳旻昊 | zh_TW |
dc.date.accessioned | 2021-06-16T05:08:24Z | - |
dc.date.available | 2016-10-09 | |
dc.date.copyright | 2014-10-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-19 | |
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(2008) Structural elements of the ubiquitin-independent proteasome degron of ornithine decarboxylase. Biochem. J. 410, 401-407 25. Ghoda, L., Phillips, M. A., Bass, K. E., Wang, C. C., and Coffino, P. (1990) Trypanosome ornithine decarboxylase is stable because it lacks sequences found in the carboxyl terminus of the mouse enzyme which target the latter for intracellular degradation. J. Biol. Chem. 265, 11823-11826 26. Murakami, Y., Ichiba, T., Matsufuji, S., and Hayashi, S. (1996) Cloning of antizyme inhibitor, a highly homologous protein to ornithine decarboxylase. J. Biol. Chem. 271, 3340-3342 27. Albeck, S., Dym, O., Unger, T., Snapir, Z., Bercovich, Z., and Kahana, C. (2008) Crystallographic and biochemical studies revealing the structural basis for antizyme inhibitor function. Protein Sci. 17, 793-802 28. Nilsson, J., Grahn, B., and Heby, O. (2000) Antizyme inhibitor is rapidly induced in growth-stimulated mouse fibroblasts and releases ornithine decarboxylase from antizyme suppression. Biochem. J. 346 Pt 3, 699-704 29. Bercovich, Z., and Kahana, C. (2004) Degradation of antizyme inhibitor, an ornithine decarboxylase homologous protein, is ubiquitin-dependent and is inhibited by antizyme. J. Biol. Chem. 279, 54097-54102 30. Heller, J. S., Fong, W. F., and Canellakis, E. S. (1976) Induction of a protein inhibitor to ornithine decarboxylase by the end products of its reaction. Proc. Natl. Acad. Sci. USA 73, 1858-1862 31. Ichiba, T., Matsufuji, S., Miyazaki, Y., Murakami, Y., Tanaka, K., Ichihara, A., and Hayashi, S. (1994) Functional regions of ornithine decarboxylase antizyme. Biochem. Biophys. Res. Commun. 200, 1721-1727 32. Li, X., and Coffino, P. (1994) Distinct domains of antizyme required for binding and proteolysis of ornithine decarboxylase. Mol. Cell. Biol. 14, 87-92 33. Mamroud-Kidron, E., Omer-Itsicovich, M., Bercovich, Z., Tobias, K. E., Rom, E., and Kahana, C. (1994) A unified pathway for the degradation of ornithine decarboxylase in reticulocyte lysate requires interaction with the polyamine-induced protein, ornithine decarboxylase antizyme. Eur. J. Biochem. 226, 547-554 34. Hoffman, D. W., Carroll, D., Martinez, N., and Hackert, M. L. (2005) Solution structure of a conserved domain of antizyme: a protein regulator of polyamines. Biochemistry 44, 11777-11785 35. Mangold, U. (2005) The antizyme family: polyamines and beyond. IUBMB life 57, 671-676 36. Mangold, U., and Leberer, E. (2005) Regulation of all members of the antizyme family by antizyme inhibitor. Biochem. J. 385, 21-28 37. Hoshino, K., Momiyama, E., Yoshida, K., Nishimura, K., Sakai, S., Toida, T., Kashiwagi, K., and Igarashi, K. (2005) Polyamine transport by mammalian cells and mitochondria: role of antizyme and glycosaminoglycans. J. Biol. Chem. 280, 42801-42808 38. Murai, N., Murakami, Y., and Matsufuji, S. (2003) Identification of nuclear export signals in antizyme-1. J. Biol. Chem. 278, 44791-44798 39. Gritli-Linde, A., Nilsson, J., Bohlooly, Y. M., Heby, O., and Linde, A. (2001) Nuclear translocation of antizyme and expression of ornithine decarboxylase and antizyme are developmentally regulated. Dev. Dyn. 220, 259-275 40. Schipper, R. G., Cuijpers, V. M., De Groot, L. H., Thio, M., and Verhofstad, A. A. (2004) Intracellular localization of ornithine decarboxylase and its regulatory protein, antizyme-1. J. Histochem. Cytochem. 52, 1259-1266 41. Ivanov, I. P., Gesteland, R. F., and Atkins, J. F. (1998) A second mammalian antizyme: conservation of programmed ribosomal frameshifting. Genomics 52, 119-129 42. Murai, N., Shimizu, A., Murakami, Y., and Matsufuji, S. (2009) Subcellular localization and phosphorylation of antizyme 2. J. Cell. Biochem. 108, 1012-1021 43. Zhu, C., Lang, D. W., and Coffino, P. (1999) Antizyme2 is a negative regulator of ornithine decarboxylase and polyamine transport. J. Biol. Chem. 274, 26425-26430 44. Chen, H., MacDonald, A., and Coffino, P. (2002) Structural elements of antizymes 1 and 2 are required for proteasomal degradation of ornithine decarboxylase. J. Biol. Chem. 277, 45957-45961 45. Snapir, Z., Keren-Paz, A., Bercovich, Z., and Kahana, C. (2009) Antizyme 3 inhibits polyamine uptake and ornithine decarboxylase (ODC) activity, but does not stimulate ODC degradation. Biochem. J. 419, 99-103, 101 p following 103 46. Ivanov, I. P., Rohrwasser, A., Terreros, D. A., Gesteland, R. F., and Atkins, J. F. (2000) Discovery of a spermatogenesis stage-specific ornithine decarboxylase antizyme: antizyme 3. Proc. Natl. Acad. Sci. USA 97, 4808-4813 47. Tosaka, Y., Tanaka, H., Yano, Y., Masai, K., Nozaki, M., Yomogida, K., Otani, S., Nojima, H., and Nishimune, Y. (2000) Identification and characterization of testis specific ornithine decarboxylase antizyme (OAZ-t) gene: expression in haploid germ cells and polyamine-induced frameshifting. Genes Cells 5, 265-276 48. Hsieh, J. Y., Yang, J. Y., Lin, C. L., Liu, G. Y., and Hung, H. C. (2011) Minimal antizyme peptide fully functioning in the binding and inhibition of ornithine decarboxylase and antizyme inhibitor. PLoS One 6, e24366 49. Shaw Stewart, P. D., Kolek, S. A., Briggs, R. A., Chayen, N. E., and Baldock, P. F. M. (2011) Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization. Cryst. Growth Des. 11, 3432-3441 50. Murai, N., Murakami, Y., and Matsufuji, S. (2011) Protocols for studying antizyme expression and function. Methods Mol. Biol. 720, 237-267 51. Solano, F., Penafiel, R., Solano, M. E., and Lozano, J. A. (1985) Equilibrium between active and inactive forms of rat liver ornithine decarboxylase mediated by L-ornithine and salts. FEBS Lett. 190, 324-328 52. Koromilas, A. E., and Kyriakidis, D. A. (1987) Reversal of Antizyme-Induced Inhibition of Ornithine Decarboxylase by Cations in Barley Seedlings. Plant Growth Regul. 6, 267-275 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55782 | - |
dc.description.abstract | 多胺(polyamine)是一群帶有正電荷的多價有機小分子,主要包含了腐胺、亞精胺與精胺。由於帶有正電荷的特性,其能夠與帶負電荷的DNA、RNA或是蛋白表面帶負電的區域產生交互作用,進而影響細胞的存活與凋亡。過去的研究也發現過量的多胺與細胞的癌化有關,因此,細胞內的多胺含量必須受到嚴密的調控。
鳥胺酸脫羧酶(ornithine decarboxylase, ODC)是5’-磷酸吡哆醛(pyridoxal 5’-phosphate, PLP)依賴型酵素,可將鳥胺酸(ornithine)催化為腐胺(putrescine),此催化作用不僅是多胺生合成途徑的第一個步驟,更是整個多胺生合成的速率決定步驟與主要的調控點。當細胞內的多胺含量過高時,會促進抗酶蛋白(antizyme, Az)的轉譯作用,Az能與ODC形成異質二聚體(heterodimer),而阻止ODC形成具有催化活性的同質二聚體(homodimer),Az與ODC所形成的複合體會被26S蛋白酶體(26S proteasome)辨認,並在不需泛素(ubiquitin)參與的情況下造成ODC的降解,另外,Az也能阻斷細胞對外來多胺的攝取,因此,Az是多胺生合成的負調控因子。人類的Az有三種亞型,第一亞型(Az1)與第二亞型(Az2)在組織中的分布很相似,但在in vitro的實驗中卻發現僅有Az1具備讓ODC降解的能力,而Az2的功能或許是用來暫時性地抑制ODC的活性。本研究希望藉由比較兩者在與ODC形成複合體的結構差異,從而釐清兩者在功能上的區別。 本研究著重於解析人類ODC-Az2蛋白複合體之晶體結構,以便與實驗室已有的ODC-Az1蛋白複合體結構進行比較。首先,建構一系列Az2重組蛋白與ODC的表達質體,以利兩者於大腸桿菌(Escherichia coli, E. coli)進行共同表現,接著利用固定性金屬離子親和層析法和分子篩層析法進行複合體的純化,最後以蒸氣擴散法進行晶體的培養。目前已獲得相當純度的蛋白複合體,並針對蛋白複合體在層析圖所展現的特性進行初步的分析,但尚未獲得適合晶體生成的條件。未來將試著搭配不同截切長度的Az2與ODC片段進行蛋白質複合體的結晶實驗。 | zh_TW |
dc.description.abstract | Polyamines are polyvalent organic cations found in all eukaryotes and most bacteria. Because of their cationic nature, polyamines can interact with acidic patches on protein surfaces and negatively charged DNA and RNA to alter their functions. Therefore, polyamines are known to regulate cell growth, survival, and proliferation, and abnormal polyamine levels may induce tumorigenesis. To control the cellular levels of polyamines, their biosynthesis is tightly regulated. Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to form putrescine as the first and step in polyamine biosynthesis. When polyamine level is high enough, the translation of antizyme (Az) is stimulated to suppress ODC function and polyamine uptake via the formation of an ODC-Az heterodimer. Moreover, Az-binding allows ODC to be recognized and degraded by the 26S proteasome in an ubiquitin-independent manner. In human, four Az isoforms have been identified to date. Among them, isoform 1 (Az1) and 2 (Az2) exhibit similar tissue distribution. Previous studies demonstrated that only Az1 can efficiently promote ODC degradation in vitro, suggesting that Az2 might act as a reservoir for transient suppression of ODC function. To understand how the ODC-Az1 and ODC-Az2 complexes diverge in function, our laboratory has determined the crystal structure of a truncated Az1 in complex with ODC. The aim of my thesis research is to obtain the crystal structure of the ODC-Az2 complex.
To this end, I first constructed plasmids for expressing different truncations of human Az2 with either N-terminal or C-terminal His tag. Then, ODC and different forms of Az2 were co-expressed in E. coli BL21 (DE3) cells. Immobilized metal affinity chromatography was used to capture the ODC-Az2 complexes. Gel filtration column was used next to remove excess Az2 in the sample. Using vapor diffusion crystallization technique, a preliminary condition for growing crystals of the ODC-Az2 was identified. However, subsequent efforts for reproducing these crystals were not successful. A retrospective examination of the purification profiles revealed the ODC-Az2 complex eluted in two peaks, indicating that crystallization may be hampered by structural heterogeneity of the protein sample. Cross-linking experiments suggested that the complex may undergo fast equilibrium between the dimeirc and tetrameric forms and that pooling protein samples of two peaks for crystallization was feasible. Moreover, a new crystallization strategy termed random microseed matrix seeding (rMMS) was performed to facilitate crystallization by using micro-crystals of the ODC-Az195-228 complex. Although a few micro-crystals were observed in some screening conditions, they likely correspond unbroken crystals of the ODC-Az195-228 complex rather than new crystals of the ODC-Az257-189 complex. Additional crystallization trials will be performed using different constructs of Az2 and ODC. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T05:08:24Z (GMT). No. of bitstreams: 1 ntu-103-R01442012-1.pdf: 5143349 bytes, checksum: 26fa642396818d8e25a371b8da14dd43 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 謝辭 i
中文摘要 ii Abstract iii 目錄 v 圖目錄 viii 表目錄 x 縮寫表 xi 一、前言 1 1.1多胺(Polyamine) 1 1.1.1多胺之生理功能 1 1.1.2多胺之生成 2 1.1.3多胺之降解 3 1.2鳥胺酸脫羧酶(Ornithine decarboxylase, ODC) 3 1.2.1鳥胺酸脫羧酶之結構 3 1.2.2鳥胺酸脫羧酶之催化機制 4 1.2.3鳥胺酸脫羧酶之降解機制 4 1.2.4抗酶抑制因子 5 1.3抗酶(Antizyme, Az) 6 1.3.1抗酶之結構 6 1.3.2抗酶之種類與特性 6 1.4研究目的 8 二、材料與方法 9 2.1表現質體之建構(Construction of expression plasmids) 9 2.1.1已建構之蛋白表現質體 9 2.1.2 pET21b-Az2 9 2.1.3 pET21b-Az257-189 13 2.1.4 pET21d-N-His- Az257-189 13 2.1.5 pET21d-N-His- Az264-189 14 2.1.6 pET21d-N-His- Az275-189 15 2.2蛋白表現量之測試 15 2.2.1 ODC-Az2蛋白複合體之共表達 15 2.2.2 ODC-Az257-189蛋白複合體之共表達 18 2.2.3 ODC1-423-Az2蛋白複合體之共表達 18 2.2.4 ODC1-423-Az257-189蛋白複合體之共表達 18 2.2.5 ODC1-423-Az264-189蛋白複合體之共表達 19 2.3蛋白純化 20 2.3.1 ODC-Az2蛋白複合體之純化 20 2.3.2 ODC-Az257-189蛋白複合體之純化 23 2.3.3 ODC1-423-Az2蛋白複合體之純化 23 2.3.4 ODC1-423-Az257-189蛋白複合體之純化 23 2.3.5 ODC1-423-Az264-189蛋白複合體之純化 23 2.4蛋白之定量 23 2.5蛋白之均質性測定 24 2.6化學交聯法 24 2.7蛋白晶體培養 25 2.7.1預結晶試劑 25 2.7.2蛋白結晶條件篩選 25 2.7.3養晶條件微調 26 2.7.4添加物測驗 26 2.7.5 Seeding 27 2.8蛋白複合體之結構模擬 27 三、結果 28 3.1 ODC-Az2蛋白複合體之結構模擬 28 3.2 Az2質體的建構 28 3.3 ODC-Az2蛋白複合體 29 3.3.1 ODC-Az2蛋白複合體的表現 29 3.3.2 ODC-Az2蛋白複合體的純化 30 3.3.3 ODC-Az2蛋白複合體的晶體培養 31 3.4 ODC-Az257-189蛋白複合體 31 3.4.1 ODC-Az257-189蛋白複合體的表現 31 3.4.2 ODC-Az257-189蛋白複合體的純化 32 3.4.3以化學交聯法分析ODC-Az257-189蛋白複合體的聚合狀態 33 3.4.4 ODC-Az257-189蛋白複合體的晶體培養 34 3.5 ODC1-423-Az2蛋白複合體 34 3.5.1 ODC1-423-Az2蛋白複合體的表現 34 3.5.2 ODC1-423-Az2蛋白複合體的純化 35 3.5.3 ODC1-423-Az2蛋白複合體的晶體培養 36 3.6 ODC1-423-Az257-189蛋白複合體 36 3.6.1 ODC1-423-Az257-189蛋白複合體的表現 36 3.6.2 ODC1-423-Az257-189蛋白複合體的純化 37 3.6.3 ODC1-423-Az257-189蛋白回收液的再純化 37 3.6.4 ODC1-423-Az257-189蛋白複合體的晶體培養 38 3.7 ODC1-423-Az264-189蛋白複合體 38 3.7.1 ODC1-423-Az264-189蛋白複合體的表現 38 3.7.2 ODC1-423-Az264-189蛋白複合體的純化 38 3.7.3 ODC1-423-Az264-189蛋白複合體的晶體培養 39 四、討論 40 圖 46 表 84 參考文獻 91 | |
dc.language.iso | zh-TW | |
dc.title | 人類第二亞型抗酶與鳥胺酸脫羧酶複合體之結構研究 | zh_TW |
dc.title | Structural study of human antizyme isoform 2 in complex with ornithine decarboxylase | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 徐駿森(Chun-Hua Hsu),曾秀如(Shiou-Ru Tzeng) | |
dc.subject.keyword | 鳥胺酸脫羧?,抗?, | zh_TW |
dc.subject.keyword | ornithine decarboxylase (ODC),antizyme (Az), | en |
dc.relation.page | 97 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-19 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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