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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 詹迺立 | |
dc.contributor.author | Fang Chou | en |
dc.contributor.author | 周芳 | zh_TW |
dc.date.accessioned | 2021-05-20T21:59:35Z | - |
dc.date.available | 2012-09-09 | |
dc.date.available | 2021-05-20T21:59:35Z | - |
dc.date.copyright | 2010-09-09 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-07-18 | |
dc.identifier.citation | 1. Agostinelli, E., Marques, M. P., Calheiros, R., Gil, F. P., Tempera, G., Viceconte, N., Battaglia, V., Grancara, S., and Toninello, A. (2010) Polyamines: fundamental characters in chemistry and biology, Amino Acids 38, 393-403.
2. Igarashi, K., and Kashiwagi, K. (2000) Polyamines: mysterious modulators of cellular functions, Biochem Biophys Res Commun 271, 559-564. 3. Williams, K. (1997) Interactions of polyamines with ion channels, Biochem J 325 ( Pt 2), 289-297. 4. Gerner, E. W., and Meyskens, F. L., Jr. (2004) Polyamines and cancer: old molecules, new understanding, Nat Rev Cancer 4, 781-792. 5. Casero, R. A., Jr., and Marton, L. J. (2007) Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases, Nat Rev Drug Discov 6, 373-390. 6. Childs, A. C., Mehta, D. J., and Gerner, E. W. (2003) Polyamine-dependent gene expression, Cell Mol Life Sci 60, 1394-1406. 7. Coffino, P. (2001) Regulation of cellular polyamines by antizyme, Nat Rev Mol Cell Biol 2, 188-194. 8. Wallace, H. M., Fraser, A. V., and Hughes, A. (2003) A perspective of polyamine metabolism, Biochem J 376, 1-14. 9. Wang, C., Delcros, J. G., Cannon, L., Konate, F., Carias, H., Biggerstaff, J., Gardner, R. A., and Phanstiel, I. V. O. t. (2003) Defining the molecular requirements for the selective delivery of polyamine conjugates into cells containing active polyamine transporters, J Med Chem 46, 5129-5138. 10. Quemener, V., Blanchard, Y., Chamaillard, L., Havouis, R., Cipolla, B., and Moulinoux, J. P. (1994) Polyamine deprivation: a new tool in cancer treatment, Anticancer Res 14, 443-448. 11. Almrud, J. J., Oliveira, M. A., Kern, A. D., Grishin, N. V., Phillips, M. A., and Hackert, M. L. (2000) Crystal structure of human ornithine decarboxylase at 2.1 A resolution: structural insights to antizyme binding, J Mol Biol 295, 7-16. 12. 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. 13. Myers, D. P., Jackson, L. K., Ipe, V. G., Murphy, G. E., and Phillips, M. A. (2001) Long-range interactions in the dimer interface of ornithine decarboxylase are important for enzyme function, Biochemistry 40, 13230-13236. 14. Su, K. L., Liao, Y. F., Hung, H. C., and Liu, G. Y. (2009) Critical factors determining dimerization of human antizyme inhibitor, J Biol Chem 284, 26768-26777. 15. Kern, A. D., Oliveira, M. A., Coffino, P., and Hackert, M. L. (1999) Structure of mammalian ornithine decarboxylase at 1.6 A resolution: stereochemical implications of PLP-dependent amino acid decarboxylases, Structure 7, 567-581. 16. Dunathan, H. C. (1966) Conformation and reaction specificity in pyridoxal phosphate enzymes, Proc Natl Acad Sci U S A 55, 712-716. 17. Pegg, A. E. (2006) Regulation of ornithine decarboxylase, J Biol Chem 281, 14529-14532. 18. Jackson, L. K., Brooks, H. B., Osterman, A. L., Goldsmith, E. J., and Phillips, M. A. (2000) Altering the reaction specificity of eukaryotic ornithine decarboxylase, Biochemistry 39, 11247-11257. 19. Hayashi, S., and Murakami, Y. (1995) Rapid and regulated degradation of ornithine decarboxylase, Biochem J 306 ( Pt 1), 1-10. 20. Moshier, J. A., Osborne, D. L., Skunca, M., Dosescu, J., Gilbert, J. D., Fitzgerald, M. C., Polidori, G., Wagner, R. L., Friezner Degen, S. J., Luk, G. D., and et al. (1992) Multiple promoter elements govern expression of the human ornithine decarboxylase gene in colon carcinoma cells, Nucleic Acids Res 20, 2581-2590. 21. O'Brien, T. G., Megosh, L. C., Gilliard, G., and Soler, A. P. (1997) Ornithine decarboxylase overexpression is a sufficient condition for tumor promotion in mouse skin, Cancer Res 57, 2630-2637. 22. Packham, G., and Cleveland, J. L. (1997) Induction of ornithine decarboxylase by IL-3 is mediated by sequential c-Myc-independent and c-Myc-dependent pathways, Oncogene 15, 1219-1232. 23. Yuspa, S. H., Lichti, U., Ben, T., Patterson, E., Hennings, H., Slaga, T. J., Colburn, N., and Kelsey, W. (1976) Phorbol esters stimulate DNA synthesis and ornithine decarboxylase activity in mouse epidermal cell cultures, Nature 262, 402-404. 24. Gilmour, S. K., Verma, A. K., Madara, T., and O'Brien, T. G. (1987) Regulation of ornithine decarboxylase gene expression in mouse epidermis and epidermal tumors during two-stage tumorigenesis, Cancer Res 47, 1221-1225. 25. Auvinen, M., Paasinen, A., Andersson, L. C., and Holtta, E. (1992) Ornithine decarboxylase activity is critical for cell transformation, Nature 360, 355-358. 26. Manni, A., and Wright, C. (1985) Polyamines as mediators of the effect of prolactin and growth hormone on the growth of N-nitroso-N-methylurea-induced rat mammary tumor cultured in vitro in soft agar, J Natl Cancer Inst 74, 941-944. 27. McCann, P. P., and Pegg, A. E. (1992) Ornithine decarboxylase as an enzyme target for therapy, Pharmacol Ther 54, 195-215. 28. Manni, A., and Wright, C. (1984) Polyamines as mediators of estrogen action on the growth of experimental breast cancer in rats, J Natl Cancer Inst 73, 511-514. 29. Wang, C. C. (1995) Molecular mechanisms and therapeutic approaches to the treatment of African trypanosomiasis, Annu Rev Pharmacol Toxicol 35, 93-127. 30. Grishin, N. V., Osterman, A. L., Brooks, H. B., Phillips, M. A., and Goldsmith, E. J. (1999) X-ray structure of ornithine decarboxylase from Trypanosoma brucei: the native structure and the structure in complex with alpha-difluoromethylornithine, Biochemistry 38, 15174-15184. 31. Jariel-Encontre, I., Bossis, G., and Piechaczyk, M. (2008) Ubiquitin-independent degradation of proteins by the proteasome, Biochim Biophys Acta 1786, 153-177. 32. Murakami, Y., Matsufuji, S., Hayashi, S., Tanahashi, N., and Tanaka, K. (2000) Degradation of ornithine decarboxylase by the 26S proteasome, Biochem Biophys Res Commun 267, 1-6. 33. 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. 34. Kahana, C. (2007) Ubiquitin dependent and independent protein degradation in the regulation of cellular polyamines, Amino Acids 33, 225-230. 35. Matsufuji, S., Matsufuji, T., Miyazaki, Y., Murakami, Y., Atkins, J. F., Gesteland, R. F., and Hayashi, S. (1995) Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme, Cell 80, 51-60. 36. Nilsson, J., Koskiniemi, S., Persson, K., Grahn, B., and Holm, I. (1997) Polyamines regulate both transcription and translation of the gene encoding ornithine decarboxylase antizyme in mouse, Eur J Biochem 250, 223-231. 37. Palanimurugan, R., Scheel, H., Hofmann, K., and Dohmen, R. J. (2004) Polyamines regulate their synthesis by inducing expression and blocking degradation of ODC antizyme, EMBO J 23, 4857-4867. 38. Kahana, C. (2009) Antizyme and antizyme inhibitor, a regulatory tango, Cell Mol Life Sci 66, 2479-2488. 39. Ivanov, I. P., and Atkins, J. F. (2007) Ribosomal frameshifting in decoding antizyme mRNAs from yeast and protists to humans: close to 300 cases reveal remarkable diversity despite underlying conservation, Nucleic Acids Res 35, 1842-1858. 40. Kitani, T., and Fujisawa, H. (1984) Purification and some properties of a protein inhibitor (antizyme) of ornithine decarboxylase from rat liver, J Biol Chem 259, 10036-10040. 41. Cohavi, O., Tobi, D., and Schreiber, G. (2009) Docking of antizyme to ornithine decarboxylase and antizyme inhibitor using experimental mutant and double-mutant cycle data, J Mol Biol 390, 503-515. 42. Li, X., and Coffino, P. (1993) Degradation of ornithine decarboxylase: exposure of the C-terminal target by a polyamine-inducible inhibitory protein, Mol Cell Biol 13, 2377-2383. 43. 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. 44. Kahana, C., Asher, G., and Shaul, Y. (2005) Mechanisms of protein degradation: an odyssey with ODC, Cell Cycle 4, 1461-1464. 45. Mangold, U. (2005) The antizyme family: polyamines and beyond, IUBMB Life 57, 671-676. 46. Ghoda, L., Sidney, D., Macrae, M., and Coffino, P. (1992) Structural elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation, Mol Cell Biol 12, 2178-2185. 47. Miyazaki, Y., Matsufuji, S., Murakami, Y., and Hayashi, S. (1993) Single amino-acid replacement is responsible for the stabilization of ornithine decarboxylase in HMOA cells, Eur J Biochem 214, 837-844. 48. Takeuchi, J., Chen, H., Hoyt, M. A., and Coffino, P. (2008) Structural elements of the ubiquitin-independent proteasome degron of ornithine decarboxylase, Biochem J 410, 401-407. 49. Takeuchi, J., Chen, H., and Coffino, P. (2007) Proteasome substrate degradation requires association plus extended peptide, EMBO J 26, 123-131. 50. Loetscher, P., Pratt, G., and Rechsteiner, M. (1991) The C terminus of mouse ornithine decarboxylase confers rapid degradation on dihydrofolate reductase. Support for the pest hypothesis, J Biol Chem 266, 11213-11220. 51. Ghoda, L., van Daalen Wetters, T., Macrae, M., Ascherman, D., and Coffino, P. (1989) Prevention of rapid intracellular degradation of ODC by a carboxyl-terminal truncation, Science 243, 1493-1495. 52. Zhang, M., Pickart, C. M., and Coffino, P. (2003) Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate, EMBO J 22, 1488-1496. 53. Coleman, C. S., Stanley, B. A., Viswanath, R., and Pegg, A. E. (1994) Rapid exchange of subunits of mammalian ornithine decarboxylase, J Biol Chem 269, 3155-3158. 54. 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. 55. Murai, N., Murakami, Y., and Matsufuji, S. (2003) Identification of nuclear export signals in antizyme-1, J Biol Chem 278, 44791-44798. 56. 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. 57. 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. 58. 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 U S A 73, 1858-1862. 59. Iwata, S., Sato, Y., Asada, M., Takagi, M., Tsujimoto, A., Inaba, T., Yamada, T., Sakamoto, S., Yata, J., Shimogori, T., Igarashi, K., and Mizutani, S. (1999) Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation, Oncogene 18, 165-172. 60. Koike, C., Chao, D. T., and Zetter, B. R. (1999) Sensitivity to polyamine-induced growth arrest correlates with antizyme induction in prostate carcinoma cells, Cancer Res 59, 6109-6112. 61. Coffino, P. (2001) Antizyme, a mediator of ubiquitin-independent proteasomal degradation, Biochimie 83, 319-323. 62. Mitchell, J. L., Judd, G. G., Bareyal-Leyser, A., and Ling, S. Y. (1994) Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells, Biochem J 299 ( Pt 1), 19-22. 63. Suzuki, T., He, Y., Kashiwagi, K., Murakami, Y., Hayashi, S., and Igarashi, K. (1994) Antizyme protects against abnormal accumulation and toxicity of polyamines in ornithine decarboxylase-overproducing cells, Proc Natl Acad Sci U S A 91, 8930-8934. 64. 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. 65. 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. 66. 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. 67. Mangold, U., and Leberer, E. (2005) Regulation of all members of the antizyme family by antizyme inhibitor, Biochem J 385, 21-28. 68. 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. 69. Li, X., and Coffino, P. (1994) Distinct domains of antizyme required for binding and proteolysis of ornithine decarboxylase, Mol Cell Biol 14, 87-92. 70. Li, X., Stebbins, B., Hoffman, L., Pratt, G., Rechsteiner, M., and Coffino, P. (1996) The N terminus of antizyme promotes degradation of heterologous proteins, J Biol Chem 271, 4441-4446. 71. Li, X., and Coffino, P. (1992) Regulated degradation of ornithine decarboxylase requires interaction with the polyamine-inducible protein antizyme, Mol Cell Biol 12, 3556-3562. 72. 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. 73. Newman, R. M., Mobascher, A., Mangold, U., Koike, C., Diah, S., Schmidt, M., Finley, D., and Zetter, B. R. (2004) Antizyme targets cyclin D1 for degradation. A novel mechanism for cell growth repression, J Biol Chem 279, 41504-41511. 74. Dulloo, I., Gopalan, G., Melino, G., and Sabapathy, K. (2010) The antiapoptotic DeltaNp73 is degraded in a c-Jun-dependent manner upon genotoxic stress through the antizyme-mediated pathway, Proc Natl Acad Sci U S A 107, 4902-4907. 75. Lim, S. K., and Gopalan, G. (2007) Aurora-A kinase interacting protein 1 (AURKAIP1) promotes Aurora-A degradation through an alternative ubiquitin-independent pathway, Biochem J 403, 119-127. 76. McFerrin, M. B., and Snell, E. H. (2002) The development and application of a method to quantify the quality of cryoprotectant solutions using standard area-detector X-ray images, J Appl Crystallogr 35, 538-545. 77. Heras, B., and Martin, J. L. (2005) Post-crystallization treatments for improving diffraction quality of protein crystals, Acta Crystallogr D 61, 1173-1180. 78. Hanson, B. L., Schall, C. A., and Bunick, G. J. (2003) New techniques in macromolecular cryocrystallography: macromolecular crystal annealing and cryogenic helium, J Struct Biol 142, 77-87. 79. 李佩螢 (2009). 人類抗酶抑制因子與抗酶複合體之結晶與初步X-ray單晶繞射分析 (國立中興大學生物化學研究所) 80. 林婉婷 (2009). 人類鳥胺酸脫羧酶與抗酶複合體之純化與晶體培養 (國立中興大學生物化學研究所) | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10796 | - |
dc.description.abstract | 多元胺 (polyamines) 為結構中帶有多個胺基的脂肪族小分子,包含腐胺 (putrescine) 、亞精胺 (spermidine) 和精胺 (spermine),此類帶正電的物質可和帶負電的DNA、RNA或蛋白質產生交互作用,參與細胞生長、分化與凋亡的過程。
人類鳥胺酸脫羧酶 (Ornithine decarboxylase, ODC;EC 4.1.1.17) 為依賴5’-磷酸吡哆醛 (pyridoxal 5’-phosphate, PLP) 輔酶之酵素,負責催化鳥胺酸 (ornithine) 之脫羧反應得到腐胺,此為胞內多元胺合成途徑之第一與速率決定的步驟,亦是多元胺合成的主要調控點。ODC結構包含兩個domains,N端為TIM-like α/β-barrel domain;C端為β-sheet domain。兩個ODC單體 (monomer) 以頭尾相連 (head-to-tail) 的方式組成雙聚體 (homodimer),其活性中心之胺基酸Lys-69會以Schiff base方式和輔酶PLP結合,形成具有催化活性的酵素。 胞內多元胺的含量會直接影響多元胺的合成,當細胞內多元胺含量過高時,會使抗酶 (antizyme;AZ) 的mRNA藉由轉譯調控合成具有功能的全長AZ蛋白。AZ為ODC之負回饋調控因子,可和ODC單體結合形成異雙聚體 (heterodimer)。AZ與ODC結合後不但會使ODC失去酵素活性,還會使ODC C端發生構型變化,露出可被26S 蛋白酶體 (proteasome) 辨認的降解訊號,進行不依賴泛素的降解路徑 (ubiquitin-independent degradation pathway),進而抑制多元胺的合成。除了上述的負調控機制外,細胞尚可藉由抗酶抑制因子 (Antizyme inhibitor, AZI) 正向調控多元胺的合成,AZI與ODC具有序列與結構上的同源性,能與AZ形成更穩定的複合體而釋放出ODC,使胞內ODC雙體的含量上升。 本篇論文的主要目的在於解析ODC-AZ蛋白複合體的晶體結構,以深入探討ODC與AZ交互作用的方式,進而解釋AZ如何促使ODC進行不依賴泛素的蛋白降解機制。我們已經成功置備ODC與多種AZ刪除突變形成之複合體,並順利得到複合體的晶體。目前最好的繞射數據其解析度約為3.2 Å,初步分析顯示此晶體屬於Primitive tetragonal 晶系,空間群為P41212,晶胞參數為 a=b=266.64 Å,c=52.37 Å,α=β=γ=90˚。未來希望能得到品質更高的晶體,以利後續結構的解析。 | zh_TW |
dc.description.abstract | Polyamines, such as putrescine, spermidine and spermine, are abundant multivalent organic cations. These compounds can interact with negatively charged molecules like DNA and RNA to participate in many cellular processes, including chromatin condensation, maintenance of DNA structure, RNA processing, translation and protein activation. Polyamines are essential for normal cell growth and apoptosis, they also play crucial roles during cell differentiation and the development of many cancers.
Ornithine decarboxylase (ODC) is involved in the first and rate-limiting step of the polyamine biosynthesis pathway, catalyzing the decarboxylation of ornithine to produce putrescine, which is subsequently converted into spermindine and then to spermine. Human ODC is a 53 kDa pyridoxal 5’-phosphate (PLP)-dependent enzyme consists of 461 amino acids. The ODC monomer consists of two domains: an N-terminal TIM-like α/β-barrel domain and a C-terminal β-sheet domain. The active form of ODC exists as a head-to-tail homodimer, and the active site Lys69 binds the PLP cofactor via a Schiff-base linkage. The cellular ODC level is tightly regulated by polyamine concentration via an antizyme (AZ) dependent protein degradation pathway. Accumulation of polyamines promotes translational frameshifting of AZ mRNA, allowing expression of the full-length 25 kDa AZ protein. AZ binds to and inactivates ODC by forming a tight non-covalent 1:1 complex. The ODC-AZ heterodimer formation induces conformation changes in the ODC C-terminal region (residues 425-461), which triggers its association and degradation by the 26S proteasome in an ubiquitin-independent fashion. Residues 117-140 of ODC are critical for its association with AZ. In addition to the AZ-mediated negative regulation, the intracellular polyamine homeostasis is also regulated by antizyme inhibitor (AZI), an enzymatically inactive ODC homolog. Because AZI binds to AZ with high affinity, ODC can be released from ODC-AZ complex in the presence of AZI, leading to the restoration of ODC activity. This study is aimed to understand how AZ recognizes ODC and how AZ binding promotes proteasomal degradation of ODC by determining the crystal structure of ODC-AZ complex. Using vapor-diffusion crystallization technique, we have successfully obtained crystals of many different forms of AZ truncation mutants in complex with both the full-length and a mutant ODC (ODCΔ299-310). A diffraction data set has been collected to 3.2 Å resolution. Preliminary diffraction analysis indicated that ODC-AZ complex belongs to space group P41212, with unit-cell parameters a=b=266.64 Å, c=52.37 Å, α=β=γ=90˚. We will continue to optimize the crystallization as well as the cryo-protection conditions to facilitate structural determination. | en |
dc.description.provenance | Made available in DSpace on 2021-05-20T21:59:35Z (GMT). No. of bitstreams: 1 ntu-99-R97442017-1.pdf: 7818665 bytes, checksum: 8041141ea224d63105c7aaf87207f6ea (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 口試委員會審定書............................................................................................................i
中文摘要...........................................................................................................................ii 英文摘要..........................................................................................................................iii 目錄................................................................................................................................v 圖目錄.............................................................................................................................ix 表目錄............................................................................................................................xii 縮寫表............................................................................................................................xiii 一、 前言............ ...........................................................................................................................1 1-1 多元胺在生理上的重要性 ................................................................................................ 1 1-2 多元胺代謝路徑 ................................................................................................................ 2 1-3 鳥胺酸脫羧酶 (ornithine decarboxylase, ODC) .............................................................. 2 1-3-1 人類鳥胺酸脫羧酶的結構 ........................................................................................ 2 1-3-2 鳥胺酸脫羧酶之催化機制 ........................................................................................ 3 1-3-3 鳥胺酸脫羧酶與疾病 ................................................................................................ 4 1-3-4 鳥胺酸脫羧酶之降解機制 ........................................................................................ 5 1-4 抗酶抑制因子 (Antizyme inhibitor, AZI) .......................................................................... 7 1-5 抗酶 (Antizyme, AZ) .......................................................................................................... 8 1-5-1 抗酶之功能 ................................................................................................................. 8 1-5-2 抗酶之結構 ................................................................................................................. 8 1-6 鳥胺酸脫羧酶與抗酶結合區域之預測............................................................................. 9 1-7 研究目的 .......................................................................................................................... 10 二、 材料與方法................................................................................................................. ........11 2-1 蛋白質表現質體之構築 .................................................................................................. 11 2-1-1 pET28a-ODC、pET28a-ODCΔ299-310、pET21b-AZ110-228、pET21b-AZ119-228與pQE30-AZ95-228. ....................................................................................................................11 2-1-2 pET15b-AZ119-228 ....................................................................................................... 11 2-1-3 pET21d-N-His -AZ119-228 ........................................................................................... 14 2-2 蛋白表現量的測試 .......................................................................................................... 15 2-2-1 ODCΔ299-310-AZ110-228蛋白複合體之共表達 ............................................................. 15 2-2-2 ODCΔ299-310-AZ119-228(I)與ODC-AZ119-228(I)蛋白複合體之共表達 ......................... 17 2-2-3 ODCΔ299-310-AZ119-228(II)與ODC-AZ119-228(II)蛋白複合體之共表達.................. .....17 2-2-4 ODCΔ299-310-AZ95-228與ODC-AZ95-228蛋白複合體之共表達....................................18 2-3 蛋白純化 ............................................................................................................................18 2-3-1 ODCΔ299-310-AZ110-228 蛋白複合體的純化 ..................................................................18 2-3-2 ODCΔ299-310-AZ119-228(I)與ODC-AZ119-228(I)蛋白複合體的純化 ..............................22 2-3-3 O DCΔ299-310-AZ119-228(II)與ODC-AZ119-228(II)蛋白複合體的純化 ..........................23 2-3-4 ODCΔ299-310-AZ95-228與ODC-AZ95-228蛋白複合體的純化........................................23 2-4蛋白質濃縮與定量................ ........................................................................................... 24 2-5蛋白質均質性測定 ........................................................................................................... 24 2-6蛋白晶體培養 ................................................................................................................... 25 2-6-1 PCT (Pre-Crystallization Test) .................................................................................. 25 2-6-2 晶體生長條件測試 ................................................................................................... 25 2-6-3 微調養晶條件 ........................................................................................................... 26 2-6-4 添加物試驗 ............................................................................................................... 26 2-6-5 Seeding ...................................................................................................................... 27 2-7 蛋白質晶體之X-ray繞射數據的分析與收集.................................................................27 2-7-1 蛋白質晶體冷凍保護 (cryo-protection) ................................................................... 27 2-7-2 單晶繞射實驗 ........................................................................................................... 28 三、 結果 ................................................................................................................................... 30 3-1 構築pET15b-AZ119-228與pET21d-N-His -AZ119-228質體 ............................................... 30 3-2 ODCΔ299-310-AZ110-228蛋白複合體 .................................................................................... 30 3-2-1 ODCΔ299-310-AZ110-228蛋白複合體的表現 ................................................................. 30 3-2-2 ODCΔ299-310-AZ110-228 蛋白複合體的純化 ................................................................. 30 3-2-3 ODCΔ299-310-AZ110-228蛋白複合體的晶體培養與繞射數據分析 ............................. 31 3-3 ODCΔ299-310-AZ119-228(I)蛋白複合體 ................................................................................. 32 3-3-1 ODCΔ299-310-AZ119-228(I)蛋白複合體的表現 .............................................................. 32 3-3-2 ODCΔ299-310-AZ119-228(I)蛋白複合體的純化 .............................................................. 32 3-3-3 ODCΔ299-310- AZ119-228(I)蛋白複合體的晶體培養 ..................................................... 33 3-3-4 ODCΔ299-310- AZ119-228(I)蛋白複合體的數據收集 ..................................................... 34 3-4 ODC-AZ119-228(I)蛋白複合體 ........................................................................................... 34 3-4-1 ODC-AZ119-228(I)蛋白複合體的表現 ........................................................................ 34 3-4-2 ODC-AZ119-228(I)蛋白複合體的純化 ........................................................................ 35 3-4-3 ODC-AZ119-228(I) 蛋白複合體的晶體培養 ............................................................... 35 3-4-4 ODC-AZ119-228(I)蛋白複合體的數據收集與初步結構解析 .................................... 36 3-4-5 含Se-Methionine標定ODC-AZ119-228(I)蛋白複合體的晶體培養 ......................... 37 3-5 ODCΔ299-310-AZ119-228(II)蛋白複合體 ............................................................................... 37 3-5-1 ODCΔ299-310-AZ119-228(II)蛋白複合體的表現 ............................................................ 37 3-5-2 ODCΔ299-310-AZ119-228(II)蛋白複合體的純化 ............................................................ 38 3-5-3 ODCΔ299-310- AZ119-228(II)蛋白複合體的晶體培養 ................................................... 38 3-6 ODC-AZ119-228(II)蛋白複合體 .......................................................................................... 39 3-6-1 ODC-AZ119-228(II)蛋白複合體的表現 ...................................................................... 39 3-6-2 ODC-AZ119-228(II) 蛋白複合體的純化 ..................................................................... 39 3-6-3 ODC-AZ119-228(II)蛋白複合體的晶體培養與繞射數據分析 .................................. 40 3-6-4 含Se-Methionine標定ODC-AZ119-228(II)蛋白複合體的晶體培養 ........................ 40 3-7 ODCΔ299-310-AZ95-228蛋白複合體...................................................................................... 41 3-7-1 ODCΔ299-310-AZ95-228蛋白複合體的表現 .................................................................. 41 3-7-2 ODCΔ299-310-AZ95-228蛋白複合體的純化 .................................................................. 41 3-7-3 ODCΔ299-310- AZ95-228蛋白複合體的晶體培養 ......................................................... 42 3-8 ODC-AZ95-228蛋白複合體 ................................................................................................ 42 3-8-1 ODC-AZ95-228蛋白複合體的表現 ............................................................................ 42 3-8-2 ODC-AZ95-228蛋白複合體的純化 ............................................................................ 43 3-8-3 ODCΔ299-310- AZ95-228蛋白複合體的晶體培養 ......................................................... 43 3-8-4 含Se-Methionine標定ODC-AZ95-228蛋白複合體的晶體培養 .............................. 44 四、 討論 ................................................................................................................................... 45 4-1 ODCΔ299-310-AZ110-228蛋白複合體 .................................................................................... 45 4-2 ODCΔ299-310-AZ119-228(I)蛋白複合體與ODC-AZ119-228(I)蛋白複合體 ............................ 45 4-3 ODCΔ299-310-AZ119-228(II)蛋白複合體與ODC-AZ119-228(II)蛋白複合體 .......................... 47 4-4 ODCΔ299-310-AZ95-228蛋白複合體與ODC-AZ95-228蛋白複合體 ...................................... 48 圖....................................................................................................................................49 表..................................................................................................................................117 參考文獻.......................................................................................................................124 附錄...............................................................................................................................131 | |
dc.language.iso | zh-TW | |
dc.title | 人類鳥胺酸脫羧酶與抗酶複合體之結構研究 | zh_TW |
dc.title | Structural study of human ornithine decarboxylase in complex with antizyme | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 洪慧芝,徐駿森 | |
dc.subject.keyword | 鳥胺酸脫羧酶,抗酶, | zh_TW |
dc.subject.keyword | ornithine decarboxylase,antizyme, | en |
dc.relation.page | 132 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2010-07-19 | |
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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