Please use this identifier to cite or link to this item:
標題: 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
Crystallization and preliminary X-ray crystallographic analysis of the human antizyme inhibitor in complex with antizyme
作者: 李佩螢
Lee, Pei-Ying
關鍵字: polyamine;鳥胺酸脫羧酶抗酶抗酶抑制因子;ornithine decarboxylase;antizyme;antizyme inhibitor
出版社: 生物化學研究所
引用: 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. 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. 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. 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. Coffino, P. (2001a). Antizyme, a mediator of ubiquitin-independent proteasomal degradation. Biochimie 83, 319-323. Coffino, P. (2001b). Regulation of cellular polyamines by antizyme. Nat Rev Mol Cell Biol 2, 188-194. 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. Derewenda, Z. S. (2004). Rational protein crystallization by mutational surface engineering. Structure 12, 529-535. Dong, A., Xu, X., Edwards, A. M., Chang, C., Chruszcz, M., Cuff, M., Cymborowski, M., Di Leo, R., Egorova, O., Evdokimova, E., et al. (2007). In situ proteolysis for protein crystallization and structure determination. Nat Methods 4, 1019-1021. Feith, D. J., Shantz, L. M., and Pegg, A. E. (2001). Targeted antizyme expression in the skin of transgenic mice reduces tumor promoter induction of ornithine decarboxylase and decreases sensitivity to chemical carcinogenesis. Cancer Res 61, 6073-6081. Fujita, K., Murakami, Y., and Hayashi, S. (1982). A macromolecular inhibitor of the antizyme to ornithine decarboxylase. Biochem J 204, 647-652. Gandre, S., Bercovich, Z., and Kahana, C. (2002). Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity. Eur J Biochem 269, 1316-1322. Gerner, E. W., and Meyskens, F. L., Jr. (2004). Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer 4, 781-792. Gosavi, R. A., Mueser, T. C., and Schall, C. A. (2008). Optimization of buffer solutions for protein crystallization. Acta Crystallogr D Biol Crystallogr 64, 506-514. Hayashi, S., and Murakami, Y. (1995). Rapid and regulated degradation of ornithine decarboxylase. Biochem J 306 (Pt 1), 1-10. 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. Heras, B., and Martin, J. L. (2005). Post-crystallization treatments for improving diffraction quality of protein crystals. Acta Crystallogr D Biol Crystallogr 61, 1173-1180. 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. 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 U S A 97, 4808-4813. Iwata, S., Sato, Y., Asada, M., Takagi, M., Tsujimoto, A., Inaba, T., Yamada, T., Sakamoto, S., Yata, J., Shimogori, T., et al. (1999). Anti-tumor activity of antizyme which targets the ornithine decarboxylase (ODC) required for cell growth and transformation. Oncogene 18, 165-172. 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. Kim, S. W., Mangold, U., Waghorne, C., Mobascher, A., Shantz, L., Banyard, J., and Zetter, B. R. (2006). Regulation of cell proliferation by the antizyme inhibitor: evidence for an antizyme-independent mechanism. J Cell Sci 119, 2583-2591. 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. Kitani, T., and Fujisawa, H. (1989). Purification and characterization of antizyme inhibitor of ornithine decarboxylase from rat liver. Biochim Biophys Acta 991, 44-49. 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. 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. 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. Lim, S. K., and Gopalan, G. (2007). Antizyme1 mediates AURKAIP1-dependent degradation of Aurora-A. Oncogene 26, 6593-6603. Lin, Y., Martin, J., Gruendler, C., Farley, J., Meng, X., Li, B. Y., Lechleider, R., Huff, C., Kim, R. H., Grasser, W. A., et al. (2002). A novel link between the proteasome pathway and the signal transduction pathway of the bone morphogenetic proteins (BMPs). BMC Cell Biol 3, 15. Liu, G. Y., Liao, Y. F., Hsu, P. C., Chang, W. H., Hsieh, M. C., Lin, C. Y., Hour, T. C., Kao, M. C., Tsay, G. J., and Hung, H. C. (2006). Antizyme, a natural ornithine decarboxylase inhibitor, induces apoptosis of haematopoietic cells through mitochondrial membrane depolarization and caspases'' cascade. Apoptosis 11, 1773-1788. Lopez-Contreras, A. J., Lopez-Garcia, C., Jimenez-Cervantes, C., Cremades, A., and Penafiel, R. (2006). Mouse ornithine decarboxylase-like gene encodes an antizyme inhibitor devoid of ornithine and arginine decarboxylating activity. J Biol Chem 281, 30896-30906. Lopez-Contreras, A. J., Ramos-Molina, B., Cremades, A., and Penafiel, R. (2008). Antizyme inhibitor 2 (AZIN2/ODCp) stimulates polyamine uptake in mammalian cells. J Biol Chem 283, 20761-20769. Mangold, U. (2006). Antizyme inhibitor: mysterious modulator of cell proliferation. Cell Mol Life Sci 63, 2095-2101. Mangold, U., Hayakawa, H., Coughlin, M., Munger, K., and Zetter, B. R. (2008). Antizyme, a mediator of ubiquitin-independent proteasomal degradation and its inhibitor localize to centrosomes and modulate centriole amplification. Oncogene 27, 604-613. Mangold, U., and Leberer, E. (2005). Regulation of all members of the antizyme family by antizyme inhibitor. Biochem J 385, 21-28. 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. McCann, P. P., and Pegg, A. E. (1992). Ornithine decarboxylase as an enzyme target for therapy. Pharmacol Ther 54, 195-215. 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. Miyazaki, Y., Matsufuji, S., and Hayashi, S. (1992). Cloning and characterization of a rat gene encoding ornithine decarboxylase antizyme. Gene 113, 191-197. 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. 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. 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. Pegg, A. E. (2006). Regulation of ornithine decarboxylase. J Biol Chem 281, 14529-14532. Pegg, A. E., Shantz, L. M., and Coleman, C. S. (1994). Ornithine decarboxylase: structure, function and translational regulation. Biochem Soc Trans 22, 846-852. Pitkanen, L. T., Heiskala, M., and Andersson, L. C. (2001). Expression of a novel human ornithine decarboxylase-like protein in the central nervous system and testes. Biochem Biophys Res Commun 287, 1051-1057. 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. 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. Wallace, H. M., Fraser, A. V., and Hughes, A. (2003). A perspective of polyamine metabolism. Biochem J 376, 1-14. Zhang, J., Wang, Y., Zhou, Y., Cao, Z., Huang, P., and Lu, B. (2005). Yeast two-hybrid screens imply that GGNBP1, GGNBP2 and OAZ3 are potential interaction partners of testicular germ cell-specific protein GGN1. FEBS Lett 579, 559-566. 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.
多元胺是一類結構中具有多個胺基的脂肪族小分子,其於細胞生長、分化以及凋亡皆扮演重要的角色。細胞中多元胺濃度的變化藉由控制鳥胺酸脫羧酶(ornithine decarboxylase, ODC)的蛋白含量而達成,以穩定細胞內的多元胺水平。當細胞內多胺濃度升高時,抗酶(antizyme, AZ)會經由mRNA之轉譯調控而被合成,能特異性地與ODC單體結合,並藉由與泛素無關的途徑(ubiquitin- independent pathway)使ODC被26S蛋白酶體辨認並降解,以抑制多元胺的合成。此外,抗酶還會與胞膜上特定的運輸蛋白結合,降低細胞對多元胺的攝取,反之,當多元胺濃度偏低時,細胞會以抗酶抑制因子(antizyme inhibitor, AZI)來拮抗AZ的功能,為多元胺合成的正調控蛋白,其序列及分子量和ODC相似,但不具有酵素活性。AZI與AZ之間的親和性極高,因而能夠經由競爭釋放與AZ結合的ODC,並恢復其酵素活性。與ODC不同的是,AZI是經由泛素參與之途徑被26S蛋白酶體降解,與AZ結合後,可抑制其與泛素連接酶(Ubiquitin E3 ligase)之間的交互作用,增加其穩定度。最近研究指出,AZ與AZI之間的拮抗作用,除了可穩定細胞內多元胺的水平外,還可調節中心粒的複製,對細胞的分裂反應極為重要。
本論文的主要目的在於解析AZI與AZ形成之蛋白複合體的晶體結構,希望藉此結構進一步了解其兩者之交互作用,並解釋AZ如何避免AZI與泛素連接酶的結合。目前已在數種結晶條件下得到hAZ-hAZI蛋白複合體的晶體。然而,由於這些晶體內部的排列並非十分規則,因而其X-ray繞射訊號的解析度僅達7Å,初步分析為Hexagonal晶系,空間群為P3,晶胞參數 a = b = 163.11 Å, c = 149.69 Å,α= β= 90°, γ =120°。雖然曾試圖以添加物試驗、改變蛋白溶液試驗、crystal annealing、In situ proteolysis等方法提升晶體繞射度,惟目前尚未成功。未來將繼續嘗試其他可能改善晶體品質的方法。

Polyamines are aliphatic cations which play critical roles during cell proliferation, cell death and differentiation. Intracellular polyamine levels are controlled by regulating the protein level of ornithine decoboxylase (ODC), the first enzyme in the polyamine biosynthesis pathway. Antizyme (AZ) is a negative regulator of cellular ODC level and polyamine transport. The elevation of cellular polyamine concentration stimulates a programmed +1 frameshift during translation of the AZ messenger RNA (mRNA), and allows the expression of functional full-length AZ. AZ binds and inactivates ODC by forming AZ-ODC heterodimer, and promotes an ubiquitin-independent degradation by the 26S proteasome, which consequently reduces polyamine synthesis. Mammalian cells contain another relevant regulatory protein termed antizyme inhibitor (AZI), which is homolous to ODC but lacks decarboxylating activity. AZI has a higher affinity for AZ than does ODC, therefore ODC may released from AZ in the presence of AZI and restores the ODC activity. While AZ promotes protolytic destruction of ODC, the degradation of AZI is ubiquitin-dependent and does not require interaction with AZ. Moreover, AZ binding actually stabilizes AZI by inhibiting its ubiquitination. Besides controlling cellular polyamines homeostasis, it has been shown that AZI may regulate the replication of centriole and participate in cell division.
To understand how AZ interacts with AZI and to explain how AZ inhibits the poly-ubiqitination of AZI, this study is aimed to determine the structure of AZ-AZI complex. A number of crystallization conditions have been indentified from which crystals of AZ-AZI complex can be obtained. However, these crystals only diffracted X-ray to ~ 7Å resolution. Preliminary analysis indicated that AZ-AZI crystals belongs to space group P3, with unit-cell parameters a = b = 163.11 Å, c = 149.69 Å, α= β= 90°, γ =120°. Additive screen、crystal annealing、optimal buffer searching and in situ proteolysis had been performed to improve crystal quality. However, a useful crystallization condition for AZ-AZI complex remains to be identified.
其他識別: U0005-1002200915120400
Appears in Collections:生物化學研究所

Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.