Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22959
標題: 影響人類與錐蟲的鳥胺酸脫羧酶上對抗酶結合力差異的重要因子
Essential Factors Determining the Differential AZ-binding Affinity of Human and Trypanosome Ornithine Decarboxylase
作者: 許登華
Hsu, Den-Hua
關鍵字: ornithine decarboxylase
鳥胺酸脫羧酶抗酶錐蟲
antizyme
trypanosome
出版社: 生命科學系所
引用: 1. Almrud, J. J., M. A. Oliveira, A. D. Kern, N. V. Grishin, M. A. Phillips, and M. L. Hackert. 2000. Crystal structure of human ornithine decarboxylase at 2.1 A resolution: structural insights to antizyme binding. J Mol Biol 295:7-16. 2. Bacchi, C. J., H. C. Nathan, S. H. Hutner, P. P. McCann, and A. Sjoerdsma. 1980. Polyamine metabolism: a potential therapeutic target in trypanosomes. Science 210:332-4. 3. Coffino, P. 2001. Regulation of cellular polyamines by antizyme. Nat Rev Mol Cell Biol 2:188-94. 4. Coleman, C. S., B. A. Stanley, R. Viswanath, and A. E. Pegg. 1994. Rapid exchange of subunits of mammalian ornithine decarboxylase. J Biol Chem 269:3155-8. 5. Gerner, E. W., and F. L. Meyskens, Jr. 2004. Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer 4:781-92. 6. Ghoda, L., D. Sidney, M. Macrae, and P. Coffino. 1992. Structural elements of ornithine decarboxylase required for intracellular degradation and polyamine-dependent regulation. Mol Cell Biol 12:2178-85. 7. Ghoda, L., T. van Daalen Wetters, M. Macrae, D. Ascherman, and P. Coffino. 1989. Prevention of rapid intracellular degradation of ODC by a carboxyl-terminal truncation. Science 243:1493-5. 8. Hayashi, S., and Y. Murakami. 1995. Rapid and regulated degradation of ornithine decarboxylase. Biochem J 306 ( Pt 1):1-10. 9. Heby, O., S. C. Roberts, and B. Ullman. 2003. Polyamine biosynthetic enzymes as drug targets in parasitic protozoa. Biochem Soc Trans 31:415-9. 10. Hoffman, D. W., D. Carroll, N. Martinez, and M. L. Hackert. 2005. Solution structure of a conserved domain of antizyme: a protein regulator of polyamines. Biochemistry 44:11777-85. 11. Ivanov, I. P., R. F. Gesteland, and J. F. Atkins. 2000. Antizyme expression: a subversion of triplet decoding, which is remarkably conserved by evolution, is a sensor for an autoregulatory circuit. Nucleic Acids Res 28:3185-96. 12. Jackson, L. K., H. B. Brooks, A. L. Osterman, E. J. Goldsmith, and M. A. Phillips. 2000. Altering the reaction specificity of eukaryotic ornithine decarboxylase. Biochemistry 39:11247-57. 13. Li, X., and P. Coffino. 1993. Degradation of ornithine decarboxylase: exposure of the C-terminal target by a polyamine-inducible inhibitory protein. Mol Cell Biol 13:2377-83. 14. Li, X., and P. Coffino. 1992. Regulated degradation of ornithine decarboxylase requires interaction with the polyamine-inducible protein antizyme. Mol Cell Biol 12:3556-62. 15. MacRae, M., D. L. Kramer, and P. Coffino. 1998. Developmental effect of polyamine depletion in Caenorhabditis elegans. Biochem J 333 ( Pt 2):309-15. 16. Mangold, U. 2005. The antizyme family: polyamines and beyond. IUBMB Life 57:671-6. 17. Matsufuji, S., T. Matsufuji, Y. Miyazaki, Y. Murakami, J. F. Atkins, R. F. Gesteland, and S. Hayashi. 1995. Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell 80:51-60. 18. Muller, S., G. H. Coombs, and R. D. Walter. 2001. Targeting polyamines of parasitic protozoa in chemotherapy. Trends Parasitol 17:242-9. 19. Murakami, Y., S. Matsufuji, T. Kameji, S. Hayashi, K. Igarashi, T. Tamura, K. Tanaka, and A. Ichihara. 1992. Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. Nature 360:597-9. 20. Pegg, A. E. 2006. Regulation of ornithine decarboxylase. J Biol Chem 281:14529-32. 21. Seiler, N., C. L. Atanassov, and F. Raul. 1998. Polyamine metabolism as target for cancer chemoprevention (review). Int J Oncol 13:993-1006. 22. Soulet, D., B. Gagnon, S. Rivest, M. Audette, and R. Poulin. 2004. A fluorescent probe of polyamine transport accumulates into intracellular acidic vesicles via a two-step mechanism. J Biol Chem 279:49355-66. 23. Van Nieuwenhove, S., P. J. Schechter, J. Declercq, G. Bone, J. Burke, and A. Sjoerdsma. 1985. Treatment of gambiense sleeping sickness in the Sudan with oral DFMO (DL-alpha-difluoromethylornithine), an inhibitor of ornithine decarboxylase; first field trial. Trans R Soc Trop Med Hyg 79:692-8. 24. Zhang, M., A. I. MacDonald, M. A. Hoyt, and P. Coffino. 2004. Proteasomes begin ornithine decarboxylase digestion at the C terminus. J Biol Chem 279:20959-65. 25. Zhang, M., C. M. Pickart, and P. Coffino. 2003. Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate. EMBO J 22:1488-96.
摘要: 人類鳥胺酸脫羧酶(human ornithine decarboxylase, hODC)是催化多元胺(polyamine) 形成的酵素,而且必須以雙聚體型式才具有催化活性。一旦體內多元胺量過多時,就會誘導抗酶的生成 (antizyme, AZ),進而與ODC結合,使其喪失活性,而讓多元胺濃度回歸穩定。目前引起非洲昏睡病 (African sleeping sickness) 的原因之一,是由於錐蟲 (trypanosome) 寄生在宿主中,錐蟲鳥胺酸脫羧酶 (trypanosome ODC, tODC) 卻因為不會與抗酶結合,所以無法受到抑制,而很多治療此種疾病的方法,都從抑制tODC活性方面下手。根據文獻指出,ODC胺基酸序列的第119到第140號位置是與AZ結合的重要區域。我們的目的是找出ODC上與AZ結合的重要胺基酸。藉由比對hODC與tODC的蛋白質序列,我們把hODC中不保留處,以tODC上對應的胺基酸取代。用光譜儀記錄ODC加入AZ後,其活性變化。若突變株無法與AZ成功地結合,那麼酵素活性就不會有明顯的下降。由單點突變的結果顯示,Q119H、Q129D、V137D、M140E活性下降趨勢較為緩慢,推測可能是因為多了額外的電荷,讓AZ無法順利結合。另外,將這7個位置同時全都改成錐蟲的胺基酸,再把每個胺基酸改回人類的,觀察加入抗酶後抑制的結果。發現119與137這兩個位置被改回人類的胺基酸後,喪失了不與抗酶結合的特性,表示在抗酶的結合上,這兩點是造成hODC與tODC有不同特性的主要原因。此外,利用分析級超高速離心機 (analytical ultracentrifugation, AUC),來分析兩種蛋白間的解離常數 (Kd, dissociation constant),亦能佐證。
Human ornithine decarboxylase (hODC) is a pyrodoxal 5'-phosphate (PLP)-dependent enzyme that involves in polyamine biosynthesis and catalyzes polyamine formation. The catalytically active form of hODC is dimeric form. AZ has been identified its role in facilitating degradation of mammalian ODC. Binding of antizyme promotes the dissociation of ODC homodimers and targets ODC for degradation by the 26S proteasomes. In contrast, trypanosomal ODC (tODC) cannot bind to AZ. In this study, we aim to identify the essential amino acid residues governing the AZ-binding affinity for ODC. Based on the multiple sequence alignments in the putative AZ-binding site of ODC, the non-conserved amino acid residues on tODC will be introduced into hODC. If the mutant decreased its AZ-binding affinity, the ODC enzyme activity will not be largely reduced in the presence of AZ. Our data indicated that the single mutant, Q119H, Q129D, V137D and M140E demonstrated a higher residual enzyme activity, suggesting that the additional charge of the three residues will disadvantage the AZ binding. Furthermore, the multiple mutants displayed insensitivity toward AZ inhibition. According to our results, we can identify the essential amino acid residues on hODC required for AZ binding. We can assess the Kd value between ODC and AZ by analytical ultracentrifugation and the results are consistent.
URI: http://hdl.handle.net/11455/22959
其他識別: U0005-2207200909592500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2207200909592500
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