Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/60846
標題: 一、果蠅Hopscotch kinase domain蛋白結晶與結構預測分析 二、產酸克雷白氏菌Klebsiella oxytoca之Fosfomycin resistance protein FosA的蛋白表現、純化與結晶
A. Crystallization and Structure Prediction of Hopscotch Kinase Domain from Drosophila melanogaster B. Expression, Purification and Crystallization of Fosfomycin Resistance Protein FosA from Klebsiella oxytoca
作者: 李佳燁
Lee, Jia-Ye
關鍵字: JAK/STAT訊息傳遞
JAK/STAT signaling pathway
果蠅
Hopscotch
產酸克雷白氏菌
FosA
Drosophila melanogaster
Hopscotch
Klebsiella oxytoca
FosA
出版社: 基因體暨生物資訊學研究所
引用: 1. Harrison, D.A., The Jak/STAT pathway. Cold Spring Harb Perspect Biol, 2012. 4(3). 2. O''Sullivan, L.A., et al., Cytokine receptor signaling through the Jak-Stat-Socs pathway in disease. Mol Immunol, 2007. 44(10): p. 2497-506. 3. Ward, A.C., I. Touw, and A. Yoshimura, The Jak-Stat pathway in normal and perturbed hematopoiesis. Blood, 2000. 95(1): p. 19-29. 4. Hou, S.X., et al., The Jak/STAT pathway in model organisms: emerging roles in cell movement. Dev Cell, 2002. 3(6): p. 765-78. 5. Yamaoka, K., et al., The Janus kinases (Jaks). Genome Biol, 2004. 5(12): p. 253. 6. Binari, R. and N. Perrimon, Stripe-specific regulation of pair-rule genes by hopscotch, a putative Jak family tyrosine kinase in Drosophila. Genes Dev, 1994. 8(3): p. 300-12. 7. Liongue, C., et al., Evolution of JAK-STAT pathway components: mechanisms and role in immune system development. PLoS One, 2012. 7(3): p. e32777. 8. Kawamura, M., et al., Molecular cloning of L-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes. Proc Natl Acad Sci U S A, 1994. 91(14): p. 6374-8. 9. Chrencik, J.E., et al., Structural and thermodynamic characterization of the TYK2 and JAK3 kinase domains in complex with CP-690550 and CMP-6. J Mol Biol, 2010. 400(3): p. 413-33. 10. Lucet, I.S., et al., The structural basis of Janus kinase 2 inhibition by a potent and specific pan-Janus kinase inhibitor. Blood, 2006. 107(1): p. 176-83. 11. Tsui, V., et al., A new regulatory switch in a JAK protein kinase. Proteins-Structure Function and Bioinformatics, 2011. 79(2): p. 393-401. 12. Williams, N.K., et al., Dissecting Specificity in the Janus Kinases: The Structures of JAK-Specific Inhibitors Complexed to the JAK1 and JAK2 Protein Tyrosine Kinase Domains. Journal of Molecular Biology, 2009. 387(1): p. 219-232. 13. Manning, G., et al., The protein kinase complement of the human genome. Science, 2002. 298(5600): p. 1912-34. 14. Saharinen, P., K. Takaluoma, and O. Silvennoinen, Regulation of the Jak2 tyrosine kinase by its pseudokinase domain. Mol Cell Biol, 2000. 20(10): p. 3387-95. 15. Baxter, E.J., et al., Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet, 2005. 365(9464): p. 1054-61. 16. James, C., et al., A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature, 2005. 434(7037): p. 1144-8. 17. Kralovics, R., et al., A gain-of-function mutation of JAK2 in myeloproliferative disorders. New England Journal of Medicine, 2005. 352(17): p. 1779-1790. 18. Levine, R.L., et al., Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell, 2005. 7(4): p. 387-97. 19. Zhou, Y.J., et al., Unexpected effects of FERM domain mutations on catalytic activity of Jak3: structural implication for Janus kinases. Mol Cell, 2001. 8(5): p. 959-69. 20. Luo, H. and C.R. Dearolf, The JAK/STAT pathway and Drosophila development. Bioessays, 2001. 23(12): p. 1138-47. 21. Perrimon, N. and A.P. Mahowald, L(1)Hopscotch, a Larval Pupal Zygotic Lethal with a Specific Maternal Effect on Segmentation in Drosophila. Developmental Biology, 1986. 118(1): p. 28-41. 22. Luo, H., et al., The Drosophila Jak kinase hopscotch is required for multiple developmental processes in the eye. Developmental Biology, 1999. 213(2): p. 432-441. 23. Gauzzi, M.C., et al., Interferon-alpha-dependent activation of Tyk2 requires phosphorylation of positive regulatory tyrosines by another kinase. Journal of Biological Chemistry, 1996. 271(34): p. 20494-20500. 24. Luo, H., et al., Mutation in the Jak kinase JH2 domain hyperactivates Drosophila and mammalian Jak-Stat pathways. Mol Cell Biol, 1997. 17(3): p. 1562-71. 25. Luo, H., W.P. Hanratty, and C.R. Dearolf, An Amino-Acid Substitution in the Drosophila Hop(Tum-L) Jak Kinase Causes Leukemia-Like Hematopoietic Defects. Embo Journal, 1995. 14(7): p. 1412-1420. 26. Hou, X.S., M.B. Melnick, and N. Perrimon, Marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the mammalian STATs. Cell, 1996. 84(3): p. 411-9. 27. Yan, R.Q., et al., Identification of a Stat gene that functions in Drosophila development. Cell, 1996. 84(3): p. 421-430. 28. Hombria, J.C., et al., Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand. Dev Biol, 2005. 288(2): p. 420-33. 29. Ke, S.H. and E.L. Madison, Rapid and efficient site-directed mutagenesis by single-tube ''megaprimer'' PCR method. Nucleic Acids Res, 1997. 25(16): p. 3371-2. 30. Carvalho, A.F., et al., High-yield expression in Escherichia coli and purification of mouse ubiquitin-activating enzyme E1. Mol Biotechnol, 2012. 51(3): p. 254-61. 31. Vuillard, L., et al., Protein crystallography with non-detergent sulfobetaines. Journal of Crystal Growth, 1996. 168(1–4): p. 150-154. 32. Vuillard, L., et al., Halophilic protein stabilization by the mild solubilizing agents nondetergent sulfobetaines. Anal Biochem, 1995. 230(2): p. 290-4. 33. Vuillard, L., T. Rabilloud, and M.E. Goldberg, Interactions of non-detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation. Eur J Biochem, 1998. 256(1): p. 128-35. 34. Vuillard, L., et al., A new additive for protein crystallization. FEBS Lett, 1994. 353(3): p. 294-6. 35. Bai, Y., T.C. Auperin, and L. Tong, The use of in situ proteolysis in the crystallization of murine CstF-77. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2007. 63(Pt 2): p. 135-8. 36. Dong, A., et al., In situ proteolysis for protein crystallization and structure determination. Nat Methods, 2007. 4(12): p. 1019-21. 37. Wernimont, A. and A. Edwards, In situ proteolysis to generate crystals for structure determination: an update. PLoS One, 2009. 4(4): p. e5094. 38. Lietha, D., et al., Structural basis for the autoinhibition of focal adhesion kinase. Cell, 2007. 129(6): p. 1177-87. 39. Kleber-Janke, T. and W.M. Becker, Use of modified BL21(DE3) Escherichia coli cells for high-level expression of recombinant peanut allergens affected by poor codon usage. Protein Expr Purif, 2000. 19(3): p. 419-24. 40. Kim, E.K., et al., Large-scale production of soluble recombinant amyloid-beta peptide 1-42 using cold-inducible expression system. Protein Expr Purif, 2012. 86(1): p. 53-7. 41. Ashkenazy, H., et al., ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res, 2010. 38(Web Server issue): p. W529-33. 42. Glaser, F., et al., ConSurf: identification of functional regions in proteins by surface-mapping of phylogenetic information. Bioinformatics, 2003. 19(1): p. 163-4. 43. Landau, M., et al., ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res, 2005. 33(Web Server issue): p. W299-302. 44. Christensen, B.G., et al., Phosphonomycin: structure and synthesis. Science, 1969. 166(3901): p. 123-5. 45. Hendlin, D., et al., Phosphonomycin, a new antibiotic produced by strains of streptomyces. Science, 1969. 166(3901): p. 122-3. 46. Kahan, F.M., et al., The mechanism of action of fosfomycin (phosphonomycin). Ann N Y Acad Sci, 1974. 235(0): p. 364-86. 47. Bugg, T.D. and C.T. Walsh, Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance. Nat Prod Rep, 1992. 9(3): p. 199-215. 48. Skarzynski, T., et al., Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, an enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin. Structure, 1996. 4(12): p. 1465-74. 49. Krcmery, S., J. Hromec, and D. Demesova, Treatment of lower urinary tract infection in pregnancy. Int J Antimicrob Agents, 2001. 17(4): p. 279-82. 50. Allerberger, F. and I. Klare, In-vitro activity of fosfomycin against vancomycin-resistant enterococci. J Antimicrob Chemother, 1999. 43(2): p. 211-7. 51. Falagas, M.E., et al., Fosfomycin for the treatment of multidrug-resistant, including extended-spectrum beta-lactamase producing, Enterobacteriaceae infections: a systematic review. Lancet Infect Dis, 2010. 10(1): p. 43-50. 52. Mendoza, C., et al., Plasmid-determined resistance to fosfomycin in Serratia marcescens. Antimicrob Agents Chemother, 1980. 18(2): p. 215-9. 53. Arca, P., C. Hardisson, and J.E. Suarez, Purification of a glutathione S-transferase that mediates fosfomycin resistance in bacteria. Antimicrob Agents Chemother, 1990. 34(5): p. 844-8. 54. Arca, P., et al., Formation of an adduct between fosfomycin and glutathione: a new mechanism of antibiotic resistance in bacteria. Antimicrob Agents Chemother, 1988. 32(10): p. 1552-6. 55. Rife, C.L., et al., Crystal structure of a genomically encoded fosfomycin resistance protein (FosA) at 1.19 A resolution by MAD phasing off the L-III edge of Tl(+). J Am Chem Soc, 2002. 124(37): p. 11001-3. 56. Cao, M., et al., FosB, a cysteine-dependent fosfomycin resistance protein under the control of sigma(W), an extracytoplasmic-function sigma factor in Bacillus subtilis. J Bacteriol, 2001. 183(7): p. 2380-3. 57. Wachino, J., et al., Prevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and identification of novel plasmid-mediated fosfomycin-modifying enzymes. Antimicrob Agents Chemother, 2010. 54(7): p. 3061-4. 58. Lee, S.Y., et al., Prevalence of acquired fosfomycin resistance among extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in Korea and IS26-composite transposon surrounding fosA3. J Antimicrob Chemother, 2012. 67(12): p. 2843-7. 59. Bernat, B.A. and R.N. Armstrong, Elementary steps in the acquisition of Mn2+ by the fosfomycin resistance protein (FosA). Biochemistry, 2001. 40(42): p. 12712-8. 60. Hogenauer, C., et al., Klebsiella oxytoca as a causative organism of antibiotic-associated hemorrhagic colitis. N Engl J Med, 2006. 355(23): p. 2418-26. 61. Liao, T.L., et al., Complete genome sequence of Klebsiella oxytoca E718, a New Delhi metallo-beta-lactamase-1-producing nosocomial strain. J Bacteriol, 2012. 194(19): p. 5454. 62. Kelley, L.A. and M.J. Sternberg, Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc, 2009. 4(3): p. 363-71. 63. Eswar, N., et al., Comparative protein structure modeling using Modeller. Curr Protoc Bioinformatics, 2006. Chapter 5: p. Unit 5 6. 64. Jatiani, S.S., et al., Jak/STAT pathways in cytokine signaling and myeloproliferative disorders: approaches for targeted therapies. Genes Cancer, 2010. 1(10): p. 979-93. 65. Baker, S.J., S.G. Rane, and E.P. Reddy, Hematopoietic cytokine receptor signaling. Oncogene, 2007. 26(47): p. 6724-37. 66. Arbouzova, N.I. and M.P. Zeidler, JAK/STAT signalling in Drosophila: insights into conserved regulatory and cellular functions. Development, 2006. 133(14): p. 2605-16. 67. Rigsby, R.E., et al., Phosphonoformate: a minimal transition state analogue inhibitor of the fosfomycin resistance protein, FosA. Biochemistry, 2004. 43(43): p. 13666-73.
摘要: 一、果蠅Hopscotch kinase domain蛋白結晶與結構預測分析 JAK/STAT訊息傳導路徑是細胞重要的調控機制,對於細胞增生、維持恆定、免疫反應與幹細胞維持扮演重要的角色,此路徑異常與許多癌症發生有關。Janus kinases (Jaks)為非受體型的酪胺酸激酶,它會將signal transducers and activators of transcription(STATs)磷酸化,以啟動轉錄作用。在哺乳動物的JAK/STAT路徑中,有四個Jaks與七個STATs,而在果蠅的JAK/STAT系統中,只有一個Jak kinase—Hopscotch (HOP)與一個轉錄因子STAT92E。由於果蠅與哺乳動物JAK/STAT訊息傳遞鍊在演化上具有高度保留性,而果蠅系統相對簡化的特色,使得果蠅成為研究JAK/STAT路徑良好的模式生物。由於目前尚未有Hopscotch的結構資訊,本實驗選殖黑腹果蠅(Drosophila melanogaster) HOP的kinase domain,預期以蛋白結晶方式解析其分子結構。目前已可成功純化出水溶性良好之HOP kinase domain,未來將持續進行結晶條件篩選。我同時以生物資訊方法,先預測出HOP kinase domain的結構,並探討其是否可能與FERM domain交互作用。我也利用分子對接預測研究kinase domain與其抑制劑的結合方式。這些結果可以幫助我們更加了解HOP蛋白結構與功能之間的關係。 二、產酸克雷白氏菌Klebsiella oxytoca之Fosfomycin resistance protein, FosA的蛋白表現、純化與結晶 Fosfomycin是自然界中廣泛存在的抗生素,對於革蘭氏陽性菌與革蘭氏陰性菌皆有作用,目前臨床上用於治療非複雜型泌尿道感染。Fosfomycin藉由干擾細菌細胞壁合成來抑制細菌的生長。然而細菌的抗藥性有逐漸增加的趨勢,例如肺炎克雷白氏菌是造成嚴重院內感染的菌種之一,其產生的Fosfomycin resistance protein(FosA)會將fosfomycin接上一個glutathione,使fosfomycin失去活性。FosA存在於許多微生物基因體中,然而目前在蛋白結構資料庫內,僅有綠膿桿菌(Pseudomonas aeruginosa) FosA結構資訊,因此,本實驗室將產酸克雷白氏菌E718的FosA利用E. coli進行蛋白質表現,篩選出具有fosfomycin抗性的菌株,並已純化出水溶性良好並且具有功能之FosA,進行蛋白結晶試驗。未來仍須持續調整結晶條件以得到蛋白質晶體。
A. Crystallization and Structure Prediction of Hopscotch Kinase Domain from Drosophila melanogaster JAK/STAT signaling pathway is an essential component regulating cell proliferation, homeostasis, immune response and stem-cell maintenance. Misregulation of the pathway is associated with multiple human malignancies such as leukemia and solid tumors. Janus kinases (Jaks) are non-receptor tyrosine kinases which are activated upon autophosphorylation. Activated Jaks then phosphorylate associated receptors and STATs, resulting in STATs translocation to the nucleus where they bind to target DNA sequence to activate transcription. In mammals, there are four Jaks and seven STATs. Whereas in Drosophila, there is only one Jak kinase known as Hopscotch (HOP) and a transcription factor STAT92E. A simpler pathway of Drosophila JAK/STAT and high level of conservation between insects and mammals make Drosophila an excellent model for studying JAK/STAT pathway. Detailed structural information on Jaks is needed to understand the mechanism of Jak activation. Therefore, we expressed and purified the kinase domain of HOP from Drosophila melanogaster for crystallization experiments. In addition, we predicted the structure of HOP kinase domain and its interaction with FERM domain, and performed docking studies with possible inhibitors. These results will improve our understanding into the structure-function relationship of this important protein. B. Expression, Purification and Crystallization of Fosfomycin Resistance Protein, FosA from Klebsiella oxytoca Fosfomycin is a natural product which has antimicrobial activities against both Gram-positive and Gram-negative bacteria. Fosfomycin inhibits bacterial growth by interfering with the first committed step in microbial cell wall biosynthesis. This antibiotic is currently used for treatment of acute uncomplicated lower urinary tract infection. However, after the use of fosfomycin in the clinic, resistance to the antibiotic began to emerge. Fosfomycin resistance protein, FosA, a 16 kDa polypeptide is responsible for the addition of glutathione (GSH) to the antibiotic, rendering it inactive. To date, structural information of FosA is limited to that encoded in Pseudomonas aeruginosa. We have generated a soluble and functional recombinant FosA from Klebsiella oxytoca E718 and carried out randomized protein crystallization trials. It is important to further improve condition that favors protein crystals formation.
URI: http://hdl.handle.net/11455/60846
其他識別: U0005-2608201316263200
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2608201316263200
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