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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王惠鈞(Andrew H.-J. Wang) | |
dc.contributor.author | Yao-Chen Tsui | en |
dc.contributor.author | 崔耀珍 | zh_TW |
dc.date.accessioned | 2021-06-13T03:26:10Z | - |
dc.date.available | 2008-07-31 | |
dc.date.copyright | 2006-07-31 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2006-07-27 | |
dc.identifier.citation | REFERENCE
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Miyashita K, Kusumi M, Utsumi R, Komano T, Satoh N: Expression and purification of recombinant 3C proteinase of Coxsackievirus B3. Biosci Biotechnol Biochem 1992, 56(5):746-750. 8. Seipelt J, Guarne A, Bergmann E, James M, Sommergruber W, Fita I, Skern T: The structures of picornaviral proteinases. Virus Res 1999, 62(2):159-168. 9. Palmenberg AC: Proteolytic processing of picornaviral polyprotein. Annu Rev Microbiol 1990, 44:603-623. 10. Kemp G, Webster A, Russell WC: Proteolysis is a key process in virus replication. Essays Biochem 1992, 27:1-16. 11. Krausslich HG, Wimmer E: Viral proteinases. Annu Rev Biochem 1988, 57:701-754. 12. Lee CK, Wimmer E: Proteolytic processing of poliovirus polyprotein: elimination of 2Apro-mediated, alternative cleavage of polypeptide 3CD by in vitro mutagenesis. Virology 1988, 166(2):405-414. 13. Yin J, Bergmann EM, Cherney MM, Lall MS, Jain RP, Vederas JC, James MN: Dual modes of modification of hepatitis A virus 3C protease by a serine-derived beta-lactone: selective crystallization and formation of a functional catalytic triad in the active site. J Mol Biol 2005, 354(4):854-871. 14. Bergmann EM, Mosimann SC, Chernaia MM, Malcolm BA, James MN: The refined crystal structure of the 3C gene product from hepatitis A virus: specific proteinase activity and RNA recognition. J Virol 1997, 71(3):2436-2448. 15. Bergmann EM, Cherney MM, McKendrick J, Frormann S, Luo C, Malcolm BA, Vederas JC, James MN: Crystal structure of an inhibitor complex of the 3C proteinase from hepatitis A virus (HAV) and implications for the polyprotein processing in HAV. Virology 1999, 265(1):153-163. 16. Allaire M, Chernaia MM, Malcolm BA, James MN: Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases. Nature 1994, 369(6475):72-76. 17. Matthews DA, Smith WW, Ferre RA, Condon B, Budahazi G, Sisson W, Villafranca JE, Janson CA, McElroy HE, Gribskov CL et al: Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein. Cell 1994, 77(5):761-771. 18. Mosimann SC, Cherney MM, Sia S, Plotch S, James MN: Refined X-ray crystallographic structure of the poliovirus 3C gene product. J Mol Biol 1997, 273(5):1032-1047. 19. Birtley JR, Knox SR, Jaulent AM, Brick P, Leatherbarrow RJ, Curry S: Crystal structure of foot-and-mouth disease virus 3C protease. New insights into catalytic mechanism and cleavage specificity. J Biol Chem 2005, 280(12):11520-11527. 20. Miyashita K, Okunishi J, Utsumi R, Tagiri S, Hotta K, Komano T, Tamura T, Satoh N: Cleavage specificity of coxsackievirus 3C proteinase for peptide substrate (2): Importance of the P2 and P4 residues. Biosci Biotechnol Biochem 1996, 60(9):1528-1529. 21. Miyashita K, Okunishi J, Utsumi R, Komano T, Tamura T, Satoh N: Cleavage specificity of coxsackievirus 3C proteinase for peptide substrate. Biosci Biotechnol Biochem 1996, 60(4):705-707. 22. Lawson MA, Dasmahapatra B, Semler BL: Species-specific substrate interaction of picornavirus 3C proteinase suballelic exchange mutants. J Biol Chem 1990, 265(26):15920-15931. 23. McKinlay MA: Recent advances in the treatment of rhinovirus infections. Curr Opin Pharmacol 2001, 1(5):477-481. 24. Otwinowski Z, and W. Minoe: Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 1997, 276:307-326. 25. Navaza J: AMoRe: an automated package for molecular replacement. Acta Crystallogr 1994, A50:157-163. 26. McRee DE: XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. J Struct Biol 1999, 125(2-3):156-165. 27. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS et al: Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 1998, 54(Pt 5):905-921. 28. Laskowski RA, M. W. MacArthur, D. S. Moss and J. M. Thornton: PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 1993, 26:283-291. 29. Petersen JF, Cherney MM, Liebig HD, Skern T, Kuechler E, James MN: The structure of the 2A proteinase from a common cold virus: a proteinase responsible for the shut-off of host-cell protein synthesis. Embo J 1999, 18(20):5463-5475. 30. Matthews DA, Dragovich PS, Webber SE, Fuhrman SA, Patick AK, Zalman LS, Hendrickson TF, Love RA, Prins TJ, Marakovits JT et al: Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes. Proc Natl Acad Sci U S A 1999, 96(20):11000-11007. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31965 | - |
dc.description.abstract | 克沙奇B3型病毒屬於腸病毒的一種,主要感染孩童造成急性或慢性心肌炎,其伴隨的病症嚴重者會導致死亡,目前除了減輕病痛及減緩病症的治療外,並沒有針對克沙奇B3型病毒的專用藥物。克沙奇病毒的3C蛋白酶屬於微小RNA病毒(picornaviridae) Cysteine蛋白酶,專門負責切割病毒轉譯的複合蛋白質,使產生各具功能的多種蛋白質,由於此3C蛋白酶在克沙奇型病毒的成熟及感染過程中不可或缺,以及它在不同克沙奇病毒株的高度一致性,使得3C蛋白酶成為抗病毒藥物發展的主要目標。在此,我們解出第一個克沙奇B3型病毒3C蛋白酶和它C147A突變株的晶體結構,且解析度分別達到1.8 Å
及1.4 Å;根據結構分析,克沙奇B3型病毒3C蛋白酶具有類似chymotrypsin的摺疊方式, 且其催化中心Cys-His-Glu與大部份Serine蛋白酶的催化中心Ser-His-Asp有著相似的構造。進一步,我們利用多種SARS類3C蛋白酶的抑制劑,分析它們對克沙奇B3型病毒3C蛋白酶的抑制能力,並且解出其中一個抑制劑與蛋白酶共結晶的結構,解析度為1.7 Å;抑制劑 TG4998 和克沙奇B3型病毒3C蛋白酶之間,透過與催化中心Cys147形成共價鍵,以及與活化區內其他氨基酸形成氫鍵而結合,在這些作用中,TG4998對於抑制效果的影響,主要透過其P1、P2及P4位置與克沙奇B3型病毒3C蛋白酶的作用。綜合我們觀察克沙奇B3型病毒3C蛋白酶所做的結構分析,可為抗病毒藥物提供設計與改良上的重要依據。 | zh_TW |
dc.description.abstract | Coxsackievirus B3 (CVB3) causes acute or chronic myocarditis, which may lead to death, especially in children. Until now, there is no specific therapy for CVB3 other than treatment to relieve pain and other symptoms. CVB3 3C protease (3Cpro), a member of the picornaviridae cysteine protease, is required to cleave the viral polyprotein into functional proteins. CVB3 3Cpro is highly conserved in different strains and essential for viral maturation and infectivity, making it an attractive target for the development of antiviral drugs. Here we report the first crystal structures of native CVB3 3Cpro and its mutant (C147A) at 1.8 Å and 1.4 Å resolution, espectively. The structure of CVB3 3Cpro adopts a chymotrypsin-like fold and possesses a catalytic triad, Cys-His-Glu, which is
similar to the configuration of the Ser-His-Asp triad in almost all serine proteases. Furthermore, we analyzed the inhibition activity of CVB3 3Cpro with several SARS 3C-like protease inhibitors and determined a structure of co-crystallized proteaseinhibitor complex at 1.7 Å resolution. The inhibitor, TG4998, interacts with CVB3 3Cpro through a covalent bond with Cys147 of the catalytic triad and hydrogen bonds in the active-site pocket. The main effect on enzyme-inhibition depends upon the interactions between CVB3 3Cpro and the P1, P2 and P4 residues of TG4998. Collectively, these structural characteristics provide valuable basis for antiviral drug design. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T03:26:10Z (GMT). No. of bitstreams: 1 ntu-94-R93b46013-1.pdf: 6132866 bytes, checksum: a3948b816bfbe63f9ed037c6c9562cce (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | CONTENTS
中文摘要………………………………………………………..…………………….….Ⅰ ABSTRACT……………………………………………………….……………...…..…Ⅱ CONTENTS………………………………………………………….………………….Ⅲ LIST OF FIGURES……………………………………………………………………..Ⅴ Inhibition Assay of CVB3 3Cpro………………………………………………….14 Crystallization and Structure Determination…………………………………….14 Overall Structure of CVB3 3Cpro and Conformation of Active Site……….……..15 Interaction between CVB3 3Cpro and TG4998……………………………….........16 Comparison of Native 3Cpro, C147A and Complex Structures……………….…….17 DISCUSSION……………………………………………………………………………18 REFERENCE………………………………..…………………………………….........20 LIST OF FIGURES Fig. 1 Consensus sequences of CVB3 3Cpro……………………………………..…..24 Fig. 2 Expression and purification of native CVB3 3Cpro……………………….…..25 Fig. 3 SDS-PAGE analysis of CVB3 3Cpro mutants……………………………...….26 Fig. 4 Mass spectrum of CVB3 3Cpro……………………………………………..…27 Fig. 5 Reverse-Phase HPLC analysis of CVB3 3Cpro activity………………………28 Fig.6 Crystal pictures and conditions of different type CVB3 3Cpro………………...29 Fig. 7 The overall structure of native CVB3 3Cpro………………………………..…30 Fig. 8 Binding of the TG4998 in the CVB3 3Cpro active site……………………..…31 Fig. 9 Surface representation of 3Cpro/TG4998 complex……………………..……..32 Fig. 10 Superposition of the structures of native 3Cpro, C147A mutant, and 3Cpro/TG4998 complex………………………………………………..….......33 Fig. 11 Multiple sequence alignment of the picornaviral 3Cpro and secondary assignment of CVB3 3Cpro……………………………………………..…….34 Fig. 12 Comparison of the active site structures of picornaviral 3C proteases.....……35 LIST OF TABLES Table 1 Members of Enteroviruses……………………………………………..……..36 Table 2 Members of Picornaviridae……………………………………..……………37 Table 3 Data collection and refinement statistics for native CVB3 3Cpro, C147A mutant, and TG4998 complex…………..……………………………………38 Table 4 The inhibitory ability of CVB3 3Cpro with different 3C(L)pro inhibitors……..39 LIST OF TABLES……………………………………………………….……………...Ⅵ INTRODUCTION……………………………………..……..…………………………..1 Coxsackieviruses and Diseases……………………………..……………………..1 Picornaviruses and Their Life Cycle……………………………..……...………...2 3C Protease (3Cpro)…………………...……………………………..……………..3 MATERIALS AND METHODS……………………………………………..……..…...6 Materials……………………………………………………………………..……..6 Plasmid Construction………………………………………………………..……..7 Expression and Purification……………………………………………………..…7 Site-directed Mutagenesis………………………………………………………….8 Crystallization and Co-crystallization……………………….……………….……9 Data Collection and Processing……………………………….…………………...9 Structure Determination and Refinement…………………….…………………..10 Peptide Synthesis………………………………………………..………………..10 Protease Activity and Inhibition Assays……………………….…………….......11 RESULTS……………………………………...………………………………….……..12 Expression and Purification of Native and Mutant 3Cpro………………….…….12 Protease Activity Assay…………………………………………………….…….13 | |
dc.language.iso | en | |
dc.title | 克沙奇B3型病毒3C蛋白酶及其抑制劑複合體結構分析--抗病毒藥物設計之應用 | zh_TW |
dc.title | Structural Analysis of Coxsackievirus B3 3C Protease and its Inhinitor Complex: Implications in Antiviral Drug Design | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張文章(Wen-Chang Chang),梁博煌(Po-Huang Liang) | |
dc.subject.keyword | 克沙奇,蛋白酶,抗病毒藥物,晶體結構, | zh_TW |
dc.subject.keyword | coxsackievirus,3C,protease,structure,antiviral drug, | en |
dc.relation.page | 39 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-29 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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