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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64712完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張崇毅(Chung-I Chang) | |
| dc.contributor.author | Cheng-Yui Yang | en |
| dc.contributor.author | 楊政諭 | zh_TW |
| dc.date.accessioned | 2021-06-16T22:57:53Z | - |
| dc.date.available | 2017-08-27 | |
| dc.date.copyright | 2012-08-27 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-09 | |
| dc.identifier.citation | 1.A L Goldberg and L Waxman,The role of ATP hydrolysis in the breakdown of proteins and peptides by protease La from Escherichia coli.,J. Biol. Chem. 1985. 260: p12029-34.
2.Tsilibaris, V., G. Maenhaut-Michel, and L. Van Melderen, Biological roles of the Lon ATP-dependent protease. Res Microbiol, 2006. 157(8): p. 701-13. 3.Bernstein, S.H., et al., The mitochondrial ATP-dependent Lon protease: a novel target in lymphoma death mediated by the synthetic triterpenoid CDDO and its derivatives. Blood, 2012. 119(14): p. 3321-9. 4.Minami, N., et al., Regulatory role of cardiolipin in the activity of an ATP-dependent protease, Lon, from Escherichia coli. J Biochem, 2011. 149(5): p. 519-27. 5.Matsushima, Y., b. a, Yu-ichi Goto, and a.L.S.K. b, Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM). PNAS, 2010. 107(43): 18410–15. 6.Andrew F. Neuwald, L.A., John L. Spouge, and Eugene V. Koonin, AAA+: A Class of Chaperone-Like ATPases Associated with the Assembly, Operation, and Disassembly of Protein Complexes. Genome Res. 1999. 9: 27-43. 7.Sauer, R.T. and T.A. Baker, AAA+ proteases: ATP-fueled machines of protein destruction. Annu Rev Biochem, 2011. 80: p. 587-612. 8.Baker, T.A. and R.T. Sauer, ATP-dependent proteases of bacteria: recognition logic and operating principles. Trends Biochem Sci, 2006. 31(12): p. 647-53. 9.Cheng, I., et al., Identification of a region in the N-terminus of Escherichia coli Lon that affects ATPase, substrate translocation and proteolytic activity. J Mol Biol, 2012. 418(3-4): p. 208-25. 10.Uversky, V.N., et al., A Lon-Like Protease with No ATP-Powered Unfolding Activity. PLoS ONE, 2012. 7(7): p. e40226. 11.Rotanova, T.V., et al., Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains. Protein Sci, 2006. 15(8): p. 1815-28. 12.Venkatesh, S., et al., Multitasking in the mitochondrion by the ATP-dependent Lon protease. Biochim Biophys Acta, 2012. 1823(1): p. 56-66. 13.Eda Bener Aksam, A.K., Stefanie Jourdan, Marten Veenhuis and Ida J. van der Klei A Peroxisomal Lon Protease and Peroxisome Degradation by Autophagy Play Key Roles in Vitality of Hansenula polymorpha Cells. 2007. 3(2): p. 96 - 105. 14.Zach Adam*, I.A., Kazumi Nakabayashi, Oren Ostersetzer, Kirsten Haussuhl, Andrea and B.Z. Manuell, Olivier Vallon, Steven R. Rodermel, Kazuo Shinozaki, and Adrian K. Clarke, Chloroplast and Mitochondrial Proteases in Arabidopsis.A Proposed Nomenclature. Plant Physiology,2001.125(4) 1912-1918, 2001. 15.Maehara, T., T. Hoshino, and A. Nakamura, Characterization of three putative Lon proteases of Thermus thermophilus HB27 and use of their defective mutants as hosts for production of heterologous proteins. Extremophiles, 2008. 12(2): p. 285-96. 16.Rotanova, T.V., et al., Classification of ATP-dependent proteases Lon and comparison of the active sites of their proteolytic domains. Eur J Biochem, 2004. 271(23-24): p. 4865-71. 17.Li, M., et al., Structure of the N-terminal fragment of Escherichia coli Lon protease. Acta Crystallogr D Biol Crystallogr, 2010. 66(Pt 8): p. 865-73. 18.Duman, R.E. and J. Lowe, Crystal structures of Bacillus subtilis Lon protease. J Mol Biol, 2010. 401(4): p. 653-70. 19.Gur, E. and R.T. Sauer, Recognition of misfolded proteins by Lon, a AAA(+) protease. Genes Dev, 2008. 22(16): p. 2267-77. 20.Gur, E. and R.T. Sauer, Degrons in protein substrates program the speed and operating efficiency of the AAA+ Lon proteolytic machine. Proc Natl Acad Sci U S A, 2009. 106(44): p. 18503-8. 21.Lee, A.Y., C.H. Hsu, and S.H. Wu, Functional domains of Brevibacillus thermoruber lon protease for oligomerization and DNA binding: role of N-terminal and sensor and substrate discrimination domains. J Biol Chem, 2004. 279(33): p. 34903-12. 22.Lin, Y.C., et al., DNA-binding specificity of the Lon protease alpha-domain from Brevibacillus thermoruber WR-249. Biochem Biophys Res Commun, 2009. 388(1): p. 62-6. 23.Botos, I., et al., Atomic-resolution crystal structure of the proteolytic domain of Archaeoglobus fulgidus lon reveals the conformational variability in the active sites of lon proteases. J Mol Biol, 2005. 351(1): p. 144-57. 24.Botos, I., et al., The catalytic domain of Escherichia coli Lon protease has a unique fold and a Ser-Lys dyad in the active site. J Biol Chem, 2004. 279(9): p. 8140-8. 25.Garcia-Nafria, J., et al., Structure of the catalytic domain of the human mitochondrial Lon protease: proposed relation of oligomer formation and activity. Protein Sci, 2010. 19(5): p. 987-99. 26.Cha, S.S., et al., Crystal structure of Lon protease: molecular architecture of gated entry to a sequestered degradation chamber. EMBO J, 2010. 29(20): p. 3520-30. 27.Koltai, E., et al., Age-associated Declines in Mitochondrial Biogenesis and Protein Quality Control Factors are Minimized by Exercise Training. Am J Physiol Regul Integr Comp Physiol, 2012. 303(2):p. 127-34. 28.Mueller, M., S. Jenni, and N. Ban, Strategies for crystallization and structure determination of very large macromolecular assemblies. Curr Opin Struct Biol, 2007. 17(5): p. 572-9. 29.Tjioe, E., et al., MultiFit: a web server for fitting multiple protein structures into their electron microscopy density map. Nucleic Acids Res, 2011. 39(Web Server issue): p. W167-70. 30.Morris, G.M., et al., AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem, 2009. 30(16): p. 2785-91. 31.Hu, X., S. Balaz, and W.H. Shelver, A practical approach to docking of zinc metalloproteinase inhibitors. J Mol Graph Model, 2004. 22(4): p. 293-307. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64712 | - |
| dc.description.abstract | 在細胞中蛋白質的合成與降解是處於一個動態的平衡狀態，有新的蛋白質合成也有失去功能與無法正常摺疊之蛋白被降解。兩者之間的平衡一但被破壞將導致細胞的環境失去恆定性，導致細胞因此產生警示性的反應而引發細胞凋亡以及細胞癌化的可能。
在眾多調控細胞蛋白質和成與降解的機制中，有一群被稱為AAA+ protease family的蛋白脢。它們共有的特徵就是在其中具有ATPase的AAA+ module可以跟ATP結合與水解，因此產生蛋白質構形改變與用此能量來unfolding目標蛋白。它們通常是辨認目標蛋白於不正常摺疊時所露出的特定序列，通常以”degron”稱呼之。在細胞中它們如同品管人員一般，進行protein quality control以及決定特定蛋白在細胞中的表現水平。在AAA+ protease family之中有一類蛋白脢名為‘'Lon protease”，依照序列比對又可再細分為LonA、LonB與LonC三類。LonA由N端之N domain以及接下來的AAA+ domain與protease domain組成；LonB與LonC較為相似，LonB相較於LonA少了N domain但AAA+ domain有多出穿膜區段，LonC其AAA+ domain則是去活性的，在AAA+ domain上有延伸出一段 coiled-coil domain。其中LonA不論是原核與真核生物的胞器都可以發現它的存在。於真核生物中它存在於粒線體、葉綠體、過氧化體這些胞器，因此它在維持細胞能量代謝以及氧化壓力水平扮演著重要的角色。 到目前關於LonA完整的結構尚未解出，目前解出的多為獨立功能域的結構。因此在本篇研究嘗試以Meiothermus taiwanensis之MtaLonA1蛋白用於長晶，希望能拼湊出MtaLonA1蛋白完整的結構。雖然我們的確有長出MtaLonA1蛋白全長晶體，X-ray繞射之最佳解析度介於3~3.5A，但是晶格某一方向的軸異常的長(468.38A)，此晶體繞射品質尚在改進中。目前另採取其他策略把全長蛋白分成兩個蛋白用於長晶，其一為涵蓋N端至AAA+ domain之α/β sub domain；另一為去除N domain的蛋白。 | zh_TW |
| dc.description.abstract | Lon proteases belong to a family of AAA+ (ATPases associated with diverse cellular activities) proteases, which perform protein quality control in bacteria and eukaryotic organisms. The Lon protease family has been divided into three subfamilies, LonA、LonB and LonC, based on sequence homology and domain organization. LonA proteases are composed of a large N-terminal domain, a central ATPase domain, and a C-terminal protease domain. LonB proteases lacked the N-terminal domain of Lon A; however, they contain transmembrane regions inserted within the ATPase domain. LonC lacked ATPase activity and the transmembrane regions in LonB was replaced by the coiled-coil domains. So far the structure of full-length LonA has remained elusive, which has limited the understanding about the molecular mechanisms for substrate recognition and processing.
Here we describe expression, purification, and crystallization of MtaLonA1 from Meiothermus taiwanensis(Mta). The largest crystals grew in 4~6 day at 22 °C to the size of 0.3 × 0.2 × 0.05 mm, with diffraction resolution reaching 3~ 3.5 A. But one of the axis in the unit cell was long (468.38A), and the data set could not be processed due to high mosaicity. As an alternative, we have made two truncated MtaLonA1 proteins for crystal growth. Mta_NAAA contains the N-terminal to the AAA + the domain of the α/β sub domain；and Mta_AAAP which contains the AAA+ domain and protease domain. Structures of these constructs may be used to assemble a full-length model of MtaLonA1 by superimposing their overlapping AAA domains. The expression, purification, and crystallization of these constructs are described. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T22:57:53Z (GMT). No. of bitstreams: 1 ntu-101-R99b46027-1.pdf: 2463936 bytes, checksum: f7837bd9b3db6bb5b5b8bb4f0f3c2440 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 口試委員會審定書……………………………………………………………… i
誌謝………………………………………………………………………………. ii 中文摘要………………………………………………………………………… i ii 英文摘要…………………………………………………………………………. iv 第一章 緒論………………………………………………………………………………………………………………1 1.1 AAA+ Protease Family的特性………………………………………………………………1 1.2 Lon Protease 分類…………………………………………………………………………………………2 1.3 Lon A各個domain的細節…………………………………………………………………………………3 1.4目前已發表之Lon的結構………………………………………………………………………………………4 1.5研究動機……………………………………………………………………………………………………………………5 第二章 材料與方法…………………………………………………………………………………………………6 2.1 MtaLonA1_S678A 基因來源………………………………………………………………………6 2.2 質體建構…………………………………………………………………………………………………………………6 2.3 抽取質體 DNA 與轉殖………………………………………………………………………………………7 2.4 大量表現…………………………………………………………………………………………………………………8 2.5 純化…………………………………………………………………………………………………………………………8 2.5.1 Ni column 親和力管柱層析…………………………………………………………………9 2.5.2 Mono QR 5/50 離子交換層析………………………………………………………………9 2.5.3 TEV protease 去除 His tag……………………………………………………………10 2.5.4 Gel-filtration…………………………………………………………………………………………10 2.5.5 純化過程加入 Nucleotides……………………………………………………………………11 2.6 長晶條件篩選…………………………………………………………………………………………………………11 2.7 微調與最佳化長晶條件………………………………………………………………………………………11 2.8 Seeding 與 Additive Screen………………………………………………………………12 2.9 Cryoprotectant 的選取………………………………………………………………………………13 2.10 Post-crystallization treatments………………………………………………14 2.11 X-ray 繞射數據收集………………………………………………………………………………………14 第三章 實驗結果 3.1 大量表現結果……………………………………………………………………………………………………………15 3.1.1 MtaLonA1_S678A C-ter His 6 -tag………………………………………………15 3.1.2 MtaLonA1_S678A N-ter His 6 -tag………………………………………………15 3.1.3 MtaLonA1_AAAP………………………………………………………………………………………………15 3.1.4 MtaLonA1_NAAA………………………………………………………………………………………………16 3.2 蛋白純化結果……………………………………………………………………………………………………………16 3.2.1 MtaLonA1_S678A C-ter His 6 -tag………………………………………………16 3.2.2 MtaLonA1_S678A N-ter His 6 -tag………………………………………………17 3.2.3 MtaLonA1_AAAP………………………………………………………………………………………………17 3.2.4 MtaLonA1_NAAA………………………………………………………………………………………………18 3.2.5 純化過程加入 Nucleotides………………………………………………………………………18 3.3 長晶條件篩選結果……………………………………………………………………………………………………19 3.4 長晶條件最佳化…………………………………………………………………………………………………………21 3.4.1 MtaLonA1_S678A C-ter His 6 -tag………………………………………………21 3.4.2 MtaLonA1_AAAP………………………………………………………………………………………………23 3.5 Cryoprotectant 的測試與 X-ray 繞射結果…………………………………………24 3.6 Post-crystallization treatments 的效果……………………………………25 第四章 討論…………………………………………………………………………………………………………………25 第五章 基於已知的 HDAC 抑制劑 SAHA 開發新型 Osthole derivatives capped HDAC8 專一性競爭型抑制劑………………………………………………………………………………………………28 參考文獻……………………………………………………………………………………………………………………………………37 附表……………………………………………………………………………………………………………………………………………40 附圖……………………………………………………………………………………………………………………………………………42 附錄……………………………………………………………………………………………………………………………………………66 | |
| dc.language.iso | zh-TW | |
| dc.title | 台灣中度嗜熱菌MtaLonA1蛋白酶之純化與其蛋白質
晶體之分析 | zh_TW |
| dc.title | Purification and crystallization of MtaLonA1 from Meiothermus
taiwanensis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳世雄(Shih-Hsiung Wu),蕭傳鐙(Chwan-Deng (David) | |
| dc.subject.keyword | AAA + protease,Lon protease,degron,protein quality control,X-ray 繞射,Meiothermus taiwanensis,MtaLonA1, | zh_TW |
| dc.subject.keyword | AAA + protease,Lon protease,degron,protein quality control,X-ray diffraction,Meiothermus taiwanensis,MtaLonA1, | en |
| dc.relation.page | 67 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-09 | |
| 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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