請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7512完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 曾秀如 | zh_TW |
| dc.contributor.author | 曾音筑 | zh_TW |
| dc.contributor.author | Yin-Chu Tseng | en |
| dc.date.accessioned | 2021-05-19T17:45:18Z | - |
| dc.date.available | 2024-02-28 | - |
| dc.date.copyright | 2018-10-09 | - |
| dc.date.issued | 2018 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | 1 Swamy, K. H. & Goldberg, A. L. E. coli contains eight soluble proteolytic activities, one being ATP dependent. Nature 292, 652-654 (1981).
2 Charette, M. F., Henderson, G. W. & Markovitz, A. ATP hydrolysis-dependent protease activity of the lon (capR) protein of Escherichia coli K-12. Proc Natl Acad Sci U S A 78, 4728-4732 (1981). 3 Gur, E. & Sauer, R. T. Recognition of misfolded proteins by Lon, a AAA(+) protease. Genes Dev 22, 2267-2277, doi:10.1101/gad.1670908 (2008). 4 Sauer, R. T. & Baker, T. A. AAA+ proteases: ATP-fueled machines of protein destruction. Annu Rev Biochem 80, 587-612, doi:10.1146/annurev-biochem-060408-172623 (2011). 5 Patterson-Ward, J., Huang, J. & Lee, I. Detection and characterization of two ATP-dependent conformational changes in proteolytically inactive Escherichia coli Lon mutants by stopped flow kinetic techniques. Biochemistry 46, 13593-13605, doi:10.1021/bi701649b (2007). 6 Waxman, L. & Goldberg, A. L. Selectivity of intracellular proteolysis: protein substrates activate the ATP-dependent protease (La). Science 232, 500-503 (1986). 7 Menon, A. S. & Goldberg, A. L. Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP. The Journal of biological chemistry 262, 14929-14934 (1987). 8 Gur, E. & Sauer, R. T. Degrons in protein substrates program the speed and operating efficiency of the AAA+ Lon proteolytic machine. Proc Natl Acad Sci U S A 106, 18503-18508, doi:10.1073/pnas.0910392106 (2009). 9 Wohlever, M. L., Baker, T. A. & Sauer, R. T. Roles of the N domain of the AAA+ Lon protease in substrate recognition, allosteric regulation and chaperone activity. Mol Microbiol 91, 66-78, doi:10.1111/mmi.12444 (2014). 10 Su, S. C. et al. Structural Basis for the Magnesium-Dependent Activation and Hexamerization of the Lon AAA+ Protease. Structure 24, 676-686, doi:10.1016/j.str.2016.03.003 (2016). 11 Hanson, P. I. & Whiteheart, S. W. AAA+ proteins: have engine, will work. Nat Rev Mol Cell Biol 6, 519-529, doi:10.1038/nrm1684 (2005). 12 Rotanova, T. V. et al. Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains. Protein Sci 15, 1815-1828, doi:10.1110/ps.052069306 (2006). 13 Chung, C. H. & Goldberg, A. L. The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La. Proc Natl Acad Sci U S A 78, 4931-4935 (1981). 14 Stahlberg, H. et al. Mitochondrial Lon of Saccharomyces cerevisiae is a ring-shaped protease with seven flexible subunits. P Natl Acad Sci USA 96, 6787-6790, doi:DOI 10.1073/pnas.96.12.6787 (1999). 15 Rudyak, S. G., Brenowitz, M. & Shrader, T. E. Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis. Biochemistry 40, 9317-9323 (2001). 16 Park, S. C. et al. Oligomeric structure of the ATP-dependent protease La (Lon) of Escherichia coli. Mol Cells 21, 129-134 (2006). 17 Kereiche, S. et al. The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease. Sci Rep 6, 33631, doi:10.1038/srep33631 (2016). 18 Waxman, L. & Goldberg, A. L. Protease La from Escherichia-Coli Hydrolyzes Atp and Proteins in a Linked Fashion. P Natl Acad Sci-Biol 79, 4883-4887, doi:DOI 10.1073/pnas.79.16.4883 (1982). 19 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. Journal of Biological Chemistry 279, 8140-8148, doi:10.1074/jbc.M312243200 (2004). 20 Rotanova, T. V. et al. Classification of ATP-dependent proteases Lon and comparison of the active sites of their proteolytic domains. Eur J Biochem 271, 4865-4871, doi:10.1111/j.1432-1033.2004.04452.x (2004). 21 Roudiak, S. G. & Shrader, T. E. Functional role of the N-terminal region of the Lon protease from Mycobacterium smegmatis. Biochemistry 37, 11255-11263, doi:10.1021/bi980945h (1998). 22 Cheng, I. et al. Identification of a Region in the N-Terminus of Escherichia coli Lon That Affects ATPase, Substrate Translocation and Proteolytic Activity. Journal of Molecular Biology 418, 208-225, doi:10.1016/j.jmb.2012.02.039 (2012). 23 Duman, R. E. & Lowe, J. Crystal structures of Bacillus subtilis Lon protease. J Mol Biol 401, 653-670, doi:10.1016/j.jmb.2010.06.030 (2010). 24 Li, M. et al. Structure of the N-terminal fragment of Escherichia coli Lon protease. Acta Crystallogr D Biol Crystallogr 66, 865-873, doi:10.1107/S0907444910019554 (2010). 25 Li, M. et al. Crystal structure of the N-terminal domain of E. coli Lon protease. Protein Sci 14, 2895-2900, doi:10.1110/ps.051736805 (2005). 26 Gur, E. The Lon AAA+ protease. Subcell Biochem 66, 35-51, doi:10.1007/978-94-007-5940-4_2 (2013). 27 Suzuki, C. K. et al. ATP-dependent proteases that also chaperone protein biogenesis. Trends in biochemical sciences 22, 118-123 (1997). 28 Wickner, S., Maurizi, M. R. & Gottesman, S. Posttranslational quality control: folding, refolding, and degrading proteins. Science 286, 1888-1893 (1999). 29 Zehnbauer, B. A., Foley, E. C., Henderson, G. W. & Markovitz, A. Identification and purification of the Lon+ (capR+) gene product, a DNA-binding protein. Proc Natl Acad Sci U S A 78, 2043-2047 (1981). 30 Fu, G. K., Smith, M. J. & Markovitz, D. M. Bacterial protease Lon is a site-specific DNA-binding protein. The Journal of biological chemistry 272, 534-538 (1997). 31 Pinti, M. et al. Mitochondrial Lon protease at the crossroads of oxidative stress, ageing and cancer. Cellular and molecular life sciences : CMLS 72, 4807-4824, doi:10.1007/s00018-015-2039-3 (2015). 32 Justice, S. S., Hunstad, D. A., Cegelski, L. & Hultgren, S. J. Morphological plasticity as a bacterial survival strategy. Nature reviews. Microbiology 6, 162-168, doi:10.1038/nrmicro1820 (2008). 33 Ishii, Y. & Amano, F. Regulation of SulA cleavage by Lon protease by the C-terminal amino acid of SulA, histidine. Biochem J 358, 473-480 (2001). 34 Torres-Cabassa, A. S. & Gottesman, S. Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis. J Bacteriol 169, 981-989 (1987). 35 Ebel, W., Skinner, M. M., Dierksen, K. P., Scott, J. M. & Trempy, J. E. A conserved domain in Escherichia coli Lon protease is involved in substrate discriminator activity. J Bacteriol 181, 2236-2243 (1999). 36 Wohlever, M. L., Baker, T. A. & Sauer, R. T. A mutation in the N domain of Escherichia coli lon stabilizes dodecamers and selectively alters degradation of model substrates. J Bacteriol 195, 5622-5628, doi:10.1128/JB.00886-13 (2013). 37 Adam, C. et al. Biological roles of the Podospora anserina mitochondrial Lon protease and the importance of its N-domain. PLoS One 7, e38138, doi:10.1371/journal.pone.0038138 (2012). 38 Chen, M. Y., Lin, G. H., Lin, Y. T. & Tsay, S. S. Meiothermus taiwanensis sp. nov., a novel filamentous, thermophilic species isolated in Taiwan. Int J Syst Evol Microbiol 52, 1647-1654, doi:10.1099/00207713-52-5-1647 (2002). 39 Lin, C. C. et al. Structural Insights into the Allosteric Operation of the Lon AAA+ Protease. Structure 24, 667-675, doi:10.1016/j.str.2016.03.001 (2016). 40 McCoy, A. J., Fucini, P., Noegel, A. A. & Stewart, M. Structural basis for dimerization of the Dictyostelium gelation factor (ABP120) rod. Nat Struct Biol 6, 836-841, doi:10.1038/12296 (1999). 41 Rutgers, D. o. C. a. C. B. o. High field NMR, <http://chem.rutgers.edu/facilities/biomolecular-nmr/biomolecular-nmr-experiments/573-triple-resonance-experiments-for-backbone-assignments> 42 Phillips, M. Triple Resonance Backbone Assignment: HNCA, <https://sites.google.com/site/nmrgenerator/home> | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7512 | - |
| dc.description.abstract | Protease la又稱為Lon AAA+蛋白酶 (LonA) 是第一個被發現的ATP依賴型蛋白酶，屬於多功能性的蛋白，在進行生物功能時會由單一種單體聚合成多聚體，在古細菌、原核生物及真核生物的胞器中都具有LonA。LonA蛋白酶在生物體中主要藉由分解不正常或是損壞的蛋白進行蛋白質的品質管理，避免廢物在細胞中累積，除此之外，LonA也參與多種生物功能的調控。先前在台灣本土嗜熱菌Meiothermus taiwanensis LonA蛋白酶 (MtaLonA) 的研究中發現，N端功能區與因為溫度影響而造成結構部分展開的受質 (Ig2) 之降解相關，然而對於LonA N端功能區到底如何辨識受質並與受質結合詳細的機制仍不清楚，因此本研究想利用核磁共振實驗以結構生物學的角度探討LonA N端功能區與受質間的交互用關係，藉此了解N端功能區所扮演的角色。在全長的MtaLonA異核核磁共振實驗中，我們得到一個有約兩百個清楚交叉峰的光譜，而這些訊號大致上與MtaLonA N端截短區塊 (NN206) 的交叉峰重疊，顯示MtaLonA的N端功能區以一個較為動態的接鏈和ATPase功能區及蛋白酶功能區連接，使得N端功能區能夠獨立的在環境中擺動，因此我們也得以利用核磁共振實驗觀察N端功能區的化學位移擾動，除此之外，利用多維異核核磁共振實驗，我們完成了NN206約95%的骨架循序判定。將受質加入NN206中並加熱使受質結構逐漸展開後，從核磁共振光譜中可以觀察到NN206的化學位移發生劇烈的改變，而根據NN206的結構及循序判定的結果也發現受到影響的殘基主要位於N端子功能區，我們也進一步對NN206進行點突變，並且發現NN206 N端子功能區暴露於外界環境中的疏水性殘基對於與未折疊的受質結合是必須的。綜合上述結果，本篇實驗發現MtaLonA藉由N端功能區中N端子功能區的疏水性殘基辨識未折疊或聚集的蛋白。 | zh_TW |
| dc.description.abstract | Lon AAA+ protease (LonA), the first ATP-dependent protease to be identified, is a multi-functional, homo-oligomeric enzyme. LonA plays a crucial role in protein quality control and regulation of diverse biological processes in prokaryotes and eukaryotic organelles. Previous studies suggest that N-terminal domain (NTD) of Meiothermus taiwanensis LonA (MtaLonA) is required for a thermally unfolded tandem immunoglobulin substrate from Dictyostelium discoideum (Ig2) binding. However, the structural basis for the substrate recognition mediated by the NTD domain has not been characterized. Here we seek to understand how the NTD of LonA recognizes and traps protein substrates by Nuclear Magnetic Resonance (NMR) spectroscopy. Heteronuclear NMR analyses show 200 well-resolved 15N-1H correlations of full-length hexameric MtaLonA at an apparent molecular weight of 0.5 MDa. Importantly, most of these correlations are superimposable to those observed in the N-terminal truncated form (NN206) spectrum. The results reveal that the NTD is loosely linked to the hexameric core via a flexible linker rendering it possible for detailed chemical shift perturbation mapping of substrate binding in the context of full-length MtaLonA. We also apply multidimensional heteronuclear NMR experiments to assign 95% of the non-proline backbone 1H and 15N resonances of NN206. Moreover, the chemical shifts of NN206 are significantly perturbed by the addition of damaged substrate and structural mapping of the chemical shift perturbations shows that the affected residues are mainly located in the N subdomain. We further show exposed hydrophobic residues are required for interaction with unfolding conformation by mutagenesis. Together, our findings demonstrate that MtaLonA recognizes unfolded or aggregated substrate via exposed hydrophobic residues located in the N subdomain of its NTD. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-19T17:45:18Z (GMT). No. of bitstreams: 1 ntu-107-R05442002-1.pdf: 8297668 bytes, checksum: 936a889fd068b3c30b5130b0b97ffaba (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 目錄
論文口試委員審定書 I 致謝 Ⅱ 中文摘要 IV Abstract V 目錄 VII 圖目錄 X 表目錄 XI 縮寫表 XII 一、 前言 1 1. Lon AAA+蛋白酶 (Lon AAA+ protease, LonA) 1 2. LonA的結構 2 3. LonA的功能 4 4. LonA的受質 5 5. LonA受質的辨識 6 6. MtaLonA (Meiothermus taiwanensis LonA) 7 7. 研究動機 8 二、 實驗材料與方法 9 2.1實驗材料 9 2.2實驗方法 9 2.2.1基因的選殖 9 2.2.2勝任細胞的製備 9 2.2.3細胞轉型 (transformation) 10 2.2.4點突變及選殖 (site directed mutagenesis and cloning) 10 2.2.4.1引子的設計 (primer design) 10 2.2.4.2聚合酶連鎖反應 (polymerase chain reaction, PCR) 10 2.2.4.3 DNA定序 (DNA sequencing) 12 2.2.5重組蛋白的表現及純化 12 2.2.5.1 重組蛋白於LB培養基的表現 12 2.2.5.2 重組蛋白於M9培養基的表現 12 2.2.5.3 重組蛋白的純化—固定化金屬離子親合層析法 (immobilized-metal affinity chromatography, IMAC) 13 2.2.5.4 蛋白質濃縮 13 2.2.5.5 膠體過濾層析 (gel-filtration chromatography) 14 2.2.5.6 蛋白質濃度的測定 15 2.2.6熱轉變分析 (thermal shift assay) 15 2.2.7 蛋白質核磁共振 (protein NMR) 15 2.2.7.1 特定的氨基酸標定策略 (specific amino acid labeled strategy) 16 2.2.7.1.1 15N、13C同位素標定的NN206 (15N,13C dual-labeled NN206) 16 2.2.7.1.2 15N-lysine標定的NN206 (15N-lysine selectively labeled NN206) 16 2.2.7.1.3 14N-arginine反標定的NN206 (14N-arginine selectively reverse labeled NN206) 16 2.2.7.2 核磁共振樣品製備 16 2.2.7.3 NN206骨架 (backbone) 的循序判定 (Sequence specific assignment) 17 2.2.7.4 核磁共振滴定實驗 (NMR titration) 17 2.2.7.5 核磁共振樣品之分析 18 2.2.7.6用核磁共振計算結構正確之Ig2D5 18 三、 實驗結果 19 3.1 重組蛋白的表現及純化 19 3.1.1 LonA的表現及純化 19 3.1.2 NN206的表現及純化 20 3.1.3 Ig2的表現及純化 21 3.2 蛋白質折疊轉變溫度中點的測定 22 3.3 LonA的N端功能區與Ig2之交互作用 22 3.3.1 WT LonA與NN206之二維TROSY-HSQC光譜比較 22 3.3.2 NN206骨架的循序判定 (Sequence specific assignment) 23 3.3.3 NN206與受損的受質 (damaged substrate) 之交互作用 24 3.4 LonA的N端與Ig2D5之交互作用 27 3.4.1 Ig2D5的表現及純化 27 3.4.2 Ig2與Ig2D5之比較 27 3.4.3 NN206與Ig2D5之交互作用 28 3.4.4 LonA N端功能區對於Ig2D5再折疊 (refolding) 的影響 28 3.5 NN206的疏水性胺基酸對於損壞的受質結合之重要性 29 3.5.1 NN206的點突變 (site directed mutagenesis) 29 3.5.2 NN206P22AM85A、NN206P22A及NN206M85的表現及純化 30 3.5.3 NN206P22AM85A與NN206之核磁共振光譜比較 30 3.5.4 NN206P22AM85A與Ig2之交互作用 31 3.5.5 NN206P22AM85A對於Ig2D5再折疊的影響 32 四、 討論 33 圖 36 表 87 參考文獻 93 附錄 97 圖目錄 圖1-1 LonA與LonB的功能區組成 36 圖1-2台灣本土嗜熱菌Meiothermus taiwanensis LonA 37 圖1-3 N端功能區對受質降解之重要性 38 圖1-4 LonA N端功能區之結構 39 圖2-1 核磁共振骨架的循序判定 (NMR backbone assignment) 40 圖3-1 WT LonA的固定化金屬離子親合層析法純化 41 圖3-2 WT LonA之陰離子交換層析法 42 圖3-3 WT LonA之分子篩層析純化 43 圖3-4 NN206的固定化金屬離子親合層析法純化 44 圖3-5 NN206之分子篩層析純化 46 圖3-6 Ig2的固定化金屬離子親合層析法純化 47 圖3-7 Ig2之分子篩層析純化 49 圖3-8 蛋白質熱轉變分析 (thermal shift assay) 50 圖3-9 WT LonA與NN206之二維1H-15N TROSY-HSQC光譜比較 51 圖3-10 NN206之部分 (partial) 循序判定光譜 (residue 42-51) 52 圖3-11 NN206的骨架循序判定 (Sequence specific assignment) 53 圖3-12 NN206的骨架循序判定結果 54 圖3-13 特殊標定的NN206核磁共振光譜 55 圖3-14 Ig2滴定NN206之二維1H-15N TROSY-HSQC光譜 56 圖3-15 Ig2滴定NN206的核磁共振樣品分析 57 圖3-16 WT LonA、Ig2滴定WT LonA與NN206之二維1H-15N TROSY-HSQC光譜比較 59 圖3-17 NN206加入Ig2之化學位移變化 60 圖3-18 Ig2之結構 61 圖3-19 Ig2D5的固定化金屬離子親合層析法純化 62 圖3-20 Ig2D5之分子篩層析純化 64 圖3-21 Ig2與Ig2D5之二維1H-15N TROSY-HSQC光譜比較 65 圖3-22 蛋白質熱轉變分析 66 圖3-23 NN206加入Ig2D5之化學位移變化 67 圖3-24 WT LonA之N端功能區對於Ig2D5結合的影響 68 圖3-25 NN206的點突變 70 圖3-26 NN206P22AM85A的固定化金屬離子親合層析法純化 71 圖3-27 NN206P22AM85A之分子篩層析純化 73 圖3-28 NN206P22A的固定化金屬離子親合層析法純化 74 圖3-29 NN206P22A之分子篩層析純化 76 圖3-30 NN206M85A的固定化金屬離子親合層析法純化 77 圖3-31 NN206M85A之分子篩層析純化 79 圖3-32 NN206與NN206P22AM85的比較 80 圖3-33 Ig2滴定NN206P22AM85A之二維1H-15N TROSY-HSQC光譜 81 圖3-34 NN206P22AM85A加入Ig2之化學位移變化 82 圖3-35 WT LonA之N端功能區對於Ig2D5結合的影響 83 圖3-36 WT LonA之N端功能區點突變對於Ig2D5結合的影響 84 圖4-1 N端功能區對α-casein降解之重要性 85 圖4-2 MtaLonA與EcLon之N端功能區比較 86 表目錄 表2-1 本實驗使用之菌種 87 表2-2 本實驗使用之質體 88 表2-3 本實驗使用之培養基、培養液 89 表2-4 本實驗使用之緩衝溶液 90 表2-5 本實驗使用之引子 91 表3-1 NN206的胺基酸組成 92 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 核磁共振 | zh_TW |
| dc.subject | Ig2 | zh_TW |
| dc.subject | N端功能區 | zh_TW |
| dc.subject | Lon蛋白? | zh_TW |
| dc.subject | Meiothermus taiwanensis | zh_TW |
| dc.subject | N subdomain | en |
| dc.subject | Meiothermus taiwanensis | en |
| dc.subject | LonA | en |
| dc.subject | Ig2 | en |
| dc.subject | NMR | en |
| dc.title | La蛋白酶N端功能區對於受質辨識所扮演的角色 | zh_TW |
| dc.title | Unraveling the roles of the N domain of protease La in substrate recognition | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 106-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 徐駿森;張崇毅 | zh_TW |
| dc.contributor.oralexamcommittee | ;; | en |
| dc.subject.keyword | Meiothermus taiwanensis,Lon蛋白?,N端功能區,Ig2,核磁共振, | zh_TW |
| dc.subject.keyword | Meiothermus taiwanensis,LonA,N subdomain,Ig2,NMR, | en |
| dc.relation.page | 99 | - |
| dc.identifier.doi | 10.6342/NTU201802703 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2018-08-08 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | - |
| dc.date.embargo-lift | 2023-10-09 | - |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-106-2.pdf 未授權公開取用 | 8.1 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。