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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 曾秀如(Shiou-Ru Tzeng) | |
| dc.contributor.author | Chia-Ying Hsu | en |
| dc.contributor.author | 許佳嫈 | zh_TW |
| dc.date.accessioned | 2022-11-25T06:35:00Z | - |
| dc.date.copyright | 2021-11-09 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-09-28 | |
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Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences 79, 4883-4887, doi:DOI 10.1073/pnas.79.16.4883 (1982). 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 279, 8140-8148, doi:10.1074/jbc.m312243200 (2004). 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). Waxman, L. Goldberg, A. L. Selectivity of intracellular proteolysis: protein substrates activate the ATP-dependent protease (La). Science 232, 500-503 (1986). Menon, A. S. Goldberg, A. L. Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP. J Biol Chem 262, 14929-14934 (1987). Gur, E. Sauer, R. T. Degrons in protein substrates program the speed and operating efficiency of the AAA+ Lon proteolytic machine. Proceedings of the National Academy of Sciences 106, 18503, doi:10.1073/pnas.0910392106 (2009). 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). 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). 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). 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). 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). Tsilibaris, V., Maenhaut-Michel, G. Van Melderen, L. Biological roles of the Lon ATP-dependent protease. Research in Microbiology 157, 701-713, doi:https://doi.org/10.1016/j.resmic.2006.05.004 (2006). Gur, E. The Lon AAA+ protease. Subcell Biochem 66, 35-51, doi:10.1007/978-94-007-5940-4_2 (2013). Suzuki, C. K. et al. ATP-dependent proteases that also chaperone protein biogenesis. Trends Biochem Sci 22, 118-123 (1997). Wickner, S., Maurizi, M. R. Gottesman, S. Posttranslational quality control: folding, refolding, and degrading proteins. Science 286, 1888-1893 (1999). Fu, G. K., Smith, M. J. Markovitz, D. M. Bacterial protease Lon is a site-specific DNA-binding protein. J Biol Chem 272, 534-538 (1997). Pinti, M. et al. Mitochondrial Lon protease at the crossroads of oxidative stress, ageing and cancer. Cell Mol Life Sci 72, 4807-4824, doi:10.1007/s00018-015-2039-3 (2015). Pinti, M. et al. Emerging role of Lon protease as a master regulator of mitochondrial functions. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1857, 1300-1306, doi:https://doi.org/10.1016/j.bbabio.2016.03.025 (2016). Wohlever, M. L., Nager, A. R., Baker, T. A. Sauer, R. T. Engineering fluorescent protein substrates for the AAA+ Lon protease. Protein Eng Des Sel 26, 299-305, doi:10.1093/protein/gzs105 (2013). 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). Torres-Cabassa, A. S. Gottesman, S. Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis. J Bacteriol 169, 981-989 (1987). 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). 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). Chin, D. T., Goff, S. A., Webster, T., Smith, T. Goldberg, A. L. Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La. Journal of Biological Chemistry 263, 11718-11728, doi:https://doi.org/10.1016/S0021-9258(18)37843-8 (1988). 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). 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). Tzeng, S. R. et al. Molecular insights into substrate recognition and discrimination by the N-terminal domain of Lon AAA+ protease. Elife 10, doi:10.7554/eLife.64056 (2021). 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). 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). 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). <https://www.ibbr.umd.edu/nmrpipe/install.html> <http://www.onemoonscientific.com> Damberger, F. Keller, R. <http://cara.nmr.ch/doku.php> Otwinowski, Z. Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276, 307-326 (1997). Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr D Biol Crystallogr 64, 61-69, doi:10.1107/s090744490705024x (2008). Fei, X., Bell, T. A., Barkow, S. R., Baker, T. A. Sauer, R. T. Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates. Elife 9, doi:10.7554/eLife.61496 (2020). Rotanova, T. V. et al. New insights into structural and functional relationships between LonA proteases and ClpB chaperones. FEBS Open Bio 9, 1536-1551, doi:10.1002/2211-5463.12691 (2019). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82285 | - |
| dc.description.abstract | 隆蛋白酶為一種腺苷三磷酸依賴型蛋白酶 (ATP-dependent protease)，隸屬於AAA+蛋白家族。隆蛋白酶除了具有蛋白酶的活性之外，還有伴護蛋白的功能。隆蛋白酶主要存在於原核生物的細胞內及真核生物的胞器中，透過降解失去正確構型的蛋白來參與細胞的蛋白質品質管理，隆蛋白酶也會透過降解帶有降解標籤的蛋白，來進行細胞生理反應的調控。隆蛋白酶的結構包含與受質辨識相關的胺基端功能區、負責腺苷三磷酸 (ATP) 水解的腺苷三磷酸酶 (ATPase) 功能區及蛋白酶功能區。先前研究利用蛋白質交聯 (cross-linking) 實驗證明大腸桿菌隆蛋白酶 (EcLonA) 需要透過其胺基端功能區來與降解標籤結合，然而目前尚無結構上的證據可以說明隆蛋白酶的胺基端功能區與降解標籤之間的交互作用。本研究利用核磁共振實驗來探討隆蛋白酶與細胞分裂抑制劑SulA的羧基端末20個胺基酸 (Sul20降解標籤) 的交互作用。首先我們以受質降解實驗證明隆蛋白酶需要透過胺基端功能區的辨識，才能降解攜帶降解標籤的蛋白受質。透過分析核磁共振光譜的變化，我們發現隆蛋白酶的胺基端功能區主要會透過其胺基端子功能區與降解標籤結合，而降解標籤上的芳香族胺基酸及疏水性胺基酸也被證實在該反應中扮演重要的角色。接著我們分別表現攜帶不同長度降解標籤的蛋白受質進行受質降解實驗，來探討降解標籤的長度是否會影響其促進隆蛋白酶降解受質的能力，結果顯示Sul20降解標籤的羧基端5個胺基酸對促進隆蛋白酶之降解速率相當重要，同時我們也發現降解標籤的長度亦會對隆蛋白酶之降解效率有一定的影響。最後我們希望透過共結晶與配體浸泡實驗，更直接地看見隆蛋白酶的胺基端功能區與合成之降解標籤胜肽的複合體結晶結構，然而截至目前為止都沒有理想的結果，其原因大致可歸納如下，分別是晶體在浸泡過程中破裂、沒破裂的晶體馬賽克性 (mosaicity) 過高或解析度過低。綜合以上，本研究發現隆蛋白酶主要透過其胺基端功能區上的胺基端子功能區與降解標籤進行交互作用，而降解標籤上的疏水性胺基酸及芳香族胺基酸亦在該作用中扮演重要的角色。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T06:35:00Z (GMT). No. of bitstreams: 1 U0001-2709202112000700.pdf: 6399406 bytes, checksum: 0d27840482837ae0386f0a4c7386b977 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "口試委員審定書 I 致謝 II 中文摘要 IV Abstract VI 目錄 VIII 圖目錄 XI 表目錄 XIII 縮寫表 XIV 一、 前言 1 1. 隆蛋白酶 (Lon protease) 1 2. LonA的結構 1 3. LonA的功能 3 4. LonA的受質 4 5. LonA對受質的辨識 5 6. 台灣本土嗜熱菌 (Meiothermus taiwanensis) LonA (MtaLonA) 6 7. 研究動機 7 二、 實驗材料與方法 9 2.1實驗材料 9 2.1.1 生物材料與試劑 9 2.2實驗方法 9 2.2.1質體建構 9 2.2.2細胞轉型 (transformation) 10 2.2.3基因點突變與缺失突變 (site directed mutagenesis and deletion mutagenesis) 10 2.2.3.1引子設計 (primer design) 10 2.2.3.2聚合酶連鎖反應 (polymerase chain reaction, PCR) 11 2.2.3.3 DNA定序 (DNA sequencing) 12 2.2.4蛋白的表現與純化 12 2.2.4.1 蛋白於LB培養基之大量表現 12 2.2.4.2 蛋白於M9培養基之大量表現 12 2.2.4.3 固定化金屬離子親合層析法 (immobilized-metal affinity chromatography, IMAC) 13 2.2.4.4 蛋白質濃縮 14 2.2.4.5 膠體過濾層析 (gel-filtration chromatography) 15 2.2.4.6 蛋白質定量 15 2.2.5 受質降解試驗 (substrate degradation assay) 16 2.2.6 蛋白質核磁共振 (protein NMR) 16 2.2.6.1 胺基酸標定 (specific amino acid labeled strategy) 17 2.2.6.2 核磁共振實驗之蛋白樣品製備 17 2.2.6.3 Ig2D5骨架 (backbone) 的循序判定 (Sequence specific assignment) 18 2.2.6.4 核磁共振滴定實驗 (NMR titration) 19 2.2.6.5 NN206滴定Sul20胜肽前後化學位移擾動 (chemical shift perturbation) 定量 19 2.2.6.6利用核磁共振光譜計算結構正確之Ig2D5、Ig2D5-S20 20 2.2.7 蛋白質結晶 20 2.2.7.1 NN206蛋白質結晶實驗 20 2.2.7.2 NN206蛋白質共結晶 (co-crystallization) 實驗 21 2.2.7.3 NN206晶體之配體浸泡 21 2.2.7.4 蛋白質晶體X-ray繞射實驗 21 三、 結果 22 3.1 蛋白的表現與純化 22 3.1.1 MtaLonA WT的表現與純化 22 3.1.2 AAAP的表現與純化 22 3.1.3 NN206的表現及純化 23 3.1.4 Ig2D5的表現與純化 23 3.1.5 Ig2D5-S20的表現與純化 24 3.2 Ig2D5-S20與Ig2D5之結構差異 24 3.3 MtaLonA的胺基端功能區與Sul20的交互作用 24 3.3.1 MtaLonA與AAAP降解Ig2D5及Ig2D5-S20的效率比較 25 3.3.2 NN206與Sul20之間的交互作用 25 3.3.3 Ig2D5-S20-Y19A/H20A的表現與純化 26 3.3.4 MtaLonA降解Ig2D5-S20以及Ig2D5-S20-Y19A/H20A的效率比較 26 3.3.5 NN206與Ig2D5-S20及Ig2D5-S20-Y19A/H20A交互作用的比較 26 3.4 Sul20降解標籤對MtaLonA降解效率的影響 27 3.4.1 Ig2D5骨架的循序判定 27 3.4.2 MtaLonA降解Ig2D5及Ig2D5-S20的速率差異 28 3.5 Sul20序列的長度對LonA降解受質的效率影響 29 3.5.1 Ig2D5-S5、Ig2D5-S15的表現與純化 29 3.5.2 MtaLonA降解Ig2D5、Ig2D5-S5、Ig2D5-S15及Ig2D5-S20的速率比較 30 3.5.3 Ig2D5-S20-Δ1-5、Ig2D5-S20-Δ1-10、Ig2D5-S20-Δ1-15的表現與純化 31 3.5.4 MtaLonA降解Ig2D5-S20-Δ1-5、Ig2D5- S20-Δ1-10及Ig2D5- S20-Δ1-15的效率比較 32 3.6 NN206蛋白質結晶 33 3.6.1 NN206晶體之配體浸泡結果 33 3.6.2 NN206共結晶實驗 34 四、討論 35 圖 39 表 96 參考文獻 104 附錄 108" | |
| dc.language.iso | zh-TW | |
| dc.subject | 降解標籤 | zh_TW |
| dc.subject | 核磁共振 | zh_TW |
| dc.subject | 芳香族胺基酸及疏水性胺基酸 | zh_TW |
| dc.subject | 胺基端功能區 | zh_TW |
| dc.subject | 隆蛋白酶 | zh_TW |
| dc.subject | hydrophobic and aromatic residues | en |
| dc.subject | MtaLonA | en |
| dc.subject | N-terminal domain | en |
| dc.subject | degron | en |
| dc.subject | magnetic resonance spectroscopy | en |
| dc.title | 探討La蛋白酶胺基端功能區與降解標籤的交互作用 | zh_TW |
| dc.title | Investigating the interaction between degrons and N-terminal domain of Lon AAA+ protease | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 詹迺立(Hsin-Tsai Liu),徐駿森(Chih-Yang Tseng) | |
| dc.subject.keyword | 隆蛋白酶,胺基端功能區,降解標籤,核磁共振,芳香族胺基酸及疏水性胺基酸, | zh_TW |
| dc.subject.keyword | MtaLonA,N-terminal domain,degron,magnetic resonance spectroscopy,hydrophobic and aromatic residues, | en |
| dc.relation.page | 109 | |
| dc.identifier.doi | 10.6342/NTU202103386 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2021-09-29 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2026-09-27 | - |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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