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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張崇毅(Chung-I Chang) | |
| dc.contributor.author | Hui-Hsin Shih | en |
| dc.contributor.author | 施惠心 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:00:34Z | - |
| dc.date.available | 2022-08-01 | |
| dc.date.copyright | 2019-08-27 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-08-24 | |
| dc.identifier.citation | 1. Bukau, B., Weissman, J., and Horwich, A. (2006). Molecular chaperones and protein quality control. Cell 125, 443-451.
2. Neuwald, A.F., Aravind, L., Spouge, J.L., and Koonin, E.V. (1999). AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res 9, 27-43. 3. Wlodawer, A., and Gustchina, A. (2004). Classification of ATP-dependent proteases Lon and comparison of the active sites of their proteolytic domains. Eur J Biochem 271, 4865-4871. 4. Sauer, R.T., and Baker, T.A. (2011). AAA+ proteases: ATP-fueled machines of protein destruction. Annu Rev Biochem 80, 587-612. 5. Wlodawer, A. (2006). Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains. Protein Sci 15, 1815-1828. 6. Maehara, T., Hoshino, T., and Nakamura, A. (2008). Characterization of three putative Lon proteases of Thermus thermophilus HB27 and use of their defective mutants as hosts for production of heterologous proteins. Extremophiles 12, 285-296. 7. Venkatesh, S., Lee, J., Singh, K., Lee, I., and Suzuki, C.K. (2012). Multitasking in the mitochondrion by the ATP-dependent Lon protease. Biochim Biophys Acta 1823, 56-66. 8. Aksam, E.B., Koek, A., Kiel, J.A., Jourdan, S., Veenhuis, M., and van der Klei, I.J. (2007). A peroxisomal lon protease and peroxisome degradation by autophagy play key roles in vitality of Hansenula polymorpha cells. Autophagy 3, 96-105. 9. Adam, Z., Adamska, I., Nakabayashi, K., Ostersetzer, O., Haussuhl, K., Manuell, A., Zheng, B., Vallon, O., Rodermel, S.R., Shinozaki, K., et al. (2001). Chloroplast and mitochondrial proteases in Arabidopsis. A proposed nomenclature. Plant Physiol 125, 1912-1918. 10. Gur, E., and Sauer, R.T. (2008). Recognition of misfolded proteins by Lon, a AAA(+) protease. Genes Dev 22, 2267-2277. 11. Gur, E., and Sauer, R.T. (2009). 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. 12. Cheng, I., Mikita, N., Fishovitz, J., Frase, H., Wintrode, P.L., and Lee, I. (2012). Identification of a Region in the N-Terminus of Escherichia coli Lon That Affects ATPase, Substrate Translocation and Proteolytic Activity. J Mol Biol 418, 208-225. 13. Su, S.C., Lin, C.C., Tai, H.C., Chang, M.Y., Ho, M.R., Babu, C.S., Liao, J.H., Wu, S.H., Chang, Y.C., Lim, C., et al. (2016). Structural Basis for the Magnesium-Dependent Activation and Hexamerization of the Lon AAA+ Protease. Structure 24, 676-686. 14. Botos, I., Melnikov, E.E., Cherry, S., Tropea, J.E., Khalatova, A.G., Rasulova, F., Dauter, Z., Maurizi, M.R., Rotanova, T.V., Wlodawer, A., et al. (2004). 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. 15. Kisselev, A.F., Akopian, T.N., Woo, K.M., and Goldberg, A.L. (1999). The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes - Implications for understanding the degradative mechanism and antigen presentation. J Biol Chem 274, 3363-3371. 16. Mia Shin, A.A., Cristina Puchades, Eric Adjei , R. Luke Wiseman, A. Wali Karzai, Gabriel C. Lander (2019). Distinct Structural Features of the Lon Protease Drive Conserved Hand-over-Hand Substrate Translocation. In BioRxiv. doi: 10.1101/617159. 17. Pinti, M., Gibellini, L., Nasi, M., De Biasi, S., Bortolotti, C.A., Iannone, A., and Cossarizza, A. (2016). Emerging role of Lon protease as a master regulator of mitochondrial functions. Bba-Bioenergetics 1857, 1300-1306. 18. Bernstein, S.H., Venkatesh, S., Li, M., Lee, J., Lu, B., Hilchey, S.P., Morse, K.M., Metcalfe, H.M., Skalska, J., Andreeff, M., et al. (2012). The mitochondrial ATP-dependent Lon protease: a novel target in lymphoma death mediated by the synthetic triterpenoid CDDO and its derivatives. Blood 119, 3321-3329. 19. Hu, J., Bianchi, F., Ferguson, M., Cesario, A., Margaritora, S., Granone, P., Goldstraw, P., Tetlow, M., Ratcliffe, C., Nicholson, A.G., et al. (2005). Gene expression signature for angiogenic and nonangiogenic non-small-cell lung cancer. Oncogene 24, 1212-1219. 20. Kita, K., Suzuki, T., and Ochi, T. (2012). Diphenylarsinic acid promotes degradation of glutaminase C by mitochondrial Lon protease. J Biol Chem 287, 18163-18172. 21. Fukuda, R., Zhang, H., Kim, J.W., Shimoda, L., Dang, C.V., and Semenza, G.L. (2007). HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells. Cell 129, 111-122. 22. Hori, O., Ichinoda, F., Tamatani, T., Yamaguchi, A., Sato, N., Ozawa, K., Kitao, Y., Miyazaki, M., Harding, H.P., Ron, D., et al. (2002). Transmission of cell stress from endoplasmic reticulum to mitochondria: enhanced expression of Lon protease. J Cell Biol 157, 1151-1160. 23. Koumenis, C. (2006). ER stress, hypoxia tolerance and tumor progression. Curr Mol Med 6, 55-69. 24. Bota, D.A., Ngo, J.K., and Davies, K.J.A. (2005). Downregulation of the human Lon protease impairs mitochondrial structure and function and causes cell death. Free Radical Bio Med 38, 665-677. 25. Bota, D.A., and Davies, K.J. (2002). Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism. Nat Cell Biol 4, 674-680. 26. Kirstein, J., Moliere, N., Dougan, D.A., and Turgay, K. (2009). Adapting the machine: adaptor proteins for Hsp100/Clp and AAA+ proteases. Nat Rev Microbiol 7, 589-599. 27. Kirstein, J., Schlothauer, T., Dougan, D.A., Lilie, H., Tischendorf, G., Mogk, A., Bukau, B., and Turgay, K. (2006). Adaptor protein controlled oligomerization activates the AAA+ protein ClpC. EMBO J 25, 1481-1491. 28. Zhou, Y., and Gottesman, S. (1998). Regulation of proteolysis of the stationary-phase sigma factor RpoS. J Bacteriol 180, 1154-1158. 29. Skorupski, K., Tomaschewski, J., Ruger, W., and Simon, L.D. (1988). A Bacteriophage-T4 Gene Which Functions to Inhibit Escherichia-Coli Lon Protease. Journal of Bacteriology 170, 3016-3024. 30. Hilliard, J.J., Maurizi, M.R., and Simon, L.D. (1998). Isolation and characterization of the phage T4 PinA protein, an inhibitor of the ATP-dependent lon protease of Escherichia coli. J Biol Chem 273, 518-523. 31. Zehnbauer, B.A., Foley, E.C., Henderson, G.W., and Markovitz, A. (1981). Identification and purification of the Lon+ (capR+) gene product, a DNA-binding protein. Proc Natl Acad Sci U S A 78, 2043-2047. 32. Matsushima, Y., Goto, Y., and Kaguni, L.S. (2010). Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM). Proc Natl Acad Sci U S A 107, 18410-18415. 33. Jonas, K., Liu, J., Chien, P., and Laub, M.T. (2013). Proteotoxic stress induces a cell-cycle arrest by stimulating Lon to degrade the replication initiator DnaA. Cell 154, 623-636. 34. Jing Liu, L.F., Peter Chien (2018). Lon recognition of the replication initiator DnaA is not confined to a single degron.The bioRxiv. doi: 10.1101/301655. 35. Rilee D. Zeinert, J.L., Qiyuan Yang, Yunguang Du, Cole M. Haynes, Peter Chien (2018). A legacy role for DNA binding of Lon protects against genotoxic stress. . The bioRxiv. doi: 10.1101/317677. 36. Lee, A.Y.L., Chen, Y.D., Chang, Y.Y., Lin, Y.C., Chang, C.F., Huang, S.J., Wu, S.H., and Hsu, C.H. (2014). Structural basis for DNA-mediated allosteric regulation facilitated by the AAA(+) module of Lon protease. Acta Crystallogr D 70, 218-230. 37. Lee, A.Y., Hsu, C.H., and Wu, S.H. (2004). 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 279, 34903-34912. 38. Karlowicz, A., Wegrzyn, K., Gross, M., Kaczynska, D., Ropelewska, M., Siemiatkowska, M., Bujnicki, J.M., and Konieczny, I. (2017). Defining the crucial domain and amino acid residues in bacterial Lon protease for DNA binding and processing of DNA-interacting substrates. J Biol Chem 292, 7507-7518. 39. Fu, G.K., Smith, M.J., and Markovitz, D.M. (1997). Bacterial protease lon is a site-specific DNA-binding protein. J Biol Chem 272, 534-538. 40. Fu, G.K., and Markovitz, D.M. (1998). The human LON protease binds to mitochondrial promoters in a single-stranded, site-specific, strand-specific manner. Biochemistry 37, 1905-1909. 41. Lu, B., Liu, T., Crosby, J.A., Thomas-Wohlever, J., Lee, I., and Suzuki, C.K. (2003). The ATP-dependent Lon protease of Mus musculus is a DNA-binding protein that is functionally conserved between yeast and mammals. Gene 306, 45-55. 42. Bittner, L.M., Arends, J., and Narberhaus, F. (2016). Mini review: ATP-dependent proteases in bacteria. Biopolymers 105, 505-517. 43. Lin, C.C., Su, S.C., Su, M.Y., Liang, P.H., Feng, C.C., Wu, S.H., and Chang, C.I. (2016). Structural Insights into the Allosteric Operation of the Lon AAA plus Protease. Structure 24, 667-675. 44. Reece, J.B., Urry, L.A., Cain, M.L., Wasserman, S.A., Minorsky, P.V., and Jackson, R.B. (2014). Campbell biology (Pearson Boston). 45. Robert K. Murray, D.A.B., Kathleen M. Botham, Peter J. Kennelly, Victor W. Rodwell,P. Anthony Weil (2009). Harper’s Illustrated Biochemistry, 29th Edition. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78501 | - |
| dc.description.abstract | Lon是高度保留存在於各個生物體中的蛋白酶，協助蛋白質的代謝及調控、維持DNA穩定正常表現，屬於AAA+ 蛋白酶家族。此蛋白酶家族共有的特徵是需要依賴ATP結合與水解，產生的能量讓蛋白酶構型改變並將蛋白質去折疊，構型改變會產生拉力將蛋白質運送至蛋白酶區域進行降解，此區域在有ATP結合情況下，構型上呈現封閉的腔體 (chamber)，避免進行非特異性結合。之前在研究LonA特殊生化學模式，MtaLonA K361A長晶時無添加ATP，但加入鎂離子時，結構中在ATP結合位發現丙二酸 (malonate)。丙二酸為長晶條件下的添加物，MtaLonA此時的結構也呈現封閉的腔體。丙二酸結構與檸檬酸循環代謝中間物琥珀酸 (succinate) 相似，與琥珀酸競爭琥珀酸去氫酶，進一步抑制其活性。因此，研究假設檸檬酸循環中代謝中間物會調節LonA的活性，LonA形成無活性的封閉狀態，研究也進一步發現部分檸檬酸循環代謝中間物會抑制LonA降解蛋白質的活性，接著進行高通量熱穩定測試 (ThermoFluor) 實驗，以確認結合強度。已有研究指出T4噬菌體利用蛋白酶抑制劑，比如PinA抑制大腸桿菌中EcLonA降解蛋白質的活性，但保留胜肽酶的活性。當LonA截去N端時，蛋白質水解活性降低，推測PinA結合LonA的N端從而抑制LonA的活性。蛋白質體外結合實驗 (protein pull-down assay) 及微量熱泳動技術同時證實PinA結合LonA的N端 (N210及N245)。DNA也被證實具調控LonA的活性，並且促進LonA降解DNA結合蛋白的活性，這些蛋白會結合在LonA的其他相鄰區域。LonA的N端已被證實會參與受質的辨識及結合，微量熱泳動技術證實DNA結合在Lon的核心區域 (3HAAAP)。綜合上述，我們從生物物理的角度去探討這些LonA調節因子的結合位，從體外實驗去推測細胞在生理狀況下如何調控LonA的活性。 | zh_TW |
| dc.description.abstract | Lon protease is a member of AAA+ protein family. Lon protease is an ATP-dependent homo-oligomeric protease which carries both ATPase and protease activities and is well known for its role in cellular functions like protein quality control and metabolic regulation. Besides, DNA binding of Lon is critical for DNA damage tolerance. In E. coli, proteolysis of abnormal proteins is inhibited when the bacteria are infected with bacteriophage T4. Previous studies have shown that the bacteriophage T4 protein PinA (Proteolysis inhibition A) inhibits the Lon protease activity without affecting the peptidase activities, but the mechanism remains unclear. PinA causes similar reduction of protease activity to N-terminal truncation of Lon, indicating that PinA may inhibit Lon via interaction with the N-terminal region. In my study, PinA and E. coli Lon protease (EcLonA) were purified with or without the N-terminal region for several protein-protein interaction assays. Pull-down and microscale thermophoresis (MST) assays were used to confirm the interaction between PinA and two N-terminal constructs of EcLonA (N245 and N210). In vitro, DNA can directly regulate Lon activity and control degradation of DNA bound proteins by adjacent bound Lon. The N-terminal domain is proposed to be involved in binding and recognition of substrate proteins. MST assays revealed the interaction between the core region (3HAAAP) of EcLonA and DNA, as it turned out. In previous study, we discovered that malonate bound with Lon at ATP binding site in the structure of MtaLonA K361A. Malonate is a competitive inhibitor of succinate dehydrogenase which has similar shape to a TCA cycle metabolic intermediate succinate. We further found that some intermediates inhibited degradation activity of Lon protease. The binding affinities between EcLonA (3HAAA) and these intermediates were determined in ThermoFluor. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:00:34Z (GMT). No. of bitstreams: 1 ntu-108-R06b46021-1.pdf: 5290103 bytes, checksum: ebe31a0c67d398d66db24c117276ede6 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 目錄
口試委員審定書 I 中文摘要 II 英文摘要 III 目錄 IV 圖目錄 VII 表目錄 IX 縮寫字對照表 X 第一章 緒論 1-1 蛋白質品質的控制 1 1-2 AAA+ 蛋白酶家族 1 1-3 Lon蛋白酶的分類 2 1-4 LonA蛋白酶的結構 2 1-5 LonA蛋白酶的功能 4 1-6 LonA蛋白酶表現量改變的影響 4 1-7 研究動機 5 1.7.1 LonA蛋白酶與檸檬酸循環代謝中間物 5 1.7.2 LonA蛋白酶與PinA 6 1.7.3 LonA蛋白酶與DNA 7 第二章 材料與方法 2-1 實驗材料 21 2.1.1 實驗儀器 21 2.1.2 實驗藥品 23 2.1.3 實驗試劑 24 2-2 實驗流程 27 2-3 實驗方法 27 2.3.1 基因來源及質體建構 27 2.3.2 抽取質體DNA與轉殖 29 2.3.3 質體DNA表現 30 2.3.3.1 Heat shock轉型作用 (Transformation) 30 2.3.3.2 小量表現 30 2.3.3.3 大量表現 30 2.3.4 蛋白質純化 30 2.3.4.1 親和性鎳離子螯合樹脂管柱層析 31 2.3.4.2 陰離子交換層析 (Ion exchange chromatography) 32 2.3.4.3 分子篩選層析 (Size-exclusion chromatography) 32 2.3.5 蛋白質濃縮 33 2.3.6 電泳分析 (SDS-PAGE) 34 2.3.7 高通量熱穩定測試實驗 (ThermoFluor) 34 2.3.8 蛋白質體外結合實驗 (Pull-down assay) 34 2.3.9 DNA黏合 (DNA annealing) 35 2.3.10 微量熱泳動技術 (Microscale thermophoresis, MST) 35 2.3.11 等溫滴定熱量儀 (Isothermal Titration Calorimetry, ITC) 36 2.3.12分子結合情形並純化兩分子結合複合體:分子篩選層析分析 37 2.3.13蛋白質結晶 (Protein crystallization) 條件篩選 37 第三章 實驗結果 3-1 Lon蛋白酶與檸檬酸循環代謝中間物 38 3.1.1 蛋白質純化結果 38 3.1.2 丙二酸與MtaLonA K361A結合的發現 38 3.1.3 檸檬酸循環代謝中間物抑制Lon的活性 38 3.1.4 檸檬酸循環代謝中間物與Lon的結合強度 39 3.1.4.1 高通量熱穩定測試實驗 39 3.1.4.2 等溫滴定熱量儀 39 3.1.4.3 微量熱泳動技術 40 3-2 Lon蛋白酶與PinA 40 3.2.1 蛋白質純化結果 40 3.2.2 分析PinA是否與LonA結合 41 3.2.2.1 分子篩選層析分析 41 3.2.2.2 蛋白質體外結合實驗 41 3.2.3 分析PinA與LonA結合強度 42 3.2.3.1 微量熱泳動技術 42 3.2.4 長晶條件篩選結果 42 3-3 Lon蛋白酶與DNA 42 3.3.1 蛋白質純化結果 42 3.3.2 分析DNA與LonA結合強度 43 3.3.2.1 微量熱泳動技術 43 第四章 討論與結論 4-1 Lon蛋白酶與檸檬酸循環代謝中間物 63 4-2 Lon蛋白酶與PinA 63 4-3 Lon蛋白酶與DNA 64 參考文獻 65 圖目錄 第一章 圖1-1、蛋白質品質控制系統 (Protein quality control system) 9 圖1-2、AAA+ 模組基本架構 10 圖1-3、ATP循環驅動AAA+ 蛋白酶水解蛋白質的機制 11 圖1-4、在細菌中AAA+ 蛋白酶家族組成 12 圖1-5、LonA、LonB、LonC架構圖比較 14 圖1-6、ClpC經由MecA活化而聚合 15 圖1-7、在人類的粒線體中Lon蛋白酶的功能 16 圖1-8、EM分析Lon與ATP結合後構型的改變 17 圖1-9、檸檬酸循環與丙二酸 18 圖1-10、噬菌體複製週期 19 圖1-11、DNA扮演支架促進Lon降解DnaA 20 第三章 圖3-1、His-tag EcLonA 3HAAA純化結果 44 圖3-2、MtaLonA K361A-丙二酸複合體之結構示意圖 45 圖3-3、檸檬酸循環代謝中間物抑制HuLonA及EcLonA活性的IC50分析 46 圖3-4、檸檬酸循環代謝中間物與EcLonA 3HAAA高通量熱穩定測試實驗結果 47 圖3-5、烏頭酸、檸檬酸與EcLonA 3HAAA高通量熱穩定測試實驗結果 48 圖3-6、丙二酸、檸檬酸與EcLonA的緩衝液進行ITC實驗 49 圖3-7、LonA與檸檬酸MST實驗結果 50 圖3-8、EcLonA 3HAAA與檸檬酸MST實驗結果 51 圖3-9、His-tag MtaLonA N206純化結果 52 圖3-10、MtaLonA N206與PinA分子篩選層析分析結果 53 圖3-11、His-tag EcLonA N245純化結果 54 圖3-12、EcLonA N245與PinA分子篩選層析分析結果 55 圖3-13、EcLonA N210與PinA蛋白質體外結合實驗結果 56 圖3-14、EcLonA N210、EcLonA N245與PinA MST實驗結果 57 圖3-15、EcLonA N245-PinA複合體晶體篩選結果 58 圖3-16、五種片段長度的His-tag EcLonA純化結果 59 圖3-17、EcLonA與DNA MST實驗結果 61 圖3-18、緩衝液加入鎂離子後EcLonA與DNA MST實驗結果 62 表目錄 表2-1、使用儀器設備一覽表 21 表2-2、使用實驗藥品一覽表 23 表2-3、使用生化試劑一覽表 24 表2-4、使用緩衝液一覽表 26 表2-5、PCR反應步驟 29 | |
| dc.language.iso | zh-TW | |
| dc.title | Lon蛋白酶與調節因子的生物物理特性之分析 | zh_TW |
| dc.title | Biophysical characterization between the Lon AAA+ protease and its regulatory factor | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王彥士,徐駿森 | |
| dc.subject.keyword | 蛋白?,Lon,噬菌體,PinA,DNA,檸檬酸循環, | zh_TW |
| dc.subject.keyword | Protease,Lon,Bacteriophage,PinA,DNA,TCA cycle, | en |
| dc.relation.page | 70 | |
| dc.identifier.doi | 10.6342/NTU201903866 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-08-26 | |
| 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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