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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 袁小琀(Hanna S. Yuan) | |
dc.contributor.author | Kuan-Wei Huang | en |
dc.contributor.author | 黃冠偉 | zh_TW |
dc.date.accessioned | 2021-05-16T16:20:18Z | - |
dc.date.available | 2015-12-31 | |
dc.date.available | 2021-05-16T16:20:18Z | - |
dc.date.copyright | 2013-09-24 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-05 | |
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Yan, N., Cherepanov, P., Daigle, J.E., Engelman, A., and Lieberman, J. (2009). The SET complex acts as a barrier to autointegration of HIV-1. Plos Pathog 5, e1000327. 32. Yan, N., and Lieberman, J. (2011). Gaining a foothold: how HIV avoids innate immune recognition. Current opinion in immunology 23, 21-28. 33. Yan, N., Regalado-Magdos, A.D., Stiggelbout, B., Lee-Kirsch, M.A., and Lieberman, J. (2010). The cytosolic exonuclease TREX1 inhibits the innate immune response to human immunodeficiency virus type 1. Nature immunology 11, 1005-1013. 34. Yang, W. (2011). Nucleases: diversity of structure, function and mechanism. Quarterly reviews of biophysics 44, 1-93. 35. Zuo, Y., and Deutscher, M.P. (2002). The physiological role of RNase T can be explained by its unusual substrate specificity. The Journal of biological chemistry 277, 29654-29661. 36. Zuo, Y., Zheng, H., Wang, Y., Chruszcz, M., Cymborowski, M., Skarina, T., Savchenko, A., Malhotra, A., and Minor, W. (2007). 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6065 | - |
dc.description.abstract | DnaQ核酸水解外切酶家族由超過萬種蛋白質成員組成,廣泛分布於原核與真核生物中。此家族成員具有一高度保留性3端至5端核酸水解外切酶結構區域,含有5個高度保留性胺基酸。此類核酸水解蛋白質在細胞中主要參與DNA或RNA的代謝,例如DNA的複製,DNA的修復、DNA的降解,或RNA成熟修飾過程。近來研究發現,部分病毒會利用DnaQ核酸水解外切酶家族成員,來幫助自身感染宿主細胞。故篩選抑制劑來抑制DnaQ核酸水解酶的活性,可能有助於抗病毒感染藥物的研發。
本論文使用DnaQ核酸水解外切酶家族成員之一的CRN-4與RNase T做為模型,用以篩選11種可能的核酸水解酶抑制劑。CRN-4核酸水解外切酶的活性實驗結果顯示,相對於其他9種可能的抑制劑而言,4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoicacid 與 5,5'-dithiobis(2-nitrobenzoate) 具有相對較好的抑制效果。而在 RNase T核酸水解外切酶活性實驗中,則是p-chloromercuriphenyl sulfonate 與Aurintricarboxylic acid 具有相對較好的抑制效果。此外根據CRN-4和2-(N-morpholino)ethanesulfonic acid的複合物之晶體結構,顯示出此化合物結合在CRN-4的活性中心位置,造成CRN-4活性中心發生構形變化,而導致CRN-4的核酸水解外切酶活性受到抑制。我們的研究結果顯示篩選的部分化合物,的確可以抑制DnaQ核酸水解外切酶家族成員的活性,然而這些化合物的抑制效果與專一性需要再加改善。 | zh_TW |
dc.description.abstract | The family of the DnaQ-like exonucleases contains more than ten thousand members widely distributed in prokaryotes and eukaryotes. These exonucleases all contain a highly conserved DEDDh domain with four acidic residues for metal ion binding and one general base residue in the active site. Members in this family play key roles in DNA or RNA metabolism, such as proofreading in DNA replication, DNA processing in DNA repair, DNA degradation in apoptosis and RNA processing in RNA maturation. Recent studies show that several exonucleases in this superfamily are important for viral infections. It is thus important to identify inhibitors for this family of nucleases that may be helpful for the development of anti-viral agents.
Here using CRN-4 and RNase T, members of DnaQ-like exonuclease, as the model system, we screened 11 inhibitor candidates. We found that two compounds, 4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)- benzoicacid and 5,5'-dithiobis(2-nitrobenzoate), could weakly inhibit the exonuclease activity of CRN-4, whereas two compounds, p-chloromercuriphenyl sulfonate and aurintricarboxylic acid, could strongly inhibit the exonuclease activity of RNase T. Moreover, we co-crystallized CRN-4 with one of the weak inhibitors, 2-(N-morpholino)ethanesulfonic acid (MES). The crystal structure of CRN-4 in complex with MES shows that MES was bound in the active site and the general base His179 was flipped out of the active site. In summary, we identified potential inhibitors for the DnaQ-like exonucleases; however, the inhibition activity and specificity of these compounds need to be further improved. | en |
dc.description.provenance | Made available in DSpace on 2021-05-16T16:20:18Z (GMT). No. of bitstreams: 1 ntu-102-R00442026-1.pdf: 3959501 bytes, checksum: e24ba52502add4c42b642f0c7f8364b7 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 目錄
中文摘要 1 英文摘要 2 壹、 序論 一、 DnaQ核酸水解外切酶家族 3 二、 DnaQ核酸水解外切酶家族在細胞內所扮演的功能 4 三、 部分病毒利用不同DnaQ核酸水解外切酶家族協助感染宿主 6 四、 研究目的 7 貳、 實驗材料方法 一、 質體及其建構 8 二、 蛋白質表現及純化 8 1. 細菌菌株 8 2. 線蟲CRN-4蛋白質小量表現測試 8 3. 線蟲CRN-4蛋白質大量表現及純化 9 4. 大腸桿菌RNase T蛋白質大量表現及純 11 三、 Thrombin活性測試 12 四、 蛋白質膠體電泳法 12 五、 西方點墨法 12 六、 5端γ-32P放射性同位素標定 13 七、 去氧核醣核酸水解酶活性分析 13 八、 蛋白質結晶與X光繞射數據收集 14 參、 實驗結果 一、 線蟲CRN-4蛋白質小量表現測試 16 二、 線蟲CRN-4蛋白質大量表現及純化 16 三、 大腸桿菌RNase T蛋白質大量表現及純化 17 四、 核酸水解酶活性分析 17 1. CRN-4核酸水解酶活性分析 18 2. RNase T核酸水解酶活性分析 18 五、 MES-CRN-4複合物之結晶與其結構 19 肆、 討論 21 伍、 圖表 表一、DnaQ核酸水解外切酶家族成員 23 表二、十一種已用於研究之核酸抑制劑 24 表三、抑制CRN-4核酸水解外切酶所需之抑制劑濃度 25 表四、抑制RNase T核酸水解外切酶所需之抑制劑濃度 26 表五、MES-CRN-4複合物晶體繞射點資料及模型建立參數 27 圖一、DnaQ核酸水解外切酶家族區域結構比對圖 28 圖二、DnaQ核酸水解外切酶家族的催化機制 29 圖三、DNA-RNase T複合物活性位結構重疊比對圖 30 圖四、HIV病毒利用人類TREX1躲避宿主免疫系統之機制 31 圖五、不同種冠狀病毒具一高度保留核酸水解外切酶區域 32 圖六、LASV病毒之NP蛋白質結構 33 圖七、CRN-4蛋白質小量表現測試 34 圖八、Thrombin切除CRN-4重組蛋白上His-tag的活性測試 35 圖九、CRN-4大量純化結果 36 圖十、RNase T大量純化結果 37 圖十一、CRN-4核酸水解外切酶活性測試 38 圖十二、RNase T核酸水解外切酶活性測試 39 圖十三、MES-CRN-4複合物蛋白質結構 41 圖十四、MES-CRN-4複合物活性位之電子密度圖 42 圖十五、MES-CRN-4複合物的構形變化 43 圖十六、MES-CRN-4與DNA-RNase T複合物結構重疊比對圖 44 陸、 參考文獻 45 | |
dc.language.iso | zh-TW | |
dc.title | DnaQ核酸水解外切酶家族之可能的抑制劑之鑑定 | zh_TW |
dc.title | Identification of the potential inhibitors for the DnaQ-like exonucleases | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 詹迺立,蕭傳鐙 | |
dc.subject.keyword | DnaQ核酸水解外切?家族,核酸水解?抑制劑,CRN-4,RNase T,蛋白質結晶結構, | zh_TW |
dc.subject.keyword | DnaQ-like exonucleases,nuclease inhibitor,CRN-4,RNase T,protein crystal structure, | en |
dc.relation.page | 49 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2013-08-05 | |
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 |
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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