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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81711完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 袁小琀(Hanna S. Yuan) | |
| dc.contributor.author | Chun-Jung Lin | en |
| dc.contributor.author | 林羣融 | zh_TW |
| dc.date.accessioned | 2022-11-24T09:26:06Z | - |
| dc.date.available | 2022-11-24T09:26:06Z | - |
| dc.date.copyright | 2022-01-26 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-01-13 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81711 | - |
| dc.description.abstract | 哺乳類的 DNA甲基化是表觀遺傳中關鍵的調控機制，而其建立和維持由 DNA 甲基轉移酶 (DNMT)負責，酵素成員包括 DNMT1、DNMT3A 和 DNMT3B。所有 DNMT 都包含一個甲基轉移酶結構域，用於將甲基從輔助因子 SAM 轉移到 CpG 位點的胞嘧啶 C5 位置。 DNMT 的失調和突變與疾病有關，包括各種癌症。目前有兩種 DNMT 抑制劑阿扎胞苷 (azacitidine) 和地西他濱 (decitabine) 被批准用於癌症的治療，然而，這兩種抑制劑會與所有 DNMT 共價結合，並不具有特異性而導致嚴重的副作用。目前已發現許多非以共價鍵結的 DNMT 抑制劑，但它們對每種 DNMT 的抑制活性、特異性以及抑制機轉仍不清楚。在本篇研究中，我們選擇了幾種結構相異的非共價鍵結 DNMT 抑製劑，並測試它們對重組人類 DNMT1、DNMT3A 和 DNMT3B 的抑制活性。從我們測試的結果得知，harmine和nanaomycin最能有效抑制DNMT3A和DNMT3B，其IC50數值介於5-12 M範圍內，而白藜蘆醇 (resveratrol) 和EGCG對於三個 DNMT的抑制活性較低，IC50數值介於40-200 M範圍內。 為了細究harmine的抑制機轉，我們獲得解析度達3.09 Å 的DNMT3B-3L催化結構域與harmine結合的晶體結構，我們從中發現harmine 結合在輔助因子SAM 結合位點，解釋了為什麼harmine 能夠抑制三種DNMT ，並由酵素動力學分析證實harmine 在抑制 DNMT3B 活性方面與 SAM 競爭。我們的研究提供harmine對DNMT3B於分子層面下的抑制機轉，根據這個結果，未來，我們將優化此非共價抑制劑以設計出對其中一種 DNMT 具有特異性的新一代非共價抑製劑，期望將其應用於癌症治療上。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T09:26:06Z (GMT). No. of bitstreams: 1 U0001-1101202216402500.pdf: 7274965 bytes, checksum: b57988b52016f5960d13d4ac9c49736c (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 中文摘要 I Abstract II Table of Contents III List of Figures V List of Tables VI Chapter 1. Introduction 1 1.1 Epigenetics modification: DNA methylation and its cellular events 1 1.2 The mechanism of CpG methylation and the roles of DNA methyltransferases (DNMTs) in mammals 1 1.2.1 Crystal structure of DNMT3B-3L catalytic domain complex 3 1.3 Expression dysregulation and carcinogenic mechanisms of DNMTs 4 1.4 FDA-approved DNMT inhibitors 4 1.5 The reported potential DNMT inhibitors 5 1.5.1 RG108 6 1.5.2 Nanaomycin A 6 1.5.3 EGCG 7 1.5.4 Harmine 7 1.5.5 Resveratrol 8 1.6 Specific aims 8 Chapter 2. Materials and methods 9 2.1 Chemicals 9 2.2 Plasmid constructs 9 2.3 Protein expression and purification 10 2.4 Protein crystallization and structure refinement 11 2.5 Determination of half maximum inhibitory concentration (IC50) 12 2.6 Differential scanning fluorimetry assay (DSF) 13 2.7 Circular dichroism (CD) spectroscopy 13 2.8 Intrinsic tryptophan fluorescence spectroscopy 14 2.9 Enzyme kinetics 14 Chapter 3. Results 16 3.1 DNMTs protein expression and purification. 16 3.2 Measurement of IC50 for non-covalent inhibitors 16 3.3 Harmine directly binds DNMT3B-3L complex 17 3.4 Crystal structure of DNMT3B-3L-harmine complex reveals harmine is bound at SAM-binding site 19 3.5 Confirmation of the competitive inhibition mechanism of harmine with SAM by kinetic assays. 20 Chapter 4. Discussion 22 Chapter 5. References 25 | |
| dc.language.iso | en | |
| dc.subject | DNA甲基轉移酶 | zh_TW |
| dc.subject | DNA甲基化 | zh_TW |
| dc.subject | DNA甲基轉移酶抑制劑 | zh_TW |
| dc.subject | DNA methyltransferase | en |
| dc.subject | DNA methylation | en |
| dc.subject | DNMT inhibitors | en |
| dc.title | 人類DNA甲基轉移酶抑制劑之生化特性與結構分析 | zh_TW |
| dc.title | Biochemical and structural insights into the inhibitors of human DNA methyltransferases | en |
| dc.date.schoolyear | 110-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 詹迺立(Jing-Fa Tsai),李明學(Chih-Wei Chang),曾秀如(Shashidhar Siddagangaiah),(Chiao-Ming Peng) | |
| dc.subject.keyword | DNA甲基化,DNA甲基轉移酶,DNA甲基轉移酶抑制劑, | zh_TW |
| dc.subject.keyword | DNA methylation,DNA methyltransferase,DNMT inhibitors, | en |
| dc.relation.page | 58 | |
| dc.identifier.doi | 10.6342/NTU202200042 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2022-01-13 | |
| 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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