臺灣博碩士論文加值系統

摘要
人類S100A12蛋白質(或鈣粒蛋白C)是屬於S100家族蛋白質之一，此系列蛋白多為小分子蛋白且水溶液中多以雙聚體存在。當鈣離子與S100A12結合時，鈣離子會接上結構中EF-hand的部分，促使蛋白構型改變並活化蛋白，可與多種蛋白作用，產生生物反應。RAGE (Receptor for Advanced glycation end products)蛋白為一種免疫球蛋白，與生物體內發炎反應、糖尿病相關，是近年備受矚目的蛋白。RAGE可接收不同配體，其中S100家族蛋白大多與其有相互作用，而近幾年研究指出，S100A12與RAGE 的結合會造成細胞增生或發炎的症狀，與癌症或糖尿病息息相關。
Tranilast是一種已商業量產之抗過敏藥物，目前主要用於治療過敏相關症狀，而在本篇研究中，發現其對於S100A12蛋白與RAGE V domain蛋白間作用，能夠產生有效的抑制，被認定為可作為抑制劑的藥物。為探討其抑制的機制，本篇論文中，利用許多生物與物理上技術，像是核磁共振儀、螢光光譜儀、HADDOCK軟體以及WST-1 細胞實驗，來得到蛋白產生結合的相關結構資訊。
本篇論文中，我們分別探討了S100A12和tranilast以及S100A12和RAGE V domain的錯合物結構，其中發現了tranilast的結合位置能夠擋住S100A12與RAGE V domain的結合位置，這樣的現象能夠幫助新藥物的開發，發展能夠抑制RAGE和S100A12結合的藥物，藉以治療相關疾病。

Abstract
S100A12 (Calgranuline C) is a small and dimeric protein which belongs to the S100 family. When calcium ions bind to the two EF-hands of S100A12, the protein structure changes and promotes the interaction with its target proteins. RAGE (receptor for advanced glycation end) is one of the target protein for S100A12. By the past research, it showed the binding between these two proteins generated a pro-inflammatory response and activated several signal pathways such as ERK and NF-κB. It is an effective way that preventing the formation of S100A12-RAGE protein complex to inhibit various cancers.
Tranilast, an anti-allergic drug that is usually used as treatment of allergic disorders, was found to have a strong interaction with S100A12 which may be a potential inhibitor. In this study, we discussed the interaction of S100A12 with RAGE and S100A12 with tranilast by their three dimension structure. We utilized several biophysical techniques, including multidimensional NMR spectroscopy, fluorescence spectroscopy, HADDOCK and WST-1 assay to characterize the structural information of protein complex.
This study describes structural properties of S100A12-tranilast and S100A12-RAGE complex. It shows that tranilast blocks the binding sites of S100A12- RAGEV. This result can be useful for the development of new drug against various diseases.

目錄
誌謝辭 ii
摘要 iii
Abstract iv
目錄 v
圖目錄 vi
表目錄 ix
縮寫表 x
第一章 前言 1
1.1 生物核磁共振技術介紹 1
1.2 核磁共振實驗蛋白質樣品製備 2
1.2.2 蛋白質骨架循序判定 2
1.2.3 蛋白質分子支鏈循序判定 5
1.3 計算蛋白質結構之限制 7
1.3.1 距離限制 (NOE distance constraints) 7
1.3.2 雙面角限制 (Dihedral angle constraints) 7
1.3.3 氫鍵限制(Hydrogen bond constraints) 8
1.4 ARIA/CNS 結構計算 9
1.5 S100家族蛋白結構與特性 12
1.6 S100A12蛋白質結構與特性 13
1.7 RAGE V 蛋白質之結構與特性 15
1.8 S100蛋白質與RAGE交互作用產生之複合物結構與生理功能 18
1.9 Tranilast結構與特性 19
1.10 實驗動機 20
第二章 實驗材料與方法 21
2.1人類S100A12蛋白質之基因 21
2.1.1 人類S100A12蛋白質表現 21
2.1.2 人類S100A12蛋白質純化 24
2.2 RAGE V domain基因取得與表現 26
2.2.1 RAGE V 蛋白質的表現 27
2.2.2 RAGE V蛋白質的純化 29
2.3 蛋白質的性質檢定 32
2.3.1 蛋白質質量測定 32
2.3.2 蛋白質之濃度測定 33
2.3.3 螢光放射光譜 34
2.3.4 二維核磁共振光譜實驗 36
2.3.5 核磁共振光譜滴定實驗 (HSQC titration) 38
2.3.6 圓二色光譜儀 (Circular dichroism) 38
2.3.7 蛋白質複合物結構計算與分析 (Complex structure calculation) 39
第三章 結果與討論 41
3.1 蛋白質表現與純化 41
3.1.1 異丙基硫化半乳糖(IPTG)對於大腸桿菌之誘導作用 41
3.1.2 S100A12蛋白質大量表現 42
3.1.3 S100A12蛋白質純化 43
3.1.4 RAGE V蛋白質純化 45
3.2 蛋白質基本性質測定 47
3.2.1 S100A12與 RAGE V蛋白質分子量鑑定 47
3.2.2 S100A12 蛋白質二維1H-15NHSQC光譜測定 49
3.2.3 RAGE V蛋白質二維1H-15NHSQC光譜測定 50
3.3 S100A12 與RAGE V蛋白質之交互作用 52
3.3.1 核磁共振滴定實驗(1H-15NHSQC titration) 52
3.4 S100A12 與tranilast 之交互作用 57
3.4.1 核磁共振滴定實驗(1H-15N HSQC titration) 57
3.5 RAGE V 蛋白質與tranilast之交互作用 59
3.5.1 核磁共振滴定實驗(1H-15N HSQC titration) 59
3.6 S100A12 與RAGE V 蛋白質複合物結構 60
3.6.1 S100A12 與RAGE V 作用之配位基團 61
3.6.2 S100A12 與RAGE V 複合物結構計算 61
3.6.3 S100A12 與RAGE V複合物之結合表面 65
3.6.4 以圓二色光譜儀測量複合物結構變化 65
3.6.5 以螢光滴定實驗研究S100A12 與RAGE V之相互作用 67
3.7 S100A12與tranilast之複合物結構 69
3.7.1 S100A12與tranilast形成複合物之配位基團 70
3.7.2 S100A12與tranilast複合物之結合表面 71
3.7.3 以圓二色光譜分析複合物結構變化 72
3.7.4 以螢光滴定實驗研究S100A12與tranilast作用 73
3.8 S100A12-tranilast-RAGE V之蛋白質結構 75
3.9 以WST-1 Assay 探討蛋白與藥物對生物活性之影響 77
結論 79
參考文獻 80
附錄 88

Abstract
S100A12 (Calgranuline C) is a small and dimeric protein which belongs to the S100 family. When calcium ions bind to the two EF-hands of S100A12, the protein structure changes and promotes the interaction with its target proteins. RAGE (receptor for advanced glycation end) is one of the target protein for S100A12. By the past research, it showed the binding between these two proteins generated a pro-inflammatory response and activated several signal pathways such as ERK and NF-κB. It is an effective way that preventing the formation of S100A12-RAGE protein complex to inhibit various cancers.
Tranilast, an anti-allergic drug that is usually used as treatment of allergic disorders, was found to have a strong interaction with S100A12 which may be a potential inhibitor. In this study, we discussed the interaction of S100A12 with RAGE and S100A12 with tranilast by their three dimension structure. We utilized several biophysical techniques, including multidimensional NMR spectroscopy, fluorescence spectroscopy, HADDOCK and WST-1 assay to characterize the structural information of protein complex.
This study describes structural properties of S100A12-tranilast and S100A12-RAGE complex. It shows that tranilast blocks the binding sites of S100A12- RAGEV. This result can be useful for the development of new drug against various diseases.