S100A4蛋白可干擾S100A1蛋白和the RAGE V domain結合抑制細胞增生

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研究生：	陳泰元 Chen, Tai-Yuan
論文名稱：	S100A4蛋白可干擾S100A1蛋白和the RAGE V domain結合抑制細胞增生 S100A4 inhibits cell proliferation by interfering with the RAGE V domain-S100A1
指導教授：	余靖 Yu, Chin
口試委員:	陳金榜 Chen, Chin-pan 蘇士哲 Sue, Shih-Che
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2019
畢業學年度：	107
語文別：	中文
論文頁數：	57
中文關鍵詞：	S100A4 蛋白質、S100A1 蛋白質、S100A4與S100A1蛋白質
外文關鍵詞：	S100A4 protein, S100A1 protein, S100A1 protein and S100A4 protein
相關次數：	點閱：1 下載：0
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人類S100蛋白質家族具有EF-hand結構(helix-loop-helix)的小分子量酸性蛋白質家族，結構上在C端具有與Ca2+結合的canonical EF-hand，在N端則具有S100特有的pseudo EF結構。該系列至少20種，存在於脊椎動物特定組織與細胞中，平均分子量大約在9-13 kDa間。S100蛋白質通常形成homodimer，也可以形成heterodimer。
研究顯示，S100A1及S100A4都可以與RAGE蛋白上的V-domain 結合，而S100蛋白質家族中所有的蛋白質在水溶液都以dimer存在，而每一個S100A1 (或S100A4)的homodimer會與2個RAGE結合，而造成2個RAGE蛋白的cytoplasmid domain 相互靠近，進而產生相互磷酶化(autophosphorylation）現象，終導致訊號傳遞發生，而造成細胞之增生（cell proliferation）。研究也顯示S100A1 與S100A4 是相互拮抗的。
本實驗藉由HADDOCK的計算，可得兩蛋白質形成的heterodimer的三維結構圖，以及兩蛋白質反應的作用面，也顯示了S100A1蛋白質與S100A4蛋白質形成heterodimer時，S100A1蛋白質與S100A4蛋白質的相互結合區域正好是S100A1與V-domain 的結合區域。再藉由螢光與等溫滴定微量熱法(ITC)測量兩蛋白質的解離常數(Kd)，最後由WST-1 Assay可得S100A1-S100A4 的heterodimer是不具細胞增生的活性，及形成heterodimer之後他就喪失了細胞增生的生物活性。也證明了S100A4蛋白質確實具有阻斷S100A1與其他蛋白質結合的能力。

S100A1 is a homodimeric protein that belongs to the S100 subfamily of EF hand of calcium binding protein. Upon Ca2+ binding to S100A1 EF-hand motifs, the conformation of S100A1 change promoting interaction with target proteins. V domain of receptors for the advanced glycation end products(V domain of RAGE) is one of the target proteins for S100A1 binding to its hydrophobic surface and further triggers signaling transduction cascades that induces cell growth, cell proliferation, and tumorigenesis. The present study describes S100A4 and S100A1 can become dimer and can mutual antagonist each other.
In this study, we elucidated the structural interactions between S100A4 and S100A1. We employed a variety of biophysical techniques, including fluorescence spectroscopy, multidimensional NMR spectroscopy, HADDOCK, isothermal titration calorimeter(ITC) mutagenesis study and functional assay to characterize the interaction between S100A4 and S100A1. The binding constant was determined from fluorescence spectroscopy and ITC. The binding interfaces upon complex formation of S100A4 and S100A1 mapping from 15N-1H HSQC titration. Further HADDOCK modeling and mutagenesis study study and functional assay indicated the role of important residues of S100A4 protein for S100A1 protein interactions.
In this study, we also find the S100A4 become blocker between S100A1 and V-domain that is also supported by WST-1 cell proliferation.

目錄
誌謝 ........ I
摘要 .......II
Abstract........ III
圖目錄 ....... IX
表目錄 ...........XII
第一章 前言 ..............1
1.1生物核磁共振技術介紹........1
1.1.1製備核磁共振實驗的樣品......1
1.1.2 蛋白質分子的骨架循序判定.......2
1.1.3 蛋白質分子的支鏈循序判定.......4
1.2 計算蛋白質結構之限制條件...........6
1.2.1 距離限制條件(NOE Distance Constraints)
............................6
1.2.2 雙面角限制條件....................6
1.2.3 氫鍵限制條件(Hydrogen Bond Constraints)...................................................7
1.3 ARIA/CNS結構計算...................7
1.4 HADOCK介紹.............................9
1.5 S100蛋白質家族結構與特性.......10
1.6 S100A4蛋白質結構與特性........... 11
1.7 S100A1蛋白質結構與特性...........12
1.8 S100家族與Ca2+結合產生構型轉變.........................................14
1.9 S100A1蛋白質誘發之RAGE訊息傳遞.........................................................15
1.10 實驗動機...................................17
第二章 實驗材料與方法 ....................................19
2.1 S100A4蛋白質之基因取得..................19
2.1.1 S100A4蛋白質之表現................19
[實驗儀器] ...........................................19
[實驗步驟] ..........................................20
2.1.2 S100A4蛋白質之純化................21
[實驗儀器] ............................................21
[實驗步驟] ............................................21
2.2 S100A1蛋白質之基因取得..................23
2.2.1 S100A1蛋白質之表現..............23
[實驗儀器] ............................................24
[實驗步驟] .............................................24
2.1.2 S100A1蛋白質之純化..............25
[實驗儀器] ............................................25
[實驗步驟] ...........................................26
2.3 蛋白質基本性質鑑定...................27
2.3.1 蛋白質質量鑑定........................27
[實驗儀器] ............................................28
[實驗步驟] ............................................28
2.3.2 蛋白質濃度測定........................28
2.3.4 二維核磁共振實驗....................29
[實驗儀器] ............................................29
[實驗步驟] .............................................29
2.3.5 二維核磁共振滴定實驗(HSQC titration) .....................................................30
2.3.6 螢光放射實驗............................30
[實驗儀器] ...........................................31
[實驗參數] ............................................31
[實驗步驟] ...........................................31
2.3.7 等溫滴定微量熱法(ITC) ..............31
[實驗儀器] .............................................32
[實驗步驟] ............................................32
2.4 蛋白質分子的結構計算..................32
第三章 結果與討論 .............................................33
3.1 蛋白質的純化................................33
3.1.1 S100A4蛋白質之大量表現.......33
3.1.2 S100A4蛋白質純化...................34
3.1.3 S100A1蛋白質純化.................35
3.2 蛋白質質量鑑定............................36
3.2.1 蛋白質除鹽後結果.....................36
3.2.2 蛋白質質量鑑定.........................38
3.3 S100A1蛋白質與S100A4蛋白質二維1H-15N HSQC 光譜.........................39
3.4 S100A1蛋白質與S100A4蛋白質交互作用................................................41
3.4.1 核磁共振滴定實驗.....................41
3.5 S100A4與S100A1之蛋白質結構....45
3.5.1 S100A4與S100A1之蛋白質結構.......................................................45
3.5.2 S100A4與S100A1之蛋白質結構.......................................................46
3.6 S100A1蛋白質與S100A4蛋白質解離常數(Kd)測定....................................48
3.6.1 以螢光實驗測量解離常數(Kd) ...........................................................48
3.6.2 以等溫滴定微量熱法(ITC)測量解離常數(Kd) ............................................50
3.7 利用WST-1 Assay研究對細胞生物活性之影響...........................................51
結論 .................................................52
參考資料 .............................................53
                                

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