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研究生： 吳崇瑋
Wu, Chung-Wei
論文名稱： 化合物GP-L1-07可阻斷S100P與p53交互作用進而抑制細胞增生
Compound GP-L1-07 Reduces Cell Proliferation by Blocking S100P-p53 Interaction
指導教授： 余靖
Yu, Chin
口試委員: 蘇士哲
Su, Shih-Che
楊立威
Yang, Lee-Wei
孫仲銘
Sun, Chung-Ming
鄒瑞煌
Chou, Ruey-Hwang
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： 2022
畢業學年度： 111
語文別： 中文
論文頁數： 63
中文關鍵詞： S100P蛋白 、p53蛋白 、核磁共振 、GP-L1-07 、WST-1細胞試驗
外文關鍵詞： S100P, p53, NMR, GP-L1-07, WST-1 assay
相關次數： 點閱：2  下載：0 
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    • S100蛋白質家族的分子量大約為10-12 kDa，多存在於脊椎動物的身體之中。S100蛋白質家族為EF-hand (helix-loop-helix)鈣離子結合家族的最大亞族之一，其EF-hand結構可與鈣離子結合，導致蛋白質構型出現變化，暴露出埋藏在結構中的疏水性區域，進而與目標分子作用。S100蛋白質通常可形成多聚體，且因S100蛋白質的結構與序列相近，部分S100蛋白質甚至可形成異質二聚體。
    本篇論文研究mutant S100P與GP-L1-07的關係，根據已發表的文獻，S100P蛋白質會與p53蛋白結合，使p53喪失抑制細胞增生的功能；而pentamidine具有阻斷S100P-p53複合體的功能，並重新活化p53，使細胞增生受到抑制。透過研究pentamidine的衍生藥物GP-L1-07與mutant S100P的結合位向，便能知曉GP-L1-07是否與pentamidine有相同的效果，抑制S100P-p53的交互作用。
    因此，本實驗首先透過大腸桿菌表現與純化mutant S100P蛋白質，並使用M9培養液將蛋白質標上15N標記，以觀察目標蛋白質的訊號；利用二維核磁共振滴定實驗，可得知GP-L1-07與mutant S100P作用時受到擾動的胺基酸位點；將受擾動的胺基酸殘基做為參數輸入至HADDOCK軟體，即可計算出最佳mutant S100P-GP-L1-07複合物。透過PyMOL軟體將mutant S100P-GP-L1-07複合物與已發表的mutant S100P-p53複合物結構疊圖分析，可發現由結構上來說，GP-L1-07有阻擋mutant S100P-p53交互作用的可能，進而抑制細胞的增生。
    最後，我們使用WST-8 Assay來量測細胞實際增生率，以驗證前述認定是否正確。實驗使用ZR-75-1 cell line進行，並分別使用三組條件：第一組為對照組，細胞內含有大量內生S100P與p53蛋白，且不加入任何藥物，第二組添加不同濃度的pentamidine (5 μM-100 μM)，第三組則改為添加GP-L1-07 (5 μM-100 μM)。透過比較這三組條件，可發現GP-L1-07同樣具阻擋mutant S100P-p53交互作用並抑制細胞增生的效果。因此，可以進一步證實GP-L1-07同樣也能做為mutant S100P-p53交互作用的抑制劑。

    S100 proteins are small-sized proteins whose molecular weight are 10-12 kDa. They own a characteristeic structure called EF-hand structure. EF-hand structure is used to bind with calcium ions. After binding with calcium ions, the structure of S100 proteins is altered, exposing the hydrophobic area. The hydrophobic area of the proteins can be used to bind with target proteins or molecules, triggering the following physiological reactions. For example, S100P protein can bind to p53 protein, inactivating it and inducing cell proliferation. S100 proteins are prone to form homodimers, some S100 proteins can even form heterodimers since they share similar structures.
    From the reported literature, S100P protein can bind to p53 protein, inactivating it and inducing cell proliferation. Also, it is already proved that pentamidine, a drug which is used to treat pneumocystis pneumonia, can interact with S100P protein and release p53 protein, inhibiting cell proliferation. The purpose of this thesis, therefore, is to find out whether GP-L1-07, pentamidine derivatives synthesized by Prof. Sun in NYCU, have the same ability to bind S100P and reduce cell proliferation.
    In this thesis, we utilize the plasmid from E. Coli as a vector, transfer the plasmid containing mutant S100P gene into E. Coli cells and induce the cell to express mutant S100P protein by adding IPTG. After the expression and purification procedures of mutant S100P protein in M9 media, we can obtain the NMR HSQC spectrum of mutant S100P protein. By processing the data, we areable to label all the residues participating in the formation of mutant S100P-GP-L1-07 complex. By inputting those residues as parameters into HADDOCK software, we can obtain the calculated structure of the complex.
    According to the complex, we draw a conclution that GP-L1-07 structurally blocks the interaction between mutant S100P and p53 by overlapping the structure of mutant S100P-GP-L1-07 complex with the structure of mutant S100P-p53 complex previously reported.
    Lastly, we use WST-8 assay to test the conditions for the cell growth in the presence of pentamidine and GP-L1-07. The result shows that the cell proliferation of ZR-75-1 cells is surpressed in the presence of both pentamidine and GP-L1-07. GP-L1-07 can also be an inhibitor of the interaction between mutant S100P and p53.

    摘要....i Abstract....ii 誌謝....iii 目錄....iv 圖目錄....vii 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 1.1.1 核磁共振光譜之樣品配製....2 1.1.2 循序判定蛋白質分子之骨架....4 1.1.3 蛋白質分子支鏈判定....7 1.2 計算蛋白質結構之限制條件....10 1.2.1 距離限制條件(NOE Distance Constrains) ....10 1.2.2 雙面角限制條件....11 1.2.3 氫鍵限制條件(Hydrogen Bond Constraints)....12 1.3 ARIA/CNS結構計算....12 1.4 HADDOCK軟體介紹....15 1.5 S100蛋白質家族的結構與其特性....17 1.6 S100蛋白質家族與Ca2+的結合....19 1.7 S100P蛋白質的結構與特性....20 1.8 p53蛋白質的結構與特性....22 1.9 Pentamidine (潘他密汀)藥物與S100蛋白質的作用....23 1.10 實驗動機....24 第二章 實驗材料與方法....26 2.1 mutant S100P蛋白質的基因取得....26 2.1.1 mutant S100P蛋白質表現....26 實驗儀器....26 實驗步驟....26 2.1.2 mutant S100P蛋白質純化....27 實驗儀器....28 實驗步驟....28 2.2 蛋白質基本性質鑑定....29 2.2.1 蛋白質電泳技術(SDS-PAGE) ....30 實驗步驟....30 2.2.2 蛋白質質量鑑定....32 實驗儀器....32 實驗步驟....32 2.2.3 mutant S100P蛋白質濃度鑑定....32 2.2.4 二維核磁共振實驗....33 實驗儀器....34 實驗步驟....34 2.2.5 二維核磁共振滴定實驗 (HSQC Titration) ....34 2.2.6 蛋白質分子的結構計算....34 2.2.7 WST-8 Cell Proliferation Assay....35 實驗步驟....35 第三章 結果與討論....37 3.1 mutant S100P蛋白質的表現與純化....37 3.1.1 異丙基硫化半乳糖(IPTG)對大腸桿菌的誘導....37 3.1.2 mutant S100P (C85S) ....38 3.1.3 mutant S100P蛋白質純化....39 3.2 mutant S100P蛋白質質量鑑定....41 3.2.1 mutant S100P蛋白質除鹽....41 3.2.2 mutant S100P蛋白質質量鑑定....42 3.3 mutant S100P蛋白質二維1H-15N HSQC光譜....43 3.4 核磁共振測量蛋白質交互作用的原理....44 3.5 mutant S100P蛋白質與GP-L1-07藥物交互作用....47 3.5.1 核磁共振滴定實驗介紹....47 3.5.2 GP-L1-07藥物滴定標定mutant S100P蛋白質....47 3.6 mutant S100P-GP-L1-07複合物討論....49 3.6.1 mutant S100P-GP-L1-07複合物結構....50 3.6.2 mutant S100P-p53複合物與mutant S100P-pentamidine複合物結構....52 3.6.3 mutant S100P-GP-L1-07複合物反應作用討論與比較....53 3.7 利用WST-8 Assay驗證和比較對細胞活性的影響....55 結論....57 參考文獻....59

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