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標題: | 探討幽門螺旋桿菌蛋白GroES羧基端和受體結合的關鍵胺基酸 Identification of the critical residues in C-terminal domain of H.pylori GroES for receptor recognition |
作者: | Feng-Tse Hsieh 謝豐澤 |
指導教授: | 周綠蘋(Lu-Ping Chow) |
關鍵字: | 幽門螺旋桿菌,熱休克蛋白10,類鐸受體4,介白素8,表面電漿共振感測, Helicobacter pylori,GroES,Hsp10,TLR4,interleukin-8,SPR, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 幽門螺旋桿菌是一個會造成胃潰瘍、胃炎、胃癌主要的危險因子。先前的研究發現其致病因子熱休克蛋白GroES羧基端含有一個特殊的功能域B (B domain)。功能域B為幽門螺旋桿菌所獨有,並不在其他菌種的熱休克蛋白GroES被發現,其為一個富含半胱氨酸和組胺酸的金屬結合域,這個結合域會結合兩個鎳金屬離子。在過去的研究結果顯示,幽門螺旋桿菌的GroES分泌至菌體外之後,會和人類胃上皮細胞的類鐸受體4 (TLR4) 結合,活化MAPK 訊息路徑誘發前發炎因子介白素8 (IL-8) 的分泌並且引發發炎反應。而過去本實驗室也研究發現,若是其功能域B被裁切掉後,蛋白將失去誘發介白素8的能力,顯見功能域B的存在似乎對於GroES結合TLR4引發發炎反應是重要的,值得我們更深入的探討。 由於功能域B是富含組胺酸的金屬結合域,我們首先使用了可以將金屬敖合去除的試劑EDTA作用蛋白,去除其鎳金屬離子後,發現其失去了誘發胃上皮細胞分泌IL-8的功能;而又有報導指出鎳金屬離子和蛋白結合主要是透過多個組胺酸,這讓我們開始著手使用點突變的方式將其蛋白上的八個組胺酸突變成丙胺酸,之後建構進質體中純化蛋白,再同樣透過檢測其誘導細胞分泌介白素8的變化將蛋白歸類分群。在我們的結果發現,H96A和H104A兩個突變蛋白仍然保有和野生型蛋白 (Wild type) 一樣完整刺激介白素8釋放的能力,另外其餘蛋白包括了H100A、H102A、108A、H113A、H115A和H118A釋放量約為野生型蛋白的50%、50%、50%、50%、80%和20%。 另外我們將使用表面電漿共振感測儀 (Surface Plasmon Resonance, SPR) 探討各類GroES蛋白和TLR4結合情況以得到親和力關係。表面電漿共振感測實驗結果顯示,野生型GroES和TLR4的解離常數 (Kd) 是0.25 μΜ,另外組胺酸突變的蛋白包括H96A、H104A和H115A的解離常數分別為0.11 μΜ、0.15 μΜ和0.38 μΜ。前面實驗發現活性較差的H100A、H102A、H108A、H113A 和H118A則是親和力過於微弱無法經由軟體計算。 根據兩者實驗的分析結果,可以發現會影響功能的胺基酸包含了H100A、H102A、H108A、H113A 和H118A,而在IL-8釋放量功能性測試完全失去活性的H118A下降最多顯見其可能最關鍵的胺基酸是H118;最後,能發現在GroES 功能域B的關鍵胺基酸對於後續要治療幽門螺旋桿菌引發的胃癌來說又多了一個新的治療策略以及新的藥物發展目標。 Helicobacter pylori (H. pylori) has been proven as a major risk factor in the development of gastric ulcer, chronic gastritis, and gastric cancer. Previous study showed that H. pylori GroES has a unique carboxyl extension (residue 91-118) called domain B , which is a cysteine- and histidine-rich metal-binding domain and not found in GroES produced by other microbes. Moreover, the domain B was reported as a nickel binding domain that each GroES monomer domain B may bind 2 nickel ions. In our previous study, we found that H. pylori GroES induced interleukin-8 (IL-8) in human gastric epithelial cells, and the truncated H.pylori GroES without domain B was unable to induce IL-8 production. Therefore, it is of our interest to investigate the importance of domain B to H.pylori GroES-induced IL-8 through receptor recognition. Considering domain B is unique to H.pylori GroES and is a histidine-rich metal-binding domain, H.pylori GroES pre-treated with chelating agent EDTA can no longer induce IL-8 release. Based on other studies, the most important residue for nikle ion binding is histidine. We further want to find the crucial residues in GroES domain B by alanine scanning site-directed mutagenesis. Our result indicated two histidine mutant proteins, H96A and H104A, had fully ability to induce IL-8 release comparing to wild type protein. And the ability of other six histidine mutants, which inculding H100A , H102A, H108A, H113A, H115A and H118A, to cause IL-8 release were 50%, 50%, 50%, 80% and 20% comparing to wild type. Next, Surface Plasmon Resonance (SPR) was used to investigate the binding of TLR4 receptor to GroES mutants, and it could determine the values of affinity (kd) to GroES mutants and TLR4. The Surface Plasmon Resonance (SPR) data showed that the values of affinity (kd) to GroES wild type and TLR4 is 0.25 μΜ. In addition, kd of H96A, H104A and H115A is 0.11 μΜ, 0.15 μΜ and 0.38 μΜ. The kd of others five low activity mutant proteins inculding H100A, H102A, H108A, H113A and H118A are too weak to be caculated. Based on the IL-8 secretion functional assay and SPR data, the critical residues for the interaction of GroES and TLR4 are H100 , H102, H108, H113 and H118. And the most cricial residue is H118, which lose the ability to cause IL-8 secretion. Finally, finding these critical residues in GroES domain B for receptor recognition can further be of contribution to new therapeutic strategies and drug development in gastric cancer treatment. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53721 |
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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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