Tranilast藥物可阻絕S100A11蛋白與RAGE V domain之交互作用並抑制細胞增生

Tranilast blocks the interaction between the protein S100A11 and Receptor for Advanced Glycation End Product (RAGE) V Domain and inhibits cell proliferation

黃彥凱(Huang, Yen Kai)

指導教授 ： 余靖

國立清華大學/理學院/化學系/碩士(2016年)

摘要

人類S100A11蛋白為S100蛋白家族中的其中一員，在水溶液下為一雙聚體蛋白質，其具有兩個可與鈣離子結合的EF-hand位置，S100A11與鈣離子結合後會發生構型上的改變，進而與目標蛋白交互作用，而RAGE(Receptor for Advanced Glycation Endproducts)中的V domain就是其中一種目標蛋白，然而研究指出，在許多癌症中發現S100A11蛋白都有異常的過量表現。RAGE屬於一種位於細胞膜上的免疫球蛋白，與不同配體結合後，進而誘導細胞產生訊息傳遞的現象，其與許多疾病有關，例如發炎現象、糖尿病等。Tranilast 最早是一種用於治療兒童支氣管哮喘的藥物，目前已被證實能夠抑制細胞的增生。
本篇論文中，我們利用NMR技術，探討S100A11和V domain在水溶液下的結構，經過HSQC滴定實驗，分別找出S100A11-V domain複合物相互作用區域及S100A11-Tranilast作用的區域，並利用HADDOCK軟體解出S100A11-V domain及S100A11-Tranilast之複合物結構模型。結果顯示S100A11用來與V domain及Tranilast作用的區域相似，故推測Tranilast能夠阻擋S100A11-V domain作用。最後利用螢光滴定求得複合物Kd，以及利用WST-1 assay 證實此訊息傳遞所產生的細胞增生確實是經由S100A11-V domain作用所造成，而Tranilast也能夠阻隔S100A11與RAGE V domain的交互作用，降低細胞訊息傳遞，進而降低疾病發生。本研究對於S100蛋白與V domain的作用有更進一步的了解，並利於往後研發有潛力的衍生藥物或新藥來達到更有效的抑制效果。

關鍵字

S100A11蛋白 ； RAGE V domain蛋白 ； Tranilast藥物 ； 核磁共振 ； 細胞增生 ； 蛋白複合物 ； 蛋白質純化

並列摘要

The human S100 calcium-binding protein A11 (S100A11) is a member of S100 protein family. Once S100A11 proteins bind to calcium ions at EF-hand motifs, S100A11 will change its conformation promoting interaction with target proteins. The receptor for advanced glycation end products (RAGE) consists of three extracellular domains, including V domain, C1 domain and C2 domain. In this case, V domain is the target for mS100A11 binding. RAGE binds to the ligands result in cell proliferation, cell growth and several signal transduction cascades.
We used NMR and fluorescence spectroscopy to demonstrate the interactions between S100A11 and V domain. The Tranilast molecule is a drug used for treating allergic disorders. We found out that V domain and Tranilast would interact with S100A11 by using 1H-15N HSQC NMR titrations. According to the results, we obtained two binary complex models from the HADDOCK program, S100A11-RAGE V domain and S100A11-Tranilast, respectively. We superimposed these two models with the same orientation of S100A11 homodimer and demonstrated that Tranilast molecule would block the binding site between S100A11 and V domain. We further utilized the WST-1 assay to indicate that Tranilast indeed can inhibit the cell proliferation which is induced by the S100A11-V domain interaction. These results will be potentially useful in the development of derivative or new anti-cancer drugs for RAGE-dependent diseases.

並列關鍵字

S100A11 protein ； RAGE V domain ； Tranilast drug ； NMR ； cell proliferation ； protein complex ； protein purification

參考文獻

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