人類S100A4蛋白質為S100蛋白質家族之一份子，此系列蛋白質在結構上具有高度相似性，在水溶液狀態下為一同質雙聚體蛋白質，並於人體中具有多樣的生理活性。而其結構中具有EF-hand的部分，可與鈣離子結合而改變構形與多種蛋白質作用，其中RAGE (Receptor for Advanced Glycation End products)蛋白在近年來引起許多科學家的關注。RAGE為一種免疫球蛋白，其與發炎現象、糖尿病等疾病相關。RAGE具有多樣化的配體，他們分別與RAGE蛋白細胞外的不同部分反應，進而誘導細胞產生訊息傳遞的現象。近期研究中，科學家利用表面電漿共振指出，S100A4蛋白會與RAGE蛋白細胞外的V-domain反應。
Amlexanox為目前已商業量產之藥物，其目前主要用於過敏及潰瘍的治療，而本實驗團隊發現其對於S100部分蛋白質具有良好的活性抑制效果。因此，我們希望了解S100A4-RAGEV、S100A4-Amlexanox及S100A4-Amlexanox-RAGEV之間的交互作用，並研究蛋白質錯合物結構及細胞生理活性的影響與變化。
本篇論文中，我們希望藉由核磁共振技術近一步了解S100A4與RAGEV之間的反應，並研究其形成的複合物結構。我們使用了三維核磁共振實驗HNCA、HN(CO)CA、HNCACB、CBCACoNH、HCCH-TOCOSY以及HCCH-COSY等完成蛋白的化學位移判定，再利用二維核磁共振滴定實驗找出S100A4-RAGEV及mS100A4-Amlexanox作用位置，並利用實驗所得之距離、雙面角、氫鍵限制條件等經由電腦計算，解出結構；最後藉由恆溫滴定熱卡計及螢光滴定等數據，藉此推測mS100A4及RAGE-V之結合比例與解離常數。
本研究幫助我們更了解S100蛋白質家族與RAGEV之反應情形，並對於抗癌藥物的研究有更進一步的幫助，然而後續仍需要更多延伸之研究分析相關結果，探討此反應是否可用於癌症及相關疾病之治療。

S100A4 is a homodimeric protein that belongs to the S100 subfamily of EF hand of Calcium binding protein. Calcium bounded S100A4 is known to activate the RAGE receptor and stimulate both ERK and NF-κB signaling. RAGE receptor and S100A4 protein involved in a wide range of inflammation-related pathological states, such as vascular diseases, diabetes, neurodegeneration and cancer. As RAGE receptor and S100A4 play an important role in tumor formation, it is clear that preventing the formation of RAGE-S100A4 multi-protein complex is an effective strategy to inhibit various cancers.
In this study, we elucidated the structural interactions between mS100A4 and RAGE V-domain. We employed a variety of biophysical techniques, including fluorescence spectroscopy, multidimensional NMR spectroscopy, HADDOCK, mutagenesis study and functional assay to characterize the interactions between mS100A4 and RAGE V-domain. The binding constant was determined from fluorescence titration. The binding interfaces upon complex formation of mS100A4 and RAGE V mapping from 15N-1H HSQC titrations. Further HADDOCK modeling and mutagenesis study and functional assay indicated the role of important residues of mS100A4 protein for RAGE V-domain protein interactions. In this study, we also identified amlexanox as a small molecule that can bind to mS100A4 and inhibit the interactions between mS100A4 and the RAGE V-domain, according to our HADDOCK binding model and functional assay studies.
The present study describes the binding properties of mS100A4-Amlexanox and provides knowledge of the binding sites at the molecular level, which facilitate the design of better drugs to disrupt the mS100A4-RAGE pathway and treat various diseases, such as cancer, metastasis, and diabetes.

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