利用CHAPS分子來隔絕S100A9 (C3S)與RAGE V domain兩種蛋白間之交互作用: 一種抑制細胞增生的新藥開發

Blocking the interaction between S100A9 (C3S) and RAGE V domain using CHAPS molecule: A new drug development against cell proliferation

張晉齊(Change, Chin Chi)

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S100A9鈣離子結合蛋白 (鈣粒蛋白B)為S100蛋白質家族的一員，通常伴隨著S100A8鈣離子結合蛋白 (鈣粒蛋白A) 出現，此系列蛋白在結構上具有高度相似性，通常S100A9以同質二聚體形式存在於骨髓細胞中，在發炎反應發生時，扮演著重要的調節角色。人類RAGE 蛋白質 (Receptors for the Advanced Glycation End products) 是一種免疫球蛋白，可以與多種配體結合 (如S100蛋白家族)，誘導細胞產生訊息傳遞，進而促進發炎反應、糖尿病及一些慢性疾病甚至是細胞增生的現象。
CHAPS為目前商業量產的藥物，在結構上具有助於結合細胞膜蛋白疏水性區域，而在本實驗發現對於mS100A9蛋白具有良好的活性抑制效果，因此我們希望進一步了解mS100A9-RAGE V domain、mS100A9-CHAPS之間的交互作用，並利用HADDOCK模擬在生物體內蛋白質錯合物的結構。
在本篇論文中，我們利用三維核磁共振實驗來完成蛋白質化學位移判定，將實驗所得距離、二面角、氫鍵限制條件經由軟體計算，計算解出mutant S100A9蛋白質在水溶液的二聚體結構，並搭配二維核磁共振滴定實驗，研究mS100A9蛋白與RAGE V domain蛋白以及與CHAPS之間的反應結合位置，並藉由二維光譜上的交叉峰位移，來推測mS100A9及RAGE V domain的解離常數(Kd)為5.7 μM。最後根據WST-1 Assay實驗的結果證實mS100A9是經由與RAGE上的 V domain結合後影響，並使細胞活性提升。同時CHAPS分子也能夠與RAGE V domain競爭mS100A9的結合，成功地抑制細胞活性。
此篇研究有助於了解S100A9蛋白質與RAGE V domain的反應情形，並對於未來的抑制細胞增生藥物發展有更進一步的幫助。然而後續仍須研究生物性的實驗，探討此反應是否可應用在癌症及相關疾病的治療。

關鍵字

S100A9 ； 醣基化終產物 V domain ； CHAPS ； 核磁共振結構 ； 蛋白質複合體 ； 細胞增生

並列摘要

Human S100A9 (Calgranulin B) is a Ca2+-binding protein from the S100 family that often accompanies human the S100A8 protein (Calgranulin A). S100A9 presents as a homodimer in myeloid cells and emerges as an important mediator during inflammation after calcium binds to its EF hand motifs. Human receptors for the advanced glycation end products (RAGE) protein is one of the target proteins for S100A9 binding to its hydrophobic surface. Interaction between these two proteins will trigger signaling transduction cascades that promote cell growth, proliferation, and tumorigenesis. Here, we solved the solution structure of the mS100A9 homodimer by conducting multi-dimensional NMR experiments. We further characterize the solution interactions between mS100A9 and the RAGE V domain as well as S100A9 with the CHAPS molecule via NMR spectroscopy. Finally, using the HADDOCK program, we demonstrate that CHAPS molecules play a crucial role in blocking the interaction between mS100A9 and the RAGE V domain. WST-1 assay results also support that CHAPS inhibit the bioactivity of mS100A9. This report will help to inform new drug development against cell proliferation.

並列關鍵字

S100A9 ； Receptor for advanced glycation end products V domain ； CHAPS ； NMR structure ； Protein complex ； Cell proliferation

參考文獻

1. Schneider M, Fu X, Keating AE. X-ray vs. Nmr structures as templates for computational protein design. Proteins 2009:77(1): 97-110.

2. Bax A, Ikura M. An efficient 3d nmr technique for correlating the proton and 15n backbone amide resonances with the alpha-carbon of the preceding residue in uniformly 15n/13c enriched proteins. J Biomol NMR 1991:1(1): 99-104.

3. Brutscher B, Cordier F, Simorre JP, Caffrey M, Marion D. High-resolution 3d hncoca experiment applied to a 28 kda paramagnetic protein. J Biomol NMR 1995:5(2): 202-206.

4. Meissner A, Sorensen OW. Sequential hncacb and cbcanh protein nmr pulse sequences. J Magn Reson 2001:151(2): 328-331.

5. Arbogast L, Majumdar A, Tolman JR. Hnco-based measurement of one-bond amide 15n-1h couplings with optimized precision. J Biomol NMR 2010:46(2): 175-189.