以結構為基礎建構RGS 藥物之篩選平台

Structure based development of fluorescently modified regulator of G protein signaling (RGS) as a strategy for RGS drug screening

謝昌泓(Chung-Hung Hsieh)

指導教授 ： 楊啟伸

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01942

摘要

異三元體 G 蛋白質系統 (heterotrimeric G-protein system)，在人體中調控超過一半以上的賀爾蒙以及多數的生理反應。接受多樣的外界訊息分子須倚賴種類繁複的 G 蛋白質耦合接受器 (G-protein coupled receptor，GPCR)，並且經由觸發細胞內部各種訊息路徑，進而產生相對應的生理反應去面對外界的改變。雖然 G 蛋白質系統是由 GPCR 啟動，但是整體系統的專一性及作用時間的長短卻是由 G 蛋白質調節蛋白質 (regulator of G-protein signaling，RGS) 負責。RGS 在整個 G 蛋白質系統中，透過加速 Gα 次單元體的 GTP 水解以達到控制訊息傳遞的結束。除了在 G 蛋白質系統中的重要性之外，RGS 在人體中的分布，主要集中在中樞神經及心血管系統，此一特點大大的增加了藥物的組織專一性。綜合以上幾點，針對 RGS 與 Gα 次單元體交互作用為標靶的藥物，十分具有開發的潛力。
為了加速 RGS 藥物的發展，本實驗的目的是希望發展出一套以螢光為基礎進行 RGS-Gα 蛋白質交互作用的觀測系統。前人的研究初步證明此系統的可行性，但是螢光訊號變化卻仍相當微弱。本研究透過以結構為基礎的一系列最佳化過程，找出適當的螢光標定觀測點。更進一步發現，α 螺旋及環狀區塊的交界處，是交互作用觀測點的特性。並且成功的由阿拉伯芥的 RGS 蛋白質證明，此概念更可推廣至具有與 RGS4 具有結構同源性的 R4 家族。綜合以上實驗結果，除了證明挑選螢光標定點策略的可行，也證明此策略的廣泛應用性。

關鍵字

藥物篩選系統螢光標定 ； RGS 藥物 ； 蛋白質蛋白質交互作用 ； α 螺旋及環狀區塊交界處

並列摘要

Heterotrimeric G-protein system mediates more than 60% of hormones in human and senses extracellular signals via G-protein coupled receptor (GPCR) which triggers cytosolic signaling cascades to respond to the environment. This signal is highly specific and regulated by a group of proteins, called regulator of G-protein signaling (RGS). RGS serves as GTPase accelerating protein (GAP) which acts specifically at corresponding Gα protein and terminates signaling pathway. Besides, RGS can distribute specifically at central nervous system, cardiovascular system or other tissues, and since it shows high specificity toward Gα protein, it becomes a promising drug target for therapeutic usage.
Our goal is to develop a fluorescence-based screening system to monitor the interactions between RGS and Gα, and our data showed that the helix-loop junction at RGS proteins are the best positions for fluorescent probe modifications to serve such a purpose. This strategy was then successfully shown by using an AtRGS1domain protein with fluorescent probe modified at helix-loop junction area and a sensitive fluorescence change can be observed upon activation. A general principle to select proper residues for monitoring protein-protein interaction (PPI) was thus proposed.

並列關鍵字

drug screening system ； flurescence probe ； RGS drug

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