以螢光修飾與嵌合蛋白質發展RGS調節因子篩選平台

G蛋白質耦合受器與異三元體G蛋白質系統是哺乳動物最常使用的訊息傳遞系統之一，異三元體Ｇ蛋白質系統由Gα與Gβγ次單元所構成，負責將Ｇ蛋白質耦合受器所接收到的胞外訊息往胞內傳遞。其中，Gα次單元具備水解GTP的能力，G蛋白質耦合受器的活化將促使Gα次單元與GTP結合，開啟下游訊息路徑；而當Gα次單元將GTP水解為GDP，則會使異三元體G蛋白質系統回到不活化的基態。細胞中有一類關鍵性的蛋白質負責調控G蛋白質系統訊息傳遞，稱為Regulator of G-protein signaling (RGS)，RGS能夠催化Gα水解GTP的速度，進而終結訊息傳遞。RGS在訊息傳遞中扮演的角色使其成為藥物標靶的可能性於近幾年來受到高度重視，然而由於其活性測定上的困難使研究受到限制，雖然可能的RGS藥物篩選平台相繼被提出，至今仍未出現任何RGS藥物。本研究利用可大量表現的Gα嵌合蛋白質與螢光修飾蛋白質的技術，發展一可簡便偵測RGS-Gα交互作用的分析方式，並以之建立RGS9與RGS4的藥物或調節因子篩選平台。在RGS9篩選平台方面，以螢光標定之Gα嵌合蛋白質偵測與RGS9交互作用，並使用96孔微量滴定盤與螢光ELISA操作大量化分析，已成功達到17%的報導訊號；RGS4篩選平台則利用螢光標定的RGS4偵測與Gαi1的交互作用，以數個RGS4內源Cysteine變異蛋白質成功辨識以往訊號中背景雜訊來源為Cys148上的螢光標定，藉由剔除背景來源，並於交互作用介面引入標定位置，希望發展報導效率更佳之變異蛋白質。此外，藉由分析序列中數個Cysteine發現Cys95提供結構上穩定性，對蛋白質表現量不可或缺；另外， Cys148可能是RGS4異位調控中的關鍵位置之一。

關鍵字

G蛋白質系統； RGS ； RGS調節因子； RGS藥物篩選；螢光標定； G-alpha嵌合蛋白質；蛋白質交互作用； RGS異位調控

並列摘要

Heterotrimeric G-protein system composed by Gα and Gβγ subunits is the most prevalent cellular signaling molecule, mediating the delivering of extracellular signal from G-protein coupled receptor (GPCR) to intracellular effectors. The ligand bound GPCR activates G-proteins and switches on the signaling cascade via the GTP bound form Gα subunit. The signaling event terminates till the GTP hydrolysis catalyzed by the intrinsic GTPase activity of Gα, rendering G-protein system return to ground state and ready for the next signal. The regulator of G-protein signaling (RGS) belongs to GTPase accelerating protein (GAP) family, which regulates signaling events downstream GPCR by promoting the GTP hydrolysis of Gα, therefore, reducing the duration and amplitude of activated signaling. The critical role on G-protein signaling implies RGS as a potential candidate for drug targeting, which attracts increasing professional and academic attention in last decade. However, the difficulty on analyzing RGS activity limits the progress of RGS physiologic research. There is no clinical RGS drug until today. In this study, we focused on development of RGS4 and RGS9 drug screening platform based on highly expressible chimeric and fluorescently labeled proteins. In RGS9 part, we used a fluorescently modified chimeric Gα to probe the interaction with RGS9. By using 96-well plate and fluorescent ELISA, high throughput, highly sensitive, real-time monitoring of Gαt-RGS9 interaction could be achieved. Also, we developed RGS4- Gαi1 interaction assay via fluorescently labeled RGS4. In this part, through analysis of cysteines in RGS4 sequence, we successfully identified the background labeling came from Cys148 which also played a role in allosteric regulation of RGS4. Furthermore, Cys95 would be an essential residue for protein stability. Based on these results, we introduced few labeling sites on RGS4- Gαi1 interface to construct mutants for signaling optimization.

並列關鍵字

G-protein system ； regulator of G-protein signaling (RGS) ； RGS modulator ； RGS drug screening ； fluorescent labeling ； chimeric G-alpha ； protein-protein interaction ； RGS allosteric regulation

參考文獻

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3. Schoneberg, T., A. Schulz, and T. Gudermann, The structural basis of G-protein-coupled receptor function and dysfunction in human diseases. Reviews of Physiology Biochemistry and Pharmacology, Vol 144, 2002. 144: p. 143-227.

4. Biddlecome, G.H., G. Berstein, and E.M. Ross, Regulation of phospholipase C-beta1 by Gq and m1 muscarinic cholinergic receptor. Steady-state balance of receptor-mediated activation and GTPase-activating protein-promoted deactivation. J Biol Chem, 1996. 271(14): p. 7999-8007.

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以螢光修飾與嵌合蛋白質發展RGS調節因子篩選平台

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