利用酵母菌雙雜合系統尋找與RhoGDIβ結合的蛋白質

Searching for proteins that bind to RhoGDIβ by yeast two-hybrid system

黃怡綸(Ie-Lun Huang)

指導教授 ： 林育誼

中山醫學大學/口腔醫學院/口腔醫學研究所/碩士(2008年)

https://doi.org/10.6834/CSMU.2008.00034

摘要

先前由中山醫學大學楊肇基老師實驗室將ZAK蛋白質利用酵母菌雙雜交系統尋找與ZAK相關之蛋白質，其中找到了與調節small GTPases功能有關的RhoGDIβ。Rho GDP-dissocation inhibitors(RhoGDIs)為一內生性(endogenous)的Rho GTPase的抑制物(inhibitor)，在調節生物活性(biological activity)上扮演著舉足輕重的角色。目前已知RhoGDIs以三種形式存在，分別為RhoGDIα (RhoGDI，RhoGDI-1)、RhoGDIβ (D4/LyGDI，RhoGDI-2)及RhoGDIγ (RhoGDI-3)而 RhoGDIβ大多表現於造血細胞(Haematopoetic cell)中。在我們實驗室中發現RhoGDIβ表現在心臟細胞時會造成心肌細胞的肥大，為了想更進一步的了解RhoGDIβ在心臟細胞中的功能便利用藉由酵母菌雙雜交系統的方式來尋找人類心臟細胞內可與RhoGDIβ結合的蛋白質來了解RhoGDIβ在心臟細胞中所扮演的角色。藉由酵母菌雙雜交系統所得到酵母菌菌落，送至定序後獲得二個與RhoGDIβ相關的蛋白質，分別為Rho A及 MYBPC3，這二個蛋白質都能與RhoGDIβ蛋白質相結合，接著我們將更進一步的去確認這二個蛋白質在in vivo中是否能真正的與RhoGDIβ結合及RhoGDIβ是否能調節此二蛋白質的功能。

關鍵字

酵母菌雙雜交系統

並列摘要

To search effectors of ZAK and to study the ZAK signaling cascade, our laboratory used a yeast two-hybrid system to isolate ZAK associated proteins from a human heart cDNA library. One of the isolated cDNAs encoded Rho GDP dissociation inhibitor beta (RhoGDIβ). RhoGDIβ, also known as Ly-GDI or D4-GDI, which belongs to a family of Rho GDP dissociation inhibitors that includes RhoGDIα, RhoGDIβ and RhoGDIγ is thought to regulate many biological activities in cells and also regulate the activity and localization of Rho family proteins. RhoGDIβ is almost exclusively expressed in hematopoietic lineages. In the lab, we discovered that RhoGDIβ induced hypertrophic growth of a cultured rat cardiac cell line, H9c2. In order to study the mechanisms of RhoGDIβ to regulate hypertrophic growth, we used a yeast two-hybrid system to determine RhoGDIβ associated protein from a human heart cDNA library. Only four yeast colonies were growing in selection plates and we isolated these cDNAs. We then identified two possible RhoGDIβ associated proteins, RhoA and MYBPC3, after sequencing these cDANs. We set to determine the association of these proteins with RhoGDIβ in vivo in this study.

並列關鍵字

RhoGDIβ ； yeast two-hybrid system

參考文獻

1. Aikawa, R., I. Komuro, et al. (1999). "Rho family small G proteins play critical roles in mechanical stress-induced hypertrophic responses in cardiac myocytes." Circ Res 84(4): 458-66.

2. Aoki, H., S. Izumo, et al. (1998). "Angiotensin II activates RhoA in cardiac myocytes: a critical role of RhoA in angiotensin II-induced premyofibril formation." Circ Res 82(6): 666-76.

3. Aznar, S., P. Fernandez-Valeron, et al. (2004). "Rho GTPases: potential candidates for anticancer therapy." Cancer Lett 206(2): 181-91.

4. Banyard, J., B. Anand-Apte, et al. (2000). "Motility and invasion are differentially modulated by Rho family GTPases." Oncogene 19(4): 580-91.

5. Bishop, A. L. and A. Hall (2000). "Rho GTPases and their effector proteins." Biochem J 348 Pt 2: 241-55.