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標題: | 鑑定參與GCM1汎素化之E2結合酵素及其特性探討 Identification and Characterization of an E2 Ubiquitin-Conjugating Enzyme in GCM1 Ubiquitination |
作者: | Meng-Hsiu Chiang 江孟修 |
指導教授: | 陳宏文 |
關鍵字: | 胎盤, GCM1, SCF複合體, FBXW2, E2-6, Placenta, GCM1, SCF complex, FBXW2, E2-6, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | GCM1是在胎盤發育中一個必需的轉錄因子,而且它的活性已經被證實受到汎素-蛋白酶體的降解系統所調控。蛋白質的汎素化需要三種酵素的參與,分別是E1活化酵素、E2結合酵素以及E3接合酵素。在我們之前的研究中指出,SCFFBXW2複合體能夠藉由人類F-box蛋白FBW2專一性地辨識GCM1並扮演其E3接合酶的角色;然而,是哪一個E2結合酵素參與在其中仍然是個待解的習題。
在這個研究中,我們首先指出HeLa S-100萃取液可以在活體外促進GCM1的汎素化;並且進一步地在一系列的E2中鑑定出一個可以專一地調控GCM1汎素化的E2蛋白(稱之為E2-6)。我們也利用活體外汎素化的實驗證實了E1、E2-6和SCFFBXW2對於GCM1的汎素化都是不可或缺的。E2-6活性區的點突變會使得它失去和汎素結合的能力,在我們的實驗中也證實了這樣的突變會導致E2-6失去和E3的交互作用而無法將GCM1汎素化。另外,在293T細胞中利用RNA干擾抑制E2-6蛋白的表現會使得GCM1較不易被汎素化而能夠被保存下來。 上述的實驗結果顯示,無論在活體內或是活體外,E2-6都在GCM1的汎素化中扮演著重要角色。 Protein ubiquitination involves E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ligase. GCM1, whose activity has been demonstrated to be regulated by the ubiquitin-proteasome system, is an essential transcription factor in placental development. Our previous studies have shown that GCM1 is a substrate for the SCFFBXW2 E3 complex, which specifically recognizes GCM1 via the human F-box protein, FBW2. However, the ubiquitin-conjugating enzyme (E2) involved in GCM1 ubiquitination remains elusive, in this study, we first showed that the HeLa S-100 extract can promote ubiquitination of GCM1 in vitro, and then identified an E2 (termed E2-6) specifically regulates GCM1 ubiquitination after screening a panel of E2s. In vitro ubiquitination assay of GCM1 also demonstrated that E1, E2-6, and SCFFBW2 are necessary for GCM1 ubiquitination. Mutation on the catalytic site of E2-6 (E2-6CA) resulted in loss of its interaction with SCFFBW2 and the ubiquitination of GCM1. Moreover, small hairpin RNA-mediated knockdown of E2-6 reduced ubiquitination of GCM1 and subsequently increased GCM1 protein stability in 293T cells. These results reveal that E2-6 is crucial for ubiquitination of GCM1 in vitro and in vivo. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26523 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
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