陳宏文臺灣大學:生化科學研究所江孟修Chiang, Meng-HsiuMeng-HsiuChiang2010-05-042018-07-062010-05-042018-07-062008U0001-2607200821034500http://ntur.lib.ntu.edu.tw//handle/246246/178850GCM1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。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。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.目錄……………………………………………………………… ……………………...I表目錄……………………………………………………………………………….III寫表………………………………………………………………………………….IV文摘要……………………………………………………………………………….VI文摘要Abstrate……………………………………………………………………..VII一章 　序論………………………………………………………………………...1.1　胎盤………………………………………………………………………..…1 .2　gcm轉錄因子基因家族……………………………………………………..3.3　GCMa的降解機制…………………………………………………………..5.4　E2結合酵素………………………………………………………………….7.5　研究動機……………………………………………………………………..8二章 　 材料及方法…………………………………………………………..9.1　重組質體的構築與增殖……………………………………………………..9.2　細胞培養……………………………………………………………………13.3　活體外汎素化反應分析…………………………………………………....15.4　西方墨點法…………………………………………………………………16.5　共同免疫沉澱法…………………………………………………………....16.6　GST融合蛋白表現………………………………………………………....17.7　活體內汎素化試驗………………………………………………………....17.8　35S脈衝追蹤試驗…………………………………………………………..18.9　反轉錄聚合酶連鎖反應…………………………………………………....18三章 實驗結果………………………………………………………………….20.1　汎素在活體外對GCM1的標定………………………………….………..20.2　E2-6可以專一性地促進GCM1汎素化…………………………………..20.3　E2-6在GCM1汎素化中扮演E2結合酵素的角色…………………….…21.4　E2-6和SCFFBW2複合體的交互作用……………………………………...22.5　利用外生性shRNA質體抑制細胞內E2-6蛋白表現………………….…23.6　E2-6對GCM1汎素化及蛋白穩定度的影響……………………………..23四章　　討論與總結……………………………………………………………….25五章 　　圖表……………………………………………………………………….28六章 　　參考文獻………………………………………………………………….36application/pdf1578875 bytesapplication/pdfen-US胎盤, GCM1, SCF複合體, FBXW2, E2-6Placenta, GCM1, SCF complex, FBXW2, E2-6http://ntur.lib.ntu.edu.tw/bitstream/246246/178850/1/ntu-97-R94b46044-1.pdf