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研究生:劉紹宇
研究生(外文):Shau-Yu Liu
論文名稱:探討Disabled-2蛋白在p75-TRAF6訊息複合體形成中所扮演的角色
論文名稱(外文):Characterization of Disabled-2 function in the formation of p75-TRAF6 signal complex
指導教授:曾慶平
指導教授(外文):C. P. Tseng
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:92
中文關鍵詞:蛋白複合體細胞凋亡
外文關鍵詞:DAB2TRAF6p75ubiquitination
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Disabled-2(DAB2)已知是細胞內一個很重要的蛋白,參與在許多訊息傳遞的路徑中。由於其具備了抑制癌細胞生長的能力,因此被視為一個腫瘤抑制因子(tumor suppressor)。利用生物資訊的預測發現,DAB2會藉由其胺基酸序列上NSPTESKDI motif以及GIPQENADH motif和tumor necrosis factor receptor-associated factor six(TRAF6)結合。由於文獻指出TRAF6是p75受體很重要的結合蛋白,同時影響p75受體所調控的細胞生長。因此我們推測DAB2可能藉由與TRAF6結合來調控p75受體所傳遞的訊息。我們利用免疫沉澱法第一次發現DAB2與TRAF蛋白家族之間的結合能力,尤其又以TRAF6的結合能力最強。進一步的分析發現,TRAF6是藉由其TRAF-C區域與DAB2結合。根據所預測的結合位置,我們修改結合位置的胺基酸修飾,不過並沒有消除兩者之間的結合情形。接著我們發現DAB2不會影響TRAF6所誘導NF-kappaB活性的改變;反之,藉由TRAF6 RING區域E3 ligase的活性,會誘導DAB2 K63-linked polyubiquitination。另外,我們也發現DAB2其中一個可能的ubiquitination位點,而尚有未知的ubiquitination位點等待進一步探討。透過TRAF6的幫助,我們發現p75與DAB2會結合在一起,進一步形成一個蛋白複合體。利用ibuprofen處理PC3細胞誘導表現p75蛋白並使其進行細胞凋亡,觀察DAB2在其中所扮演的角色。我們利用siRNA抑制內生性DAB2的表現,會促使ibuprofen所誘導的細胞凋亡,顯示DAB2的存在會抑制由ibuprofen所調控的細胞凋亡過程。綜合以上結果,DAB2可能藉由與p75-TRAF6形成一個蛋白複合體,進一步去調控攝護腺癌細胞的細胞凋亡。
Disabled-2(DAB2)has already known as an important cellular adaptor protein that is involved in various signaling pathways and cell functions. DAB2 is a candidate of tumor-suppressor based on its ability for cancer cell suppression. Bioinformatics analysis revealed that tumor necrosis factor receptor-associated factor six (TRAF6) may interact with the NSPTESKDI motif and GIPQENADH motif of DAB2. It has been shown that TRAF6 is an important interacting protein of p75, and can transmit the signaling of p75 receptor to affect cell survival. Hence, DAB2 may be involved in p75 signaling by interacting with TRAF6. We demonstrated for the first time that DAB2 can interact with TRAF6 and other TRAF family proteins by co-immunoprecipitation analysis in 293T cells, with the strongest interaction occurred between DAB2 and TRAF6. Further analysis revealed that DAB2 interacted with the TRAF6 TRAFC domain. Based on the predict binding motif, we constructed the point mutation and deletion mutation of DAB2, but each construct can’t eliminate the interaction between DAB2 and TRAF6. Moreover, we found that DAB2 can’t affect TRAF6-mediated NF-B activity. By the TRAF6 RING domain E3 ligase activity, DAB2 serves as a substrate for TRAF6-mediated K63-linked polyubiquitination. We also found that one of DAB2 ubiquitination site, but we thought there still have other ubiquitination site. The interaction of DAB2 and TRAF6 enhanced the novel association between DAB2 and p75 receptor in a TRAF6 dose-dependent manner. We used ibuprofen to induce PC3 cell apoptosis and p75 expression to observe the function of DAB2 in p75 signaling. If we used siRNA to knock down DAB2 expression, the cell apoptosis effect of ibuprofen was increased, demonstrating that the formation of this complex regulates the process of cell apoptosis. Together, these findings suggest that DAB2 may play an important role in prostate cancer cell apoptosis by forming a signaling complex.
目 錄
指導教授推薦書………………………………………………………………..……
口試委員會審定書…………………………………………………………..………
授權書……………………………………………………………………..….iii
誌謝……………………………………………………………………………...iv
中文摘要……………………………………………………………………………v
英文摘要……………………………………………………………………..….vi
目錄………………………………………………………………………….….vii
第一章、背景介紹……………………………………………..………………..1
一、Disabled-2(DAB2)……………………………………..………………1
二、p75 neurotrophin receptor………………………………........3
三、TNF receptor-associated factor family(TRAF)….……...5
四、Ubiquitin system......................................7
五、Research aim..........................................10
第二章、材料與方法……………………………………………………………..12
一、實驗材料…………………………………………………………..……..12
二、實驗方法…..…………………………………………………….…………14
第三章、實驗結果…………………………………………………………....21
第四章、討論…….………………………………………………………..….34
第五章、參考文獻……………………………………………………………….45
圖表…………………………………………………………………….………….51
附表……………………………………………………..…………………………81

圖 表 目 錄
圖一、p75蛋白的結構示意圖…………………………………………………..51
圖二、TRAF6蛋白不同domain deletion constructs的結構示意圖…..52
圖三、DAB2上所預測的TRAF6 binding motif…………………….…….53
圖四、TRAF family蛋白的驗證與表現的結果……………………………..54
圖五、DAB2與TRAF6蛋白結合的情形…………………………………....…55
圖六、不同domain deletion之TRAF6與DAB2接合能力的探討………….57
圖七、不同的TRAFs family蛋白與DAB2之間結合的情形………………….58
圖八、DAB2不同constructs之示意圖以及TRAF6蛋白預測的binding motif…….59
圖九、不同constructs的DAB2與TRAF6之間接合能力的探討…………..…60
圖十、DAB2 mutation constructs示意圖……………………………….61
圖十一、DAB2 point mutation與TRAF6接合能力的探討…………………62
圖十二、DAB2 deletion mutation與TRAF6接合能力的探討……...……63
圖十三、DAB2對於TRAF6-induced NF-kappaB activity的探討…….…64
圖十四、TRAF6對於DAB2 ubiquitination的探討………………...…..65
圖十五、抑制endogenous TRAF6的效果………………………………………67
圖十六、Endogenous TRAF6對DAB2 ubiquitination的影響……………69
圖十七、DAB2 ubiquitination機制的探討…………………………..……70
圖十八、DAB2 ubiquitination位點的探討…………………………..……71
圖十九、TRAF6不同domain對於DAB2 ubiquitination的影響……………72
圖二十、p75與TRAF6蛋白的結合情形………………………………...…..73
圖二十一、觀察p75、TRAF6與DAB2蛋白之間的結合情形……………………74
圖二十二、表現量不同的TRAF6對於p75與DAB2之間結合的影響……….…75
圖二十三、不同TRAF蛋白對於p75與DAB2之間結合的影響…………..…….76
圖二十四、Ibuprofen誘導p75蛋白表現的情形.....................77
圖二十五、DAB2對於ibuprofen-induced細胞凋亡的探討………….…..78
圖二十六、DAB2在p75-TRAF6訊息複合體中可能扮演的角色...........80
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