cIAP1結合蛋白質及cIAP1泛素化之研究

The studies on the cIAP1-associated proteins and cIAP1 ubiquitination

張永福(Yung-Fu Chang)

指導教授 ： 游仲逸

共同指導教授 ： 鐘育志(Yuh-Jyh Jong)

高雄醫學大學/醫學院/醫學研究所/博士(2006年)

https://doi.org/10.6832/KMU.2006.00136

摘要

cIAP1(cellular inhibitor of apoptosis protein 1)是人類八個抑制細胞凋亡蛋白質家族成員之一，在結構上，包含了N端的三個BIR (baculovirus IAP repeat) domains、中間一個CARD domain(caspase recruitment domain)及C端的一個Ring domain。我們以人類抑制細胞凋亡蛋白質cIAP1著手進行細胞凋亡的分子機制之研究。首先，我們以酵母雙雜交篩選方法試圖找出cIAP1調節過程中參與的蛋白質。實驗進行中，我們發現人類抑制細胞凋亡蛋白質cIAP1分子中具有轉錄活化區域 (transcriptional activation domain) 進一步找出cIAP1分子中具有轉錄活化(transcriptional activity)的區域 介於第一個BIR domain和第二個BIR domain之間。接著我們以cIAP1不含轉錄活化區域的片段為釣餌，用酵母菌雙雜交篩選法來選殖人類細胞中與cIAP1結合的蛋白質之基因。先以cIAP1第二個BIR domain到C端點的片段為釣餌，得到一株帶有Fbxo7基因的菌株。我們以共同免疫沉澱法驗證cIAP1與Fbxo7的結合性。接著以共軛焦顯微鏡分析二者同存在時所座落於細胞中的位置，結果明顯的看出cIAP1和Fbxo7共同坐落的位置，不論細胞核和細胞質均有。而且發現，大量表現Fbxo7會促進cIAP1進行泛素化(ubiquitination) 。我們的結果認為Fbxo7會經由SCF ubiquitin protein ligase 調節cIAP1的 ubiquitination，這樣的結果提供了研究cIAP1調節機制的新方向。接著再以cIAP1同樣不含轉錄活化區域的N端到第一個BIR domain的片段為釣餌，結果我們釣出AUP1(ancient ubiquitous protein 1)基因，於是我們重新建構在C端點帶有myc與His標記的pcDNA3.1/myc-His-AUP1，並證實此質體可以使人類細胞大量表現AUP1。接下來希望經由共同免疫沉澱法(Co-immunoprecipitation assay)確認AUP1與cIAP1的結合性。最後，我們找出cIAP1分子中被泛素化的位置(ubiquitination site)位於第三個BIR domain到C端點之間。這些研究結果，讓我們對cIAP1蛋白質有更進一步的認識。cIAP1的異常表現量被認為與許多癌症的形成有關。因此，我們希望這部份的研究結果不僅能深入探討cIAP1在細胞凋亡過程中所扮演的角色，也希望能解釋部份疾病形成原因，甚至提供藥品開發等臨床應用的可能性。

關鍵字

cIAP1 ； 抑制細胞凋亡蛋白質 ； 泛素化

並列摘要

cIAP1 is one of eight human inhibitor of apoptosis proteins (IAPs). It contains three BIR (baculovirus IAP repeat) domains in N-terminal, one CARD domain (caspase recruitment domain) in the middle and one Ring domain in C-terminal. In order to get insight into the mechanism of apoptosis, we started with cIAP1. First of all, we performed yeast two-hybrid assays to isolate the human genes encoding cIAP1-associated proteins. DuRing performing yeast two-hybrid assays, we found that cIAP1 contains a transcriptional activation domain. And the transcriptional activation domain of cIAP1 was found to locate between first and second BIR domain. We then used different cIAP1 deleted fragments without transcriptional activation domain as bait to perform yeast two-hybrid assays. We expected to get the clones of cIAP1-associated proteins from human cDNA library. When we used cIAP1 fragment containing from the second BIR domain to the C-terminus as bait, one clone encodes full-length of Fbxo7 protein was isolated from this screen. We performed co-immmunoprecipitation assay to demonstrate that cIAP1 associated with Fbxo7 in mammalian cells. We also showed that cIAP1 co-localized with Fbxo7 in mammalian cells in both cytoplasm and nucleus by confocal fluorescence microscope. Furthermore, we found that overexpressed Fbxo7 promotes the ubiquitination of cIAP1. Since F-box proteins are specificity determining subunits of SCF ubiquitin ligases, our results are the first report showing that the ubiquitination of one IAP protein can be mediated by SCF ubiquitin ligase and suggesting additional mechanism for negative regulation of cIAP1 expression. When we used cIAP1 fragment containing from N-terminus to the end of first BIR domain as bait, one clone encodes ancient ubiquitous protein 1, AUP1, was isolated from yeast two-hybrid screen. In order to test the interaction of AUP1 and first BIR domain of cIAP1 proteins, we constructed pCDNA3.1-myc-His-AUP1 and pEGFP-cIAP1-B1 for co-immmunoprecipitation. cIAP1-B1 contained cIAP1 fragment from N-terminus to the end of first BIR domain. Western blot analysis indicated that both GFP-cIAP1-B1 and AUP1-myc-His were detected from mammalian cells. The co-immmunoprecipitation assay is still under studied. Finally, we identified the ubiquitination region of cIAP1 located between the third BIR domain and C-terminus. cIAP1 was reported to have some roles involved in cancers and diseases. We hope our results will provide insights into the mechanism of diseases and even the clinical utilization in the future.

並列關鍵字

cIAP1 ； inhibitor of apoptosis proteins (IAPs) ； ubiquitination

參考文獻

參考文獻

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