臺灣博碩士論文加值系統

BGLF4是EB病毒目前被發現唯一的serine/threonine蛋白質激酶，為了進一步了解BGLF4可能參與的生物功能，先前實驗室利用yeast two-hybrid找到了許多可能與BGLF4有連結的細胞蛋白質。本篇的研究目標便是其中一個已知位在中心體且與中心體結構完整性息息相關的細胞蛋白質ART27。在暫時轉染實驗中發現，BGLF4會造成ART27蛋白質降解。 經nocodazole處理後，觀察同步在有絲分裂時期的細胞，則發現ART27的蛋白質表現有上升的情形。在細胞週期進行的過程中，細胞常利用ubiquitin-26S proteasome的機制調控蛋白質的半生期，其中Anaphase promoting complex/cyclosome (APC/C) 便是參與調控有絲分裂到G1時期的一個重要的E3-ligase，APC/C利用分別與Cdc20或Cdh1蛋白質的結合決定受質的特異性。在暫時轉染的實驗中，共同表現Cdc20會造成ART27的蛋白質降解，共同免疫沉澱實驗的結果則證明在in vivo情況下，ART27可以與Cdc20交互作用，顯示ART27的蛋白質穩定性可能受到APC/C調控。Cdc20已知會辨認受質蛋白質的destruction motif (D-box)，但將ART27胺基酸序列上的D-box之RXXL點突變後，仍能觀察到共同表現Cdc20所造成的蛋白質降解，亦不影響兩者間的交互作用，顯示可能有其他的胺基酸序列或分子參與ART27與Cdc20結合。 為了探討BGLF4是否透過APC/C參與ART27的半生期調控，利用siRNA阻斷Cdc20表現的策略，發現BGLF4需要透過APC/C-Cdc20造成ART27蛋白質的降解。共同免疫沉澱實驗的結果亦顯示，BGLF4可以與Cdc20交互作用，共同表現BGLF4則造成Cdc20蛋白質量上升。文獻指出負責調控APC/C最重要的激酶是細胞中的Cdc2，而已知BGLF4會磷酸化數個Cdc2受質(例如轉譯延遲因子1 -δ)相同的序列，暗示兩者在生物功能上的保留性。本篇研究提出BGLF4的作用模式，即BGLF4可能類似細胞中Cdc2，藉由磷酸化誘使APC/C的活化，並造成APC/C的受質ART27的蛋白質降解。

BGLF4 is the only serine/threonine protein kinase identified in Epstein-Barr virus (EBV). In order to understand the biological function of BGLF4, our lab established yeast-two hybrid screening system and identified ART27 as a cellular binding partner of BGLF4. Here we found that overexpression of BGLF4 induced ART27 degradation. ART27 has been suggested to be a component of centrosome and essential for cell viability. Since centrosome maturation is the key event during mitosis, we observed the protein expression pattern of ART27 during cell cycle progression after releasing from mitotic arrest. We found that the protein amount of ART27 increased at mitosis and decreased at G1. The anaphase-promoting complex/Cyclosome (APC/C) is the most important E3-ligase that regulates the protein half-life during mitosis. We demonstrated that overexpression of APC/C activator Cdc20 but not Cdh1 disrupted ART27 protein expression. From immunoprecipitation assay, we demonstrated that ART27 can interact with Cdc20 in vivo. However besides the destruction box (D box), the Cdc20 binding motif of already known APC/C substrates, there seems to be other amino acid sequences or factors involved in ART27 binding with Cdc20. Furthermore, abrogation of Cdc20 protein expression by small interfering RNA blocked ART27 degradation induced by BGLF4. The interaction between BGLF4 and Cdc20 was demonstrated by immunoprecipitation assay. In transient transfection, BGLF4 enhanced protein stability of APC/C activator Cdc20. In this study, ART27 was identified as a novel substrate of APC/C. Since BGLF4 was found to phosphorylate several substrates at Cdc2 targeting site, we speculate that BGLF4 might regulate ART27 protein stability through activating APC/C activity similary to Cdc2 does.

目錄 --------------------------------------------------1
中文摘要 ----------------------------------------------2
英文摘要 --------------------------------------------- 3
前言 ------------------------------------------------- 4
材料與方法 -------------------------------------------- 20
結果 ------------------------------------------------ 28
討論 ------------------------------------------------ 32
圖表 ----------------------------------------------- 35
參考文獻 -------------------------------------------- 48

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