RanBPM扮演SUMO E3 Ligase的角色促進EB病毒Rta蛋白質的SUMOylation

RanBPM acting as a SUMO E3 Ligase enhances Rta sumoylation in Epstein-Barr virus

郭仲文(Chung-Wen Kuo)

指導教授 ： 洪義人

高雄醫學大學/醫學院/生物化學研究所/碩士(2003年)

摘要

EB病毒（Epstein-Barr virus，EBV）是一種人類疱疹病毒（herpesvirus），可以感染淋巴細胞與表皮細胞，具有兩種不同的生活史。當EB病毒感染B淋巴細胞後，會造成宿主細胞的不死化（immortalization）並且潛伏在細胞內，伴隨著細胞分裂而複製。而一旦當宿主受到外來刺激，例如加入12-ο-tetradecanoyl-13-phorbol acetate、sodium butyrate或trichostatin A時， EB病毒則會由潛伏循環進入到溶裂循環。許多研究顯示，EB病毒由潛伏循環進入溶裂循環，與BZLF1和BRLF1這兩個極早期基因的表現有關，而此兩個基因的產物則分別是Zta和Rta蛋白質，都是一種轉錄因子。本研究主要的目的，是在探討RanBPM如何調控Rta的轉錄活性。在酵母菌雙雜交系統與共免疫沉澱法中發現，RanBPM會與Rta結合。RanBPM是一個90-kDa且在細胞質與細胞核皆會分佈的蛋白質。此外，由於RanBPM與RanBP2一樣都是Ran的結合蛋白質，因此我推測RanBPM可能和RanBP2一樣，具有SUMO E3 ligase的活性。從我的研究顯示，Rta在in vivo的狀態下會進行sumoylation修飾作用，且觀察到RanBPM會促進此修飾作用的進行。進一步的研究結果發現，RanBPM不僅會促進Sp3的sumoylation，而且還會與Ubc9及SUMO-1相互結合，而這些現象也符合作為SUMO E3 ligase的特性。此外，細胞轉染分析的實驗顯示，RanBPM會使Rta的轉錄活性增加為2倍。而將會表現Ubc9、SUMO-1與RanBPM的質體轉染到細胞中，結果發現會增加Rta和Gal4-Rta融合蛋白質對含有Rta反應區域與Gal4結合位置的啟動子的轉錄活性，顯示RanBPM可能是藉由扮演SUMO E3 ligase的角色，進而促進Rta的轉錄活性。

關鍵字

轉錄後修飾作用 ； EB病毒 ； 溶裂循環

並列摘要

Epstein-Barr virus (EBV), a human herpesvirus that infects lymphoid and epithelial cells, has two distinct life cycles. After infecting B-lymphocytes, the virus immortalizes the host cells and is maintained under latent conditions. However, the viral latency is disrupted if cells are exposed to external stimuli such as 12-ο-tetradecanoyl-13-phorbol acetate, sodium butyrate, and trichostatin A. As is well know, EBV lytic activation is closely associated with the expressing of two EBV immediate-early genes, BRLF1 and BZLF1, which encode Rta and Zta, respectively. The aim of this study is to elucidate how RanBPM regulates the transactivation activity of Rta. RanBPM, a 90-kDa protein which is distributed in both cytoplasm and nucleus, interacts with Rta by yeast two-hybrid analysis and coimmunoprecipitation. Due to the fact that RanBPM is a Ran-binding protein like RanBP2, I hypothesized that RanBPM may function as a SUMO E3 ligase. My work reveals that Rta is sumoylated in vivo and the sumoylation process is enhanced by RanBPM. Moreover, RanBPM not only enhances Sp3 sumoylation but also interacts with Ubc9 and SUMO-1, which is consistent with the characteristics of SUMO E3 ligases. Additionally, transient tranfection analysis reveals that RanBPM enhances Rta transactivation activity by two fold. Transfecting plasmids expressing Ubc9, SUMO-1, and RanBPM, increases the ability of Rta and Gal4-Rta fusion protein to transactivate promoters, that contain an Rta-response element and Gal4-binding sites, respectively, showing that RanBPM functions as SUMO E3 ligase to enhance Rta transactivation.

並列關鍵字

RanBPM ； Rta ； EB virus ； sumoylation

參考文獻

Adamson, A. L., and Kenney, S. (2001). Epstein-barr virus immediate-early protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia bodies. J Virol 75, 2388-2399.

Allday, M. J., Crawford, D. H., and Griffin, B. E. (1989). Epstein-Barr virus latent gene expression during the initiation of B cell immortalization. J Gen Virol 70, 1755-1764.

Arora, T., Liu, B., He, H., Kim, J., Murphy, T. L., Murphy, K. M., Modlin, R. L., and Shuai, K. (2003). PIASx is a transcriptional co-repressor of signal transducer and activator of transcription 4. J Biol Chem 278, 21327-21330.

Asai, S., Namikawa, I., and Ito, Y. (1984). Ultrastructural studies on human lymphoblastoid cells treated with n-butyrate and 12-O-tetradecanoylphorbol-13-acetate. Eur J Cancer Clin Oncol 20, 1533-1542.

Bai, D., Chen, H., and Huang, B. R. (2003). RanBPM is a novel binding protein for p75NTR. Biochem Biophys Res Commun 309, 552-557.