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標題: | EBV BGLF4蛋白質激酶對於PML核體與 PML小類泛素修飾的調控作用 The regulatory effects of EBV BGLF4 kinase on PML nuclear body organization and PML sumoylation |
作者: | Chu-Yuan Hsu 徐祝元 |
指導教授: | 陳美如 |
關鍵字: | EB病毒,PML核體, EBV,BGLF4,PML nuclear body, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | EB病毒的蛋白質激酶BGLF4是病毒溶裂期表現的蛋白質。BGLF4會改變細胞核內的結構,引發細胞內的染色體濃縮。觀察BGLF4對於細胞核內其他構造的影響,發現BGLF4會造成PML核體改變,其數目有顯著的減少。PML核體的功能為調控細胞中的基因表現、細胞凋亡以及作為細胞中抗病毒的機制,所以進一步探討BGLF4對於PML核體的影響以及調控的機制。BGLF4在HeLa細胞以及鼻咽癌細胞中皆會引起PML核體數目的減少,不是透過降解PML核體中的必需蛋白質PML。經由先前的酵母菌雙雜交實驗得知BGLF4會與SUMO1有交互作用,進一步以共同免疫沉澱確認兩者有交互作用。因為PML的小類泛素修飾會調控PML核體的組成,進而探討BGLF4對於PML的小類泛素修飾之影響。在細胞中轉染BGLF4之表現質體可以抑制PML VI的小類泛素修飾,而且細胞中蛋白質的小類泛素修飾並未受到太大的影響,顯示BGLF4對於PML的小類泛素修飾有特異性。分離萃取細胞核基質發現BGLF4減少PML在細胞核基質的含量。BGLF4表現的細胞中,可以觀察到PML VI分子量較高,以磷酸酶處理過後,PML VI分子量較高的現象消失,顯示BGLF4可增強PML VI的磷酸化。因此可推想BGLF4是透過磷酸化PML VI而抑制其小類泛素修飾,但仍需有其他實驗佐證。PML核體會因外界環境變化而重組,召集轉錄活化因子至PML核體內,進而影響細胞內的基因調控,包含p53。利用螢光酵素報導基因檢測觀察到BGLF4促進p53調控之p21啟動子活性,可能機制是透過PML核體重組,但不能排除BGLF4會透過引發DNA損壞的訊息傳遞,進而促進p53轉錄活性,詳細機制仍需進一步釐清。本篇研究中發現BGLF4會造成PML核體數目減少以及PML的小類泛素修飾減少,推測其可能的影響為促進EB病毒的DNA複製或是抑制細胞中的抗病毒機制。 Epstein-Barr virus BGLF4 kinase is a lytic protein which can reorganize nuclear architecture and induce the condensation of host chromatin. While investigating the regulatory effects of BGLF4 on other nuclear structure, BGLF4 was found to cause the reorganization of promyelocytic leukemia nuclear body (PML NB). Promyelocytic leukemia protein (PML) is the major organizer of PML NBs, which are nuclear structures that regulate gene expression, apoptosis and function as an antiviral defense mechanism. Therefore, the effects of BGLF4 on PML NBs were further studied. BGLF4 induced the reorganization of PML NBs in HeLa cells and NPC-TW01 cells without PML degradation. The interaction between BGLF4 and SUMO1 was confirmed by co-immunoprecipitation. Transient expression of BGLF4 inhibited the sumoylation of PML and decreased the amounts of PML VI in nuclear matrix. BGLF4 enhanced the phosphorylation of PML. Furthermore, BGLF4 enhanced the p53-mediated p21 promoter in reporter assay. It is possible that BGLF4 promoted p53-mediated p21 promoter activity via PML reorganization. However, BGLF4 induced DNA damage response may also participate in the p21 promoter activation. Together, these findings suggest that BGLF4 induced PML reorganization and inhibited PML VI sumoylation. It remains to be studied wheather BGLF4 induced PML reorganization may facilitate EBV DNA replication and suppress the antiviral mechanism. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25607 |
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顯示於系所單位: | 微生物學科所 |
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