臺灣博碩士論文加值系統

卡波西氏肉瘤疱疹病毒或稱為人類第8號疱疹病毒為gammaherpesvirus的一員，已知和三種人類惡性腫瘤的形成有關，包括了卡波西氏肉瘤、原發性滲出性淋巴瘤、和多發性Castleman氏病。像所有的疱疹病毒一樣，卡波西氏肉瘤病毒具有潛伏與溶裂複製兩個不同的生活史。此病毒由潛伏生活史過渡至溶裂複製生活史的轉換，對於病毒的繁衍和病毒的致病性皆極為重要。在生活史轉換過程中，一個多功能的病毒蛋白質，稱之為ORF50蛋白質，是負責整個啟動的關鍵。雖然ORF50蛋白質是控制病毒進入複製期的重要蛋白質，可是目前對於ORF50蛋白質穩定性的調控機制仍不清楚。先前實驗室的研究結果顯示ORF50蛋白質C端含有兩個能調控ORF50蛋白質表現的PARS (protein abundance regulatory signal)， PARS-I位在ORF50蛋白質第495個胺基酸到第535個胺基酸，而 PARS-II 位於ORF50蛋白質第590個胺基酸到第650個胺基酸，當PARS-I或是PARS-II有突變，都會使得ORF50的蛋白質表現遠高於wild-type ORF50蛋白質表現。在本篇論文中我們主要是要探討PARS-I和PARS-II對於ORF50蛋白質表現所扮演的角色以及研究ORF50蛋白質穩定性調控機制。目前結果顯示PARS-I為 ORF50蛋白質主要進核訊號，能透過影響ORF50蛋白質的進核來影響蛋白質的穩定性，而進到細胞核之後ORF50蛋白質的PARS-II對調控蛋白質的穩定性很重要，其中發現具有E3 ligase功能的致癌蛋白質MDM2能與PARS區域結合而促進ORF50蛋白質的降解， MDM2蛋白質能將ORF50蛋白質泛素化修飾，而泛素化修飾的位置可能位在N端的第152與154個胺基酸位置的lysine。我們目前所有的研究結果顯示MDM2蛋白質為一個新的KSHV溶裂期的負調控子，而MDM2 蛋白質與ORF50蛋白質之間表現的平衡可能是決定KSHV能否由潛伏感染期走向溶裂期的關鍵之一。

Kaposi’s sarcoma-associated herpesvirus (KSHV) or called human herpesvirus-8 (HHV-8) is a gammaherpesvirus associated with at least three human malignancies, including Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. Like all herpesviruses, KSHV manifests two distinct phases of its life cycle, latency and lytic replication. The switch between latency and the lytic cycle is important not only for viral propagation but also for viral pathogenesis. The switch from viral latency to lytic replication is initiated by the expression of a transcriptional activator encoded in open reading frame 50 (ORF50) of viral genome. Although the ORF50 protein is the key controller for viral reactivation, the mechanism underlying the regulation of its protein stability remains largely unknown. Previously, our laboratory has shown that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates the abundance of ORF50 protein. The PARS region consists of two critical components: one component is located between aa 490 and 535 (PARS-I), and the other component is located between aa 590 and 650 (PARS-II). Mutations of either component lead to abundant expression of ORF50 protein. In this study, we characterized the functional role of each PARS component in controlling the ORF50 abundance and determined the molecular mechanism underling the protein stability of ORF50. We show here that the PARS-I motif mainly serves as a nuclear localization signal in the control of ORF50 stability, and the PARS-II motif is required for ORF50 degradation after the protein enters the nucleus. Importantly, we find that cellular oncoprotein MDM2, an E3 ubiquitin ligase, binds to the PARS region of ORF50 and promotes ORF50 degradation. The putative ubiquitin acceptor sites targeted by MDM2 are mapped to K152 and K154 in the N-terminal region of ORF50. Overall, our findings suggest that MDM2 may be a novel negative regulator of KSHV reactivation and the balance between ORF50 and MDM2 expression may be critical to determining the switch between latency and the lytic cycle.

目錄
指導教授推薦書
口試委員會審定書
誌謝 iii
中文摘要 iv
英文摘要 v
第壹章、緒論 1
一、卡波西氏肉瘤疱疹病毒（KSHV） 1
二、卡波西氏肉瘤疱疹病毒的結構與生活史 6
三、ORF50蛋白質 9
(一) ORF50蛋白質的功能 9
(二) ORF50蛋白質的DNA結合能力和蛋白質表現 12
(三)與ORF50蛋白質有交互作用的蛋白質 13
(四) ORF50蛋白質的轉譯後修飾 14
四、MDM2蛋白質 17

第貳章、研究目的 20

第參章、材料與方法 23
一、細胞培養與轉染 23
二、質體的構築 23
三、免疫螢光染色 24
四、西方墨點法 25
五、ORF50蛋白質泛素分析 26
六、蛋白質穩定性分析 27
七、共免疫沉澱 27
八、GST pull down 28
九、In vitro泛素分析 29
十、以慢病毒(Lentivirus)載體介導RNAi (RNA interference) 29

第肆章、結果 32
一、ORF50蛋白質的A form為高度磷酸化蛋白質，B form為低度磷酸化蛋 白質 32
二、ORF50蛋白質的PARS -I能影響蛋白質進核能力而影響蛋白質表現 35
三、ORF50會形成聚合子再進到細胞核，之後在細胞核中降解 42
四、ORF50蛋白質PARS-II區域會影響ORF50蛋白質在細胞核中的表現.......46
五、ORF50蛋白質為泛素化修飾的蛋白質 59
六、ORF50蛋白質有兩個區域能影響ORF50蛋白質的泛素化 63
七、MDM2蛋白質能透過ORF50蛋白質的C端來降低ORF50蛋白質的表現 68
八、在in vivo的條件下，MDM2蛋白質能與ORF50蛋白質結合.......................74
九、MDM2蛋白質第1-220胺基酸區能與ORF50蛋白質C端第490-691胺基酸區域結合 78
十、MDM2蛋白質為ORF50蛋白質泛素化的E3 ligase.....................................86
十一、MDM2蛋白質會透過ORF50蛋白質N端第152個與第154個胺基酸來調控ORF50蛋白質的穩定性 88
十二、在KSHV溶裂期中MDM2蛋白質與ORF50蛋白質的表現 93

第伍章、討論 96
一、ORF50蛋白質的降解有兩個重要的步驟 96
二、MDM2蛋白質能與ORF50蛋白質結合且會促進ORF50蛋白質的降解.....99
三、MDM2蛋白質在KSHV潛伏感染期與溶裂期的角色..................................102

第陸章、未來工作與展望 106
一、確認ORF50蛋白質被泛素修飾的鍵結位置與類型......................................106
二、LANA-MDM2-p53複合體是否會影響ORF50蛋白質的穩定性.................107
三、當KSHV走向溶裂期時MDM2蛋白質的負調控因子.................................108

第柒章、結論 111
一、 ORF50蛋白質C端PARS區域會透過影響蛋白質穩定性進而影響 ORF50蛋白質的表現 111
二、ORF50蛋白質是會被泛素修飾而透過蛋白酶體降解..................................111
三、MDM2蛋白質為ORF50蛋白質泛素化的E3 ligase.....................................112
四、MDM2蛋白質為ORF50蛋白質的負調控因子............................................112

參考文獻 114


圖目錄

圖1-1、 KSHV的基因體圖譜 4
圖1-2、 KSHV的兩階段生活史 8
圖1-3、 ORF50蛋白質以及其相關蛋白質的結合位 11
圖1-4、 ORF50蛋白質 16
圖1-5、 MDM2蛋白質 19
圖2-1、 ORF50蛋白質的表現會被PARS-I與PARS-II所調控...............................22
圖4-1、 ORF50蛋白質A form為高度磷酸化，B form為低度磷酸化.....................34
圖4-2、 PARS-I中的KKRK motif為ORF50蛋白質的主要進核訊號...................38
圖4-3、 ORF50(KK/EE)由細胞質進到細胞核後，蛋白質表現會恢復到wild-type ORF50的程度 41
圖4-4、 ORF50(KK/EE)由細胞質進到細胞核後，蛋白質表現會恢復到wild-type ORF50的程度 44
圖4-5、 ORF50蛋白質的PARS-II不影響ORF50蛋白質進核能力.......................49
圖4-6、 ORF50蛋白質的NLS1為較無效率的進核訊號.........................................51
圖4-7、 ORF50(KK/EE)由細胞質進到細胞核後，蛋白質表現會恢復到wild-type ORF50的程度 56
圖4-8、 ORF50蛋白質N端1-490能和ORF50(KK/EE)結合.................................58
圖4-9、 ORF50蛋白質是被泛素化修飾的蛋白質 61
圖4-10、 ORF50蛋白質的PARS-II以及N端第1個胺基酸到第356個胺基酸對於ORF50蛋白質被泛素修飾很重要 66
圖4-11、 泛素化相關蛋白質與ORF50蛋白質 71
圖4-12、 MDM2 蛋白質會降低ORF50蛋白質的表現 73
圖4-13、 MDM2蛋白質會和ORF50蛋白質結合 76
圖4-14、 MDM2蛋白質會和ORF50蛋白質直接結合 80
圖4-15、 確認His-ORF50蛋白質、GST-MDM2蛋白質與GST-MDM2刪除株的完整性 84
圖4-16、 在in vitro的條件下，MDM2蛋白質能促進ORF50蛋白質的泛素化......87
圖4-17、 ORF50蛋白質第152與154個胺基酸lysine為影響ORF50蛋白質穩定性的重要胺基酸 91
圖4-18、 在被KSHV感染的細胞中，MDM2蛋白質能負調控ORF50蛋白質......95
圖5-1、 PARS-I與PARS-II調控ORF50蛋白質表現的模式..............................105
圖6-1、 ORF50蛋白第152個與第154個胺基酸可能為K48-linked泛素化修飾的位置 109


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