王惠鈞臺灣大學:生化科學研究所王皓青Wang, Hao-ChingHao-ChingWang2010-05-042018-07-062010-05-042018-07-062008U0001-2407200814073600http://ntur.lib.ntu.edu.tw//handle/246246/178842為了更進一步瞭解蝦白點病毒的致病機制，及鑑定何種細胞反應途徑會在病毒感染後受到影響，故利用二維蛋白質膠體電泳呈現正常以及病毒感染 48 小時後的白蝦胃組織之蛋白質表現圖譜。在經過分析比較後，挑選了 75 個具有明顯改變或是保持穩定高表現量的蛋白質點，利用液態層析質譜儀以及生物資訊資料庫進行蛋白質身份的鑑定。最終成功鑑定了 53 個蛋白質的身份，這些蛋白質的功能包含了能量的產生，維持鈣離子的恆定，核酸的合成，訊息傳導，攜帶氧氣以及 SUMO 相關後修飾作用等。其中一個病毒感染後高表現量的蛋白質，經質譜儀鑑定為蝦白點病毒之非結構性蛋白質 ICP11，此蛋白質至今功能未知，但很有可能在病毒感染複製過程中扮演重要的角色，因此利用結構生物學的方法針對此蛋白質功能作更深入的研究。在利用多波長異常繞射法 (MAD method) 解析出ICP11 的蛋白質結構後發現，此一蛋白質具有模仿 DNA 的功能。在蛋白質晶體內，ICP11 的雙聚體可以形成多聚體，而此多聚體上具有類似雙股 DNA 的負電荷分佈，顯示 ICP11 為一 DNA 擬態蛋白質。再藉由遠西方點墨法證明 ICP11 主要是作用在宿主的 histone 蛋白質上。之後利用 histone-DNA 結合實驗進一步證明 ICP11 可有效的防止 histone H2A，H2A.x，H2B以及 H3 與 DNA 之間的結合。在免疫螢光測試方面，ICP11 也可在蝦血球內與 histone H3 以及被活化的 histone H2A.x 同存，並將 histone 蛋白質保留在細胞質中，使之無法進入細胞核進行核仁小體的裝配或進行 DNA 的修復作用。另外也發現 ICP11 具有意料之外的 DNA 水解酵素的活性，而此活性可以在添加鎂離子的情況下有效增加。因此 ICP11 的多功能性結合成新穎的抗宿主策略，為一科學界的新發現。White spot syndrome virus (WSSV) is a pathogen responsible for population collapse in intensive white shrimp (Litopenaeus vannamei) culture. This research was initiated to better understand WSSV pathogenesis and cell pathways affected in the infection process. Initially two-dimensional gel electrophoresis (2-DE) was used to create protein expression profiles for specific pathogen free and infected shrimp at 48 hours post-infection (48 hpi). Seventy-five protein spots consistently showed either a marked change in accumulated levels (>50%) or else were highly expressed throughout the course of WSSV infection. These were considered to be important in pathogenesis and were selected for further study. Bioinformatics databases were searched for matches with the results of LC-nanoESI-MS/MS. Of the 75 proteins, 53 were identified in this way. Of these, all but one had widely varying known functions, including energy production, calcium homeostasis, nucleic acid synthesis, signaling/communication, oxygen carrier/transportation, and SUMO related modification. The remaining one was an altered and highly expressed nonstructural protein which we named ICP11. We then studied this protein in detail to determine its function. The crystal structure was determined using the MAD phasing method. ICP11 is dimer. It has two rows of negatively-charged spots. Interestingly, in crystal, ICP11 dimer forms a polymer where the distribution of negative charges approximates the duplex arrangement of the phosphate groups in DNA. This suggests that ICP11 is a DNA mimic protein. By using far western analysis we found that ICP11 can interact with histone proteins (H2A, H2B, H3 and H2A.x). ICP11 out-competed DNA in binding to these proteins. Further, ICP11 was found to colocalize with histone H3 and activated H2A.x in the hemocytes of WSSV infected shrimp. This suggests that ICP11 may interfere with DNA packaging and prevent H2A.x from fulfilling its critical function of repairing DNA double strand breaks (DSBs). In addition, ICP11 exhibited an unexpected endonuclease activity in the presence of Mg2+ ions. ICP11 therefore demonstrates a multifunctional anti-host strategy never before reported.中文摘要...............1文摘要...............2. 前言................4. 材料與方法..........8. 結果...............19. 討論...............30. 參考文獻...........35. 表格...............39. 圖.................45. 附錄...............69. 參與發表論文.......76application/pdf10918660 bytesapplication/pdfen-US蝦白點病毒ICP11 結構ICP11 生理功能white spot syndrome virusICP11 structurebiological function of ICP11http://ntur.lib.ntu.edu.tw/bitstream/246246/178842/1/ntu-97-D92b46001-1.pdf