人類巨細胞病毒調控泛素蛋白質酶體系統

Alteration of ubiquitin-proteasome system by human cytomegalovirus

林新榮(Shin-Rung Lin)

指導教授 ： 王雙桂

高雄醫學大學/醫學院/醫學研究所/碩士(2012年)

摘要

本研究在探討人類巨細胞病毒(Human cytomegalovirus,HCMV) UL76表現於核內呈堆疊聚集(aggregate)的機轉。先前的研究顯示為長期表現UL76的腦瘤細胞(glioblastoma,U373-MG)出現DNA斷裂、微核、染色體異常。本篇的結果顯示UL76同樣會增加正常細胞(primary cells) DNA的斷裂，提高細胞分裂時Metaphase (M phase)紡錘體的異常數量，且證實會改變細胞週期的行進，加快細胞週期，由G1進入S的時間並延長G2/M phase的運行。實驗室先前以酵母菌雙雜交法得知UL76標的細胞的泛素蛋白酶體系統(ubiquitin-proteasome pathway,UPS)。此路徑掌控著細胞許多生化路徑的調控，其主要的功能在降解特異蛋白質。其中26S proteoasome 的S5a 是UPS與被多個ubiquitin (UB)結合的蛋白(UBn-protein)結合的主要受體(receptor)。而我們觀察到在有UL76存在下，大部份於細胞質的S5a會在細胞核內與UL76形成同位的聚集。再者經由FRET實驗顯示這兩個蛋白間的距離有交互作用的極可能性。就進一步探討發現UL76會累積細胞內生性UBn-protein的表現，由轉染S5a和UB於存有UL76之下出現累積UBn-protein、多個UB結合的S5a (UBn-S5a)的表現。接下來以蛋白酶體抑制劑MG-132處理後UL76更加強UBn-protein、S5a和UBn-S5a的累積。而這個累積的能力是需要完整全長的UL76和S5a交互作用下才有顯著的效果。另外我們以RNA干擾技術證實，降低S5a的表現時，進而使UL76誘導的核內堆疊聚集的細胞減少。因此我們證實UL76透過與S5a的交互作用使細胞內UBn-protein累積。

關鍵字

人類巨細胞病毒 ； 泛素 ； 蛋白酶體

並列摘要

We provide sufficient evidence to reveal the mechanism governing the induction of nuclear aggregate by human cytomegalovirus (HCMV) UL76. Previous reports document that UL76 is able to cause DNA damage, induce micronuclei, and accumulate chromosome abnormalities in glioblastoma (U373-MG) cells stably expressing UL76. In this study, our data demonstrate that UL76 is responsible for DNA damage. We also confirm that expressing UL76 in primary cell alters cell cycle progression by accelerating G1 into S phase, however, prolonging G2/M phase retaining time. Previous study in Dr. Wang’s Lab demonstrated that HCMV UL76 targets to ubiquitin-proteasome system (UPS) by using yeast two–hybrid screening system. UPS regulates several important cellular pathways and implicates in the degradation of specific protein. S5a of 26S proteoasome is the major receptor for ubiquitin (UB)-conjugated proteins in UPS. By employing immunofluorescence cell staining, we observed that S5a was concentrated in the nucleus and colocalized with UL76 in nuclear aggregate.Furthermore, FRET experiment suggested the distances between these two proteins allowed biological interactions to occur. We also provide evidence to prove that UL76 expression can enhance the accumulateion of endogenous UBn-proteins, S5a and UBn-S5a. The proteasome inhibitor MG-132 inhibits the degradeative activity of 20S proteasome, resulting in the accumulation of UBn-protein, S5a and UBn-S5a. In the presence of UL76 and MG-132, the levels of UBn-proteins were significantly enhanced. This ability of accumulation UBn-protein requires full-length proteins of UL76 and S5a. Furthermore, we demonstrated that knockdown endogenous S5a protein by RNA interference technique reduced the cell numbers containing UL76-induced aggregates. Taken together, we conclude that UL76 by interacting with S5a induces the accumulation of UBn-proteins at the nuclear aggregate.

並列關鍵字

human cytomegalovirus ； ubiquitin ； proteasome

參考文獻

1. Kalejta RF (2008) Tegument proteins of human cytomegalovirus. Microbiol Mol Biol Rev 72: 249-265.

2. Jackson SE, Mason GM, Wills MR (2011) Human cytomegalovirus immunity and immune evasion. Virus Res 157: 151-160.

3. Bego MG, St Jeor S (2006) Human cytomegalovirus infection of cells of hematopoietic origin: HCMV-induced immunosuppression, immune evasion, and latency. Exp Hematol 34: 555-570.

4. Streblow DN, Nelson JA (2003) Models of HCMV latency and reactivation. Trends Microbiol 11: 293-295.

5. Wang SK, Duh CY, Chang TT (2000) Cloning and identification of regulatory gene UL76 of human cytomegalovirus. Journal of General Virology 81: 2407-2416.