探討人類巨細胞病毒UL112-113 p34蛋白的後轉譯修飾

Post-translational modification of the p34 encoded by human cytomegalovirus UL112-113

陳美吟(Mei-Yin Chen)

指導教授 ： 王雙桂

高雄醫學大學/醫學院/醫學研究所/碩士(2010年)

摘要

UL112-113為人類巨細胞病毒 (Human cytomegalovirus；HCMV) oriLyt 依賴複製 (oriLyt-dependent replication) 的必要基因之一。其功能僅知參與前複製體的形成，並輔助核心複製體的組合。但其參與的蛋白功能並未釐清。已知UL112-113基因會產生出四個磷酸化蛋白，分別為p34、p43、p50和p84。另本實驗室證實於 HCMV感染期UL112-113會產生三個蛋白p20、 p26和p28。在王雙桂老師實驗室先前的研究結果指出，在加入蛋白酶抑制劑處理後，UL112-113蛋白質被降解的情形有減少的趨勢。UL112-113 表現的蛋白序列包含顯著的 PEST區域，而已知含有PEST序列的蛋白容易被降解掉，其降解可經由ubiquitin-proteasome system、calpain或是caspase代謝路徑。因此本研究想要釐清HCMV UL112-113所產生出來三個的新穎蛋白，所造成蛋白被降解的路徑、參與的蛋白酶、及是否與PEST序列有關。本次研究使用原核生物E. coli表現系統純化UL112-113蛋白，探討in vitro的蛋白酶反應下此組蛋白被蛋白酶降解修飾的情形。綜合結果顯示，p34 (含PEST的序列) 在in vitro的蛋白酶反應下，可被calpain及caspase切割；p28 (刪除p34 PEST的序列)，則僅可被caspase切割。顯示calpain切割蛋白主要是辨識PEST的結構，而caspase主要是辨識特異性的切位。

關鍵字

人類巨細胞病毒

並列摘要

Human cytomegalovirus (HCMV) UL112-113 is one of essential genes involved in viral genome oriLyt-dependent replication. Previous reports have shown that UL112-113 encodes four phosphoproteins, p34, p43, p50, and p84. Recently, three novel proteins p20, p26, and p28 are identified, Additionally, Dr. Wang and students showed that protease inhibitors reduced the levels of these three novels proteins. Taken together these results, we suggested that these novel proteins are most likely derived form cellular proteolytic modification. We further noticed that proteins encoded by UL112-113 contain significant PEST motif by using a ePESTfind program. Recent studies have revealed that degradation of PEST-containing proteins is mediated by three molecular pathways: ubiquitin-proteasome system, calpain and caspase. Therefore, this study aimed to investigate the pathways involved in the protein degradation, responsible protease(s) and implications of PEST motif. In this research, protein was produced in prokaryotic system. Highly purified p34 protein was used to identify the specific proteases-mediated these cleavages. We found that calpain and caspase cleaved p34 (contain PEST region). However, only caspase cleaved p28 (with deletion in PEST region). Therefore, calpain recognized PEST motif for cleavage. Caspase recognizes specific sequence for cutting.

並列關鍵字

human cytomegalovirus

參考文獻

Ahn, J. H., Jang, W. J. & Hayward, G. S. (1999). The human cytomegalovirus IE2 and UL112-113 proteins accumulate in viral DNA replication compartments that initiate from the periphery of promyelocytic leukemia protein-associated nuclear bodies (PODs or ND10). J Virol 73, 10458-10471.

Arlt, H., Lang, D., Gebert, S. & Stamminger, T. (1994). Identification of binding sites for the 86-kilodalton IE2 protein of human cytomegalovirus within an IE2-responsive viral early promoter. J Virol 68, 4117-4125.

Belizario, J. E., Alves, J., Garay-Malpartida, M. & Occhiucci, J. M. (2008). Coupling caspase cleavage and proteasomal degradation of proteins carrying PEST motif. Curr Protein Pept Sci 9, 210-220.

Chen, Z., Knutson, E., Kurosky, A. & Albrecht, T. (2001). Degradation of p21cip1 in cells productively infected with human cytomegalovirus. J Virol 75, 3613-3625.

Chua, B. T., Guo, K. & Li, P. (2000). Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases. J Biol Chem 275, 5131-5135.