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研究生(外文):Ying Ru Shih
論文名稱(外文):Analysis of protein modifications and abundance of Kaposi’s sarcoma-associated herpesvirus ORF50
指導教授(外文):S. D. Liu
外文關鍵詞:Kaposi’s sarcoma-associated herpesvirusKaposi’s sarcomaORF50
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ORF50是KSHV極早期表現的轉錄活化子,可以誘發潛伏感染的KSHV進入溶裂循環。ORF50由691個胺基酸所組成,包括N端的DNA結合區 (胺基酸1到390)及C端轉錄活化區 (胺基酸486到691),已知在細胞中ORF50是個高度磷酸化的蛋白質,但並不清楚ORF50是否還有其他轉錄後修飾。在本研究中,藉由西方點墨法分析發現,sodium butyrate (SB)會使Cytomegalovirus immediate-early (CMV-IE)啟動子所調控的ORF50表現量增加,再藉由 ORF50的突變株進一步證實,SB是透過C端357到691胺基酸造成此一現象。冷光酶活性分析也發現,只包含ORF50 C端胺基酸357到691的突變株和全長的ORF50都具有抑制CMV-IE及p21啟動子活性的能力。雖然目前仍然無法證實ORF50是否為乙醯化修飾的蛋白質,但實驗中發現,ORF50 C端胺基酸也是調控其蛋白質磷酸化修飾的重要區域。本研究顯示,ORF50 C端胺基酸不僅僅是ORF50的轉錄活化區,也是影響ORF50在細胞中的含量、轉錄活性及轉譯後修釋重要的調控區域。
The ORF50 protein of Kapsosi’s sarcoma-associated herpesvirus (KSHV) is a multifunctional transcriptional activator that drives the viral lytic cycle. It is a 691-amino acid (aa) polypeptide that is extensively phosphorylated and may be subject to other posttranslational modifications. The DNA binding domain of ORF50 protein is located in the N-terminal portion (aa 1 to 390) and an acidic activation domain is found in the C-terminal portion (aa 486 to 691). This study finds that sodium butyrate treatment markedly enhances the expression level of the full-length ORF50 protein, but not the C-terminal truncated mutants, encoded from the expression vector with a cytomegalovirus immediate-early (CMV-IE) promoter. Extensive deletion analyses showed that the C-terminal region from aa 357 to 691 of the ORF50 protein is sufficient to mediate an abundant enhancement in response to sodium butyrate. Furthermore, transient transfection assay revealed that the C-terminal ORF50 (aa 357-691), similar to its full-length protein, possesses an unexpectedly repressive effect on the CMV-IE promoter and a cellular p21 promoter. Despite the lack of evidence showing that whether ORF50 protein is an acetylated protein, the critical region conferring ORF50 hyperphosphorylation may be also located in the C-terminal region. Taken together, these findings suggest that the C-terminal region of ORF50 protein acts as not only a general transactivation domain, but also an important regulatory motif for protein abundance, transcriptional repression and posttranslational modifications.

四、Sodium butyrate對ORF50 基因調控的影響
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