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Title: | C型肝炎病毒非結構性蛋白質NS5A透過組蛋白甲基轉移酶SMYD3調控dusp1基因之表現 Hepatitis C Virus NS5A Protein Regulates the Expression of dusp1 Gene via Histone Methyltransferase SMYD3 |
Authors: | Yung-Chun Ko 柯詠純 |
Advisor: | 張明富 |
Keyword: | C型肝炎病毒,非結構性蛋白質NS5A,組蛋白甲基轉移?SMYD3,dusp1基因, hepatitis C virus,NS5A,SMYD3,dusp1, |
Publication Year : | 2015 |
Degree: | 碩士 |
Abstract: | C型肝炎病毒是造成嚴重肝疾病的主要病毒之一。近期研究指出C型肝炎病毒感染可能會造成基因體不穩定,而基因體或表觀基因體平衡改變可能導致肝癌的發生。C型肝炎病毒的致癌蛋白質NS5A會引發染色體不穩定,影響細胞的生長及訊息傳遞。在與NS5A結合的蛋白質資料庫中,發現一個組蛋白甲基轉移酶SMYD3會與NS5A蛋白質結合。過去研究指出SMYD3蛋白質在肝癌等癌症組織及細胞中有過量表現的現象。SMYD3具有H3K4及H4K20三甲基化的酵素活性,分別扮演轉錄活化與抑制的角色。在本研究中發現,SMYD3蛋白質在細胞內的分布情形會因NS5A蛋白質存在而改變。在C型肝炎病毒subgenomic replicon HCVR細胞發現NS5A蛋白質與SMYD3蛋白質會在細胞核周圍有共位現象,以免疫共沉澱法證實SMYD3與NS5A蛋白質於細胞質中有交互作用。為探討SMYD3蛋白質與NS5A蛋白質交互作用的重要片段,本研究利用大腸桿菌表現多個不同片段且帶有His-tag的SMYD3蛋白質,將其與HCVR細胞蛋白質進行pull-down分析,結果發現MYND domain及第二個SET domain可能是負責與NS5A蛋白質交互作用的區域。基因序列分析顯示,在dusp1基因啟動子上有多個SMYD3蛋白質結合序列5’-CCCTCC-3’及5’-GGAGGG-3’,本研究利用ChIP assay證明SMYD3蛋白質會結合在dusp1啟動子(-107~+76)上。knockdown實驗也發現,SMYD3蛋白質在調控dusp1基因表現上扮演抑制的角色。當NS5A蛋白質存在時,會降低SMYD3蛋白質結合到dusp1啟動子的能力。本研究結果發現,dusp1基因為SMYD3蛋白質的目標基因,且NS5A蛋白質與SMYD3蛋白質之間的交互作用會降低SMYD3蛋白質對於dusp1基因的抑制效果,造成dusp1基因表現量上升。 Hepatitis C virus (HCV) is a major cause of serious liver disease worldwide. HCV infection induces genome instability, leading to further genetic and epigenetic alterations which contribute to the full development of hepatocellular carcinoma. The viral oncoprotein NS5A is likely to be involved in the induction of genome instability and in dysregulating cellular control of growth and signal transduction. Proteomics study revealed a potential interaction of the NS5A protein with the epigenetic factor SMYD3 (SET and MYND domain containing protein 3), a histone methyltransferase. SMYD3 is over-expressed in several cancers including hepatocellular carcinoma. In addition, SMYD3 has histone 3-lysine 4 (H3K4) and histone 4-lysine 20 (H4K20) histone methyltransferase activities that function as transcriptional activator and repressor, respectively. In this study, subcellular localization of endogenous SMYD3 in the presence of NS5A was examined. SMYD3 were mainly colocalized with NS5A at the perinucleus foci in HCV subgenomic replicon HCVR cells. To further analyze which domains of SMYD3 are responsible for interacting with NS5A, various His-tagged truncated SMYD3 protein were ecxpressed and subjected to pull-down analysis with HCVR cell lysates. Results demonstrated involvement of that the MYND domain and the second SET domain in the interaction of SMYD3 with NS5A. Biological significance of the interaction was further studied. Sequence analysis identified several putative SMYD3 binding motifs, 5’-CCCTCC-3’ and 5’-GGAGGG-3’, in the promoter region of dusp1. ChIP assay further demonstrated the binding of SMYD3 to the dusp1 promoter (-107~+76). In addition, NS5A reduced the ability of SMYD3 in binding to dusp1 promoter. Knocked-down experiment indicated a negative role of SMYD3 in regulating dusp1 expression. Taken together, these results indicate that dusp1 is a target gene of SMYD3 and the interaction between NS5A and SMYD3 can reduce the repression function of SMYD3 on dusp1 expression. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52122 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生物化學暨分子生物學科研究所 |
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