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標題: | 小型D型肝炎抗原調節D型肝炎病毒複製之生物學與蛋白質體學的研究 Biological and Proteomic Study of Small Hepatitis Delta Antigen in Regulating HDV Replication |
作者: | Yen-Shun Chen 陳延順 |
指導教授: | 陳培哲(Pei-Jer Chen) |
關鍵字: | D型肝炎病毒,ERK1/2磷酸激脢,RNA病毒複製,磷酸化,蛋白質體學, HDV,ERK1/2,RNA virus replication,phosphorylation,proteomic study, |
出版年 : | 2008 |
學位: | 博士 |
摘要: | 小型D型肝炎病毒抗原在病毒螺旋環狀複製機制中扮演重要的角色,此抗原表面有很多蛋白質後轉譯修飾,包含磷酸化,乙醯化,以及甲基化都已經被鑑定。本論文將研究細胞內D型肝炎病毒的複製機轉,探討重點在研究小型D型肝炎抗原結合的細胞內蛋白質,以及抗原表面的後轉譯修飾。第一部分,我們確認細胞內ERK1/2激脢可以磷酸化小型抗原絲胺酸-177位置,進而調控病毒複製。首先透過共同免疫沉澱分析的方法,我們證明細胞內的ERK1/2會結合在含有Flag探針的小型抗原S177C點突變的蛋白質上(絲胺酸-177置換成半胱氨酸-177)。當透過HA-AcMEK1持續活化狀態的表現,會活化細胞內的ERK1/2,同時增加小型D型肝炎抗原的絲胺酸-177磷酸化修飾,此磷酸化修飾可以透過抗體採取西方轉運法偵測,也可以被質譜分析儀確認。在細胞體外激脢實驗的狀態下,Flag-ERK1 和 Flag-ERK2可以磷酸化絲胺酸-177的位置。有趣的是,此抗原絲胺酸-177磷酸化會促進D型肝炎病毒從反基因股複製到基因股的方向,但是降低基因股到反基因股方向的複製。本研究發現絲胺酸-177是小型D型肝炎抗原與RNAPII聚合脢結合的重要位置。目前RNAPII被認為是進行反基因股RNA複製的酵素。我們研究結果證明ERK1/2磷酸化小型D型肝炎抗原絲胺酸-177可能透過RNAPII調控D型肝炎病毒的反基因股RNA複製。
第二部分,我們採用蛋白質體學方法來鑑定小型D型肝炎抗原的後轉譯修飾。使用會持續表現小型D型肝炎抗原的 HeLa S3 細胞株,我們建立抗原純化系統,首先將細胞分離出三個部分(核仁,核質和細胞質),接著透過陰離子交換樹酯和單相聚丙烯醯氨凝膠電泳分離技術,,純化出小型D型肝炎抗原。位於膠體的小型抗原,經由胰蛋白酶或者Asp-N蛋白酶分解後,透過液相色層質譜分析儀鑑定蛋白質的後轉譯修飾訊號。我們研究小型D型肝炎抗原的後轉譯修飾調控病毒複製的機制,將顯現其重要性。 The small hepatitis delta virus antigen (SHDAg) plays an essential role in processing HDV RNA double rolling-circle replication. Several post-translational modifications (PTMs) of HDAgs, including phosphorylation, acetylation, and methylation, have been characterized. The study is focus on the investigation of cellular SHDAg-associated kinase and PTMs on SHDAg for visualizing HDV replication machinery. In the part I, we clarify the ERK1/2 kinase phosphorylates SHDAg at Ser-177 for modulating replication machinery. Using coimmunoprecipitation analysis, the cellular kinases ERK1/2 are found to associate with the Flag-tagged SHDAg S177C mutant (Ser-177 replaced with Cys-177). Activation of endogenous ERK1/2 by a constitutively active MEK1 (HA-AcMEK1) increased phosphorylation of SHDAg at Ser-177, which was confirmed by immunoblotting using anti-phosphorylated Ser-177 antibody (αpS177) and mass spectrometric analysis. In the in vitro kinase assay, SHDAg Ser-177 was directly phosphorylated by Flag-ERK1 or Flag-ERK2. Interestingly, over-expression of Flag ERK1/2 could increase HDV replication from antigenomic RNA to genomic RNA, but reduced that from genomic RNA to antigenomic RNA. We also observed that the Ser-177 residue was critical for SHDAg interaction with RNA polymerase II (RNAPII), the enzyme proposed to regulate antigenomic RNA replication. These results demonstrate the role of ERK1/2-mediated Ser-177 phosphorylation in modulating HDV antigenomic RNA replication, possibly through RNAPII regulation. In the part II study, we use the proteomic approach for investigating the PTMs of SHDAg. Using the HeLa S3 SHDAg cell line, we set up a method for the purification of SHDAg from different compartments (nucleolus, nucleoplasm, and cytoplasm) by using cation chromatography coupled with SDS-PAGE analysis. Following the purified SHDAg in gel, the trypsin and Asp N digested SHDAg was analyzed with liquid chromatography-tandem mass spectrometry for identifying PTMs. All of our results might shed light on the PTM study of SHDAg in regulating HDV replication. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9815 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 微生物學科所 |
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