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研究生:洪靜穎
研究生(外文):Chin-Yin Hung
論文名稱:K562細胞分化過程中甲基蛋白之鑑定
論文名稱(外文):Identification of methylated proteins during K562 differentiation
指導教授:林蔚靖
指導教授(外文):Wey-Jinq Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:75
中文關鍵詞:甲基化
外文關鍵詞:methylationdifferentiation
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中文摘要
共價性轉譯後修飾 (covalent post- translactional modifications)如蛋白質醣化、磷酸化、甲基化等,已知在蛋白質的活性以及功能上扮演重要的角色。就蛋白質甲基化作用而言,越來越多的證據顯示此修飾作用參與在細胞生長、基因表現和訊息傳導調控機制中。根據本實驗室先前的研究發現鈣離子會選擇性地影響不同蛋白的甲基化,而且在Ara-C誘導K562細胞走向紅血球之早期先驅細胞的分化過程中,蛋白之甲基化扮演關鍵性的角色。因此,本論文以蛋白質體學方式,嘗試找出在K562細胞內受到這二種機制所調控的甲基化蛋白,以為日後進一步探討蛋白甲基化的功能。PRMT1 (protein arginine methyltransferas 1)為哺乳動物中最主要的蛋白質精胺酸甲基轉移脢。因此在此研究中我們亦建立大量表現PRMT1 (protein arginine methyltransfrase 1)或去除PRMT1的細胞模式,以做為日後探討其功能研究的基礎。
本篇論文已成功地以二維膠體電泳展開甲基化蛋白,發現K562細胞以Ara-C處理分化前後,蛋白質的表現及甲基化作用的情形有明顯差異。這些甲基化蛋白並以質譜儀分析鑑定,目前由質譜儀 (Q-TOF)分析結果得知其中之一為可與RNA結合的蛋白質heterogeneous nuclear ribonucleoprotein K (hnRNP K),分析其序列具有精胺酸甲基轉移脢 (PRMT1)所辨識之RGG或RXR重複區域。於是進一步自人類胎盤互補DNA選殖出此基因,於大腸桿菌中大量表現並純化hnRNP K融合蛋白,利用in vitro methylation assay首先確認hnRNP K為PRMT1的受質。此外本論文亦以二維膠體成功的展現鈣離子對蛋白甲基化的顯著影響。另一方面,我們已在人類腎臟纖維母細胞 (HEK293)及K562細胞中大量表現PRMT1,經轉染的HEK293及K562其PRMT1活性明顯提升5到8倍。利用此方式可成功地於細胞中表現PRMT1的活性,此技術之建立將有助於進一步探討PRMT1在K562細胞分化過程所扮演的角色。
Abstract
In the complicated cellular processes, post-translational modifications such as phosphorylation, glycosylation, and methylation play significant roles. Accumulating evidence shows that protein methylation participates in various mechanisms controling cell growth, gene expression and signal transduction. Our previous studies showed that calcium selectively modulated methylation of different proteins. In addition, our data showed that protein methylation played a crucial role in Ara-C-induced erythroid differentiation of K562. In this study, we intended to identify proteins whose methylation state was modulated during K562 differentiation or by calcium ion using proteomic approaches.
In this study, methylated proteins were successfully displayed by two-dimensional gel electrophoresis and the identify of these methylated proteins was further determined by mass spectrometry. Heterogeneous nuclear ribonucleoprotein K (hnRNP K), a RNA-binding protein, was identified by Q-TOF mass spectrometry. hnRNP K contains RGG and RXR motifs, consensus recognition sequences for protein arginine methyltransferase 1 (PRMT1). hnRNP K was methylated readily by PRMT1 in the in vitro methylation assay suggesting that hnRNP K was a substrate of PRMT1. The methyltransferase activity in K562 and HEK293 cells thansfected with PRMT1 increased by 5-to 8-fold when measured with hnRNP A2 as a substrate. This result suggested that the overexpressed PRMT1 was enzymatically active. This system will be used to examine the role of PRMT1 in K562 differentiation.
目錄
頁次
目 錄 ………………………………………………………………………… Ⅰ
縮 寫 表 …………….……………………………………………………………. Ⅱ
圖次目錄 ………………………………………………………………………….. Ⅳ
中文摘要 ………………………………………………………………………….….1
英文摘要 ……………………………………………………………………………..2
序 論 ……………………………………………………………………………..3
研究目標 ……………………………………………………………………………10
實驗材料 …………………………………………………………………………....11
實驗方法 ……………………………………………………………………………17
實驗結果 ……………………………………………………………………………30
討 論 ……………………………………………………………………………39
參考文獻 ……………………………………………………………………………45
圖 ……………………………………………………………………………51
參考文獻
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