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研究生:柳鈞翔
研究生(外文):Jiun-Shyang Leou
論文名稱:TPA刺激K-562細胞分化過程中蛋白質甲基化之研究
論文名稱(外文):Studies on Protein Methylation During K-562 Differentiation Induced by TPA
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:2004
畢業學年度:92
語文別:中文
論文頁數:87
中文關鍵詞:蛋白質甲基化
外文關鍵詞:protein methylation
相關次數:
  • 被引用被引用:1
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目前有越來越多的證據顯示蛋白質甲基化作用參與在細胞生長、基因表現和訊息傳導調控機制中,然而目前對蛋白質甲基化的瞭解仍有限。本研究中,我們探討蛋白質甲基化,特別是精胺酸的甲基化,在K562細胞受TPA誘導分化過程中可能參與的角色。我們發現當利用TPA誘導K-562細胞走向顆粒細胞的分化時,一些內生性受質的甲基化會受到調控,而以Trx-hnRNP A2作受質測試細胞中Trx-hnRNP A2甲基轉移酶活性,可見其活性在分化24~96小時會較0小時低,推測在K-562細胞走向單核球、巨核細胞的分化時,特定蛋白質出現動態的甲基化及去甲基化。且利用GR peptide競爭之實驗結果發現大部分受質的甲基化會被抑制,因此可知甲基化主要可能是發生在精胺酸上。由於PRMT1是哺乳類動物中最主要的蛋白質精胺酸甲基轉移酶,為探討PRMT1在TPA誘導K-562細胞分化的過程中扮演著何種角色,我們以Tat protein transduction system成功地將TAT-PRMT1送入細胞當中,利用西方墨點法發現TAT-PRMT1可以進入細胞當中並且利用免疫螢光染色的方式時有高達80-90%的細胞是TAT-PRMT1 positive,而這些細胞並表現較高之蛋白質精胺酸甲基轉移酶活性;此外,我們實驗結果也顯示TAT-PRMT1進入細胞且增加蛋白質精胺酸甲基轉移酶活性是具有濃度依賴性的。而在以TAT-PRMT1處理後之K-562細胞,我們發現其對於TPA誘導之顆粒球分化有明顯降低的現象;此外,當處理TAT-PRMT1時同樣會抑制AraC誘導K-562細胞走向紅血球分化。並且我們的實驗結果顯示TAT-PRMT1處理的時間點與其是否影響K-562細胞走向紅血球分化有著重要關聯;我們發現在K-562走向紅血球分化初期處理TAT-PRMT1才會抑制其分化,若在分化後期處理則無此現象;可見K-562細胞分化初期中PRMT1的活性有著極重要之影響。
Protein arginine methylation has been recently implicated as a novel mechanism in cell growth control, gene express regulation, signal transduction and protein intracellular localization. In this study, we examined whether protein methylation, with particular focus on arginine methylation, played a role in the differentiation of K562 cells induced by TPA. Our results showed that methylation of selective endogenous protein substrates were modulated during differentiation. Methylation of a number of the endogenous substrates was inhibited in the presence of the GR peptide but not GA peptide whose arginines were replaced by alanines. This indicated that the methyl groups were incorporated predominantly into arginine residues. Moreover AdOx, a potent inhibitor of methyltransferases, suppressed megakaryotic differentiation of K562 cells as measeured by Liu’s staining. Our results suggested a crucial role of protein arginine methylation in K562 cells differentiation into megakaryocyte and monocyte. PRMT1 (protein arginine methyltransferase 1) is the first protein arginine methyltransfease cloned and represents the predominant protein arginine methyltransferase activity in cells. A protein transduction system mediated by the protein transduction domain (PTD) of TAT peptide has been established in this study to efficiently deliver PRMT1 into K562 cells. Transduction of K-562 with TAT-PRMT1 suppressed megakaryocytic differentiation induced by TPA and erythrocytic differentiation induced by Ara-C, suggesting the involvement of PRMT1 in TPA and Ara-C-induced differentiation.
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