探討內質網壓力誘導AP-endonuclease (APE1/Ref-1) 過量表現及其所扮演的角色

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標題:	探討內質網壓力誘導AP-endonuclease (APE1/Ref-1) 過量表現及其所扮演的角色 To Investigate the Mechanism and Role of Endoplasmic Reticulum-Induced Human Apurinic Endonuclease 1 Overexpression
作者:	洪瑞祥賴明德
貢獻者:	嘉南藥理科技大學生物科技系（所）
關鍵字:	內質網壓力 NF-kappaB Apurinic / apyrimidinic endonuclease B 型肝炎表面突變蛋白反轉錄聚合酶連鎖反應人類肝腫瘤組織 Endoplasmic reticulum stress NF-kappaB Apurinic / apyrimidinic endonuclease RT-PCR HBV large surface mutant protein pre-S2 hepatocellular carcinoma
日期:	2012
上傳時間:	2014-10-20 21:49:05 (UTC+8)
摘要:	內質網是一個主要用來決定蛋白質要被送至細胞內或細胞外的胞器，主要功能為蛋白質合成、修飾及透過分泌路徑來把蛋白質運送到目的地。當大量累積未正常摺疊的蛋白質所造成的內質網壓力會透過活化ATF6、PERK 及Ire1 訊息傳遞路徑。另外，許多的轉錄因子如NF-B、AP1、CHOP 及ATF4 等會參與其中來調節內質網壓力。研究顯示在一些生理狀況或疾病已經被證實跟內質網壓力有關如B 細胞分化成漿細胞、腫瘤、病毒感染、退化性神經疾病及第二型糖尿病。APE1 是一個多功能蛋白質並參與DNA 修補及調控轉錄因子的氧化還原狀態，APE1 現今已知的可以透過調控氧化還原狀態來增強轉錄因子對DNA 結合的能力如NF-B、AP1 及p53 等。在過去研究顯示在腫瘤組織中常可觀察到APE1 過量表現，並且APE1 表現量與抗藥性有直接的關係。在我們的初步結果顯示內質網壓力可誘導APE1 蛋白質及mRNA 的表現。當利用 APE1 shRNA 來降低細胞中APE1 表現量時，發現可以增加細胞死亡情形。同時，內質網壓力下可誘導NF-B 磷酸化及NF-B 結合能力的活性。更進一步，內質網壓力可以誘導p53 的表現且我們成功的發現NF-B 參與內質網壓力所誘導p53 的表現。除此之外，我們利用在正常人類肝細胞株NeHepLxHT 讓其表現B 型肝炎表面突變蛋白來誘導內質網壓力，結果顯示也可以觀察到APE1 的過量表現，進一步我們在人類肝腫瘤組織裡也可觀察到APE1 表現量與內質網壓力有關係。在本計畫中我們將著重於三個部份: (一) 分析APE1 在內質網壓力所扮演的角色。 (二) 確認在內質網壓力下APE1 的誘導機制。(三) 探討APE1 對NF-B 誘導p53 表現的影響。我們希望利用內質網壓力的環境來探討APE1 所扮演角色及對NF-B 及p53 分子活化的關係，這樣的結果可以提供一個重要的資訊可以提供未來應用在腫瘤生成及化學治療方面。總結而論，這樣的機制研究可以提供一個分子基礎來研發具有潛力的臨床用藥。 The endoplasmic reticulum (ER) is a major compartment for proteins destined to the endo/exocytotic pathway. It is responsible for the synthesis, modification and delivery of proteins to their proper target sites within the secretory pathway and the extracellular space. Accumulation of unfolded protein will activate ATF6, PERK and Ire1 signaling pathways in response to ER stress. Otherwise, many transcription factors are involved in regulation of ER stress, such as NF-B, AP1, CHOP and ATF4. Many studies have shown many physiological conditions or disease were identified in which related to ER stress, e.g. differentiation of B-cells into plasma cells, tumor, viral infection, degenerative neuronal disorders and type II diabetes. On the other hand, Human Apurinic / apyrimidinic endonuclease (APE1) is a multifunctional protein involved in both base excision DNA repair and redox regulation of transcription factors. APE1 is now known to enhance DNA-binding activity of several transcription factors, including NF-B, AP1, and p53, by regulating their redox states. Our preliminary results indicated that, APE1 mRNA expression level is induced by ER stress as determined with RT-PCR western blotting, and induction of APE protein level is significantly increased by ER stress. Furthermore, eIF2inhibitor could attenuate the induction in MCF-7 cells. Meanwhile, NF-B phosphorylation, NF-B-binding activity, ATF6 activation and eIF2 phosphorylation were also enhanced by ER stress. Moreover, p53 expression is enhanced by ER stress and the induction was regulated by NF-B. Furthermore, Induction of ER stress by HBV large surface mutant protein pre-S2in human primary hepatocyte cell line NeHepLxHT. The result has shown that increased ER stress by pre-S2 protein was significantly enhanced APE1 expression. We also observe that overexpression of APE1 was corrected with ER stress in human hepatocellular carcinoma tumor sample. In this project, we will focus on three topics: (1) To analyze the role of APE1 in response to ER stress. (2) To determined the mechanism of APE1 overexpression during ER stress. (3) To investigate the effect of APE1 on NF-B-regulated p53 expression. We hope to identify relationship between APE1 and NF-B activity or other redox-related transcription factors under ER stress. These results could provide an important insight in tumorgenesis and chemotherapy. Consequently, this mechanistic research may provide a molecular basis to develop a potent pharmaceutical agent in clinical use.
關聯:	計畫編號：NSC101-2320-B041-004 計畫年度：101；起迄日期：2012-08-01~2013-07-31
显示于类别:	[生物科技系(所)] 科技部計畫

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