| 摘要: | APE1 在內質網壓力下所扮演的角色及NF-kappaB 活性及 NF-kappaB 所調控基因表現之影響。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。許多的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。研究也顯示內質網壓力可以誘導活性氧化物的產生，當細胞處於氧化壓力下會嚴重干擾到不同特性的蛋白質如轉錄因子NF-B 及造成DNA 受損。另一方面，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。初步結果顯示我們利用RT-PCR 來分析APE1 mRNA，APE1 mRNA 的表現量可以被內質網壓力所誘導，而內質網壓力可誘導APE1 蛋白質的表現。更進一步的發現eIF2抑制劑可以減弱 MCF-7 細胞中APE1 mRNA 的表現量。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 及NF-B 的關係。(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確認APE1 及NF-B 活化的關係，這樣的結果可以提供一個重要的資訊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 role of Human apurinic endonuclease 1 under endoplasmic reticulum stress, and investigation of the effect of Human apurinic endonuclease 1 on NF-B activity and NF-B-regulated gene expression during ER stress. 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. Recent studies have shown that reactive oxygen species (ROS) production was increased by ER stress. The oxidative stress will greatly influence the various properties of protein, such transcription factor NF-B-binding activity and cause DNA damage. 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, 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 eIF2phosphorylation were also enhanced by ER stress. Moreover, p53 expression is enhanced by ER stress and the induction was probably regulated by NF-B. In addition, we also observe that expression 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) Identification of relationship between APE1 and NF-B-binding activity 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. |
