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標題: | 以非CCS活化阿拉伯芥銅鋅超氧歧化酶所需因子之功能性研究 Functional Study of Factors Required for CuZnSOD Activation Independent of CCS in Arabidopsis |
作者: | Jun Shi 石峻 |
指導教授: | 靳宗洛(Tsung-Luo Jinn) |
關鍵字: | 阿拉伯芥,銅鋅超氧歧化?,銅鑲嵌輔助蛋白, Arabidopsis,copper/zinc superoxide dismutase,CCS-independent activation, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 超氧歧化酶(superoxide dismutase; SOD)能夠將O2-轉變為H2O2及O2,進而保護細胞免於氧化傷害,這樣的歧化作用則需要依賴其催化中心的過度金屬離子。銅鑲嵌輔助蛋白(copper chaperone for SOD; CCS)已經被證實負責將銅離子嵌入銅鋅超氧歧化酶(CuZnSOD; CSD)中並使之活化,另外,也觀察到有另一條非CCS的CSD活化機制。在阿拉伯芥中,細胞質型CSD1能夠經由上述的任一機制活化。本篇研究再一次確認了穀胱甘肽與銅離子的複合物(GSH-Cu),以及一些還未發現的未知因子參與了非CCS的活化路徑,因此,我們利用了包括硫酸銨沈澱及逆相高效液態色層分析等方法,試圖將這些未知因子從缺少CCS的細胞萃取物中分離出來。篩選出的蛋白質中,3-磷酸甘油醛去氫脢(glyceraldehyde-3-phosphate dehydrogenase; GAPC)以及重金屬結合蛋白20 (heavy metal-associated isoprenylated plant protein 20; HIPP20)能夠與CSD1進行交互作用,並幫助其活化。此外,已有許多報告指出SOD具有除了清除自由基以外的其他生理功能,我們的結果提出了CSD1可能也參與訊息傳導、轉錄調控及重金屬代謝等機制,藉此或許能夠提供一些研究SOD功能的新方向。最後,我們也提出植物螯合素(phytochelatin; PC)如同GSH-Cu,能夠在非CCS活化機制中攜帶銅離子,以利CSD1進行活化。 Superoxide dismutase (SOD) catalyzes the conversion of O2- into H2O2 and O2 protecting cells from oxidative damages. This dismutating activity depends on the transition metal ion on its catalytic site. It has been shown that CCS (copper chaperone for SOD) is responsible for the Cu incorporation and the activation of CuZnSOD; meanwhile, an alternative CCS-independent pathway was also observed. Arabidopsis thaliana cytosolic CuZnSOD, CSD1, can be activated by either pathway. In this study, we confirmed that glutathione (GSH)-Cu complex and some unidentified factors were involved in the CCS-independent activation. Fractionation approaches were conducted, including ammonium sulfate precipitation and reverse-phase HPLC for the purpose to isolate the factors facilitating the activation of CSD1 from the CCS-knockout (ccs) cellular extract. Candidates such as glyceraldehyde-3-phosphate dehydrogenase (GAPC) and heavy metal-associated isoprenylated plant protein 20 (HIPP20) were confirmed interacting with CSD1 in the BiFC assay and mediating CSD1 activation. SOD has been suggested participating in multiple vital physiological functions in addition to its known functions in ROS scavenging. Our results proposed the possible functions of CSD1 in signal transduction, transcriptional regulation or heavy metal detoxification and, thus, may provide some new aspects regarding SOD function. Moreover, we demonstrated a potential Cu carrier with functions similar to GSH-Cu complex, phytochelatin, delivering Cu during CSD1 activation in the absence of CCS. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51189 |
全文授權: | 有償授權 |
顯示於系所單位: | 植物科學研究所 |
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