不同阿拉伯芥銅鋅超氧歧化酶對其活化機制偏好之研究

Copper Chaperone-Dependent and -Independent Activation of Three Copper-Zinc Superoxide Dismutase Homologs Localized in Different Cellular Compartments in Arabidopsis

黃建勛(Chien-Hsun Huang)

指導教授 ： 靳宗洛

國立臺灣大學/生命科學院/植物科學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.02867

摘要

超氧歧化酶(Superoxide Dismutase；SOD)，可將超氧分子轉變為過氧化氫及氧分子，具有解除氧化逆境的功能。對於銅鋅超氧歧化酶(CuZnSOD；CSD)的活化機制，目前已知有兩條路徑，一者是藉由銅鑲嵌輔助蛋白(Copper Chaperone of SOD1；CCS)的幫助，達到銅離子鑲嵌與形成內生性雙硫鍵的活化型態；另一者則是在人類及老鼠中發現，在沒有CCS情況下，CuZnSOD仍然具有少量的活性，只是其活化途徑與機制仍未明朗。本論文，將阿拉伯芥三個不同的CuZnSOD基因表現於酵母菌及阿拉伯芥原生質體中，我們證實阿拉伯芥不同CuZnSOD的活化具有不同的偏好：CSD1位在細胞質中，可藉由兩條活化機制達成活化，在無CCS的情況下，仍保有~36%的活性，類似人類的CuZnSOD；CSD2位在葉綠體中，只能經由CCS來達成活化，與酵母菌的CuZnSOD類似；CSD3位在過氧化體中，只經由非CCS的路徑來達成活化，類似線蟲的CuZnSOD。我們證實在AtCCS-knockout突變株中，此殘存的CuZnSOD活性量就足以提供正常生理功能之所需。最後，我們也證實了還原態的榖胱甘肽(Glutathione；GSH)參與在非CCS的活化CuZnSOD的路徑上，並且需有一未知功能的因子共同合作才能完成。我們綜合前人之研究及本實驗之證據，提出兩種不需經由CCS而達成活化的可能作用機制，以及提出CuZnSOD蛋白質之C端具有與未知因子交互作用，而達成促進活化的功用。綜上而言，我們的研究提出植物體之複雜精密的抗氧化途徑，是在其他物種中從所未見的，而其詳細的機制，則仍有待後續的研究加以釐清。

關鍵字

銅鋅超氧歧化酶活化機制

並列摘要

Superoxide dismutases (SODs) are enzymes that protect cells from oxidative damage. The major pathway for CuZnSOD activation involves the function of a Copper Chaperone for SOD (CCS), whereas an additional, minor CCS-independent pathway that has been observed in mammals. Through overexpression of three Arabidopsis CuZnSOD genes (CSDs) in yeast and Arabidopsis protoplasts, we demonstrate the existence of a CCS-independent activation pathway in Arabidopsis thaliana. Interestingly, the three Arabidopsis CSDs show strongly different preference for the two activation pathways: the main activation pathway for CSD1 in the cytoplasm involved a CCS-dependent and -independent pathway, which was similar to that for human CSD. Activation of CSD2 in chloroplasts depended totally on CCS similar to yeast (Saccharomyces cerevisiae) CSD. Peroxisome-localized CSD3 via a CCS-independent pathway was similar to nematode (Caenorhabditis elegans) CSD in retaining activity in the absence of CCS. The residual SOD activity detected in AtCCS knockout plants is sufficient for seed germination and root growth, confirming that this alternative pathway is physiologically functional. Through a series of glutathione manipulation experiments, we further confirmed that glutathione plays a role in CCS-independnet pathway but must cooperate with an unknown factor for SOD activation. According to previous publications and our finding, two models of the CCS-independent mechanism are proposed. We also suggest that the CSD protein conformation at C-terminal is important in providing a docking site for unknown factor to interact with. Our findings reveal a complex system underlying CSD activation which ensures a highly specific and sophisticated regulation of antioxidant pathways in plants and has not been reported in other organisms. However, the clear and definite mechanism needs further investigation.

並列關鍵字

CuZnSOD CCS-independent activation

參考文獻

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