阿拉伯芥伴護蛋白CPN20活化鐵超氧歧化酶所需的功能區塊之研究

超氧岐化酶(SOD)的抗氧化活性需要金屬輔酶的協助，將超氧自由基轉化為氧氣與過氧化氫以對抗氧化逆境。在先前的研究認為SOD的活化機制是藉由特殊金屬鑲嵌蛋白，將金屬離子與SOD結合後而具有活性。阿拉伯芥葉綠體中具有三個鐵超氧歧化酶(FeSOD; FSD1，FSD2，FSD3)，其中FSD1是目前唯一可偵測出活性之FeSOD。已知位於葉綠體中CPN60之伴護蛋白CPN20具FeSOD的鐵離子鑲嵌能力，能直接與FeSOD作用進而活化FeSOD。然而，CPN20活化FeSOD所需之功能性區域仍然未清楚。由於，CPN20為種子發育所必需，故無法取得基因剔除突變體。因此，本研究藉由刪除葉綠體導引訊息序列(ΔTP)之FSD1 (ΔTP-FSD1)與一系列經刪除部分結構之ΔTP-CPN20，共同表現於fsd1 (FSD1基因剔除株)原生質體之細胞質中，進行雙分子螢光互補實驗(BiFC)及FSD1活性檢測(in-gel SOD activity assay)。結果顯示，透過雙分子螢光互補實驗，在去除部分特定結構後的ΔTP-CPN20將部分與ΔTP-FSD1進行交互作用。值得注意的是從FSD1活性檢測分析得知去除部分之mobile loop之CPN20-ΔdM3，會明顯降低活化FSD1的能力。另一方面，有五個核酸結合蛋白(RNA-biding proteins)在細胞質也具有活化FSD1的能力，特別是GRP7(富含甘氨酸核酸結合蛋白7)和ΔTP-CPN20可以共同增加ΔTP-FSD1的活性。因此我們認為CPN20的dm3是活化FeSOD的重要功能區塊。

關鍵字

活性氧類；超氧岐化酶；鐵超氧岐化酶；伴護蛋白；核酸結合蛋白

並列摘要

Superoxide dismutases (SOD) require specific metallochaperones for activation of defense against oxidative stress, which converts a superoxide anion to hydrogen peroxide and oxygen. There are three iron superoxide dismutases (FeSOD; FSD1, FSD2, and FSD3) in Arabidopsis chloroplasts, and FSD1 is the only detectable FeSOD actively analyzed by in-gel activity assay. It has been shown that CPN20 functions as the iron chaperone that facilitates FeSOD activation by direct interaction. Even so, the functional domain of CPN20 required for FeSODs activation remained unclear. Since CPN20 is crucial for seed development, it is impossible to obtain a stable knockout mutant in Arabidopsis. Thus, in this study we constructed chloroplastic transit peptide-deleted FSD1 (ΔTP-FSD1; cytosolic format) and a series of domain-truncated variants of ΔTP-CPN20 (cytosolic format), and then co-expressed them in fsd1 (FSD1-defective mutant) protoplasts. We conducted bimolecular fluorescence complementation (BiFC) and in-gel FSD1 activity assays to look for the functional domains of CPN20 required for FSD1 activation. The results reveal that the CPN20-Δdm3 (the key amino acids, Gly32/130 and Leu35/133, in mobile loops) mutation significantly decreased the enhancement of FSD1 activation. In addition, the cytosolic GRP7 (glycine-rich RNA binding protein 7) itself can cooperate with CPN20 synergistically to facilitate FSD1 activation. Thus, we conclude that plant RNA-binding protein serves a new function as an iron chaperone, the dm3 of CPN20 required for FeSOD activation.

並列關鍵字

ROS ； SOD ； FeSOD ； CPN20 ； RBP

參考文獻

Pan SM, Yau YY (1992) Characterization of superoxide dismutase in Arabidopsis. Plant Cell Physiol 37: 58-66

Abdel-Ghany SE, Muller-Moule P, Niyogi KK, Pilon M, Shikanai T (2005) Two p-type ATPases are required for copper delivery in Arabidopsis thaliana chloroplasts. Plant Cell 17: 1233-1251

Alscher RG, Erturk N, Heath LS (2002) Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J Exp Bot 53: 1331-1341

Anantharaman V, Koonin EV, Aravind L (2002) Comparative genomics and evolution of proteins involved in RNA metabolism. Nucleic Acids Res 30: 1427-1464

Baneyx F, Bertsch U, Kalbach CE, van der Vies SM, Soll J, Gatenby AA (1995) Spinach chloroplast cpn21 co-chaperonin possesses two functional domains fused together in a toroidal structure and exhibits nucleotide-dependent binding to plastid chaperonin 60. J Biol Chem 270: 10695-10702

國際替代計量

阿拉伯芥伴護蛋白CPN20活化鐵超氧歧化酶所需的功能區塊之研究

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