阿拉伯芥銅鋅超氧歧化?與銅離子鑲嵌蛋白質交互作用之研究

Abstract

在人類和酵母菌中已證實，CCS是一種銅離子鑲嵌蛋白質，負責將細胞中的遊離銅離子嵌入apo-SOD，使其轉變成具正常酵素活性的CuZnSOD。在植物體中，CCS真正的功能尚未被清楚證實，故本篇論文以阿拉伯芥為材料，探討植物CCS和CuZnSOD的交互作用。在野生型阿拉伯芥（WT）大量表現阿拉伯芥的CCS（AtCCS）時，其內生CuZnSOD的活性並沒有增加，推測對WT而言其內生AtCCS可能已足夠；而利用AtCCS knockout轉殖株觀察到，缺失AtCCS蛋白質表現的轉殖株，其內生的CuZnSOD活性明顯下降，顯示AtCCS基因的確能調控CuZnSOD的活性。 CZ5是在阿拉伯芥中大量表現水稻CuZnSOD的轉殖株，比起WT多出兩個新增的水稻CuZnSOD活性區：rSODI和rSODII，推測rSODI應該是完整地鑲嵌了兩個銅離子的水稻CuZnSOD，具有正常功能；而rSODII可能是只鑲嵌一個銅離子的異常型式的水稻CuZnSOD。於CZ5中大量表現AtCCS，觀察到rSODII的活性和蛋白質量減少，而rSODI的活性和蛋白質量明顯增加，顯示AtCCS能促使銅離子鑲嵌入rSODII，使其轉換成具正常活性的rSODI。為了進一步研究AtCCS主要的功能區域，於CZ5中大量表現AtCCS的Domain II及Domain III（AtCCS-D2D3），結果顯示rSODI的活性及蛋白質量無明顯差異，推測對阿拉伯芥而言，AtCCS的Domain I可能是功能上必需的序列。此外，利用RNA幹擾技術（RNAi）抑制CZ5內AtCCS的表現，觀察到轉殖株內rSODI和rSODII活性明顯下降，顯示AtCCS基因能調控SOD金屬離子鑲嵌系統。 另外，選殖出水稻CCS基因（OsCCS），並將其轉殖入CZ5內大量表現，觀察到轉殖株之rSODI和rSODII的活性及蛋白質量無明顯變化，推測OsCCS基因可能對阿拉伯芥的系統不相容，無法形成具功能的OsCCS蛋白質。

The copper chaperone for superoxide dismutase (CCS) as a factor responsible for copper ion incorporation into CuZnSOD in yeast and human had been identified. In this study we showed that AtCCS cloned from Arabidopsis also involves in the regulation of CuZnSOD activity. That overexpression of AtCCS in wild type Arabidopsis did not enhance CuZnSOD activity suggested endogenous AtCCS is sufficient. In contrast, AtCCS null mutants resulted in significant reduction of endogenous CuZnSOD. It implied that AtCCS did regulate CuZnSOD activity. CZ5, a rice CuZnSOD overexpressor in Arabidopsis, had two active forms of CuZnSOD, including rSODI (the normal form) and rSODII (one-copper-lacked form). Overexpression of AtCCS in CZ5 background could enhance rSODI enzyme activity by reducing amount of rSODII, indicating AtCCS is responsible for copper ion incorporation into rice CuZnSOD. Otherwise, overexpression of truncated AtCCS containing Domain II and Domain III (AtCCS-D2D3) in CZ5 indicated that Domain I in AtCCS may function essentially for copper chaperone activity. Inhibition of AtCCS expression in CZ5 with RNA interference technique suppressed both rSODI and rSODII activity. These results revealed that AtCCS could mediate metal ion incorporation system of SOD in Arabidopsis. The rice CCS gene (OsCCS) was cloned, and then overexpressed in CZ5, but it had no effect on rSODI and rSODII activity. It is possible that there is some incompatibility between OsCCS and Arabidopsis, and OsCCS could not fold into a functional protein.