阿拉伯芥核糖核酸結合蛋白GRP7和GRP8具活化細胞質鐵超氧歧化酶之功能性研究

Abstract

鐵作為許多重要蛋白質和酶的輔助因子是所有生物體中不可缺少的元素。金屬伴護蛋白質(Metallochaperones)透過直接的蛋白質間相互作用將金屬離子遞送給特定的生理夥伴。目前只有三個已被證實為鐵伴護蛋白質(Fe chaperones)，人類粒線體蛋白質Frataxin、細胞質的Poly(C)-binding protein及阿拉伯芥葉綠體伴護蛋白質Chaperonin 20。本研究找到四個阿拉伯芥中核醣核酸結合蛋白質(RNA binding proteins; RBPs)分別為RBP1, RBP2, GRP7, GRP8對於位在細胞質的鐵超氧歧化酶(ΔTP-FeSOD1)具有潛在的鐵伴護蛋白質功用。在先前的研究，藉由阿拉伯芥原生質體暫時性表現，我們發現這些核醣核酸結合蛋白質都具有活化細胞質鐵超氧歧化酶的能力。為了分析鐵超氧歧化酶與核醣核酸結合蛋白質之間的交互作用: 利用(1) 酵母菌雙雜合系統發現GRP7, GRP8會與鐵超氧歧化酶有交互作用；(2) 雙分子螢光互補實驗 (BiFC) 發現核醣核酸結合蛋白質於細胞質中都會與的鐵超氧歧化酶有交互作用。經由體外超氧歧化酶活性測試，當加入GRP7或 GRP8後可提升FSD1的活性。確認對細胞質型的鐵超氧歧化酶來說，核醣核酸結合蛋白質極有可能是其鐵伴護蛋白質，因此，認為核醣核酸結合蛋白質對於細胞質中需要鐵的蛋白質具有給予鐵的能力。

Iron (Fe) is an essential element in plants and animals, while excess Fe accumulation causes cytotoxic through the production of reactive oxygen species. Metallochaperones deliver ion cofactors to the specific physiological partners through direct protein-protein interactions. Only three Fe chaperones are known, i.e. human mitochondrial frataxin, cytoplasmic poly(C)-binding proteins, and Arabidopsis chloroplastic chaperonin 20. We identified four RNA binding proteins (RBPs) RBP1, RBP2, GRP7, and GRP8 which could be functioned as a Fe chaperone for cytoplasmic-destined Fe superoxide dismutase 1 (ΔTP-FSD1) activation in Arabidopsis (Arabidopsis thaliana). In this study, using yeast two-hybrid and bimolecular fluorescence complementation assay confirmed the interaction relationship between RBPs and FSD1. In vitro SOD activity assay showed that GRP7 and GRP8 enhanced the FSD1 activity. In conclusion, our results demonstrated a critical role for RBPs to be Fe chaperones for the cytosolic FeSOD activation in Arabidopsis. Our results also suggested that RBP plays a role in Fe metalation of cytoplasmic Fe-containing proteins.