阿拉伯芥泛素化修飾分析方法之建立與非生物性逆境相關 RING-型泛素黏合酶選殖

Abstract

在阿拉伯芥中參與泛素化修飾作用的 E3 ligase 約有 1000 種。許多重要的植物訊息傳遞作用直接或間接與 E3 ligase 及其受質蛋白質之間的辨識作用有關。本實驗室先前酵母菌雙雜合實驗結果顯示，阿拉伯芥中 AtMAPR5 可能與一個含有 RING 區塊的蛋白質 At3g58030 (RING3g) 有交互作用。經序列比對發現另一個與 RING3g 具有高度相似性的蛋白質 At2g42030 (RING2g)。本論文建立本實驗室之胞外泛素化修飾分析方法，試圖研究 AtMAPR5 是否為 RING3g 之生理基質。胞外泛素化修飾分析顯示 RING2g 及 RING3g 具有 E3 ligase 的活性，但是進一步研究顯示 RING3g 無法有效將 AtMAPR5 進行泛素化修飾。推測 AtMAPR5 可能不是 RING3g E3 ligase 之受質蛋白質。將泛素化修飾分析方法擴展，利用微陣列分析資料庫挑選並選殖出 8 個與非生物逆境相關的 RING 蛋白質 (R1 ~ R8)，以胞外泛素化修飾分析發現其中 7 個具有 E3 ligase 之活性。這些 RING-type E3 ligase 生理功能仍有待進一步研究。此外，進行蛋白質體之胞外泛素化修飾，以 2D 電泳分析全蛋白質，尋找參與高鹽非生物逆境之 RING-type E3 ligase 的方法也在本論文中討論。

There are about 1000 E3 ligases in Arabidopsis, which recognize specific protein substrate and catalyze the ubiquitination reaction to these proteins. The recognition between E3 ligases and their substrate proteins occurs in response to many plant signaling pathways directly or indirectly. Previous yeast two-hybrid experiments indicated that AtMAPR5 might interact with a RING protein, RING3g, which is encoded by At3g58030. In vitro ubiquitination assay was established to find out whether AtMAPR5 is the substrate protein of RING3g. RING3g and RING2g (encoded by At2g42030), a close homolog of RING3g, showed the E3 ligase activity in the in vitro ubiquitination assay. However, RING3g did not show the ability to modify AtMAPR5 in vitro and might not be the E3 ligase of AtMAPR5. To expand our studies to find out novel E3 ligases involved in the abiotic stress signaling, eight RING proteins (R1 ~ R8) were cloned based on microarray database. Seven recombinant proteins among them possessed the activity of autoubiquination using in vitro ubiquitination assay. The physiological roles of the RING proteins still await to determine. Finally, the possibility of how to employ in vitro ubiquitination assay in the discovery of novel RING-type E3 ligases involved in salt stress signaling is also discussed.