阿拉伯芥中 AtMAPR 家族蛋白質與其潛在自身泛素化修飾功能之研究

Abstract

阿拉伯芥中的 AtMAPR 家族蛋白質由 4 個同源蛋白質所構成，分別為 AtMAPR2、AtMAPR3、AtMAPR4 與 AtMAPR5。由序列比對結果發現 AtMAPRs 具有一段與 UIM (ubiquitin-interacting motif) 相似度極高的區域。UIM 與 UBA、UEV、NZF、CUE、A20 ZnF、ZnF UBP、Ubc、GAT 等區塊皆屬於 UBDs (ubiquitin-binding domains)，研究指出許多具有 UBDs 之蛋白質會進行自身泛素化修飾 (autoubiquitination)。本論文利用大腸桿菌表現胞外泛素化修飾分析方法 (in vitro ubiquitination assay) 所需要之重組蛋白質 E1 (HsUBE1)、E2 (AtUBC8)、與一個非生物性逆境相關之 RING protein (Rg, At1g74870)，建立胞外泛素化修飾分析方法。透過此胞外泛素化修飾分析研究 Rg、AtMAPR3 與 AtMAPR5，發現它們具有自身泛素化修飾之活性。進一步以 LC-MS/MS 分析，發現 Rg 會催化由泛素 Lys6、Lys11 及 Lys48 相連接而形成的聚泛素鏈。另一方面，透過 LC-MS/MS 之分析，找到多個 AtMAPR3 與 AtMAPR5 上被泛素標定的 Lys 位置，也發現可能存在之聚泛素鏈。由以上結果推測 Rg 可能具有 E3 ligase 之活性，而 AtMAPR3 與 AtMAPR5 可能具有一種新穎之 E3-independent 泛素化修飾的能力。

AtMAPR family consists of four homologs in Arabidopsis, which are AtMAPR2, AtMAPR3, AtMAPR4 and AtMAPR5. A putative ubiquitin-interacting motif (UIM) was detected from a multiple sequence alignment of AtMAPRs and UIMs. UIM is a member of UBDs (ubiquitin-binding domains) which also include UBA, UEV, NZF, CUE, A20 ZnF, ZnF UBP, Ubc, GAT domains. It has been demonstrated that UBD-containing proteins might have autoubiquitination activity. In this study, recombinant proteins required for the in vitro ubiquitination assay, E1 (HsUBE1), E2 (AtUBC8) and an abiotic stress-related RING protein (Rg, At1g74870), are produced in E.coli. In vitro ubiquitination assays indicated that Rg, AtMAPR3 and AtMAPR5 have autoubiquitination activity. By LC-MS/MS analysis, Rg was found to catalyze the formation of polyubiquitin chain via Lys6, Lys11 and Lys48 on ubiquitin. Besides, we also identified several target ubiquitination sites in AtMAPR3 and AtMAPR5. Polyubiquitination was also confirmed by detecting the presence of modified ubiquitin fragments. These results indicated that Rg may have E3 ligase activity, and AtMAPR3 and AtMAPR5 might have a novel E3-independent ubiquitination activity.