探討Dap1與25-Dx是否具有自身泛素化修飾能力之研究

Abstract

酵母菌中damage reponse protein 1 (Dap1) 及大鼠中25-Dx與阿拉伯芥中AtMAPRs同屬MAPR家族成員，此家族蛋白質具有保守性區塊cytochrome b5 like heme/steroid binding domain。本實驗室對阿拉伯芥之MAPR同源蛋白質AtMAPRs進行研究，發現重組之AtMAPRs蛋白質在胞外具有不需E3 ligase參與的「自身泛素化修飾能力」(E3-independent autoubiquitination)，序列比對結果指出AtMAPRs之cytochrome b5 like heme/steroid binding domain上可能具有類似UIM (ubiquitin-interacting motif) 的序列，其為一種泛素結合區塊UBDs (ubiquitin binding domains)，而研究指出帶有UBD之蛋白質可能會進行自身泛素化修飾。本論文利用大腸桿菌表現Dap1、25-Dx及其他參與泛素化修飾之E2，以胞外泛素化修飾實驗 (in vitro ubiquitination assay) 分析Dap1與25-Dx是否具有自身泛素化修飾之能力，發現兩者皆不具有自身泛素化修飾能力。而序列比對結果指出AtMAPR2與Dap1及25-Dx有一段較不相似之序列，此序列可能為影響Dap1及25-Dx不具自身泛素化修飾能力之主要因素。因此自身泛素化修飾能力可能為阿拉伯芥中AtMAPRs特有之能力，此推論有待進一步實驗來證實。

AtMAPRs in Arabidopsis are homologous to Dap1 in Saccharomyces cerevisiae and 25-Dx in Rattus norvegicus. They are members of MAPR (membrane-associated progesterone receptor) family which characterized by a conserved cytochrome b5 like heme/steroid binding domain. In previous study, it has been demonstrated that the recombinant proteins of AtMAPRs, the MAPR homologs in Arabidopsis thaliana, have E3-independent autoubiquitination activity in vitro. A putative ubiquitin-interacting motif (UIM) was detected in the cytochrome b5 like heme/steroid binding domain of AtMAPRs from a multiple sequence alignment. UIM is a member of UBDs (ubiquitin-binding domains), and the UBD-containing proteins have shown autoubiquitination activity. In this study, recombinant proteins required for the in vitro ubiquitination assay, E2 (UBC4, UBE2D2) and substrate (Dap1, 25-Dx), were produced in E. coli. In vitro ubiquitination assays indicated that Dap1 and 25-Dx did not have autoubiquitination activity. There are some differences in Dap1 and 25-Dx sequences compared with AtMAPR2. This may explain that Dap1 and 25-Dx did not have autoubiquitination activity. These findings suggested that E3-independent autoubiquitination might be an unique character of AtMAPRs in Arabidopsis. Further studies are needed to support this assumption.