Nakayama Tomofumi, Yuasa Eri, Kanemaru Ayumi, Saito Masayuki, Saitoh Hisato
Department of Biological Sciences; Graduate School of Science and Technology; Kumamoto University; Kumamoto, Japan.
Department of Biological Sciences; Graduate School of Science and Technology; Kumamoto University; Kumamoto, Japan; Department of New Frontier Sciences; Graduate School of Science and Technology; Kumamoto University; Kumamoto, Japan.
Bioengineered. 2014 Mar-Apr;5(2):133-7. doi: 10.4161/bioe.27544. Epub 2014 Jan 13.
Small ubiquitin-related modifier (SUMO) is a highly conserved protein that is covalently attached to target proteins. This posttranslational modification, designated SUMOylation, is a major protein-conjugation-driven strategy designed to regulate structure and function of cellular proteins. SUMOylation consists of an enzymatic cascade involving the E1-activating enzyme and the E2-conjugating enzyme. The SUMO-E1 enzyme consists of two subunits, a heterodimer of activation of Smt3p 1 (Aos1) and ubiquitin activating enzyme 2 (Uba2), which resembles the N- and C-terminal halves of ubiquitin E1 (Uba1). Herein, we describe the rational design of a single polypeptide version of SUMO-E1, a chimera of mouse Aos1 and Uba2 subunits, termed mAU, in which the functional domains appear to be arranged in a fashion similar to Uba1. We also describe the construction of a mAU plasmid for expression in a baculovirus-insect cell system and present an in situ SUMOylation assay using the recombinant mAU. Our results showed that mAU has SUMO-E1 activity, thereby indicating that mAU can be expressed in baculovirus-insect cells and represents a suitable source of SUMO-E1.
小泛素相关修饰物（SUMO）是一种高度保守的蛋白质，可共价连接到靶蛋白上。这种翻译后修饰，称为SUMO化，是一种主要的由蛋白质共轭驱动的策略，旨在调节细胞蛋白质的结构和功能。SUMO化由一个涉及E1激活酶和E2共轭酶的酶促级联反应组成。SUMO-E1酶由两个亚基组成，即Smt3p 1激活异二聚体（Aos1）和泛素激活酶2（Uba2），它类似于泛素E1（Uba1）的N端和C端部分。在此，我们描述了SUMO-E1单多肽版本的合理设计，即小鼠Aos1和Uba2亚基的嵌合体，称为mAU，其中功能域的排列方式似乎与Uba1相似。我们还描述了用于在杆状病毒-昆虫细胞系统中表达的mAU质粒的构建，并展示了使用重组mAU的原位SUMO化测定。我们的结果表明mAU具有SUMO-E1活性，从而表明mAU可以在杆状病毒-昆虫细胞中表达，是SUMO-E1的合适来源。
