Berndt Anja, Wilkinson Kevin A, Heimann Michaela J, Bishop Paul, Henley Jeremy M
*School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, U.K.
Biochem J. 2013 Dec 15;456(3):385-95. doi: 10.1042/BJ20130241.
Monobodies are small recombinant proteins designed to bind with high affinity to target proteins. Monobodies have been generated to mimic the SIM [SUMO (small ubiquitin-like modifier)-interacting motif] present in many SUMO target proteins, but their properties have not been determined in cells. In the present study we characterize the properties of two SUMO1-specific monobodies (hS1MB4 and hS1MB5) in HEK (human embyronic kidney)-293 and HeLa cells and examine their ability to purify SUMO substrates from cell lines and rat brain. Both hS1MB4 and hS1MB5 compared favourably with commercially available antibodies and were highly selective for binding to SUMO1 over SUMO2/3 in pull-down assays against endogenous and overexpressed SUMO and SUMOylated proteins. Monobodies expressed in HeLa cells displayed a nuclear and cytosolic distribution that overlaps with SUMO1. Expression of the monobodies effectively inhibited protein SUMOylation by SUMO1 and, surprisingly, by SUMO2/3, but were not cytotoxic for at least 36 h. We attribute the effects on SUMO2/3 to the role of SUMO1 in chain termination and/or monobody inhibition of the SUMO-conjugating E1 enzyme complex. Taken together, these data provide the first demonstration that monobodies represent useful new tools both to isolate SUMO conjugates and to probe cell SUMOylation pathways in vivo.
单域抗体是一种小型重组蛋白，设计用于与靶蛋白高亲和力结合。已生成单域抗体来模拟许多SUMO（小泛素样修饰物）靶蛋白中存在的SIM（SUMO相互作用基序），但其特性尚未在细胞中确定。在本研究中，我们表征了两种SUMO1特异性单域抗体（hS1MB4和hS1MB5）在人胚肾（HEK）-293和HeLa细胞中的特性，并检测了它们从细胞系和大鼠脑中纯化SUMO底物的能力。在针对内源性和过表达的SUMO及SUMO化蛋白的下拉试验中，hS1MB4和hS1MB5与市售抗体相比表现良好，并且对SUMO1的结合具有高度选择性，而不是SUMO2/3。在HeLa细胞中表达的单域抗体显示出与SUMO1重叠的核和胞质分布。单域抗体的表达有效抑制了SUMO1介导的蛋白质SUMO化，令人惊讶的是，也抑制了SUMO2/3介导的蛋白质SUMO化，但至少36小时内没有细胞毒性。我们将对SUMO2/3的影响归因于SUMO1在链终止和/或单域抗体对SUMO缀合E1酶复合物的抑制作用中的作用。综上所述，这些数据首次证明单域抗体是分离SUMO缀合物和体内探测细胞SUMO化途径的有用新工具。
