Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation
Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation
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DOI：
10.1074/jbc.m117.794255
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发表时间：
2017-09-15
影响因子：
4.8
通讯作者：
Ernst, Andreas
中科院分区：
文献类型：
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作者：
Wiechmann, Svenja;Gaertner, Anne;Ernst, Andreas
小泛素样修饰物（SUMO）进行的翻译后修饰可调节许多细胞过程，包括基因组完整性、基因表达和核糖体生物发生。E2结合酶Ubc9催化SUMO与靶蛋白中赖氨酸残基的ε - 氨基结合。SUMO部分连接到Ubc9自身内部的赖氨酸上可进一步导致多聚SUMO链的形成。靶蛋白的单SUMO化和多SUMO化可为不同的衔接蛋白和效应蛋白提供对接位点，这对调节不同的SUMO调控通路非常重要。然而，用于区分依赖单SUMO化或多SUMO化通路的分子工具在很大程度上是缺失的。通过蛋白质工程方法，我们利用噬菌体展示技术产生了高亲和力的SUMO2变体，它们结合Ubc9的背面结合位点，并作为基于SUMO的Ubc9抑制剂（SUBIN）发挥作用。重要的是，我们发现不同的SUBIN主要抑制多聚SUMO链的形成，而单SUMO化不受影响。原理验证实验表明，在细胞环境中，SUBIN在很大程度上阻止热休克触发的多聚SUMO化。此外，SUBIN消除了砷诱导的早幼粒细胞白血病蛋白的降解。我们认为，本文报道的新型链选择性SUMO抑制剂的出现将使人们能够深入研究多聚SUMO介导的细胞过程，如DNA损伤应答和细胞周期进程。
Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) regulate many cellular processes, including genome integrity, gene expression, and ribosome biogenesis. The E2-conjugating enzyme Ubc9 catalyzes the conjugation of SUMOs to epsilon-amino groups of lysine residues in target proteins. Attachment of SUMO moieties to internal lysines in Ubc9 itself can further lead to the formation of polymeric SUMO chains. Mono-and poly-SUMOylations of target proteins provide docking sites for distinct adapter and effector proteins important for regulating discrete SUMO-regulated pathways. However, molecular tools to dissect pathways depending on either mono-or poly-SUMOylation are largely missing. Using a protein-engineering approach, we generated high-affinity SUMO2 variants by phage display that bind the back side binding site of Ubc9 and function as SUMO-based Ubc9 inhibitors (SUBINs). Importantly, we found that distinct SUBINs primarily inhibit poly-SUMO chain formation, whereas mono-SUMOylation was not impaired. Proof-of-principle experiments demonstrated that in a cellular context, SUBINs largely prevent heat shock-triggered polySUMOylation. Moreover, SUBINs abrogated arsenic-induced degradation of promyelocytic leukemia protein. We propose that the availability of the new chain-selective SUMO inhibitors reported here will enable a thorough investigation of poly-SUMO-mediated cellular processes, such as DNA damage responses and cell cycle progression.