From the Leibniz Institute of Plant Biochemistry, Independent Junior Research Group, Weinberg 3, 06120 Halle (Saale) and.
From the Leibniz Institute of Plant Biochemistry, Independent Junior Research Group, Weinberg 3, 06120 Halle (Saale) and
J Biol Chem. 2018 Oct 19;293(42):16324-16336. doi: 10.1074/jbc.RA118.004226. Epub 2018 Sep 5.
Ubiquitination is a prevalent post-translational modification involved in all aspects of cell physiology. It is mediated by an enzymatic cascade and the E2 ubiquitin-conjugating enzymes (UBCs) lie at its heart. Even though E3 ubiquitin ligases determine the specificity of the reaction, E2s catalyze the attachment of ubiquitin and have emerged as key mediators of chain assembly. They are largely responsible for the type of linkage between ubiquitin moieties and thus, the fate endowed onto the modified substrate. However, E2-E3 pairing remains largely unexplored. We therefore interrogated the interaction selectivity between 37 E2s and PUB22, a U-box type E3 ubiquitin ligase that is involved in the dampening of immune signaling. We show that whereas the U-box domain, which mediates E2 docking, is able to interact with 18 of 37 tested E2s, the substrate interacting armadillo (ARM) repeats impose a second layer of specificity, allowing the interaction with 11 E2s. activity assayed by autoubiquitination only partially recapitulated the selectivity. Moreover, pairing was modulated during the immune response; pairing with group VI UBC30 was inhibited, whereas interaction with the K63 chain-building UBC35 was increased. Functional analysis of mutants shows that they partially mimic enhanced activation of immune responses. Together, our work provides a framework to interrogate E2-E3 pairing and reveals a multi-tiered and dynamic E2-E3 network.
泛素化是一种普遍存在的翻译后修饰，涉及细胞生理学的各个方面。它是由酶级联介导的，而 E2 泛素缀合酶 (UBCs) 处于其核心。尽管 E3 泛素连接酶决定了反应的特异性，但 E2 催化泛素的附着，并已成为链组装的关键介质。它们在很大程度上负责连接泛素部分之间的类型，从而赋予修饰底物命运。然而，E2-E3 配对仍然在很大程度上未被探索。因此，我们研究了 37 种 E2 与 PUB22 之间的相互作用选择性，PUB22 是一种 U 盒型 E3 泛素连接酶，参与免疫信号的衰减。我们表明，介导 E2 对接的 U 盒结构域能够与 37 种测试 E2 中的 18 种相互作用，而与底物相互作用的臂突重复序列则施加了第二层特异性，允许与 11 种 E2 相互作用。通过自动泛素化测定的活性仅部分重现了选择性。此外，配对在免疫反应期间受到调节；与第 VI 组 UBC30 的配对受到抑制，而与 K63 链构建 UBC35 的相互作用增加。突变体的功能分析表明，它们部分模拟了增强的免疫反应激活。总之，我们的工作为研究 E2-E3 配对提供了一个框架，并揭示了一个多层次和动态的 E2-E3 网络。
