|
Author:
Date:
2017-01-05
[Abstract] The process of protein ubiquitination typically consists of three sequential steps to add an ubiquitin (Ub) or Ub chain to a substrate protein, requiring three different enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3). Most E2s possess the classical E2 activity in forming E2-Ub complex through a thioester linkage, in presence of an E1 and Ub. Additionally, some E2s have the ability of catalyzing the formation of free Ub dimer. Such activity indicates an important role of these E2s in ubiquitination pathway. Thus, we developed an in vitro Ub dimer formation assay to determine the activity of certain E2s. Moreover, by using Ub mutants, in which different lysine residues are mutated, the specific linkage of dimer can ...
[摘要] 蛋白质泛素化的过程通常包括三个顺序步骤：向底物蛋白添加泛素（Ub）或Ub链，需要三种不同的酶，泛素活化酶（E1），泛素缀合酶（E2）和泛素蛋白连接酶E3）。大多数E2在E1和Ub的存在下具有通过硫酯键形成E2-Ub复合物的经典E2活性。另外，一些E2具有催化游离Ub二聚体形成的能力。这种活性表明这些E2在泛素化途径中的重要作用。因此，我们开发了体外的Ub二聚体形成测定来确定某些E2的活性。此外，通过使用不同赖氨酸残基突变的Ub突变体，也可以确定二聚体的特异性连接。
背景 E2共轭启动试验（不添加E3和底物）的现有方案旨在检测硫酯键（E2-S-Ub）。我们的方法着重于催化游离Ub二聚体形成（Ub-Ub）的E2活性。提供了一种检测不同物种E2重要生化特征的便捷方式。此外，二聚体的特异性连接可以通过使用不同的Ub突变体来确定。
|