National Institute for Medical Research, MRC, The Ridgeway, London, United Kingdom.
FEBS Open Bio. 2013 Oct 12;3:453-8. doi: 10.1016/j.fob.2013.10.005. eCollection 2013.
Protein ubiquitination occurs through formation of an isopeptide bond between the C-terminal glycine of ubiquitin (Ub) and the ɛ-amino group of a substrate lysine residue. This post-translational modification, which occurs through the attachment of single and/or multiple copies of mono-ubiquitin and poly-ubiquitin chains, is involved in crucial cellular events such as protein degradation, cell-cycle regulation and DNA repair. The abnormal functioning of ubiquitin pathways is also implicated in the pathogenesis of several human diseases ranging from cancer to neurodegeneration. However, despite the undoubted biological importance, understanding the molecular basis of how ubiquitination regulates different pathways has up to now been strongly limited by the difficulty of producing the amounts of highly homogeneous samples that are needed for a structural characterization by X-ray crystallography and/or NMR. Here, we report on the production of milligrams of highly pure Josephin mono-ubiquitinated on lysine 117 through large scale in vitro enzymatic ubiquitination. Josephin is the catalytic domain of ataxin-3, a protein responsible for spinocerebellar ataxia type 3. Ataxin-3 is the first deubiquitinating enzyme (DUB) reported to be activated by mono-ubiquitination. We demonstrate that the samples produced with the described method are correctly folded and suitable for structural studies. The protocol allows facile selective labelling of the components. Our results provide an important proof-of-concept that may pave the way to new approaches to the in vitro study of ubiquitinated proteins.
蛋白质的泛素化是通过泛素（Ub）C 末端甘氨酸与底物赖氨酸残基的ε-氨基之间形成异肽键来实现的。这种翻译后修饰通过单和/或多个泛素和多泛素链的附着发生，参与了许多关键的细胞事件，如蛋白质降解、细胞周期调控和 DNA 修复。泛素途径的异常功能也与从癌症到神经退行性疾病等多种人类疾病的发病机制有关。然而，尽管泛素化在生物学上具有重要意义，但到目前为止，对其如何调节不同途径的分子基础的理解受到了极大的限制，因为难以生产出用于 X 射线晶体学和/或 NMR 结构表征所需的大量高度均一的样品。在这里，我们报告了通过大规模体外酶促泛素化，在体外生产毫克级高度纯的赖氨酸 117 单泛素化的 Josephin。Josephin 是负责脊髓小脑共济失调 3 型的 ataxin-3 的催化结构域。Ataxin-3 是第一个被报道被单泛素化激活的去泛素化酶（DUB）。我们证明，用所描述的方法产生的样品是正确折叠的，适合结构研究。该方案允许方便地对组件进行选择性标记。我们的结果提供了一个重要的概念验证，可能为体外研究泛素化蛋白的新方法铺平道路。
