Hewings David S, Flygare John A, Bogyo Matthew, Wertz Ingrid E
Discovery Chemistry, Genentech, South San Francisco, CA, USA.
Early Discovery Biochemistry, Genentech, South San Francisco, CA, USA.
FEBS J. 2017 May;284(10):1555-1576. doi: 10.1111/febs.14039. Epub 2017 Mar 10.
The reversible post-translational modification of proteins by ubiquitin and ubiquitin-like proteins regulates almost all cellular processes, by affecting protein degradation, localization, and complex formation. Deubiquitinases (DUBs) are proteases that remove ubiquitin modifications or cleave ubiquitin chains. Most DUBs are cysteine proteases, which makes them well suited for study by activity-based probes. These DUB probes report on deubiquitinase activity by reacting covalently with the active site in an enzyme-catalyzed manner. They have proven to be important tools to study DUB selectivity and proteolytic activity in different settings, to identify novel DUBs, and to characterize deubiquitinase inhibitors. Inspired by the efficacy of activity-based probes for DUBs, several groups have recently reported probes for the ubiquitin conjugation machinery (E1, E2, and E3 enzymes). Many of these enzymes, while not proteases, also posses active site cysteine residues and can be targeted by covalent probes. In this review, we will discuss how features of the probe (cysteine-reactive group, recognition element, and reporter tag) affect reactivity and suitability for certain experimental applications. We will also review the diverse applications of the current probes, and discuss the need for new probe types to study emerging aspects of ubiquitin biology.
泛素及泛素样蛋白对蛋白质进行的可逆翻译后修饰，通过影响蛋白质降解、定位和复合物形成，几乎调控了所有细胞过程。去泛素化酶（DUBs）是去除泛素修饰或切割泛素链的蛋白酶。大多数DUBs是半胱氨酸蛋白酶，这使得它们非常适合用基于活性的探针进行研究。这些DUB探针通过以酶催化的方式与活性位点共价反应来报告去泛素化酶的活性。它们已被证明是研究不同环境中DUB选择性和蛋白水解活性、鉴定新型DUBs以及表征去泛素化酶抑制剂的重要工具。受基于活性的DUB探针功效的启发，几个研究小组最近报道了针对泛素缀合机制（E1、E2和E3酶）的探针。这些酶中的许多虽然不是蛋白酶，但也具有活性位点半胱氨酸残基，并且可以被共价探针靶向。在这篇综述中，我们将讨论探针的特性（半胱氨酸反应基团、识别元件和报告标签）如何影响反应性以及对某些实验应用的适用性。我们还将综述当前探针的各种应用，并讨论研究泛素生物学新出现方面所需的新型探针类型。
