The covalent attachment of Ub (ubiquitin) to target proteins (ubiquitylation) represents one of the most versatile PTMs (post-translational modifications) in eukaryotic cells. Substrate modifications range from a single Ub moiety being attached to a target protein to complex Ub chains that can also contain Ubls (Ub-like proteins). Ubiquitylation plays pivotal roles in most aspects of eukaryotic biology, and cells dedicate an orchestrated arsenal of enzymes to install, translate, and reverse these modifications. The entirety of this complex system is coined the Ub code. Deciphering the Ub code is challenging due to the difficulty in reconstituting enzymatic machineries and generating defined Ub/Ubl–protein conjugates. This Review provides a comprehensive overview of recent advances in using GCE (genetic code expansion) techniques to study the Ub code. We highlight strategies to site-specifically ubiquitylate target proteins and discuss their advantages and disadvantages, as well as their various applications. Additionally, we review the potential of small chemical PTMs targeting Ub/Ubls and present GCE-based approaches to study this additional layer of complexity. Furthermore, we explore methods that rely on GCE to develop tools to probe interactors of the Ub system and offer insights into how future GCE-based tools could help unravel the complexity of the Ub code.

中文翻译：

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破译泛素密码的遗传密码扩展方法


Ub（泛素）与靶蛋白（泛素化）的共价连接是真核细胞中用途最广泛的 PTM（翻译后修饰）之一。底物修饰范围从附着在靶蛋白上的单个 Ub 部分到也可以包含 Ubls（Ub 样蛋白）的复杂 Ub 链。泛素化在真核生物生物学的大多数方面都起着关键作用，细胞专门使用精心编排的酶库来安装、翻译和逆转这些修饰。这个复杂的系统的全部被称为 Ub 代码。破译 Ub 码具有挑战性，因为难以重构酶机制和生成确定的 Ub/Ubl-蛋白质偶联物。本综述全面概述了使用 GCE （遗传密码扩展） 技术研究 Ub 密码的最新进展。我们重点介绍了位点特异性泛素化靶蛋白的策略，并讨论了它们的优缺点，以及它们的各种应用。此外，我们回顾了靶向 Ub/Ubls 的小型化学 PTM 的潜力，并提出了基于 GCE 的方法来研究这一额外的复杂性层。此外，我们探索了依靠 GCE 开发工具来探测 Ub 系统交互器的方法，并提供了关于未来基于 GCE 的工具如何帮助解开 Ub 代码复杂性的见解。