Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Lab for Synthetic Biochemistry, Technical University of Munich, Institute for Advanced Study, TUM-IAS, Garching, Germany.
Institute of Biochemistry II, Goethe University Frankfurt-Medical Faculty, University Hospital, Frankfurt am Main, Germany.
Nat Chem Biol. 2019 Mar;15(3):276-284. doi: 10.1038/s41589-019-0227-4. Epub 2019 Feb 15.
Post-translational modification of proteins with ubiquitin and ubiquitin-like proteins (Ubls) is central to the regulation of eukaryotic cellular processes. Our ability to study the effects of ubiquitylation, however, is limited by the difficulty to prepare homogenously modified proteins in vitro and by the impossibility to selectively trigger specific ubiquitylation events in living cells. Here we combine genetic-code expansion, bioorthogonal Staudinger reduction and sortase-mediated transpeptidation to develop a general tool to ubiquitylate proteins in an inducible fashion. The generated ubiquitin conjugates display a native isopeptide bond and bear two point mutations in the ubiquitin C terminus that confer resistance toward deubiquitinases. Nevertheless, physiological integrity of sortase-generated diubiquitins in decoding cellular functions via recognition by ubiquitin-binding domains is retained. Our approach allows the site-specific attachment of Ubls to nonrefoldable, multidomain proteins and enables inducible and ubiquitin-ligase-independent ubiquitylation of proteins in mammalian cells, providing a powerful tool to dissect the biological functions of ubiquitylation with temporal control.
蛋白质的泛素化和泛素样蛋白（Ubls）的翻译后修饰是真核细胞过程调节的核心。然而，我们研究泛素化影响的能力受到在体外均匀修饰蛋白质的困难以及在活细胞中选择性触发特定泛素化事件的不可能性的限制。在这里，我们结合遗传密码扩展、生物正交的 Staudinger 还原和连接酶介导的转肽作用，开发了一种通用工具，以诱导方式泛素化蛋白质。生成的泛素缀合物显示天然的异肽键，并在泛素 C 末端带有两个点突变，赋予对去泛素酶的抗性。尽管如此，通过泛素结合结构域识别，连接酶生成的二泛素在解码细胞功能中的生理完整性得以保留。我们的方法允许将 Ubls 定点连接到不可重折叠的多结构域蛋白质上，并能够在哺乳动物细胞中诱导和泛素连接酶独立的蛋白质泛素化，为具有时间控制的泛素化生物学功能提供了强大的工具。
