A modular toolbox to generate complex polymeric ubiquitin architectures using orthogonal sortase enzymes

• Published in: Nature communications Vol. 12; no. 1; p. 6515
• Main Authors: Fottner, Maximilian, Weyh, Maria, Gaussmann, Stefan, Schwarz, Dominic, Sattler, Michael, Lang, Kathrin
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 11.11.2021; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 14; 631/337/1427; 631/45/612/645; 631/92/458/582; 631/92/603; 82/6; 82/80; 82/83; Chain branching; Control stability; Deoxyribonucleic acid; DNA; DNA damage; DNA Damage - genetics; DNA Damage - physiology; DNA repair; Enzymes; Humanities and Social Sciences; Humans; Localization; multidisciplinary; Polymers - chemistry; Post-translation; Protein Binding; Protein Processing, Post-Translational; Proteins; Repair; Science; Science (multidisciplinary); Sortase; Topology; Ubiquitin; Ubiquitin - genetics; Ubiquitin - metabolism; Ubiquitination - genetics; Ubiquitination - physiology
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-26812-9

The post-translational modification of proteins with ubiquitin (Ub) and Ub-like modifiers (Ubls) represents one of the most important regulators in eukaryotic biology. Polymeric Ub/Ubl chains of distinct topologies control the activity, stability, interaction and localization of almost all cellular proteins and elicit a variety of biological outputs. Our ability to characterize the roles of distinct Ub/Ubl topologies and to identify enzymes and receptors that create, recognize and remove these modifications is however hampered by the difficulty to prepare them. Here we introduce a modular toolbox (Ubl-tools) that allows the stepwise assembly of Ub/Ubl chains in a flexible and user-defined manner facilitated by orthogonal sortase enzymes. We demonstrate the universality and applicability of Ubl-tools by generating distinctly linked Ub/Ubl hybrid chains, and investigate their role in DNA damage repair. Importantly, Ubl-tools guarantees straightforward access to target proteins, site-specifically modified with distinct homo- and heterotypic (including branched) Ub chains, providing a powerful approach for studying the functional impact of these complex modifications on cellular processes. Ubiquitin (Ub) and Ub-like modifiers (Ubls) can form chains of various topologies, but preparing defined chains for functional studies remains challenging. Here, the authors develop chemoenzymatic tools to tailormake Ub/Ubl chains and study the involvement of specific Ub/SUMO chains in DNA repair.