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

ABSTRACT: 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.

SUBMITTER: Fottner M

PROVIDER: S-EPMC8585875 | biostudies-literature |

REPOSITORIES: biostudies-literature

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