PuSH - Publikationsserver des Helmholtz Zentrums München: A modular toolbox to generate complex polymeric ubiquitin architectures using orthogonal sortase enzymes.

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Fottner, M.* ; Weyh, M.* ; Gaussmann, S. ; Schwarz, D.* ; Sattler, M. ; Lang, K.*

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

Nat. Commun. 12:6515 (2021)

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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.

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