Comprehensive approach to study branched ubiquitinchains reveals roles for K48-K63 branches inVCP/p97-related processes

Lange, S. M.; McFarland, M. R.; Lamoliatte, F.; Kwasna, D.; Shen, L.; Wallace, I.; Cole, I.; Armstrong, L. A.; Knebel, A.; Johnson, C.; De Cesare, V.; Kulathu, Y.

doi:10.1101/2023.01.10.523363

bioRxiv · 10.1101/2023.01.10.523363

Comprehensive approach to study branched ubiquitinchains reveals roles for K48-K63 branches inVCP/p97-related processes

Lange, S. M.·McFarland, M. R.·Lamoliatte, F.·Kwasna, D.·Shen, L.·Wallace, I.·Cole, I.·Armstrong, L. A.·Knebel, A.·Johnson, C.·De Cesare, V.·Kulathu, Y.

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Abstract

Branched ubiquitin (Ub) chains make up a significant proportion of Ub polymers in human cells and are formed when two or more sites on a single Ub molecule are modified with Ub creating bifurcated architectures. Despite their abundance, we have a poor understanding of the cellular functions of branched Ub signals that stems from a lack of facile tools and methods to study them. Here we develop a comprehensive pipeline to define branched Ub function, using K48-K63-branched chains as a case study. We discover branch-specific binders and, by developing a method that monitors cleavage of linkages within complex polyUb, we discover the VCP/p97-associated ATXN3, and MINDY family deubiquitinases to act as debranching enzymes. By engineering and utilizing a branched K48-K63-Ub chain-specific nanobody, we reveal roles for these chains in VCP/p97-related processes. In summary, we provide a blueprint to investigate branched Ub function that can be readily applied to study other branched chain types. HighlightsO_LIAssembly of defined branched ubiquitin chains enables identification of specific binding proteins C_LIO_LIDevelopment of quantitative DUB assay monitoring cleavage of individual Ub linkages within complex ubiquitin chains identifies debranching enzymes C_LIO_LIEngineering specific, high-affinity nanobody against branched K48-K63 ubiquitin reveals roles in VCP/p97 related processes and DNA damage responses C_LIO_LIGeneral blueprint of new methods and tools for in-depth characterization of branched ubiquitin chains and their underlying biology C_LI

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Lange, S. M., McFarland, M. R., Lamoliatte, F., Kwasna, D., Shen, L., Wallace, I., Cole, I., Armstrong, L. A., Knebel, A., Johnson, C., De Cesare, V., Kulathu, Y.. 2023-01-10. Comprehensive approach to study branched ubiquitinchains reveals roles for K48-K63 branches inVCP/p97-related processes. https://doi.org/10.1101/2023.01.10.523363

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