Comprehensive approach to study branched ubiquitin chains reveals roles for K48-K63 branches in VCP/p97-related processes

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. Highlights Assembly of defined branched ubiquitin chains enables identification of specific binding proteins Development of quantitative DUB assay monitoring cleavage of individual Ub linkages within complex ubiquitin chains identifies debranching enzymes Engineering specific, high-affinity nanobody against branched K48-K63 ubiquitin reveals roles in VCP/p97 related processes and DNA damage responses General blueprint of new methods and tools for in-depth characterization of branched ubiquitin chains and their underlying biology