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.

One-Pot, Photocontrolled Enzymatic Assembly of the Structure-Defined Heterotypic Polyubiquitin Chain

“…Several tens of microgram of polyubiquitin is generally sufficient for biochemical experiments including the gel-based evaluation of DUB-mediated cleavage and the pull-down assay for studies on protein−ubiquitin interactions. 13,20,53,54 The synthesized K63/K48 Ub 4 with a K48-linked branch at the middle site was supplied to partial trypsin digestion under nondenaturing conditions to validate the branch structure. Trypsin treatment under nondenaturing conditions leads to the cleavage of peptide bonds between the 74th arginine and 75th glycine in the polyubiquitin chain, thereby allowing the characterization of distal and branched ubiquitins as ubiquitin units lacking C-terminal GG (Ub 1−74 ) and harboring multiple GG remnants on lysine residues engaged in the linkage formation, respectively (Figure 4e, left).…”

“…The target K63/K48 Ub 4 synthesized on a larger scale was isolated by cation-exchange chromatography (52 μg, 4.3% isolated yield) (Figure S10) and characterized by MALDI-TOF MS (Figure d). Several tens of microgram of polyubiquitin is generally sufficient for biochemical experiments including the gel-based evaluation of DUB-mediated cleavage and the pull-down assay for studies on protein–ubiquitin interactions. ,,, …”

“…The time traces of biotinmodified ubiquitin species during AMSH processing of synthesized K63/K48 Ub 4 indicated the preference of AMSH for the distal side over the proximal side of K63-linked isopeptide bonds at the K63/K48 branching position, as suggested recently (Figure S12). 54 The DUB-mediated trimming of polyubiquitin is one of the essential aspects of the structural regulation of polyubiquitin chains underlying the precise control of ubiquitin functions. 59 Thus, the structuredefined heterotypic chains would offer a robust platform for evaluating how the structure of heterotypic polyubiquitin chains is controlled by enzymes including ubiquitin ligases and DUBs for precise modulation of ubiquitin signaling.…”

Biocompatible Lysine Protecting Groups for the Chemoenzymatic Synthesis of K48/K63 Heterotypic and Branched Ubiquitin Chains

“…The synthesis of Ub chains requires sequential conjugation and deprotection as illustrated in Figure A. We adopted the recently proposed nomenclatures for branched ubiquitin from Kulath et al, which provides a method of describing heterotypic ubiquitin chains . In our synthesis, the removal of excess Ub D and the E2-conjugating enzymes after the conjugation as well as buffer exchange before each conjugation/deprotection are inevitable.…”

Biocompatible lysine protecting groups for the chemoenzymatic synthesis of K48/K63 heterotypic and branched ubiquitin chains

“…We adopted the recently proposed nomenclatures for branched ubiquitin from Kulath et al, which provides a method of describing heterotypic ubiquitin chains. 50 In our synthesis, the removal of excess Ub D and the E2 conjugating enzymes after the conjugation, as well as buffer exchange before each conjugation/deprotection, is inevitable. To facilitate the synthesis and avoid chromatographic purification after each conjugation or deprotection, we implemented an on-resin synthesis of ubiquitin This facilitated the removal of non-tagged Ub D and E2s from the mixture and enabled buffer exchange by simple washing of the resin, analogous to SPPS.…”