Triubiquitin Probes for Identification of Reader and Eraser Proteins of Branched Polyubiquitin Chains

The important roles played by branched polyubiquitin chains were recently uncovered in proteasomal protein degradation, mitotic regulation, and NF-κB signaling. With the new realization of a wide presence of branched ubiquitin chains in mammalian cells, there is an urgent need of identifying the reader and eraser proteins of the various branched ubiquitin chains. In this work, we report the generation of noncleavable branched triubiquitin probes with combinations of K11-, K48-, and K63-linkages. Through a pulldown approach using the branched triUb probes, we identified human proteins that recognize branched triubiquitin structures including ubiquitin-binding proteins and deubiquitinases (DUBs). Proteomics analysis of the identified proteins enriched by the branched triubiquitin probes points to possible roles of branched ubiquitin chains in cellular processes including DNA damage response, autophagy, and receptor endocytosis. In vitro characterization of several identified UIM-containing proteins demonstrated their binding to branch triubiquitin chains with moderate to high affinities. Availability of this new class of branched triubiquitin probes will enable future investigation into the roles of branched polyubiquitin chains through identification of specific reader and eraser proteins, and the modes of branched ubiquitin chain recognition and processing using biochemical and biophysical methods.