Identification of Proteins Interacting with Ubiquitin Chains

Abstract

Ubiquitylation, the modification of proteins with ubiquitin (Ub), is one of the most versatile post-translational modifications in eukaryotic cells. Since Ub also serves as its own substrate, proteins can be modified by numerous different Ub chains, in which the individual moieties are linked via one or several of the seven lysines of Ub. Homogeneous Ub chains, in which the moieties are sequentially linked via the same residue, have been most extensively studied. However, due to their restricted availability, the functions of Ub chains linked via K27, K29, or K33 are poorly understood. We have developed an approach that, for the first time, allows the generation of all seven homogeneous Ub chains in large quantities. The potential of our approach is demonstrated by the identification of previously unknown interaction partners of K27-, K29-, and K33-linked Ub chains by affinity-based proteomics.

Keywords: affinity-based profiling; click chemistry; post-translational modification; proteomic profiling; ubiquitylation.