Biochemical characterisation of a novel E3 ubiquitin ligase which assembles mixed ubiquitin chains

Ubiquitin is a small and highly conserved protein (76 amino acids), best known for its role in mediating protein degradation as part of the Ubiquitin Proteasome System (UPS). Ubiquitin is conjugated to lysines on a protein target through an enzymatic cascade involving an E1-activating enzyme, E2-conjugating enzymes and E3 ubiquitin ligases. Ubiquitin can also be assembled into chains, forming an isopeptide bond between any of the seven lysines (K6, K11, K27, K29, K33, K48 and K63) or the N- terminus of an acceptor ubiquitin and the C-terminus of a donor ubiquitin. Therefore, as many as eight linkage types can be used to assemble homotypic, heterotypic, mixed and branched ubiquitin chains. Although the biochemical properties and cellular functions of homotypic chains are fairly well understood, much less is known regarding the more complex mixed and branched ubiquitin chains. Seminal work by the Varshavsky and Pickart labs in the mid-to-late 1990’s first led to the identification of mixed K29/K48 ubiquitin chains. I will present data on the biochemical characterisation of a novel E3 ubiquitin ligase which catalyses the formation of mixed K29/K48 ubiquitin chains and discuss these findings in the context of the increasing complexity of the ubiquitin code.