Characterisation of a new role for the UBA domain in ubiquitin chain synthesis by Ube2K

Abstract

Ubiquitylation is a post-translational modification in which the protein Ubiquitin (Ub) is attached to other proteins, dictating their fate. Ubiquitylation requires the action of three groups of enzymes, Ub activating (E1) enzymes, Ub conjugating (E2) enzymes, and Ub ligases (E3). Multiple Ub molecules can be linked onto a substrate, with chains attached when ubiquitin is linked by one of the seven lysines or N-terminal methionine . The residue that links the Ub molecules in a chain impacts the fate of the modified substrate, with K48 linkages leading to proteasomal degradation and K63 linkages involved in downstream signalling (e.g. NF-κB). Ube2K is an E2 enzyme which rapidly forms K48 degradative chains and has a C-terminal ubiquitin associated (UBA) domain which can bind Ub, but its specific function in ubiquitylation is unclear. Ube2K has been linked to conditions including Alzheimer’s disease, but the function of Ube2K is not yet well understood. Therefore, to better understand the link between Ube2K and disease, this work aimed to uncover the function of the UBA domain. The UBA domain was initially proposed to enhance Ub transfer and to possibly specify a K48 linkage during ubiquitylation. However, neither of these models were supported. It was then investigated whether the UBA domain binds K63 chains, to form K48/K63 mixed Ub chains. It was identified that Ube2K efficiently synthesised K48/K63 mixed Ub chains, with long K63-linked chains (of four Ub peptides or more) binding to the UBA domain more tightly and appearing to be more efficiently modified. This revealed a novel mechanism by which Ube2K can influence signalling within the cell. When recruited to K63 signalling scaffolds by the UBA domain, Ube2K can dynamically influence signalling by branching the K63 chain with degradative K48 linkages. While the downstream effects of this K48/K63 mixed chain modification will be difficult to characterise, this new method of signalling modulation by Ube2K may be the elusive link between Ube2K and disease.