It has recently been demonstrated that ubiquitin can be attached to non-protein substrates. Ubiquitin may be added to the C6-hydroxyl group of glucose and the 3’-hydroxyl group of single-stranded DNA and RNA in vitro. Ubiquitination of lipids has also been shown in cellulo to play important roles in control of infection, xenophagy and endocytic trafficking. Ubiquitin can be attached to the outer membrane of invading Salmonella via the lipid A moiety of lipopolysaccharide (LPS) by the E3 ligase, RNF213. Furthermore, attachment of ubiquitin to phosphatidylethanolamine (PE) in endosomes and lysosomes suggests ubiquitination of lipids is likely far more widespread than currently understood. However, the breadth and significance of this new form of cellular signalling remains limited by a lack of tools to detect it.
The Komander group has developed a sensitive, mass spectrometry-based approach to identify non-proteinaceous ubiquitination events within cells and tissues. We are adapting this workflow to assess lipid ubiquitination in vitro and in cells. Using ubiquitinated phospholipids generated in vitro we could successfully detect ubiquitinated-PE via this approach. Now, we intend to apply this method to fully grasp the unexplored roles of lipid ubiquitination in cell intrinsic pathways.