Ubiquitination is one of the most abundant protein modifications in cellular signaling, controlling numerous cellular pathways such as transcription, translation, vesicle transport and apoptosis. Ubiquitin labels substrate proteins via a highly ordered multi-step enzymatic cascade, with specific differences in the length and topology of poly-Ub chains signalling a range of signalling outcomes or proteolytic degradation via the proteasome. Identification of E3 ubiquitin ligase substrates is key to defining their biological function and understanding their roles in disease. However, even with advances in proteomics and in vitro assays, substrate identification remains a significant challenge.
We have developed an integrated approach to define the E3 ligase substrates, combining genetic mouse and cellular models with orthogonal proteomics approaches to identify interacting proteins and ubiquitylation targets and high-throughput BiFC to validate substrates in situ. We have now applied this approach to identify substrates of a number of E3 ligases, including UBR5 - which has been implicated in cancer progression and chemoresistance.
UBR5-interacting proteins were isolated using GFP-Trap affinity purification followed by nanoLC-MS/MS identification and label-free quantitation. We identified ~300 UBR5-interacting proteins, many dependent on the UBR and HECT functional domains for binding. Analysis of differentially ubiquitylated proteins in breast cancer cells depleted of UBR5 by shRNA identified ~1100 proteins with altered ubiquitylation when UBR5 is depleted. Intriguingly, a large number of Ubiquitin-Proteasome System components have altered ubiquitylation in UBR5-depleted cells, suggesting co-ordinate regulation of this system.
Integration of the UBR5 interactome and ubiquitome generates a set of 25 high-confidence ligase substrates. We have validated a number of these using BiFC and have mapped the role of novel UBR5 functional domains in mediating these interactions using disease-specific mutants. These orthogonal but complementary approaches are providing interesting new insights into the function UBR5, suggesting a role in mediating crosstalk between DNA damage response and transcriptional regulation.