Poster Presentation 26th Lorne Cancer Conference 2014

UBR5 (EDD) is a ~320kDa protein belonging to the HECT family of E3 ubiquitin (Ub) ligases. Altered expression and somatic mutations of UBR5 have been observed in numerous cancer types, and UBR5 expression modulates chemoresistance in ovarian cancer, likely through regulation of the DNA damage response. Identification of E3 ubiquitin ligase substrates is key to defining their biological function and understanding their role in disease. However, even with advances in proteomics and in vitro assays, substrate identification remains a significant challenge. To this end, we have developed an integrated approach combining LC/MS-MS proteomics and Bimolecular Fluorescence Complementation (BiFC) to define the substrates of UBR5 by identifying interacting proteins and ubiquitylation targets in situ.

UBR5 interacting proteins were isolated using GFP-Trap affinity purification followed by tandem MS based identification and label free quantitation. We identified 198 putative UBR5-interacting proteins with very good (>85%) reproducibility. Initial analysis of differentially ubiquitylated peptides following UBR5 depletion by shRNA in a breast cancer cell line using di-Gly affinity purification identified approximately 400 individual ubiquitlyation sites. We are now extending these studies using BiFC to characterize putative interactions and ubiquitylation in situ, and investigating the role of various functional domains in UBR5 using disease-specific mutants. One validated interaction between UBR5 and histone H2B is of particular interest in understanding potential crosstalk between DNA damage response and control of transcription.

These orthogonal but complementary approaches are providing interesting new insights into the function of E3 ligases in cellular signaling, with implications for understanding regulation of DNA damage response, metabolic reprogramming and other key pathways important in tumour cells.