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 from HEK293T cell lysates using GFP-Trap affinity purification following expression of a GFP-UBR5 fusion protein. Proteins were trypsin digested, followed by offline strong anion exchange pre-fractionation and C18 StageTip desalting. Tandem MS based identification was performed on C18 reverse-phase LC coupled to a LTQ Orbitrap Velos (LC-MS/MS) system. MaxQuant algorithms were used for label free quantitation and database searching. We observed good reproducibility over multiple replicates and identified 198 putative UBR5-interacting proteins. We have initiated validation of several of our proteomics hits, including histone H2B, using BiFC and reciprocal IP experiments. We hypothesise this novel UBR5-H2B interaction may be regulating the cross talk between DNA damage response and transcription.We are now extending these studies using high-throughput BiFC to further characterise putative interactions and ubiquitylation in situ, and investigating the role of various functional domains in UBR5 using disease-specific mutants. Identification of Ub ligase substrates will not only elucidate UBR5 function relevant to cancer but also identify novel molecular targets for cancer therapy.