Structural basis of PROTAC-induced target ubiquitination mechanism - identification of BRD4 ubiquitination sites

Proteolysis Targeting Chimeras (PROTACs) hijack the ubiquitin proteasome system to ubiquitinate target proteins, targeting them for degradation by the proteasome. An understudied process that is essential to the PROTAC mechanism of action is ubiquitination of the target protein and ubiquitin (Ub) chain elongation. To elucidate the mechanism of PROTAC-induced ubiquitination of BRD4(BD2) ubiquitination sites on Brd4BD2 were mapped by mass spectrometry following an in vitro ubiquitination assay containing neddylated-CRL2VHL, Brd4BD2, MZ1, Uba1 and either UBE2R1 or UBE2D2. Time points were taken at 0 minutes, 10 minutes, 0.5 hours, 1 hour and 3 hours. Proteins were separated by SDS-page analysis. To visualize proteins the gel was stained with Coomassie blue and gel slices containing ubiquitin modified Brd4BD2 across timepoints and increasing molecular weight were excised from the gel and were analyzed by mass spectrometry. To identify sites of ubiquitination a search for GG-K peptides was performed in MaxQuant. Based on existing structures the modified lysine residues were all situated on the same face of BD2.