74P Characterizing the beta-catenin interactome using inhibitor screens and novel interaction proteomics techniques

Wnt signalling is a critical developmental pathway and associated aberrant signaling drives oncogenesis including colorectal bone cancer. β-catenin, central protein forms range of protein-protein interactions(PPIs), fundamental to driving the transcription oncogenes inducing proliferation tumour formation. To further elucidate these interactions, we have sought i) identify inhibitors β-catenin PPIs ii) employ proximity dependent labelling technique (MiniTurbo) characterize novel PPIs. MiniTurbo-Mutant Biotin ligase BirA genetically fused upon addition Biotin, tagging induced proteins within 20nm in as quick 10 minutes enabling streptavidin pull down Mass spectrometry. CTNNB1 gene was successfully inserted into Miniturbo plasmid construct using SLICE cloning. DH5alpha bacteria were transformed with single colony selected Ampicillin. Following purification, it added Retroviral components for viral transfection production HEK293 cells. U2OS adenocarcinoma cells transduced puromycin. Validated western blot immunofluorescence. 24-hour application MSAB derivatives following induction Doxycycline. Streptavidin pulldown carried out samples subjected Spectrometry on-bead trypsin digestion. Proteomic analysis PPI. -MSAB influence levels target genes at transcriptional level. -Successful introduction selection miniturbo Western blots immunofluorescence verification. -Novel networks Beta-catenin interactions The results illustrate dynamic PPI network 2D cancer model how those are modulated presence small molecule therapeutic indications. Targeting directly Oncotherapy has been difficult very little success. Further characterization understanding interactome may pave an avenue addressing this challenge.