Ubiquitinated proteome analysis by mass spectrometry. Effect of R505C mutation in FBXW7 E3 ligase on protein ubiquitination.

Abstract

Ubiquitination is a reversible post-translational modification that governs many critical processes in eukaryotes. In turn, defects in the ubiquitin system can lead to various diseases. Targeted strategies to correct aberrations in the ubiquitin pathway could provide new therapeutic opportunities. The specificity of ubiquitination is determined by the substrate preference of E3 ligases. Here we investigated R505C mutation in a substrate receptor subunit FBXW7 of E3 ligase in colon organoids. We identified ubiquitin sites and FBXW7 substrates using mass spectrometry with ubiquitin enrichment by a commercial antibody recognizing di-Gly remnant left after trypsin digestion. A number of experimentally-identified FBXW7 targets were cross-validated by an established degron prediction method. By compiling evidence from the transcriptome, proteome, phosphoproteome, ubiquitinome and cell-surface staining, we demonstrated that impaired degradation via FBXW7 is the mechanism behind elevated EGFR level, MAPK signalling, and EGF-independent proliferative survival. Collectively, these illustrate the utility of our ubi-site identification workflow and further implicate FBXW7 R505C mutation in highly-proliferative cancers.