Pierre Thibault (Université de Montréal/IRIC), Chongyang Li (Université de Montréal/IRIC), Frédéric Lamoliatte (Université de Montréal/IRIC), Francis P. McManus (Université de Montréal/IRIC), Cédric Plutoni (Université de Montréal/IRIC), Cristina Mirela Pascariu (Université de Montréal/IRIC), Trent Nelson (Université de Montréal/IRIC), Ghizlane Maarifi (Université Paris-Descartes), Mounira K. Chelbi-Alix (Université Paris-Descartes), Gregory Emery (Université de Montréal/IRIC)
Immunoaffinity enrichment methods are presented for the study of protein ubiquitylation and SUMOylation. The sequential use of SUMO and ubiquitin remnant immunoaffinity purification enabled the dynamic profiling of more than 3000 SUMOylated and ubiquitylated proteins upon proteasome inhibition and revealed the unexpected regulation of deubiquitinase enzymes by ubiquitin-like modifiers and the SUMOylation of proteasome subunits, a modification necessary for the recruitment proteasomes to PML nuclear bodies. Several PIAS SUMO E3 ligases are overexpressed in various human malignancies, including prostate and lung cancers. To investigate the mechanism of action of PIAS E3 SUMO ligase, we used gene editing and quantitative SUMO proteomics to identify potential substrates of PIAS in a system-wide manner. These substrates were found to be involved in different cellular processes, including transcriptional regulation, DNA binding and cytoskeleton dynamics. Functional studies on Vimentin (VIM), a type III intermediate filament protein involved in cytoskeleton organization and cell motility, revealed that PIAS1 exerts its effects on cell migration and cell invasion through the SUMOylation of specific VIM C-terminus residues.
