Discovery and characterization of non-canonical E2 conjugating enzymes

Abstract

E2 conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the 20 attachment of ubiquitin to a substrate. This process, termed ubiquitylation is fundamental for 21 maintaining cellular homeostasis and impacts almost all cellular process. By interacting with 22 multiple E3 ligases, E2s direct the ubiquitylation landscape within the cell. Since its discovery, 23 ubiquitylation has been regarded as a post-translational modification that specifically targets lysine 24 side chains (canonical ubiquitylation). We used MALDI-TOF Mass Spectrometry to discover and 25 characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. 26 We employed protein modelling and prediction tools to identify the catalytic determinants that these 27 E2s use to interact with ubiquitin as well as their substrates.