E2 ubiquitin-conjugating enzymes (E2s) are critical components of the ubiquitination cascade, playing the central role of handing over the activated ubiquitin from an E1 ubiquitin-activating enzyme to an E3 ubiquitin ligase that attaches ubiquitin to cellular substrates. E2s are often regarded as mere middlemen, carrying ubiquitin to E3s. However, they are much more important as they often dictate the chain specificity that an E3 executes. While the terminal enzymatic reaction of ubiquitination typically results in the modification of a substrate recognised by an E3 ligase, E2s themselves are targets of ubiquitination events.
In our study, we investigate an internal ubiquitination site that is conserved in 25% of human E2s and is adjacent to their catalytic cysteine. We show that this site is autoubiquitinated in vitro, independent of an E3 ligase. Furthermore, this ubiquitination site can modulate the activity of E2s, affecting the rate at which an interacting E3 ligase can build ubiquitin chains. We show this effect both in vitro and in cells which leads to modulating the cellular response to inflammation stimuli whose initiation is dependent on ubiquitination.