Regulation of SUMO enzymes by oxidative stress

Sumoylation is an essential posttranslational mechanism, which regulates hundreds of proteins in all eukaryotic species. Modification requires ATP, a SUMO activating enzyme, a SUMO E2 conjugating enzyme, and usually one of several E3 ligases; isopeptidases ensure reversibility. Modification usually is a highly dynamic process that requires additional cues such as a specific time in the cell cycle, developmental stage, DNA damage, or heat shock. A number of years ago, we found that the reactive oxygen species hydrogen peroxide causes rapid disappearance of SUMO conjugates when added to tissue culture cells. This is due to disulfide bond formation between the catalytic cysteines of the SUMO E1 subunit Uba2 and the E2-conjugating enzyme Ubc9. Importantly, enzyme inactivation by disulfide bond formation was also observed upon induction of endogenous ROS production, i.e., the respiratory burst in macrophages. These findings add SUMO enzymes to the small list of specific direct effectors of H₂O₂ and implicate ROS as key regulators of the sumoylation pathway. To begin to understand the role of SUMO enzyme oxidation, we embarked on a search for redox insensitive Ubc9. Using random mutagenesis and in vitro sumoylation, we identified a single point mutant in the E2 conjugating enzyme Ubc9 that is as active as wt Ubc9, but insensitive to oxidation. Experiments with this mutant in mammalian tissue culture cells suggest that SUMO enzyme oxidation contributes to oxidative stress response.