Global Shifts in Protein Sumoylation in Response to Electrophile and Oxidative Stress

Abstract

Human small ubiquitin-like modifier (sumo) proteins include sumo-1 and the less studied, nearly identical sumo-2 and sumo-3 proteins. Whereas the structurally related ubiquitin molecule targets proteins for degradation, sumo provides a distinct, yet poorly understood regulatory signal. Protein sumoylation is sensitive to diverse cellular stresses, yet the targets of sumoylation in stress are unknown. We studied protein sumoylation in HEK293 cells exposed to hydrogen peroxide, alkylating agents, and the lipid oxidation-derived electrophile 4-hydroxynonenal, which is an ubiquitous product of lipid oxidation associated with oxidative stress. Confocal immunofluorescence microscopy indicated that in unstressed cells sumo-1 targeted nuclear proteins, whereas sumo-2/3 targeted proteins in both nuclei and cytoplasm. Western blot analyses revealed changes in sumo-1 and sumo-2/3 targeting patterns with stress. We used immunoaffinity chromatography to harvest sumo-associated proteins from HA-sumo-1- and HA-sumo-3-expressing HEK293 cells both before and after treatment with 4-hydroxynonenal. Multidimensional liquid chromatography-tandem mass spectrometry analyses identified 54 HA-sumo-1-associated proteins and 38 HA-sumo-3-associated proteins. Major protein targets included RNA binding and processing proteins, transcription factors, metabolic enzymes, and cytoskeletal regulators. Treatment with 4-hydroxynonenal caused a near-complete redistribution of sumo-1 and sumo-3 to different protein targets, which included chaperones, antioxidant, and DNA damage signaling proteins. A 10-15% overlap of sumo-1 and sumo-3 targets before and after stress suggests that sumo proteins target distinct protein groups. The results suggest that reactive electrophiles not only directly modify proteins but also lead to indirect changes in endogenous protein modifications that regulate protein functions.