Characterization of Protein Sumoylation in Response to Alkylation Stress in HEK 293 Cells

Abstract

Stress conditions such as heat shock, UV, alkylating agents, and H2O2 have been shown to result in the modification of a variety of protein targets via the production of reactive electrophiles. These modifications can directly impact protein function or can alter posttranslational modifications, thus leading to a disruption of cellular regulatory processes. Recent studies have shown that stress-induced protein modifications can modulate posttranslational modification by the small ubiquitin related modifier (SUMO) family of proteins. Unlike ubiquitination, which primarily targets proteins for proteasomal degradation, sumoylation exerts a variety of effects including protein stabilization, subcellular localization, and the alteration of protein-protein interactions and transcriptional activity. To investigate the effects of alkylation and oxidative stress on sumoylation, HEK293 cells were treated with iodoacetamide, hydroquinone, benzoquinone, Texas Red C5 bromoacetamide, hydrogen peroxide, and 4-hydroxynonenal (HNE), a highly reactive product of lipid peroxidation associated with oxidative stress. Western blot analysis revealed that the agents tested resulted in concentration-dependent changes in the patterns of SUMO-1 and SUMO-2/3 protein conjugation. Localization studies using western blot analysis and confocal immunofluorescence microscopy demonstrated that SUMO-1 protein conjugates were located primarily in the nucleus, whereas SUMO-2/3 protein conjugates were more equally distributed between the nucleus and the cytoplasm. SUMO-associated proteins were harvested from vehicle- and HNE-treated non-transfected HEK293 cells using agarose conjugated anti-SUMO-1 antibodies or from HA-SUMO-1- and HA-SUMO-3-expressing HEK293 cells using immunoaffinity chromatography. Multidimensional liquid chromatography-tandem mass spectrometry analyses resulted in the identification of 54 HA-SUMO-1-associated proteins and 37 HA-SUMO-3-associated proteins in vehicle-treated cells and 21 HA-SUMO-1- and HA-SUMO-3-associated proteins in HNE treated cells. Additionally, 27 SUMO-1-associated proteins were identified in the HNE-treated non-transfected cells. The functional classes of proteins targeted included RNA binding and processing proteins, metabolic enzymes, cytoskeletal regulators, and chaperone proteins. HNE treatment resulted in a near complete redistribution of both SUMO-1 and SUMO-3 to different targets. There was a 15% overlap in SUMO-1 and SUMO-3 associated proteins in vehicle-treated cells and a 10% overlap in HNE-treated cells indicating that SUMO proteins target distinct protein groups. These results indicate that protein modifying reactive electrophiles can regulate protein functions through the indirect alteration of endogenous posttranslational modifications.