Abstract
The NRF2 transcription factor regulates a major environmental and oxidative stress response. NRF2 is itself negatively regulated by KEAP1, the adaptor of a Cul3-ubiquitin ligase complex that marks NRF2 for proteasomal degradation by ubiquitination. Electrophilic compounds activate NRF2 primarily by inhibiting KEAP1-dependent NRF2 degradation, through alkylation of specific cysteines. We have examined the impact on KEAP1 of reactive oxygen and nitrogen species, which are also NRF2 inducers. We found that in untreated cells, a fraction of KEAP1 carried a long range disulfide linking Cys(226) and Cys(613). Exposing cells to hydrogen peroxide, to the nitric oxide donor spermine NONOate, to hypochlorous acid, or to S-nitrosocysteine further increased this disulfide and promoted formation of a disulfide linking two KEAP1 molecules via Cys(151). None of these oxidants, except S-nitrocysteine, caused KEAP1 S-nitrosylation. A cysteine mutant preventing KEAP1 intermolecular disulfide formation also prevented NRF2 stabilization in response to oxidants, whereas those preventing intramolecular disulfide formation were functionally silent. Further, simultaneously inactivating the thioredoxin and glutathione pathways led both to major constitutive KEAP1 oxidation and NRF2 stabilization. We propose that KEAP1 intermolecular disulfide formation via Cys(151) underlies the activation of NRF2 by reactive oxygen and nitrogen species.
Highlights
Primarily sensed at the level of KEAP1, causing NRF2 protein stabilization [7,8,9] by inhibiting KEAP1-mediated NRF2 ubiquitination [10, 11]
Oxidation of KEAP1 in Cells Exposed to H2O2—We evaluated whether H2O2 could oxidize KEAP1 at Cys residues by analyzing the redox state of HA-KEAP1 ectopically expressed in HeLa cells
In this study we have questioned whether regulation of KEAP1, the inhibitor of the electrophilic and oxidative stress response regulator NRF2, involves disulfide bond formation, a post-translational control mechanism often used by oxidative stress regulators [39]
Summary
Cell Culture and Reagents—HeLa cells were grown at 37 °C, 5% CO2, in Dulbecco’s modified Eagle’s medium containing 1 g/liter of glucose, 110 mg/ml sodium pyruvate, 4 mM GlutaMAX (Invitrogen), complemented with 10% fetal calf serum (Sigma). ShRNA sequences were designed with the DSIR program that operates an exact similarity search algorithm for potential off target detection [34] These shRNA were expressed under the control of the H1 promoter from the Epstein-Barr virus-based replicative plasmid that contains an oriP, the EBNA open reading frame, and a hygromycin B selection cassette [35]. Protein Pulldown Assays—For pulling down His tag-containing polypeptides, the cells (106/sample) were washed in ice-cold phosphate-buffered saline containing NEM (40 mM) and lysed in precipitation buffer (0.1 M Tris-HCl, pH 8, 1% Nonidet P-40, 2% glycerol, 0.3 M NaCl, 0.2 mM phenylmethylsulfonyl fluoride, Roche-Complete mini protease Inhibitor mixture, 40 mM NEM).
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