Activation of NRF2 by nitrosative agents and H2O2 involves KEAP1 disulfide formation

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.