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. 2010 Mar 12;285(11):8463-71.
doi: 10.1074/jbc.M109.051714. Epub 2010 Jan 8.

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

Simon Fourquet  1 , Raphaël Guerois, Denis Biard, Michel B Toledano
Affiliations

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

Simon Fourquet et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
KEAP1 becomes oxidized in cells treated with oxidants. A–C, HeLa cells transfected with pcDNA-HA-KEAP1 were exposed to H2O2 (0.2 mm) for the indicated time (A) or for 5 min in the presence of different concentrations of H2O2 as indicated (B), or to SpNO (1 mm) for the indicated time (C). The cells were lysed in the presence of NEM (40 mm) to block free sulfhydryls (see “Experimental Procedures”). The lysates were separated by nonreducing (upper panels) or reducing (lower panels) SDS-PAGE, and KEAP1 was revealed by Western blot using an anti-HA antibody. The arrows indicate the oxidized slow (OxIR1 and OxIR2) and fast (OxIM) and reduced (Red) KEAP1 species. D, HeLa cells expressing pcDNA-HA-KEAP1were either left untreated or were exposed to H2O2 (0.2 mm for 5 min), to CysNO (0.5 mm) for 10 min, or to SpNO (2 mm) for 45 min. KEAP1 S-nitrosylation was evaluated by the biotin-switch method as described under “Experimental Procedures.” KEAP1 was revealed by anti-HA Western blot of the streptavidin eluate (upper panel) and as control of the corresponding whole cell extracts (lower panel). βME, β-mercaptoethanol.
FIGURE 2.
FIGURE 2.
Identification of oxidized Keap1 cysteine residues. A, domain organization of the human KEAP1 protein. Positions of the KEAP1 cysteine residues are mapped in red. B, HeLa cells transfected with pcDNA expressing wild type (wt) HA-KEAP1 or the cysteine substitution derivatives C151S, C226S, or C613S were left untreated or exposed to H2O2 (0.2 mm) for 5 min, as indicated. The lysates were processed as described for Fig. 1. The arrowed band denoted cov corresponds to a noninducible, redox-insensitive KEAP1 modification (see text).
FIGURE 3.
FIGURE 3.
KEAP1 forms disulfide-linked homodimers. HeLa cells transfected with pcDNA-HA-KEAP1 and pcDNA-Myc-His-KEAP1 as indicated, were exposed to H2O2 (0.2 mm) for 5 min. Whole cell lysates (WCE) or the eluate of lysates adsorbed onto a nickel column (Ni2+ p.d.) were separated by nonreducing (upper panel) or reducing (lower panel) SDS-PAGE. HA-KEAP1 and Myc-His-KEAP1 were then revealed by Western blot (WB) with anti-HA or anti-Myc antibodies, as indicated. The black arrows indicate the bands corresponding to the disulfide-based homodimers of HA-KEAP1 or Myc-His-KEAP1 (OxIR1 and OxIR2) and intramolecular disulfide forms of these proteins (OxIM). The red arrows indicate the bands corresponding to the disulfide-based HA-KEAP1-Myc-His-KEAP1 heterodimer. Lane M, molecular mass markers; βME, β-mercaptoethanol.
FIGURE 4.
FIGURE 4.
Oxidation of KEAP1 parallels NRF2 stabilization. HeLa cells transfected with pcDNA-Myc-His-KEAP1 or Cys-mutants derivatives (1 μg), pCI-HA-NRF2 (1.5 μg), and peYFP-N1 (0.3 μg), as indicated, were treated with H2O2 (A, 0.2 mm) for the indicated time or left untreated or treated with t-BHQ (80 μm) or SpNO (2 mm) as indicated (B). The lysates were processed as for Fig. 1. KEAP1, NRF2, and YFP were revealed by Western blot using anti-Myc, anti-NRF2, or anti-YFP antibodies, as indicated. NRF2 abundance was quantified and normalized by the value of the YFP signal (lower panel). βME, β-mercaptoethanol.
FIGURE 5.
FIGURE 5.
The effect of inactivating thiol-reductase pathways on KEAP1 oxidation. A, TRxR1 protein abundance in HeLa cells stably expressing a control (Ctrl) nonhairpin RNA (RNA control) or shRNAs targeting the TRxR1 message (sh1, sh2, and sh3), evaluated by Western blot using an anti-TrxR1 antibody. B, HeLa cells stably expressing sh2 were transfected with pcDNA3-Myc-His-mKEAP1 (1 μg) and pCI-HA-mNRF2 (1.5 μg). The cells were incubated or not with buthionine sulfoximine (BSO, 0.1 mm) for 24 h as indicated and were then treated or not with H2O2 (0.1 mm) for the indicated time. The redox state of KEAP1 was evaluated by redox Western blot as in Fig. 1, and the abundance of NRF2 by Western blot was determined.
FIGURE 6.
FIGURE 6.
Prediction structure of the KEAP1 BTB domain indicating the location of Cys151. The KEAP1 BTB domain structure (delimitations 44–183) was modeled using the Bach1 BTB domain structure (Protein Data Bank code 2Z8H) as template (51). These two BTB sequences share 30% identity with no sequence insertions surrounding KEAP1 Cys151 within the alignment, thus ensuring the reliability of the structural model in this region. In this model the Cys151 side chain (magenta stick) appears buried by four surrounding positively charged amino acids (labeled in blue; Lys131, Arg135, Lys150, and His154). These four residues side chains can be modeled under different conformers, but irrespective of them, the accessibility of Cys151 never exceeds 15%, emphasizing the buried character of this residue. The position of Cys151 with regard to Cul3 was modeled using the structural similarity between Skp1 and BTB domains and the structure of the Skp1-Cul1 complex (Protein Data Bank code 1LDK) (49). These models show that Cys151 is remote from both Cul3 and the BTB interface.
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