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. 2009 Dec;76(6):1265-78.
doi: 10.1124/mol.109.058453. Epub 2009 Sep 28.

NRF2 cysteine residues are critical for oxidant/electrophile-sensing, Kelch-like ECH-associated protein-1-dependent ubiquitination-proteasomal degradation, and transcription activation

Xiaoqing He  1 , Qiang Ma
Affiliations

NRF2 cysteine residues are critical for oxidant/electrophile-sensing, Kelch-like ECH-associated protein-1-dependent ubiquitination-proteasomal degradation, and transcription activation

Xiaoqing He et al. Mol Pharmacol. 2009 Dec.

Abstract

Cells respond to oxidants and electrophiles by activating receptor/transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) to coordinate the induction of cytoprotective genes critical for defense against oxidative and other stresses. Activation involves blocking the ubiquitination-proteasomal degradation of Nrf2. Modification of cysteine thiol groups by inducers in the linker region of Kelch-like ECH-associated protein-1 (Keap1), which congregates Nrf2 into the Keap1/Cul3 E3 complex for ubiquitination, is important but not sufficient for activation of Nrf2. Here we show that evolutionarily conserved cysteine residues of Nrf2 are critical for Nrf2 regulation. FlAsH (an arsenic-based fluorophore) and phenylarsine oxide (PAO) potently induce Nrf2 target genes and bind to Nrf2 in vitro and in vivo. Binding is inhibited by prototypical inducers arsenic and tert-butylhydroquinone. PAO affinity pull-down and mutation of individual cysteine to alanine reveal that Cys235, Cys311, Cys316, Cys414, and Cys506 are critical for binding, and binding is modulated by intramolecular interactions. To corroborate the functions of cysteine residues, Nrf2 wild-type or mutants are expressed in Nrf2 knockout cells to reconstitute Nrf2 regulation. Nrf2 mutants have reduced t(1/2) that inversely correlates with increased binding to Keap1 and polyubiquitination of mutant proteins. It is remarkable that the mutants fail to respond to arsenic for Nrf2 activation and gene induction. Furthermore, mutations at Cys119, Cys235, and Cys506 impede binding of Nrf2 to endogenous antioxidant response element and to coactivator cAMP response element-binding protein-binding protein/p300. The findings demonstrate that Nrf2 cysteine residues critically regulate oxidant/electrophile sensing, repress Keap1-dependent ubiquitination-proteasomal degradation, and promote recruitment of coactivators, such that chemical sensing, receptor activation, and transcription activation are integrated at the receptor molecule.

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Figures

Fig. 1.
Fig. 1.
Cysteine residues in Nrf2 are conserved across species. A, cysteine residues and domain structure of Nrf2. B, conservation of Nrf2 cysteine residues across mouse, human, rat, and chicken. Numbers at the top represent cysteine positions in mouse Nrf2. Cysteine residues are boxed and shaded: rat Nrf2 has seven cysteine residues at the same positions as for mouse Nrf2; human Nrf2 has six cysteine residues at 119, 199, 242, 326, 422, and 515 positions; and chicken Nrf2 has four cysteine residues at 115, 181, 224, and 492.
Fig. 2.
Fig. 2.
Binding of FlAsH to purified Nrf2. A, structure of FlAsH. B, activation of Nrf2 by FlAsH. Hepa1c1c7 cells were treated with tBHQ (30 μM), As3+ (10 μM), and FlAsH (10 μM) for 5 h. Cell lysates were immunoblotted for Nrf2, HO1 and actin. C, purification of Nrf2. Mouse Nrf2 was expressed from pNrf2/ET28c in BL21(DE3) cells and purified using nickel beads. Proteins were fractionated in 10% SDS-PAGE and stained with Coomassie blue. D, competition for binding to Nrf2 among FlAsH, arsenic, and Bal. FlAsH (2 μM) was incubated with PBS, purified Nrf2 (10 μg), or purified Nrf2 preincubated for 30 min with As3+ (50 μM) or Bal (50 μM). Fluorescent intensity was measured using a fluorescence spectrophotometer with excitation at 508 nm.
Fig. 3.
Fig. 3.
Activation of Nrf2 by PAO. A, structure of PAO and 4-amino-phenylarsine oxide. B, activation of Nrf2. Hepa1c1c7 cells were treated with arsenic or PAO at concentrations indicated for 5 h. Cell lysates were immunoblotted for Nrf2 and actin. C, induction of Ho1 and Nqo1. Cells were treated with tBHQ (30 μM), PAO, or arsenic for 5 h. Total RNA was blotted for mRNA expression of Ho1, Nqo1, and Actin.
Fig. 4.
Fig. 4.
Binding of PAO to free thiol groups of Nrf2. A, binding of PAO to purified Keap1 or Nrf2. Purified Keap1 and Nrf2 were incubated with PAO for 2 h. The proteins were precipitated with trichloroacetic acid, and protein-free thiol groups were measured with the Ellman's reagent at λ412 nm. B, concentration-dependence. Keap1 or Nrf2 was incubated with increasing concentrations of PAO, and free cysteine thiol groups were measured. C, binding to endogenous Nrf2. Hepa1c1c7 cells were treated with tBHQ (30 μM), PAO (1 μM), or arsenic (10 μM) for 5 h. Nrf2 was immunoprecipitated with anti-Nrf2 antibody. The precipitate was treated with Ellman's reagent, and free thiol groups of Nrf2 were measured at λ412 nm. Data represent means and standard deviations from three samples. ∗, p < 0.05.
Fig. 5.
Fig. 5.
PAO pull-down of Nrf2 in vitro. A, pull-down of Nrf2 in vitro. Biotinylated Nrf2 was produced in vitro with TnT reticulocyte lysate and was pulled down by PAO beads. B, efficiency of PAO pull-down. Nrf2 in 1 or 10 μl of TnT lysate was pulled down by PAO; >90% of input Nrf2 was pulled down by PAO beads. C, elution by β-ME. Nrf2 in 10 μl of TnT lysate was precipitated by PAO beads. The pellet was incubated with β-ME from 0.01 to 1 M to elute Nrf2. D, elution by free PAO. Nrf2 was precipitated by PAO beads and was eluted from the beads by free PAO at concentrations from 0.01 to 100 mM. E, inhibition of PAO pull-down by tBHQ, PAO, and As3+ in vitro. Nrf2 in 10 μl of TnT lysate was incubated with MG132 (15 μM), tBHQ (30 μM), PAO (1 μM), or As3+ (10 μM) for 2 h, followed by incubation with PAO beads overnight. Precipitated Nrf2 was immunoblotted with anti-Nrf2 antibody.
Fig. 6.
Fig. 6.
PAO pull-down of Nrf2 in vivo. A, pull-down of endogenous Nrf2. Hepa1c1c7 cells were treated with MG132 (15 μM) for 2 h to increase the protein level of Nrf2. Cell lysate (1 mg) was precleaned with Affigel without PAO and then incubated with PAO beads at 4°C overnight. PAO pull-down precipitates and supernatants were immunoblotted for Nrf2 and Keap1. B, inhibition of PAO pull-down by tBHQ, PAO, and As3+ in vivo. Cells were treated with MG132 (15 μM), As3+ (1 or 10 μM), or tBHQ (30 μM) for 5 h. Cell lysate was blotted for Nrf2 or actin (top and middle), and PAO pull-down blotted for Nrf2 (bottom).
Fig. 7.
Fig. 7.
PAO pulldown of deletion mutants. A, N-terminal deletion of Nrf2. B, binding of PAO to deletion mutants. Nrf2 and deletion mutants were expressed in Cos7 cells and were pulled down by PAO beads. Top, immunoblotting of cell lysate with anti-HA antibody; bottom, immunoblotting of PAO pull-down with anti-HA. C, binding of PAO to cysteine mutants of Nrf2 ND2 fragment. D, binding of PAO to cysteine mutants of full-length Nrf2.
Fig. 8.
Fig. 8.
Effect of cysteine mutations on the half-life of Nrf2. A, characterization of immortalized Nrf2 KO cell line. Embryonic fibroblasts derived from Nrf2 WT and KO mice, respectively, were immortalized by expressing the SV40 T-antigen. The cells were treated with As3+ (10 μM) or MG132 (15 μM) for 5 h. Stabilization of Nrf2 protein was examined by immunoblotting with anti-Nrf2 antibody. B, half-life of cysteine point mutants. Nrf2 WT and cysteine point mutants were expressed in Nrf2 KO cells. The cells were treated with MG132 (15 μM) for 2 h, followed by CHX chase for 0, 30, 60, 90, and 120 min. Cell lysate was blotted for expression of Nrf2. Actin was blotted as loading control.
Fig. 9.
Fig. 9.
Cysteine mutants are degraded through the Keap1-dependent ubiquitin-proteasome pathway. A, stabilization of cysteine mutants by MG132. Nrf2 WT and cysteine point mutants were expressed in Nrf2 KO cells. Cells were treated with MG132 (15 μM, 2 h). Cytoplasmic and nuclear fractions were blotted for Nrf2 expression. B, binding of cysteine mutants with Keap1. V5-Keap1 and Nrf2 WT or Nrf2 cysteine mutant were coexpressed in Nrf2 KO cells. The cells were treated with vehicle or MG132 (15 μM) for 4 h. Cell lysate was blotted with anti-Nrf2 or anti-V5 (top and middle) or immunoprecipitated with anti-V5 followed by immunoblotting with anti-Nrf2 (bottom). C, quantification of coimmunoprecipitated Nrf2 in B.
Fig. 10.
Fig. 10.
Increased ubiquitination of cysteine mutants. A, ubiquitination of Nrf2 mutants. Nrf2 WT or cysteine mutants were coexpressed with c-myc-ubiquitin in Nrf2 KO cells. The cells were then treated with vehicle or MG132 (15 μM) for 4 h. Cell lysate was blotted with anti-Nrf2 to show Nrf2 protein expression (top) or immunoprecipitated with anti-Nrf2 and then immunoblotted with anti-ubiquitin. B, quantification of ubiquitinated Nrf2 in A.
Fig. 11.
Fig. 11.
Loss of arsenic-responsiveness in cysteine mutants. Nrf2 WT and cysteine mutants were expressed in Nrf2 KO cells. Cells were treated with As3+ (10 μM, 5 h). A, cytoplasmic and nuclear fractions were immunoblotted with anti-Nrf2 and anti-actin. B, induction of Nqo1 was examined by Northern blotting.
Fig. 12.
Fig. 12.
Effect of cysteine mutations on transcription activity of Nrf2. A, induction of Nqo1. Nrf2 WT and cysteine point mutants were expressed in Nrf2 KO cells. Induction of Nqo1 was examined by treating the cells with As3+ plus MG132 (15 μM) for 5 h. Northern blotting was performed for Nqo1, Nrf2, and Actin mRNA expressions, and immunoblotting was done for Nrf2 and actin protein expression. B, binding of cysteine mutants to endogenous ARE. Nrf2 and cysteine mutants were expressed in Nrf2 KO cells. After treatment with MG132 for 4 h, chromatin immunoprecipitation assay was performed. Nrf2-bound Nqo1 ARE was quantitated by real-time PCR. Data represent mean ± S.D. (n = 3). ∗, p > 0.05; ∗∗, p < 0.05; ∗∗∗, p < 0.001. C, coimmunoprecipitation of Nrf2 and mutants with endogenous CBP/p300. WT or cysteine mutants were expressed in KO cells treated with MG132. Cell lysate was immunoblotted with anti-CBP/p300 to show the protein expression (top) or immunoprecipitated with anti-Nrf2 and then blotted with anti-CBP/p300. Arrow, CBP/p300 protein band.
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