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. 2009 Jan;29(2):493-502.
doi: 10.1128/MCB.01080-08. Epub 2008 Nov 10.

The antioxidant defense system Keap1-Nrf2 comprises a multiple sensing mechanism for responding to a wide range of chemical compounds

Makoto Kobayashi  1 , Li Li, Noriko Iwamoto, Yaeko Nakajima-Takagi, Hiroshi Kaneko, Yuko Nakayama, Masami Eguchi, Yoshiko Wada, Yoshito Kumagai, Masayuki Yamamoto
Affiliations

The antioxidant defense system Keap1-Nrf2 comprises a multiple sensing mechanism for responding to a wide range of chemical compounds

Makoto Kobayashi et al. Mol Cell Biol. 2009 Jan.

Abstract

Animals have evolved defense systems for surviving in a chemically diverse environment. Such systems should demonstrate plasticity, such as adaptive immunity, enabling a response to even unknown chemicals. The antioxidant transcription factor Nrf2 is activated in response to various electrophiles and induces cytoprotective enzymes that detoxify them. We report here the discovery of a multiple sensing mechanism for Nrf2 activation using zebrafish and 11 Nrf2-activating compounds. First, we showed that six of the compounds tested specifically target Cys-151 in Keap1, the ubiquitin ligase for Nrf2, while two compounds target Cys-273. Second, in addition to Nrf2 and Keap1, a third factor was deemed necessary for responding to three of the compounds. Finally, we isolated a zebrafish mutant defective in its response to seven compounds but not in response to the remaining four. These results led us to categorize Nrf2 activators into six classes and hypothesize that multiple sensing allows enhanced plasticity in the system.

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Figures

FIG. 1.
FIG. 1.
Induction of zebrafish gstp1 by a variety of Nrf2 activators. (A) Structural representation of Nrf2-activating compounds. (B) Induction of gstp1 expression in adult gills was analyzed by RT-PCR after 6 h of treatment with compounds. The expression of ef1α was used to standardize the amount of cDNA. (C) Experimental scheme for zebrafish Nrf2 gene knockdown. (D) Induction of gstp1 expression in 4-day-old larvae. Strong gstp1 induction was observed in larvae treated with all three compounds, especially in the gills (red arrowheads). It was greatly reduced when nrf2MO was injected at the embryonic stage (blue arrowheads).
FIG. 2.
FIG. 2.
Chemical-specific induction of gstp1 in zebrafish embryos. (A) Experimental scheme for gstp1 induction in zebrafish embryos. (B) Induction of gstp1 expression in 8-hour-old embryos. Nrf2 and Keap1 mRNAs were coinjected into embryos at the one-cell stage. After 2 h, embryos were treated with the compounds indicated for 6 h. The expression of gstp1 was analyzed by whole-mount in situ hybridization. (C) Structure of Nrf2-GFP fusion protein. The N-terminal half of Nrf2 protein and enhanced GFP (eGFP) protein were connected by two copies of simian virus 40 nuclear localizing signal (NLS). (D) Expression analysis of Nrf2-GFP fusion protein. Nrf2NTnGFP mRNA was injected with or without mRNA encoding Keap1 proteins into one-cell stage embryos. After 2 h, embryos were treated with the compounds indicated for 6 h, and GFP expression was analyzed.
FIG. 3.
FIG. 3.
Identification of a sensor cysteine for the K1b-type compounds. Induction of gstp1 was carried out as shown in Fig. 2A, and its expression was analyzed by RT-PCR. FLAG-tagged Keap1 proteins were used instead of nontagged proteins. The amount of mRNA was normalized to that of Keap1 protein with a comparable activity in repressing Nrf2-induced gstp1 expression in uninduced conditions, except for in panel G. The expression of ef1α was used to standardize the amount of cDNA. (A) Keap1a-Keap1b chimeric proteins used in the analysis. (B) DEM-induced expression of gstp1 in embryos overexpressing chimeric proteins. Note that gstp1 induction was observed in embryos overexpressing C1 but not C2 or C3 proteins. (C) Alignments of three cysteine residues and their adjacent amino acid residues in the BTB domains of various Keap1 proteins. (D) DEM-induced expression of gstp1 in embryos overexpressing Keap1bK124T and Keap1bC125S. (E) Electrophile-induced expression of gstp1 and γgcsh in embryos overexpressing mouse Keap1 and its C151S mutation. (F) Similarity between a Cys-to-Trp mutation and covalent binding of Nrf2-activating compounds to Cys. (G) DEM-induced expression of gstp1 in embryos overexpressing Keap1bC125W. The amount of Keap1 mRNA was standardized by the expression level of FLAG-tagged Keap1 protein analyzed by immunoblotting with anti-FLAG antibody.
FIG. 4.
FIG. 4.
Identification of sensor cysteines for the K1a/b-type compounds. (A) MS analysis of peptides from mouse Keap1 digested with trypsin after incubation with (right) or without (left) 15d-PGJ2. Keap1 protein (10 μg) was incubated at 25°C for 60 min in the absence (left) or presence (right) of 100 μM 15d-PGJ2 in a total volume of 10 μl containing 20 mM Tris-HCl (pH 8.5). (B) Modification sites in mouse Keap1 for Nrf2-activating compounds were determined by MS analysis. (C) Nrf2-induced expression of gstp1 in embryos overexpressing mKeap1C273S or mKeap1C288S. The amount of Keap1 mRNA was standardized by the expression level of FLAG-tagged Keap1 protein analyzed by immunoblotting using anti-FLAG antibody. (D) Electrophile-induced expression of gstp1 in embryos overexpressing mouse Keap1 and its C288S mutation.
FIG. 5.
FIG. 5.
Screen for zebrafish mutants with abnormal gstp1 expression. (A) Summary of zebrafish mutagenesis. (B) Phenotypic classes of mutations isolated. (C) Chemical-specific gstp1 induction in it275 larvae 4 days after fertilization. The strong gstp1 induction seen in the gills of wild-type fish (red arrowheads) treated with certain Nrf2-activating compounds was greatly reduced in it275 larvae (blue arrowheads).
FIG. 6.
FIG. 6.
Hypothetical models. (A) A cascade-based classification of Nrf2-activating compounds. Chemical compounds belonging to class 3 have not been identified. (B) A cysteine code hypothesis. Electrophilic compounds that target different sets of cysteines may produce distinct biological effects.
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