Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants

Purification of Keap1.

Initial attempts to purify Keap1 were hampered because the overexpressed protein formed insoluble inclusion bodies in LB medium at 37°C. However, growth at 15°C in an inorganic medium with glucose increased the amount of soluble protein substantially. Keap1 was purified easily from crude extracts by precipitation when frozen in the presence of 100–300 mM ammonium sulfate without DTT. The precipitated protein was readily soluble in Tris-Cl buffer, pH 8.0, containing DTT. Below pH 7.0, Keap1 precipitated even in the presence of DTT. Reducing SDS/PAGE of the purified protein showed essentially one band with a molecular weight of ≈64,000 (calculated 69,552) (Fig. 4a). In contrast, in nonreducing SDS/PAGE, Keap1 migrated faster, possibly because of formation of disulfide bridges (26). The N-terminal sequence by Edman degradation was MQPEPKLSGAP-, in perfect agreement with the sequence of Keap1 deduced from its cDNA (11). Electrophoresis of purified Keap1 labeled with [³H]Dex-mes on reducing SDS/PAGE gel gave a single sharp band of radioactivity coinciding with the protein (Fig. 4b).

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Figure 4

(a) SDS/PAGE of purified Keap1. (b) SDS/PAGE of Keap1 after labeling with [³H]Dex-mes: 5 μg of Keap1 was incubated with 50 pmol of [³H]Dex-mes for 60 min at 25°C, and unbound Dex-mes was removed by gel filtration. Labeled Keap1 was separated on a 10% gel and stained with Coomassie blue. The gel lane was cut into 2-mm slices, each digested with 30% H₂O₂, and radioactivity was counted.

Competition of Phase 2 Inducers with [³H]Dex-mes Binding to Keap1.

Several inducers of different chemical types and potencies competed with the binding of [³H]Dex-mes to Keap1 (Fig. 5). The approximate concentrations of each competitor required to reduce binding by 50% were: phenylarsine oxide (3.5 μM); 1-nitro-2-cyclohexene (5.8 μM); 1-chloro-2,4-dinitrobenzene (26.5 μM); 2-cyclohexenone (110 μM); and α-methylene-γ-butyrolactone (250 μM). The order of potencies of these compounds as competitors for binding of Dex-mes to Keap1 parallels their potencies for induction of NQO1 in intact cells (4–6). Thus, phenylarsine oxide and 1-nitrocyclohexene are the most potent competitors and inducers, strongly supporting that Dex-mes reacts with Keap1 at sites relevant to the inducer mechanism.

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Figure 5

Inhibition of [³H]Dex-mes binding to Keap1. Each reaction mixture contained 10 μl (3.5 μg, 50 pmol) of Keap1 and 80 μl of 25 mM Tris-Cl/2 mM EDTA/0.01% Tween 20, pH 8.0. A range of concentrations of competitors (10 μl in 50% acetonitrile/50% water) was added, and the mixture was incubated for 30 min at 25°C. Then 10 μl of [³H]Dex-mes (27 pmol) was added, and incubation was continued for an additional 60 min. [³H]Dex-mes bound to protein was separated from unbound steroid by gel filtration, and radioactivity in the protein fraction was determined.

Stoichiometry and Kinetics of Reaction of Thiol Groups of Keap1 with Dipyridyl Disulfides.

2,2′-Dipyridyl disulfide and 4,4′-dipyridyl disulfide were chosen as spectroscopic titration reagents for the thiol groups of Keap1, because their reductions by thiols are unidirectional and relatively pH-insensitive. The resulting thiones have distinct UV absorptions and are produced in stoichiometric amounts (21). Thus, sequential titration of Keap1 with multiple 1-equivalent (per cysteine residue) additions of each dipyridyl disulfide revealed that at pH 7.0 two thiol groups were titrated at each of the first three steps, whereas only a single thiol group was titrated at the next step (Fig. 6). The reaction rates for the first three steps, calculated with GRAFIT software, showed pseudo-first order kinetics, with observed rate (k) values of 0.3607, 0.2097, and 0.1306 min⁻¹ for the reaction with 2,2′-dipyridyl disulfide and 0.9858, 0.4533, and 0.2019 min⁻¹ for reaction with 4,4′-dipyridyl disulfide, respectively. Standard errors were <5%. Rates of reaction of the two disulfides with 2-mercaptoethanol under identical conditions showed similar differences. Reaction of Keap1 with a 1,000-fold excess of 4,4′-dipyridyl disulfide was complete in <1 min, and 21–24.5 equivalents (depending on the method of protein determination) of pyridinethione were formed, suggesting that most of the cysteines were titrated and not initially involved in disulfide linkages in DTT-treated Keap1. Assuming that these measurements reflect sequential reactivities of cysteine residues, the experiments demonstrate that (i) a single cysteine residue in Keap1 is most reactive, and there is a hierarchy of less reactive cysteines, and (ii) the addition of a single equivalent of dipyridyl disulfide leads to formation of two molecules of the pyridinethione. This finding suggests that once the Keap1-pyridyl mixed disulfide is formed, the linkage is attacked rapidly by another cysteine thiolate ion of the protein to form either an intra- or intermolecular protein disulfide. The 25 cysteines of Keap1 provide many opportunities for sulfhydryl–disulfide interchange.

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Figure 6

Reaction of thiol groups of Keap1 with 4,4′-dipyridyl disulfide. To a solution of 0.5 μM Keap1 in 0.3 ml of 5 mM potassium phosphate buffer, pH 7.0, at 25°C, was added 0.5 μM (1 equivalent) 4,4′-dipyridyl disulfide in four sequential steps at times indicated with the arrows. Immediately after mixing, the increase in absorbance at 325 nm was monitored at 0.1-s intervals.

Spectroscopic Evidence for Reactivity of Thiol Groups of Keap1 with Sulforaphane, 2-HBA, and 4-HBA.

The chemistry of the reaction of the thiol groups of Keap1 with inducers was established by spectroscopic observation with two types of inducers: an isothiocyanate and two Michael reaction acceptors. Isothiocyanates such as sulforaphane (λ_max 240 nm, ɛ 950 M⁻¹⋅cm⁻¹) react with mercaptans to give rise to dithiocarbamates with characteristic absorptions near 270 nm (ɛ ≈ 10,000 M⁻¹⋅cm⁻¹). When the potent inducer sulforaphane (CD value 0.2 μM) was added to Keap1, absorbance at 270 nm was increased. Although overlapping the aromatic residues of the protein, this absorption was sufficiently intense to be quantified easily (Fig. 7a), and the initial reaction velocity was proportional to the concentration of sulforaphane (Fig. 7b). No reaction was observed if Keap1 was boiled or treated with excess of the potent inducer 2-HBA.

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Figure 7

Reaction of Keap1 with sulforaphane. (a) Absorption spectra of 50 μM sulforaphane (SF), the reaction mixture of 50 μM sulforaphane after it has been incubated with 0.7 μM Keap1 for 90 min in 5 mM potassium phosphate buffer, pH 8.0, at 25°C against a Keap1 blank (SF+Keap1), and their difference spectrum (DF). (b) Plot of the reaction rates of sulforaphane with 0.7 μM Keap1 under the same reaction conditions as a function of the concentration of sulforaphane. (c and d) Absorption spectrum kinetics of the reaction of 2-HBA with Keap1 in the absence (c) or presence (d) of 20 mM sulforaphane.

The favorable absorption characteristics of 2-HBA and 4-HBA were used next to measure the reactivity of thiol groups of Keap1 with these phase 2 enzyme inducers. Whereas 2-HBA is a powerful inducer (CD value 0.14 μM) and its absorbance at 370 nm (ɛ 16,000 M⁻¹⋅cm⁻¹ in this buffer) is bleached by the addition of thiols, 4-HBA is a much weaker inducer (CD value 14 μM) and absorbs maximally at 320 nm. We monitored the decrease in absorbance at 370 nm after the addition of Keap1 to 2-HBA (Fig. 7c). The rate of this reaction was reduced markedly if Keap1 was incubated first with sulforaphane (Fig. 7d). In contrast, when 4-HBA was used as a ligand under identical conditions, absorbance at 320 nm was not decreased, in agreement with its much lower reactivity with thiol reagents and its much weaker NQO1 inducer potency (9).

Dex-mes Binding to Cysteine Residues of Keap1.

Initially Keap1 (0.5 μM) was labeled extensively with [³H]Dex-mes (75 μM or 150-fold molar excess) at 25°C, pH 8.0, for 21 h. Measurements of the incorporated radioactivity showed that ≈23 cysteine residues had been labeled with the ligand. In contrast, a much smaller excess (10-fold) of [³H]Dex-mes and shorter incubation time (2 h) resulted in labeling of only 3–4 cysteines. Therefore, we then attempted to identify the most reactive cysteine residues of Keap1. The protein was incubated with a 33-fold excess of Dex-mes for 1 h at pH 8.0 and then treated with N-ethylmaleimide to alkylate the unreacted thiols. After tryptic digestion and peptide separation by reversed-phase HPLC, the resulting fractions were analyzed by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. All except three of the predicted tryptic peptides (i.e., 39, 51, and 53; Fig. 2) were recovered in the fractions. Most important, 18 of the 19 cysteine-containing tryptic peptides were observed. The following Dex-mes-labeled cysteine residues were identified in peptides that increased in mass by 374.19 (the mass added by the steroid; Table 1): C²⁵⁷ (peptides 22–23), C²⁷³ (peptides 25–28), C²⁸⁸ (peptides 25–28, 28–29, and 27–28), C²⁹⁷ (peptides 28–29), and C⁶¹³ (peptides 55–56). Peptides containing C²³, C³⁸, C⁷⁷, C¹⁵¹, C²²⁶, C²⁴¹, C²⁴⁹, C³¹⁹, C³⁹⁵, C⁴⁰⁶, C⁴³⁴, C⁴⁸⁹, C⁵¹³, and C⁵¹⁸ were alkylated by N-ethylmaleimide, thereby increasing the cysteine mass by 125.05. Peptides containing C¹⁷¹, C¹⁹⁶, C⁶²², and C⁶²⁴ were unmodified. Only cysteine-containing peptide 53 was not detected. The sequences and positions of Dex-mes-modified cysteines in peptides 22–23 and 27–28 were confirmed by liquid chromatography tandem mass spectrometry coupled with electrospray ionization and fragmentation by collision-induced dissociation. The predicted b (100%) and y (88%) ions of these peptides, including the Dex-mes-modified cysteine-containing ions, matched 71 and 77%, respectively, of the most intense masses in the fragmentation spectra. Fragmentation patterns of dexamethasone-modified peptides were more complex than those of unmodified peptides probably because of partial fragmentation of Dex-mes itself during ionization.

Binding of Keap1 and Neh2 Domain of Nrf2: Dissociation by Inducers.

When Keap1 was incubated with the Neh2 domain of Nrf2, native PAGE revealed a band migrating more slowly than Keap1; its formation depended on the DTT concentration and increased in intensity with the Neh2 concentration (Fig. 8 a and b). This band presumably is a complex between the two proteins. If solutions containing the Keap1–Neh2 complexes were exposed to increasing concentrations of sulforaphane, a concentration-dependent loss of this complex was observed (Fig. 8c). Similar experiments with 2-HBA and 4-HBA revealed that the ortho isomer, a 100-fold more potent inducer of NQO1 and more than 100-fold more reactive with glutathione (9) than the para isomer, also much more potently disrupted the complex between the two proteins (Fig. 8d). These experiments establish that the formation of complexes between Keap1 and Nrf2 and their disruption by inducers in vivo can be modeled in isolated systems with pure Keap1 and the Neh2 domain of Nrf2.

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Figure 8

Native gel electrophoresis showing complex formation between Keap1 (100 pmol) and the Neh2 domain (50 pmol) of Nrf2, and their dissociation. Concentration-dependent effects of DTT (a), Neh2 (b), sulforaphane (c), and 2-(ortho) and 4-(para) hydroxylated bis-(benzylidene)acetones (d) are shown. The binding reactions and native gel electrophoresis were carried out as described in Experimental Procedures. The 11.9-kDa Neh2 fragment migrates rapidly as a single band and is not shown in these gels.