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. 2016 Nov 28;36(24):3100–3112. doi: 10.1128/MCB.00389-16

Absolute Amounts and Status of the Nrf2-Keap1-Cul3 Complex within Cells

Tatsuro Iso a, Takafumi Suzuki a,✉, Liam Baird a, Masayuki Yamamoto a,b,✉
PMCID: PMC5126296  PMID: 27697860

Abstract

The transcription factor Nrf2 (NF-E2-related-factor 2) is essential for the oxidative and electrophilic stress responses. Keap1 (Kelch-like-ECH-associated-protein 1), an adaptor for a cullin-3 (Cul3)-based ubiquitin ligase, regulates Nrf2 activity through proteasomal degradation, and acts as a sensor for oxidative and electrophilic stresses. The Keap1-Cul3 complex is a critical regulator of the cellular Nrf2 level, and yet quantitative information regarding their endogenous intracellular concentrations in homeostatic conditions and during stress responses is unknown. We analyzed the absolute amounts of the Nrf2, Keap1, and Cul3 proteins in five murine cell lines by comparison with serial dilutions of purified recombinant protein standards in combination with quantitative immunoblot analyses. In the basal state, the amount of Nrf2 was maintained at lower levels than those of Keap1 and Cul3 proteins, whereas the electrophilic agent diethylmaleate dramatically increased Nrf2 to a level greater than that of Keap1 and Cul3, resulting in the accumulation of Nrf2 in the nucleus. In contrast, Keap1 and Cul3 did not display any changes in their abundance, subcellular localization, or interaction in response to electrophilic stimuli. Our results demonstrate that the regulation of the Nrf2 protein level during stress responses is mediated by the activity but not the composition of the Nrf2-Keap1-Cul3 complex.

INTRODUCTION

Nrf2 (NF-E2-related-factor 2) is a basic leucine-zipper type transcription factor that belongs to the CNC (cap'n'collar) family (1). Nrf2 dimerizes with one of the small Maf proteins (sMaf), and the Nrf2-sMaf heterodimer recognizes a specific DNA sequence, known as the antioxidant/electrophile response element (ARE/EpRE) (2, 3). Nrf2 target genes include those encoding enzymes that act in antioxidant and detoxification pathways, which together regulate cellular adaptation to oxidative and xenobiotic stresses (4). The Nrf2 knockout mouse clearly demonstrates that Nrf2 plays an important role in the response to oxidative and electrophilic stresses (5, 6).

One of the important characteristics of Nrf2 is the inducible nature of its function in response to oxidative and electrophilic stresses (6). Under homeostatic and stress-free conditions, cellular Nrf2 abundance is maintained at a very low level; however, exposure to electrophiles/oxidants or proteasome inhibitors increases Nrf2 abundance rapidly (7), indicating that Nrf2 is degraded by the proteasome and that this degradation is inhibited by electrophiles and reactive oxygen species. Therefore, a key regulatory characteristic of Nrf2 activity is the control of the abundance of the Nrf2 protein (7).

In the basal state, Keap1 (Kelch-like-ECH-associated-protein 1), an adaptor component of a Cul3 (cullin 3)-based ubiquitin E3 ligase complex, promotes ubiquitination and proteasomal degradation of Nrf2 (8–12). In Keap1-deficient cells in which the Nrf2 protein is highly accumulated, expression of Nrf2 target genes is strongly activated (13, 14), indicating that Keap1 targets Nrf2 for degradation via the ubiquitin-proteasome pathway. The abundance ratio of Keap1, Nrf2, and Cul3 must be critical for the regulation of Nrf2 activity. However, the absolute abundance of these molecules within cells has not been examined.

Localization of endogenous Keap1 and Cul3 is mainly observed in the cytoplasm, with a small amount present in the nucleus and endoplasmic reticulum (ER) of mouse liver cells (15). The development of a reliable antibody for the detection of endogenous Keap1 clearly demonstrates that oxidative and electrophilic stresses provoke nuclear accumulation of Nrf2 without altering the cytoplasmic localization of Keap1 (15). It has been proposed that oxidative and electrophilic stresses inhibit Keap1-based E3 ubiquitin ligase activity and reduce Nrf2 ubiquitination, which leads to the stabilization and nuclear accumulation of de novo-synthesized Nrf2 (16). This idea allows us to assume that overflowed Nrf2 escapes from Keap1 and translocates to the nucleus. However, to date no study has tried to examine the cytoplasmic concentrations of Keap1 and Nrf2 in the basal and induced states.

The stoichiometry of Keap1 and Nrf2 within the Keap1-Nrf2 complex is thought to be 2:1 (17, 18). A Keap1 homodimer binds to a single Nrf2 protein via a high-affinity ETGE motif and low-affinity DLG motif. The two-site recognition of Nrf2 by the Keap1 dimer is essential for ubiquitination of Nrf2 (17, 18). In contrast, there have been conflicting reports regarding the stoichiometry of Keap1 and Cul3 (18, 19). While one study reports that one Cul3 protein binds to a homodimer of Keap1 (18), another report shows that two Cul3 proteins bind to the Keap1 homodimer (19). Cys151 of Keap1 is required for the cellular response to typical Nrf2-inducing electrophiles (20–22). Cys151 is located in the BTB domain of Keap1, which is responsible for the Keap1 interaction with Cul3. Several reports have shown that modification of Cys151 inhibits the Keap1-Cul3 interaction and prevents ubiquitination of Nrf2 (12, 23–25). However, other reports disagree with this model (26, 27).

In this study, we have calculated the amounts of Nrf2, Keap1, and Cul3 proteins in murine cell lines by means of comparison to serial dilutions of recombinant protein standards in combination with quantitative immunoblot analysis. The results demonstrate that the regulation of the Nrf2 protein level during stress responses is mediated by the activity but not the composition of the Nrf2-Keap1-Cul3 complex.

MATERIALS AND METHODS

Chemical reagents.

Diethylmaleate (DEM), disodium hydrogenarsenate [Na2HAsO4(V)], sodium metaarsenite [NaAsO2(III)], cadmium chloride (CdCl2), and zinc chloride (ZnCl2) were purchased from Wako Pure Chemicals. Dimethyl sulfoxide (DMSO), 3H-1,2-dithiole-3-thione (D3T), sulforaphane, tert-butylhydroquinone (tBHQ), 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), 1,2-naphthoquinone (1,2-NQ), andrographlide, ebselen, flavone, and t-chalcone (trans-chalcone) were purchased from Sigma-Aldrich. Prostaglandin A2, diallyltrisulfide, carnosol, and isoliquilitigenin were purchased from Cayman Chemicals. Dimethylfumarate (DMF), 2-phenylethyl isothiocyanate, and curcumin were purchased from Cayman Chemicals. Chlorgenic acid, oltiplaz, and MG132 were purchased from Peptide Institute, Inc. (LKT Laboratories). 4-Hydroxynonenal and quercetin were purchased from Santa Cruz Biotechnology. Caffeic acid phenethyl ester, (±)-S-nitroso-N-acetylpenicillamine (SNAP), and xanthohumol were purchased from MP Biomedicals, Biomol, and Tokiwa Phytochemical, respectively.

Expression constructs.

Mouse Nrf2 cDNA was cloned into the pET15b vector (Novagen). Mouse Keap1 cDNA was cloned into the pET21a vector (Novagen). 6×His-TEV-tagged Keap1 was cloned into the pCold III vector (TaKaRa Bio) and used for the pulldown assay. Both the mouse Cul3-His and glutathione S-transferase (GST)–Roc1 were cloned into a pET-Duet-1 vector (Novagen). The N-terminal domain (amino acids 1 to 384, I342R/L346D) of mouse Cul3 (Cul3NTD) was cloned into the pET101 vector (Invitrogen).

Recombinant proteins.

6×His-tagged proteins of Nrf2, Keap1, and Cul3NTD were expressed in bacteria and purified as described previously (18, 19, 28, 29). All of the proteins were purified using His-Trap FF crude (GE Healthcare) and were further purified with MonoQ or Superdex 200 column chromatography (GE Healthcare). The Cul3-His/GST-Roc1 complex was coexpressed and sequentially copurified using His-trap FF crude and GSTrap FF (GE Healthcare). All of the proteins were near homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amounts of purified proteins were determined by a comparison with a bovine serum albumin standard.

Cell culture.

RAW 264.7 and 3LL cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics. Murine embryonic fibroblasts (MEFs) were prepared from E13.5 embryos and spontaneously immortalized. MEF and Hepa-1c1c7 (Hepa-1) cells were maintained in Dulbecco modified Eagle medium (DMEM) with 10% FBS and supplemented with antibiotics. mIMCD3 cells were maintained in DMEM plus Ham's F-12 medium supplemented with 10% FBS and antibiotics.

Immunoblot analysis.

Whole-cell or fractionated extracts were prepared in a lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0.1% SDS, protease inhibitor cocktail [Roche Diagnostic], and 1 μM phenylmethylsulfonyl fluoride) and subjected to immunoblot analysis with anti-Nrf2 (30), anti-Keap1 (15), anti-Cul3 (BD Bioscience; catalog no. 611848), anti-MEK1/2 (Cell Signaling Technology; catalog no. 9122), anti-CD45 (Abcam; catalog no. 10558), anti-histone H3 (Cell Signaling Technology; catalog no. 26500S), Calreticulin (BD Bioscience; catalog no. 612136), and antivimentin (Cell Signaling Technology; catalog no. 5741). For quantitative immunoblot analysis, the cells were counted before harvesting and lysed in the lysis buffer. Lysates from the equivalent number of cells and serial dilutions of protein standards containing Nrf2, Keap1, or Cul3 recombinant proteins were subjected to SDS-PAGE and immunoblot analysis. The chemiluminescent blots were imaged with a ChemiDox XRS imager (Bio-Rad), and the intensities of the bands were quantified by using Quantity One software (Bio-Rad). A standard curve was generated by serial dilutions of the recombinant proteins, and only the bands that lay within the linear range were used in the calculations.

Subcellular fractionation.

Subcellular fractionation experiments were performed using the ProteoExtract subcellular proteome extraction kit (Calbiochem, catalog no. 539790) according to the manufacturer's instructions, with 5 × 106 cells per sample. For Triton X-100 fractionation, the cells were lysed using 1% Triton X-100 on ice for 30 min and then centrifuged for 15 min at maximum speed. The supernatant and the remaining pellet constituted the Triton X-100 soluble and insoluble fractions, respectively.

Analytical ultracentrifugation analysis.

Sedimentation was carried out with a Beckman XL-A analytical ultracentrifuge, using a two-channel, Epon centerpiece. For stoichiometry analysis of the Keap1-Cul3 complex, 6×His-Keap1 and 6×His-Cul3NTD were mixed at six different concentrations: 6:0, 6:1.5, 6:3, 6:6, 6:12, and 0:6 μM. For Nrf2 inducer-dependent complex formation analysis, Keap1 was mixed or not mixed with CDDO-Im or DEM. After 1 h of incubation at 4°C, Cul3NTD was added, with the final concentrations of Keap1 and Cul3NTD being 6 and 6 μM, respectively. For stoichiometry analysis of the Keap1-Nrf2 complex, 6×His-Keap1 and 6×His-Nrf2 were mixed at six different concentrations: 6:0, 6:1.5, 6:3, 6:6, 6:12, and 0:6 μM. For Nrf2 inducer-dependent complex formation analysis, Keap1 was mixed or not mixed with CDDO-Im or DEM. After 1 h of incubation at 4°C, Nrf2 was added, with the final concentrations of Keap1 and Nrf2 being 6 and 3 μM, respectively. Samples were run at 42,000 rpm, with scans taken every 5 min at 280 nm. All scans were performed at 20°C. The sedimentation coefficient distribution [c(s)] was plotted in the program SEDFIT (31). The buffer density and viscosity were calculated from the buffer composition as 1.0083 g/ml and 0.010474 P by using the program SEDNTERP (32). A value of 0.73 ml/g was used for the partial specific volume (33). The buffer conditions were 50 mM Na-HEPES, 150 mM NaCl, and 1 mM TCEP at pH 7.8.

Pulldown analysis.

6×His-Keap1 proteins were digested with TurboTEV protease (MobiTec), and both the His tags and the protease were removed by using Ni-nitrilotriacetic acid (NTA)-agarose (Qiagen). The purified Keap1 protein (1 μΜ) was mixed with Nrf2-inducing chemicals (10 μΜ) or DMSO vehicle in washing buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 10 mM glutathione [GSH] at pH 8.0). After incubation for 30 min at 20°C, the mixture was mixed with purified 6×His-Cul3NTD (1 μΜ) in a final 50-μl reaction volume. After incubation for an additional 30 min at 20°C, the mixture was incubated with 20 μl of Ni-NTA-magnetic agarose beads (Qiagen) for 45 min at 20°C, washed two times with the above-described washing buffer, and eluted with elution buffer (20 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, and 10 mM GSH at pH 8.0). Bound proteins were analyzed by SDS-PAGE, followed by staining with Oriole fluorescent gel stain (Bio-Rad).

RESULTS

Estimation of the absolute amounts of Nrf2, Keap1, and Cul3 proteins within cells.

To clarify how abundantly Nrf2, Keap1, and Cul3 exist within cells, we decided to measure the absolute amount of the proteins in cells by using quantitative immunoblot analyses and to compare these levels to serial dilutions of recombinant protein standards (Fig. 1A). To this end, recombinant proteins of mouse Nrf2, Keap1, and Cul3 were expressed in bacteria and purified (Fig. 1B to D). 6×His-tagged Nrf2 and Keap1 proteins were expressed in bacteria and purified by Ni2+ affinity column and gel filtration chromatography (Fig. 1B and C). 6×His-tagged Cul3 protein was coexpressed with GST-tagged Roc1 (also called Rbx1) to obtain significant yields of purified protein. The Cul3-His/GST-Roc1 complex was purified sequentially using Ni2+ and GST affinity columns (Fig. 1D). In the present study, different reactivities between the recombinant protein and endogenous protein due to epitope modification were ignored.

FIG 1.

FIG 1

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Absolute amounts of Nrf2, Keap1, and Cul3 proteins in cells. (A) Scheme for the measurement of absolute amounts of endogenous Nrf2, Keap1, and Cul3 in cells. Recombinant proteins of Nrf2, Keap1, and Cul3 were expressed in bacteria and purified. The amounts of the proteins were determined by serial dilutions of recombinant protein standards in combination with quantitative immunoblot analysis. (B to D) Purification of recombinant proteins for 6×His-tagged Nrf2 (B), Keap1 (C), and Cul3 (D). (B and C) 6×His-tagged Nrf2 and Keap1 proteins were expressed in bacteria and purified by Ni2+ affinity column chromatography and gel filtration chromatography. Collected fractions were indicated with bottom bars. (D) 6×His-tagged Cul3 was coexpressed with GST-tagged Roc1 and purified sequentially by Ni2+ and GST affinity column chromatography. M, molecular marker; L, lysate; FT, flowthrough; W, wash; E, elution; I, insoluble fraction of lysate; S, soluble fraction of lysate. The molecular mass standards (in kilodaltons) are shown on the left.

Since many studies have revealed critical roles that the Keap1-Nrf2 system plays in macrophages (5, 34–36), we adopted the murine macrophage cell line RAW 264.7 as an experimental system. Consistent with previous studies (7, 16), the Nrf2 protein level was increased by treatment with DEM, a well-known electrophilic Nrf2 inducer which modifies reactive cysteine residues of Keap1 (Fig. 2A), and by treatment with MG132, a proteasome inhibitor (Fig. 2B). The levels of Keap1 and Cul3 proteins were not changed significantly by treatment with DEM or MG132. The absolute numbers of Nrf2, Keap1, and Cul3 molecules were calculated from the densitometric traces of the immunoblot results (Fig. 2C).

FIG 2.

FIG 2

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Absolute amounts of Nrf2, Keap1, and Cul3 proteins in RAW 264.7 cells. (A and B) Immunoblot analysis of Nrf2, Keap1, and Cul3 protein in RAW 264.7 cells treated or not treated with 100 μM DEM (A) or 10 μM MG132 (B) for 3 h and their recombinant proteins. (C) Graphical representation of the calculated molecule numbers of Nrf2, Keap1, and Cul3 protein in RAW 264.7 cells treated or not treated with 100 μM DEM or 10 μM MG132 for 3 h. Data are means ± the standard deviations (n = 3 or 4).

Unlike the cases for several electrophiles, such as tert-butylhydroquinone (tBHQ) (37), DEM treatment did not increase the high-molecular-mass form of Keap1 (∼140 kDa) (Fig. 3A). Therefore, the major band of Keap1 (∼70 kDa) was used for the measurement. Standard curves were generated using a serial dilution of the recombinant proteins, and the amounts of cellular protein in RAW 264.7 cells were calculated using the standard curves (Fig. 3B). In the basal state in RAW 264.7 cells, the total amount of Nrf2 protein was 49,000 molecules/cell, which increased 7- and 11-fold by treatment with 100 μM DEM and 10 μM MG132 for 3 h, respectively. The higher accumulation of Nrf2 by MG132 compared to DEM is likely due to the inhibition of proteasomal degradation of Nrf2 regulated by other mechanisms, such as Cul1/β-TrCP (38) and Cul2/Hrd1 (39), in addition to Cul3/Keap1.

FIG 3.

FIG 3

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Calculation of Nrf2, Keap1, and Cul3 protein amounts in RAW 264.7 cells. (A) Full-scanned image of immunoblot analysis of Keap1 in RAW 264.7 cells, treated or not treated with 100 μM DEM for 3 h, and the recombinant proteins. Note that the major band of Keap1 (∼70 kDa) was used for the calculation. HMW, high molecular weight. The molecular mass standards (in kilodaltons) are shown on the left. (B) Standard curves for the calculation of the amounts of Nrf2, Keap1, and Cul3 protein in RAW 264.7 cells treated or not treated with 100 μM DEM or 10 μM MG132 for 3 h. The calculated molecular amounts are shown in Fig. 2C. Standard curves were generated using serially diluted recombinant proteins (circles) and determined using linear regression through the origin. The R2 value is shown. Triangles indicate untreated RAW 264.7 cells, and squares indicate RAW 264.7 cells treated with 100 μM DEM or 10 μM MG132.

The amounts of Keap1 and Cul3 proteins in RAW 264.7 cells were 174,000 and 170,000 molecules/cell, respectively (Fig. 2C). Neither protein was significantly affected by treatment with DEM or MG132, implying that the stability of both Keap1 and Cul3 is not regulated by a proteasome-dependent pathway. These results indicate that in the basal state, the Nrf2 protein is maintained at a lower level compared to the Keap1 and Cul3 proteins, while in the induced state, the amount of Nrf2 protein reached a higher level than that of Keap1 and Cul3.

In addition to RAW 264.7 cells, we also examined Nrf2, Keap1, and Cul3 protein levels in other murine cell lines, i.e., MEF, lung cancer (3LL), hepatoma (Hepa-1), and inner medullary collecting duct (mIMCD3) cell lines (Fig. 4). As with RAW 264.7 cells, standard curves were generated, and the amounts of cellular proteins in each cell line were calculated using the standard curves (Fig. 5). Although there were some variations in the level of each protein in the basal state, the results from all of the cell lines consistently demonstrated that treatment with 100 μM DEM for 3 h increased Nrf2 but did not upregulate Keap1 or Cul3 protein levels.

FIG 4.

FIG 4

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Absolute amounts of Nrf2, Keap1, and Cul3 proteins in five cell lines treated with DEM. (A to D) Immunoblot analysis of Nrf2, Keap1, and Cul3 proteins in MEF (A), 3LL (B), Hepa-1 (C), and mIMCD3 (D) cells treated with DEM. (E) Number of Nrf2 molecules in cells treated or not treated with 100 μM DEM for 3 h. (F) Number of Keap1 molecules in cells treated or not treated with 100 μM DEM for 3 h. (G) Number of Cul3 molecules in cells treated or not treated with 100 μM DEM for 3 h.

FIG 5.

FIG 5

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Standard curves for the calculation of Nrf2, Keap1, and Cul3 protein amounts in MEF, 3LL, Hepa-1, and mIMCD3 cells treated or not treated with 100 μM DEM for 3 h. The calculated molecular amounts are shown in Fig. 4E to G. Standard curves were generated using serially diluted recombinant proteins (circles) and determined using linear regression through the origin. The R2 values are shown. Triangles indicate untreated cells, and squares indicate cells treated with 100 μM DEM.

Subcellular fractionation of Nrf2, Keap1, and Cul3 in RAW 264.7 cells.

It has been shown by means of sucrose gradient methods that Keap1 is mainly present in the cytoplasm of mouse liver cells (15). To verify in more detail the distributions of Nrf2, Keap1, and Cul3, we performed Triton X-100 fractionation of RAW 264.7 cells. In response to DEM, Nrf2 mainly increased in a detergent-insoluble fraction containing the nucleus and cytoskeleton (Fig. 6). In contrast, Keap1 is mainly present in the detergent-soluble fraction, which contained the cytoplasm, plasma membrane, and organelles (Fig. 6). Of note, DEM treatment slightly increased Keap1 in the detergent-insoluble fraction. We surmise that this may be due to electrophile-thiol modification of Keap1 that enhances Keap1 degradation through the autophagy pathway (40). Cul3 was also predominantly found in the detergent-soluble fraction.

FIG 6.

FIG 6

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Subcellular distribution of Nrf2, Keap1, and Cul3 proteins in RAW 264.7 cells treated or not treated with 100 μM DEM as determined by 1% Triton X-100 fractionation. (A) Immunoblot analysis of 1% Triton X-100-fractionated RAW 264.7 cells treated or not treated with 100 μM DEM for 3 h. (B) Graphical representation of the calculated molecule numbers of soluble or insoluble Nrf2, Keap1, and Cul3 proteins in RAW 264.7 cells treated or not treated with 100 μM DEM for 3 h.

To further examine the distribution of Nrf2, Keap1, and Cul3 within cells, subcellular fractionation experiments were performed using the ProteoExtract subcellular proteome extraction system. The results of the fractionation are shown in Fig. 7A. To further confirm the fractionation quality, we examined all four fractions using the fraction markers MEK (cytoplasm), calreticulin (endoplasmic reticulum [ER]), histone H3 (nucleus), and vimentin (cytoskeleton). Since histone H3 was detected in fraction 3 (Fr 3), as well as fraction 4 (Fr 4), but vimentin was highly accumulated in Fr 4, we concluded that Fr 3 harbored the nuclear components, whereas Fr 4 contained nuclear components, including heterochromatin, and the cytoskeleton. In contrast, MEK and calreticulin were highly recovered in fraction 1 (Fr 1) and fraction 2 (Fr 2), respectively, indicating that Fr 1 corresponded to the cytoplasm and Fr 2 corresponded to the organelles.

FIG 7.

FIG 7

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Subcellular fractionation of RAW 264.7 cells treated or not treated with DEM for 3 h as determined by the ProteoExtract subcellular proteome extraction system. (A) Immunoblot analysis of RAW 264.7 cells treated or not treated with 100 μM DEM for 3 h. (B and C) Graphical representation of the calculated concentrations of Nrf2, Keap1, and Cul3 proteins in the cytoplasm/organelles (B) or nuclei (C) of RAW 264.7 cells treated or not treated with 100 μM DEM. Data are the means ± the standard deviations (n = 3 or 4).

In response to DEM treatment, Nrf2 protein was slightly increased in Fr 1 and 2 (cytoplasm and organelles, respectively) and highly accumulated in Fr 3 and 4 (nucleus and/or cytoskeleton). On the contrary, Keap1 and Cul3 localized in both Fr 1 (cytoplasm) and Fr 2 (organelles). These results are consistent with those of the sucrose density gradient centrifugation experiments (15), which show that a small portion (14%) of Keap1 is also present in the ER. The localizations of Keap1 and Cul3 in the cytoplasm/organelle were not significantly affected by DEM treatment.

Subcellular concentration of Nrf2, Keap1, and Cul3 in RAW 264.7 cells.

Based on the calculated amounts of total Nrf2, Keap1, and Cul3 proteins in RAW 264.7 cells, the concentrations in the cytoplasm/organelles or nuclei were calculated using an estimate of the cell volume to be 460 femtoliters, and a nucleus/cytoplasm ratio of 0.54 for mouse peritoneal macrophages (41, 42). In the cytoplasm/organelles of RAW 264.7 cells, Nrf2 was present at a low level in the basal state (149 nM) and increased up to 619 nM in the DEM-induced state. Keap1 and Cul3 proteins were present at 1,060 and 941 nM in the cytoplasm/organelles, respectively, and this was not affected by DEM treatment (Fig. 7B). In the nucleus, Nrf2 was present at 278 nM in the basal state, but DEM treatment upregulated the Nrf2 protein level up to 2,733 nM (Fig. 7C). The concentrations of Keap1 and Cul3 in the nuclei were significantly lower than those of the cytoplasm/organelles and again were not affected by DEM treatment.

Stoichiometry of Keap1 and Cul3 within the Keap1-Cul3 complex.

Although the Keap1-Cul3 complex is thought to be a major regulator of Nrf2 protein abundance, there have been conflicting reports on the stoichiometry and regulation of Keap1-Cul3 activity (19, 29). To examine Keap1-Cul3 stoichiometry using analytical ultracentrifugation, we purified proteins of full-length Keap1 and the Cul3 N terminus (Cul3NTD), which is the domain responsible for the interaction with Keap1 (19) (Fig. 8A). The molecular masses of a Keap1 monomer and a Cul3NTD monomer are estimated to be approximately 70 and 45 kDa, respectively. As shown from the data in Fig. 8B, when Keap1 alone was applied, a single peak of Keap1 was observed at 5.4S, which corresponds to a molecular mass of ∼140 kDa. This means that Keap1 forms a stable homodimer, which is consistent with previous observations (15, 17). When Keap1 and Cul3NTD were mixed in a 4:1 or 2:1 molecular ratio, a new peak in between 6.0S and 7.0S appeared, in addition to the peak for the Keap1 homodimer, which corresponds to a Keap1-Cul3NTD complex in a 2:1 or 2:2 molecular ratio (Fig. 8B). When Keap1 and Cul3NTD were mixed in 1:1 molecular ratio, the Keap1 homodimer peak disappeared and a single peak of the Keap1-Cul3NTD complex appeared that showed a slightly larger S (sedimentation coefficient) value and higher S distribution than those observed for the mixture of 4:1 and 2:1 molecular ratios (Fig. 8B). When Keap1 (6 μΜ) and a twice-larger amount of Cul3NTD (12 μΜ) were mixed, a peak with an even larger S value and with higher S distribution peak was observed, in addition to a peak at 2.8S (corresponding to ∼40 kDa) that represented an excess of free Cul3NTD. The emergence of the excess Cul3NTD at the 1:2 molecular ratio of Keap1 and Cul3, and the disappearance of the free Keap1 homodimer at a 1:1 molecular ratio supports the notion that two molecules of Cul3 bind to a Keap1 homodimer. Thus, these results conclusively demonstrate a 2:2 binding stoichiometry in which two molecules of Cul3 bind to one Keap1 homodimer.

FIG 8.

FIG 8

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Stoichiometry of Keap1 and Cul3 within the Keap1-Cul3 complex. (A) Purification of recombinant proteins for N terminus of Cul3. 6×His-tagged Cul3NTD protein was expressed in bacteria and purified by Ni2+ affinity column. M, molecular marker; L, lysate; FT, flowthrough; W, wash; E, elution; I, insoluble fraction of lysate; S, soluble fraction of lysate. The molecular mass standards (in kilodaltons) are shown on the left. (B) Sedimentation coefficient distribution of Keap1 and Cul3NTD proteins mixed in different ratios as shown in the figure.

Effect of Nrf2-inducing chemicals on Keap1-Cul3 interaction.

It has previously been shown that the interaction between Keap1 and Cul3 depends on the presence of Cys151, since Nrf2-inducing chemicals that interact with Cys151 inhibit the coimmunoprecipitation of Keap1 and Cul3. These chemical inducers include tert-butylhydroquinone (tBHQ), sulforaphane (12), eicosapentanoic acid (24), and N-iodoacetyl-N-biotinylhexylenediaime (IAB) (25). However, another study has shown that Keap1-Cul3 does not dissociate upon exposure to 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) and sulforaphane (27). Similarly, a live-cell imaging analysis showed no dissociation of the Keap1-Cul3 complex by CDDO, sulforaphane, sulfoxythiocarbamate alkyne (STCA), or hydrogen peroxide (26). Since we have verified that Cys151 of Keap1 is the functional sensor for DEM (21, 22), we decided to examine the effect of DEM on the interaction between Keap1 and Cul3 in solution using sedimentation velocity experiments.

As shown in Fig. 9A and B, a Keap1-Cul3NTD complex was observed when Keap1 (6 μΜ) and Cul3NTD (6 μΜ) were mixed and applied for analytical ultracentrifugation. Importantly, the addition of 6 or 18 μΜ CDDO-Im clearly reduced the Keap1-Cul3NTD complex peak and increased the peaks of free Keap1 and Cul3NTD (Fig. 9A), indicating that CDDO-Im disrupts the interaction between Keap1 and Cul3. In contrast, the addition of DEM did not affect the Keap1-Cul3NTD complex (Fig. 9B). In addition to the sedimentation velocity experiment, we also carried out His tag pulldown experiments using purified 6×His-tagged Cul3NTD and Keap1 proteins with or without Nrf2 inducing chemicals (Fig. 9C). With the exception of CDDO-Im, none of the Nrf2-inducing chemicals affected the interaction between Keap1 and Cul3 (Fig. 9C), despite the fact that D3T, DEM, DMF, SNAP, sulforaphane, and tBHQ have all been shown as Cys151-interacting inducers (17, 20–22).

FIG 9.

FIG 9

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Effect of Nrf2-inducing chemicals on the Keap1-Cul3 interaction. (A and B) Sedimentation coefficient distribution of His-Keap1 (6 μM) and His-Cul3NTD (6 μM) proteins mixed or not mixed with 6 or 18 μM CDDO-Im (A) or 6 or 18 μM DEM (B). (C) His tag pulldown assay using 6×His-tagged Cul3NTD (1 μM) and tag-free Keap1 (1 μM) preincubated with or without various Nrf2-inducing chemicals (10 μM). The pulled-down complex was eluted by imidazole, subjected to SDS-PAGE, and visualized with fluorescent staining. (D) His tag pulldown assay using 6×His-tagged Cul3NTD (1 μM) and tag-free Keap1WT, Keap1C151S, or Keap1C226S (1 μM) preincubated with CDDO-Im (0, 1, 2, 4, or 8 μM).

It seemed quite unique that only CDDO-Im reduces the interaction between Keap1 and Cul3NTD, and therefore we verified this observation utilizing an alternative approach. To this end, we introduced a serine mutation into Cys151 of Keap1 and also into Cys226 as a control and examined the effect of CDDO-Im on the Keap1-Cul3 interaction. Pulldown experiments using the His tag of Cul3 showed that pulldown of Keap1 with the Cys151 mutation was not inhibited by CDDO-Im, while, in contrast, pulldown of wild-type Keap1, as well as Keap1 with the Cys226 mutation, was effectively inhibited by CDDO-Im (Fig. 9D). In this regard, it is interesting that cocrystal analysis of the Keap1 BTB domain with CDDO has shown that CDDO can modify the Cys151 residue (23). These results strongly support our contention that modification of Cys151 by CDDO-Im disrupts the Keap1-Cul3 interaction. Our results also suggest that unlike the case for CDDO-Im many Nrf2-inducing chemicals activate Nrf2 signaling without dissociating Keap1 from Cul3.

Stoichiometry of Keap1 and Nrf2 within the Keap1-Nrf2 complex.

Although the stoichiometry of Keap1 and Nrf2 within the Keap1-Nrf2 complex in a 2:1 molecular ratio has been shown by isothermal calorimetry analysis (iTC) and nuclear magnetic resonance (NMR) titration (18), we decided to further verify the stoichiometry of Keap1 and Nrf2 within the Keap1-Nrf2 complex using an analytical ultracentrifugation method. Although previous studies utilized the Keap1 DC (double glycine repeat and C-terminal) domain, which is responsible for Nrf2 binding, in this study we decided to use full-length recombinant proteins to elucidate the assembly of the Keap1 and Nrf2 complex.

The molecular masses of both Keap1 and Nrf2 are both estimated to be ∼70 kDa. As shown in Fig. 10A, when Nrf2 alone was applied, a single peak of Nrf2 was observed at 4.4S, which corresponds to a molecular mass of ∼70 kDa. When Keap1 alone was applied, as shown in Fig. 8B, a single peak of Keap1 was observed at 5.4S, which corresponds to a Keap1 homodimer (∼140 kDa). Note that the Nrf2 signal is weaker than the Keap1 signal although the same amounts of protein were applied. This is likely because the UV sensitivity of Nrf2 is lower than that of Keap1 due to the fact that Nrf2 and Keap1 contain different numbers of tryptophan (2 and 8, respectively) and tyrosine (10 and 27, respectively) residues.

FIG 10.

FIG 10

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Stoichiometry of Keap1 and Nrf2 within the Keap1-Nrf2 complex. (A) Sedimentation coefficient distribution of Keap1 and Nrf2 proteins mixed in different ratios as shown in the figure. (B and C) Effect of Nrf2-inducing chemicals on Keap1-Nrf2 interaction. The sedimentation coefficient distributions of His-Keap1 (6 μM) and His-Nrf2 (3 μM) proteins mixed or not mixed with 6 or 18 μM CDDO-Im (B) or 6 or 18 μM DEM (C) were determined.

When Keap1 and Nrf2 were mixed in a 4:1 molecular ratio, bimodal peaks between 8S and 10S were newly observed (Fig. 10A). The major peak at 8S corresponds to a molecular mass of ∼200 kDa, indicating that most of the Keap1-Nrf2 complex is in a 2:1 molecular ratio of Keap1 to Nrf2. Another minor peak at 10S corresponds to a molecular mass of ∼280 kDa, indicating that some of the Keap1-Nrf2 complex is in 2:2 molecular ratio. Importantly, when Keap1 and Nrf2 were mixed in a 2:1 molecular ratio, the Keap1 homodimer peak disappeared, confirming that the Keap1-Nrf2 complex is in a 2:1 molecular ratio of Keap1 to Nrf2. When Keap1 and Nrf2 were mixed in 1:1 or 1:2 molecular ratio, the peak at 4S appeared, which corresponds to free Nrf2. These observations are consistent with the previous studies which found that the Keap1-Nrf2 complex is mainly in a 2:1 molecular ratio.

Effect of Nrf2-inducing chemicals on the Keap1-Nrf2 interaction.

Utilizing an immunoprecipitation approach, it has previously been shown that Keap1-Nrf2 binding is not affected by electrophilic exposure (16). To verify this notion, we examined the effect of Nrf2-inducing chemicals on the Keap1-Nrf2 interaction utilizing analytical ultracentrifugation analysis. As shown in Fig. 10B and C, a Keap1-Nrf2 complex peak was observed when Keap1 (6 μΜ) and Nrf2 (3 μΜ) were mixed and applied for the analytical ultracentrifugation. The addition of CDDO-Im to the complex did not induce the appearance of free Keap1 (5.4S) or Nrf2 (4.4S), although it did cause a slight decrease in the 8S peak, corresponding to the Keap1-Nrf2 complex at 2:1 molecular ratio, and an increase in the 15S peak, corresponding to the aggregated complex (Fig. 10B). The addition of DEM did not affect the Keap1-Nrf2 complex (Fig. 10C). These results support the notion that Nrf2-inducing chemicals activate the Nrf2 pathway without affecting the stoichiometry of the Keap1-Nrf2 complex.

DISCUSSION

In this study, we determined the absolute abundance of Nrf2, Keap1, and Cul3 proteins within cells, utilizing five different cell lines. As summarized in Fig. 11, our quantitative analyses revealed that in the basal state Nrf2 is maintained at a level significantly lower than that of Keap1 and Cul3, so that a portion of Keap1 functions as a floodgate to trap and ubiquitinate Nrf2. In the induced state, the cytoplasmic and nuclear levels of Nrf2 increase significantly and exceed the levels of Keap1 and Cul3 proteins. Upon challenge with Nrf2 inducers, Keap1 loses its ubiquitin ligase activity (and floodgate function) due to the cysteine-thiol modification, which allows newly synthesized Nrf2 to pass through the gate and translocate into the nucleus (15). Similarly, the Nrf2 protein level is drastically increased within cells in response to proteasome inhibition (7). In contrast, we found in this study that the abundance of Keap1 and Cul3 proteins does not change upon challenge with electrophilic stimuli or proteasome inhibitors. We also examined the characteristics of the Keap1-Cul3 interaction. The stoichiometry of the Keap1-Cul3 complex is two Cul3 monomers bound to one Keap1 homodimer (2:2). Many of the Cys151-targeting electrophilic inducers provoke loss-of-Keap1 function by promoting conformational changes in Keap1 but not the dissociation of the Nrf2-Keap1-Cul3 complex. Taken together, these results support the notion that the Nrf2 protein level in homeostatic conditions, and during stress responses, is regulated by the activity but not the composition of the Keap1-Cul3 complex.

FIG 11.

FIG 11

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Molecular dynamics of the Nrf2-Keap1-Cul3 complex in cells. Under basal conditions, a portion of the Keap1-Cul3 ubiquitin ligase complex acts as a floodgate and degrades Nrf2 by the proteasome system. In response to oxidative and electrophilic stimuli, Nrf2 massively accumulates in the nucleus, whereas the stimuli do not affect the abundance and subcellular localization of Keap1 and Cul3, as well as the Keap1 interaction with Cul3 and Nrf2. Accumulated Nrf2 dimerizes with one of the small Maf proteins (sMaf), and the Nrf2-sMaf heterodimer recognizes ARE (antioxidant response element) and activates expression of its target genes.

We found here that the total number of Nrf2 proteins in the basal state is 49,000 to 190,000 molecules per cell. The total number of Nrf2 proteins increases to approximately 330,000 to 710,000 molecules per cell upon treatment with electrophilic inducers. Since it has been reported that most transcription factors in animal cells are expressed at a level between 10,000 and 300,000 molecules per cell (43), the number of Nrf2 proteins in the induced state greatly exceeds those of normal transcription factors. For example, the total number of p53 proteins in MCF7 cells is estimated to be 15,000 and 67,000 molecules per cell under basal and DNA damage conditions, respectively (44). Thus, although Nrf2 is present at an approximately 5-fold-greater level than p53 in the basal state, Nrf2 under oxidative and electrophilic stress conditions is induced to a much greater degree (∼13-fold more) than that of p53 under DNA damage induction.

We found that the abundance of Keap1 and Cul3 proteins does not change upon challenge with electrophilic stimuli, although there are some variations in the total number of Keap1 and Cul3 proteins among the five cell lines that were analyzed (numbers of Keap1 and Cul3 proteins ranges between 50,000 to 300,000 and 70,000 to 540,000 molecules per cell, respectively). In contrast, the amount of Mdm2, a component of the E3 ubiquitin ligase which targets p53 for degradation, is 81,000 molecules per cell under basal conditions and is induced to 167,000 molecules per cell in response to genotoxic agent treatment (44). This induction of Mdm2 is known as fast-feedback regulation of p53 (45). These observations indicate that, unlike the Mdm2-p53 pathway, the Keap1 and Cul3 proteins are not regulated by Nrf2 or electrophilic stimuli, at least in the short term. In contrast, available data suggest that the level of Keap1 protein may be downregulated under conditions of long-term stress. It has been reported that the half-life of Keap1 in HepG2 cells under normal conditions is 12.7 h but that electrophiles accelerate the degradation of Keap1 (40). Therefore, in the long term, the degradation of electrophile-modified Keap1 by autophagy is enhanced, and Keap1 function can be recovered by de novo synthesis of Keap1.

Quantitative and fractionation analysis in the present study revealed that ∼1 μM Keap1 is present in the cytoplasm of RAW 264.7 cells. It has been reported that potent Nrf2-inducing chemicals such as CDDO derivatives are effective in the nanomolar range (46). This means that the concentration of potent inducers is more than 10 times lower than the cytoplasmic concentration of Keap1, even assuming that the inducers are able to freely diffuse through the plasma membrane and are not eliminated by antioxidants, such as glutathione. This suggests that only a portion of Keap1 is inactivated upon the challenge of Nrf2 inducers and that the inactivation of a subset of the cellular Keap1 pool is sufficient for the stabilization and induction of Nrf2.

We have determined the stoichiometry of the Keap1-Cul3 complex by means of analytical centrifugation, which shows that two Cul3 monomers bind to one Keap1 homodimer or two Keap1 monomers (2:2). This result is consistent with reports that show two Cul3 molecules bind to one homodimer of KLHL11 and KLHL3, both of which are BTB-Kelch family members (19, 47), and SPOP, a MATH-BTB family member (48). This observation suggests that the 2:2 stoichiometry of the Keap1-Cul3 complex follows the conserved association pattern of Cul3 and BTB-containing family-based E3 ubiquitin ligase complexes.

Nrf2 activation by DEM, a representative Nrf2-inducing chemical, has been shown to require Cys151 of Keap1 (20–22). It has been postulated that bulky modification of Cys151 by Nrf2-inducing chemicals disrupts the Keap1-Cul3 interaction, since the substitution mutation of the Cys151 residue to tryptophan, which has the largest partial molar volume, with the aim of mimicking the modification of Cys151 by electrophilic molecules, decreases the association between Keap1 and Cul3 (49). However, we found that the addition of DEM did not affect the association between Keap1 and Cul3 in the sedimentation velocity experiment. In addition, pulldown experiments showed that the majority of Nrf2 inducers, including Cys151-preferable compounds (D3T, DMF, sulforaphane, and tBHQ), did not affect the interaction between Keap1 and Cul3. One exception to this observation is CDDO-Im, since modification of Cys151 by CDDO-Im disrupts the interaction of Keap1 with Cul3. This special effect of CDDO-Im on Keap1-Cul3 association is consistent with the cocrystal analysis of CDDO and the BTB domain of Keap1, showing that modification of Cys151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1 (23). Overall, our data suggest that, unlike CDDO-Im, most Nrf2-inducing chemicals activate Nrf2 signaling without dissociating Keap1 from Cul3. Even though Keap1 Cys151 senses many Nrf2-inducing chemicals, the mechanism leading to Nrf2 accumulation may be distinct among the different inducing compounds.

This study unequivocally verified that DEM treatment does not affect the abundance or localization of Keap1. Since conformational changes in Keap1 caused by inducer chemicals have been observed using a hydrophobicity probe (50), we surmise that cysteine modification elicits structural alterations in Keap1 that do not bring about the dissociation of Cul3 but affect the complex status of Keap1 and Cul3, which consequently prevents Nrf2 ubiquitination. There remains the possibility that modification of Cys151 in the BTB domain affects the orientation angle of association between Keap1 and Cul3, resulting in a change in the distance from ubiquitin to the target lysine residues in the Neh2 domain of Nrf2.

In summary, we have determined here the absolute molecule number and subcellular distribution of Nrf2, Keap1, and Cul3 in the basal and induced states. In response to oxidative and electrophilic stimuli, Nrf2 massively accumulates in the nucleus, whereas the stimuli do not affect the abundance and subcellular localization of Keap1 and Cul3 or the Nrf2-Keap1-Cul3 interaction. Delineation of the molecular basis of this pathway is a critical step toward better understanding stress response mechanisms in humans.

ACKNOWLEDGMENTS

We thank Eri Kobayashi for discussion and advice and the Biomedical Research Core of Tohoku University Graduate School of Medicine for technical support.

This study was supported in part by Grants-in-Aid for Creative Scientific Research and Scientific Research from the Japan Society for the Promotion of Science (JSPS; grants 24249015 and 26111002 to M.Y. and grants 25112502 and 26111010 to T.S.), CREST (chronic inflammation) from AMED, the NAITO Foundation, and the Takeda Foundation.

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