The regulation mechanisms of AhR by molecular chaperone complex

The regulation mechanisms of AhR by molecular chaperone complex Abstract The AhR, so called the dioxin receptor, is a member of the nuclear receptor superfamily. The ligand-free AhR forms a cytosolic protein complex with the molecular chaperone HSP90, co-chaperone p23, and XAP2 in the cytoplasm. Following ligand binding like 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), the AhR translocates into the nucleus. Although it has been reported that HSP90 regulates the translocation of the AhR to the nucleus, the precise activation mechanisms of the AhR have not yet been fully understood. AhR consists of the N-terminal bHLH domain containing NLS and NES, the middle PAS domain and the C-terminal transactivation domain. The PAS domain is familiar as a ligand and HSP90 binding domain. In this study, we focused on the bHLH domain that was thought to be a HSP90 binding domain. We investigated the binding properties of bHLH to HSP90. We analyzed the direct interaction of bHLH with HSP90, p23 and XAP2 using purified proteins. We found that not only the PAS domain but also the bHLH domain bound to HSP90. The bHLH domain forms complex with HSP90, p23 and XAP2. We also determined the bHLH binding domain was HSP90 N-domain. The bHLH domain makes a complex with HSP90, p23 and XAP2 via the HSP90 N-domain. Although the NLS is closed in the absence of a ligand, the structure of AhR will be changed in the presence of a ligand, which leads to NLS open, result in the nuclear translocation of AhR. AhR, aryl hydrocarbon receptor, co-chaperone, HSP90, molecular chaperone The aryl hydrocarbon receptor (AhR) is a member of the basic-helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family and has the function as a transcription factor (1, 2). AhR is the ligand-activated transcription regulator binding to environmental toxins with a high affinity such as 2, 3, 7, 8-tetrachlorodibenzeno-p-dioxin (TCDD) (3). In the absence of a ligand, the AhR exists with a dimer of the molecular chaperone heat shock protein 90 (HSP90), the HSP90-interacting protein p23 and hepatitis B virus X-associated protein 2 (XAP2) in the cytosol as a complex (4–6). Molecular chaperone HSP90 is abundant in eukaryotic cells and has the function of regulating more than 300 target substrates (7). HSP90 regulates AhR to maintain the AhR complex stably in ligand-free state in the cytoplasm (8, 9). In the presence of a ligand, the AhR translocates into the nucleus forming a heterodimer with the Aryl hydrocarbon receptor nuclear translocator (Arnt), and binds with xenobiotic responsible element (XRE) to act as a transcription factor inducing the toxicant metabolizing enzyme cytochrome P450 1A1 (CYP1A1) (10, 11). However, dioxin such as TCDD is accumulated in vivo without being metabolized because of the potent toxicity. The AhR is constituted by some functional domains. The PAS (per-arnt-sim) domain of the middle region has the PASA and PASB domains. The PASB domain includes a ligand binding site and the HSP90 binding sites (12). The bHLH (basic helix-loop-helix) domain near the N-terminal region has the DNA binding site, nuclear localization signal (NLS) and nuclear export signal (NES). The PAS domain has been thought to be a HSP90 binding domain (13, 14). Therefore, the bHLH domain is essential for nuclear translocation and interacts with XRE. The transcriptional activation domain is near the C-terminal region including glutamine rich site (1, 15). There are some reports about the association of AhR and HSP90 using in vitro transcription and translation system in rabbit reticulocyte lysate or wheat germ lysate (13, 14, 16). There are few reports about direct interaction of AhR complex due to the difficulty of the purification of AhR. We have recently reported the direct interaction between the AhR-PAS domain and HSP90 using purified proteins (17). HSP90 was dissociated from bHLH in the presence of 17-DMAG. After ligand binding, HSP90 and AhR translocate from cytoplasm to nucleus while maintaining complex. Although the AhR-bHLH domain is known as a HSP90 binding domain (18), this direct protein–protein interaction has not yet been confirmed using purified proteins. In the present study, we investigated the direct interaction of AhR and HSP90 and/or co-chaperones using purified bHLH, HSP90, XAP2, and p23. We determined the bHLH binding domain of HSP90 using purified HSP90 domains. We also investigated the influence of HSP90 inhibitor on the interactions. Methods and Materials Cell culture Cervical tumor-derived HeLa cells were obtained from ATCC. Cells were cultured in plastic dishes (Greiner, Germany) containing DMEM medium (Sigma-Aldrich Japan) supplemented with 5% fetal bovine serum (FBS) at 37 °C under 5% CO2 and 95% humidity. Materials 17-(Dimethylaminoethylamino)-17-demethoxygeldanamysin (17-DMAG) as the inhibitor of HSP90 was purchased from Invitrogen (San Diego, U.S.A.). Isopropyl-1-thio-β-D-galactopyranoside (IPTG) was purchased from Nakarai Tesque (Kyoto, Japan). 3-Methylcolanthrene (3MC) was purchased from Sigma-Aldrich Japan. An anti-HSP90β antibody (SPA-843) was purchased from Assay Designs (New York, U.S.A.), anti-AhR antibody (sc-5579) was purchased from Santa Cruz Biotechnology (Texas, U.S.A.), Alexa Fluor 488 conjugated anti-mouse antibody and Alexa Fluor 546 conjugated anti-rabbit antibody were purchased from Invitrogen. Immunofluorescence HeLa cells grown on a cover slip were treated with the vehicle or 3 µM 3MC for 2 h, fixed in ice-cold methanol at 4 °C for 15 min, washed three times with PBS, and incubated with 1% BSA in PBS at room temperature for 1 h. After washing with PBS, the primary antibody against HSP90β, AhR, p23, or XAP2 (diluted in 1% BSA/PBS) was mounted on a cover slip at 4 °C for 18 h. The cells were washed three times with PBS and incubated with an Alexa488- or Alexa546-conjugated secondary antibody for 3 h at room temperature. Finally, the cells were washed three times with PBS, incubated with DAPI (4′, 6-diamidino-2-phenylindole) for 30 min and mounted onto a slide glass with ProLong Gold antifade reagent (Invitrogen). Immunofluorescence images were obtained by the confocal laser microscopy (LSM780, Zeiss). Plasmid constructions PCR cycle was previously described. cDNA of bHLH was amplified by PCR using the forward primer 5′-GTCGACATGGCTGAAGGAATCAAGTCAA-3′ and reverse primer 5′-GCGGCCGCTCAATCAAAGAAGCTCTTGGCTCT-3′. The resulting PCR products were inserted into the Sal I/Not I sites of the pGEX-5X-3 vector. The constructs were confirmed by DNA sequencing. The cDNA of p23 was amplified by PCR using the forward primer 5′-CATATGCAGCCTGCTTCTGCAAAGTG-3′ and reverse primer 5′-GAATTCTTACTCCAGATCTGGCATTTTT-3′. The resulting PCR products were inserted into the Nde I/EcoR I sites of the pET21a vector. The constructs were confirmed by DNA sequencing. cDNA of XAP2 was amplified by PCR using the forward primer 5′-CATATGGCGGATATCATCGCAAG-3′ and reverse primer 5′-GAATTCTCAATGGGAGAAGATCCCC-3′. The resulting PCR products were inserted into the Nde I/EcoR I sites of the pET21a vector. The constructs were confirmed by DNA sequencing. HSP90, HSP90 N-, M-, C-, ΔN-, ΔM-andΔC-domains were amplified by PCR using the following primers: HSP90 and HSP90 N-domain N 5′-GGATCCATGCCTGAGGAAACCCAGACC-3′, HSP90 and HSP90 C-domain C 5′’-TCTAGATTAGTCTACTTCCATGCGTGA-3′, HSP90 N-domain C 5′-TCTAGATTCAGCCTCATCATCGCGTGA-3′, HSP90 C-domain N 5′-GGATCCGGTTACATGGCAG-3′, HSP90 M-domain N 5′-CATATGCTCAACAAAACAAAGCCCATC-3′, and HSP90 M-domain C 5′-CTCGAGTTCCAGGCCTTCTTTGGT-3′. The PCR products of HSP90, HSP90 N- and C-domains were digested with BamH I and Xba I restriction enzymes and cloned into the pCold I vector (TAKARA BIO, Inc. Japan). The PCR product of the HSP90 M-domain was digested with Nde I and Xho I restriction enzymes and cloned into the pET15b vector (Novagen, Inc. Japan). Recombinant protein expression and purification The bHLH was expressed in an Escherichia coli BL21 (DE3) arctic competent cell. The cells were grown at 37 °C, 250 rpm in LB BROTH medium supplemented with 100 μg/ml ampicillin until the OD600 reached 0.5. The cells were then induced by the addition of 0.1 mM IPTG, and the culture medium was incubated at 37 °C, 250 rpm for an additional 3 h. The cells were harvested by centrifugation at 4 °C, 13,000 rpm for 15 min, and cell pellets were suspended in 10 mM Tris-HCl pH 7.4. The cells were sonicated, centrifuged at 4 °C, 15,000 rpm for 15 min and the formed pellets were collected. The collected pellets were suspended in buffer (1 M Arginine, 10 mM Tris-HCl pH 7.4), then dialyzed with 10 mM Tris-HCl pH7.4, overnight to remove the Arginine. After dialysis, the lysates were cleared by centrifugation at 4 °C, 15,000 rpm for 15 min. The supernatant was applied to glutathione columns (Glutathione Sepharose 4B), washed with 10 mM Tris-HCl pH 7.4, and then eluted with elution buffer (20 mM Glutathione/10 mM Tris-HCl pH 7.4). Finally, the eluted proteins were concentrated by ultrafiltration. The p23 was expressed in an E. coli BL21(DE3) pLysS competent cell. The cells were grown at 37 °C, 250 rpm in LB BROTH medium supplemented with 100 μg/ml ampicillin and 30 μg/l chloramphenicol until the OD600 reached 0.5. The cells were then induced by the addition of 0.5 mM IPTG, and the culture medium was incubated at 37 °C, 250 rpm for an additional 3 h. The cells were harvested by centrifugation at 4 °C, 13,000 rpm for 15 min, and the cell pellets were suspended in 10 mM sodium acetate pH 6.0. The cells were sonicated, centrifuged at 4 °C, 15,000 rpm for 15 min, then the supernatants were collected. Proteins were applied to the DEAE column, and then washed with 10 mM sodium acetate buffer pH 6.0/0.17 M NaCl. After washing, the proteins were eluted with a linear gradient of 0.17–0.6 M NaCl in 10 mM sodium acetate buffer pH 6.0. The p23 fractions were dialyzed overnight with 10 mM Tris-HCl pH 7.4. After dialysis, the lysates were applied to the Q-sepharose column. Then, washed with 10 mM Tris-HCl pH 7.4/0.1 M NaCl and the proteins were eluted with linear gradient of 0.1–0.6 M NaCl in 10 mM Tris-HCl pH 7.4. The collected protein fractions were concentrated by ammonium sulphate fractionation and 60–90% fractions were collected. The protein fractions were applied to the sephacryl column and eluted with buffer (10 mM Tris-HCl pH 7.4/5% Glycerol/0.1 M NaCl). Finally, the collected fractions were concentrated by ultrafiltration. The XAP2 was expressed in an E. coli BL21 (DE3) arctic competent cell. The cells were grown at 37 °C, 250 rpm in LB BROTH medium supplemented with 100 μg/ml ampicillin until the OD600 reached 0.5. The culture medium was cooled for 30 min to 15 °C. The cells were then induced by the addition of 0.5 mM IPTG and incubated at 15 °C, 250 rpm for an additional 24 hr. The cells were harvested by centrifugation at 4 °C, 13,000 rpm for 15 min, and cell pellets were suspended in 10 mM Tris-HCl pH 7.4. The cells were sonicated for two cycles, centrifuged at 4 °C, 15,000 rpm for 15 min, then the supernatants were collected. Proteins were applied to the Heparin column, then washed with 10 mM Tris-HCl pH 7.4. After washing, the proteins were eluted with a linear gradient of 0-0.5 M NaCl in 10 mM Tris-HCl pH 7.4. The XAP2 fractions were dialyzed overnight with 10 mM Tris-HCl pH 7.4. After dialysis, collected proteins were applied to the Q-sepharose column, washed with 10 mM Tris-HCl pH 7.4. And then, proteins were eluted with a linear gradient of 0–0.6 M NaCl in 10 mM Tris-HCl pH 7.4. Finally, the eluted proteins were concentrated by ultrafiltration. The HSP90 N-, M-, C-, ΔN-, ΔM-, and ΔC-domains were expressed as a 6 × His fusion protein from the expression vector pCold I in the BL21 E. coli cells. The HSP90 M-domain was expressed as a 6 × His fusion protein from the expression vector pET15b in the arctic express (DE3) E. coli cells. The expression of the HSP90 N-, M-, C-, ΔN-, ΔM-, and ΔC-domains were induced by 0.5 mM IPTG. The cells were collected and each of cell extracts was applied to the Ni-NTA column, washed and eluted the same as the other chaperones. The AhR-ΔAD domain was amplified by PCR (iCycler, BioRad) using the forward primer 5′-GTCGACATGAACAGCAGCAGCC GCCAAC-3′ and reverse primer 5′-CTCGAGC TATTTTCGTAAATGCTCTGTTCC-3′. The resulting PCR products were inserted into the SalI/XhoI sites of the pGEX-5X-3 vector (Takara Bio, Japan). The constructs were confirmed by DNA sequencing (PRISM 3100, ABI). The GST-tagged AhR-ΔAD domain was expressed in an Escherichia coli BL21 (DE3) Arctic Competent Cell (Stratagene). The cells were grown at 30 °C in LB BROTH medium (Invitrogen) supplemented with 100μg/ml ampicillin for 3 hr, then cultured at 10 °C for 30 min. The cells were next induced by the addition of 0.5 mM IPTG and the cultures were incubated at 10 °C for an additional 24 hr. The cells were harvested by centrifugation at 20,000xg for 15 min at 4 °C, and the cell pellets were suspended in 10mM Tris-HCl, pH 7.4. The cells were sonicated, centrifuged at 20,000xg for 10 min at 4 °C and the formed pellets collected. The collected pellets were suspended in buffer (1M Arginine, 10 mM Tris-HCl, pH 7.4), then dialyzed with 10 mM Tris-HCl, pH 7.4, overnight to remove the Arginine. After dialysis, the lysates were cleared by centrifugation at 20,000 xg for 10 min at 4 °C. The supernatant was applied to glutathione columns (Glutathione Sepharose 4B, GE Healthcare Life Science), washed with 10 mM Tris-HCl, pH 7.4, and then eluted with elution buffer (20 mM Glutathione, 10 mM Tris-HCl, pH 7.4). The eluted proteins were concentrated by ultrafiltration. Antibody production An anti-p23 and anti-XAP2 antibody were produced by intramuscular injection into a rabbit of 1 mg of the purified each protein emulsified in complete Freund's adjuvant. Booster shots were given 3 times in the same manner as the original injection at 2-week intervals. The rabbit was bled 10 days after the last injection. The protocols for animal experimentation described in this paper were previously approved by the Animal Research Committee, Akita University School of Medicine; the “Guidelines for Animal Experimentation” of the University were completely adhered to in all subsequent animal experiments. XRE affinity chromatography and gel-shift assay The synthetic oligonucleotide which consist of four-tandem repeats of human XRE or CY3-XRE [10mer; (CY3) 5′-TTGCGTGCGG-3′] (19) were prepared (Fasmac Co., Ltd., Atsugi, Japan). XRE-Sepharose was prepared by coupling of XRE and Epoxy-activated Sepharose 6B (GE Healthcare Life Science) according to the manufacturer’s instructions. The purified GST-bHLH or GST were added to XRE-Sepharose or Mock (without XRE)-Sepharose column equilibrated with buffer (25 mM HEPES-KOH pH 7.4/5% Glycerol/0.1% NP-40/5 mM MgCl2) and incubated with gentle rotation using a rotator for 30 min at 4 °C. After washing with the same buffer three times, the bound proteins were separated by SDS-PAGE. Purified GST-bHLH (0.5 µM) and CY3-XRE (0.5 µM) were incubated with Binding Buffer (15 mM Tris-HCl pH 7.4, 75 mM NaCl, 1.5 mM EDTA, 1.5 mM DTT, 7.5% Glycerol, 0.5% NP-40) for 30 min at 4 °C. Gel-shift assay was performed using 4% gel and gel-shift mobility was detected using ChemiDoc XRS+ (BioRad). GST pull-down assay and 6 × His pull-down assay For the GST pull-down assay, 2.5 μM GST-bHLH, GST-ΔAD, or GST protein was added to a solution of 2.5 μM HSP90, p23, XAP2, 1 mM ATP and 150 μl buffer A (0.1 M KCl/10 mM MgCl2/20 mM Na2MoO4/0.6 M NaCl/5% Glycerol/0.1% NP-40 in 25 mM HEPES-KOH pH 7.4). The total volume of the sample was 300 μl by adding buffer B (5% Glycerol/0.1% NP-40 in 25 mM HEPES-KOH pH 7.4) and incubated using a rotator with gentle rotation at 37 °C for 15 min. The samples were loaded onto a GST resin equilibrated with buffer C (50 mM KCl/5 mM MgCl2/10 mM Na2MoO4/0.3 M NaCl/5% Glycerol/0.1% NP-40 in 25 mM HEPES-KOH pH 7.4) and incubated for 15 min at 4 °C with gentle rotation followed by centrifuged at 4 °C, 5,000 rpm for 10 s to remove the supernatant. The beads were washed three times with buffer C and eluted by boiling at 100 °C for 5 min in SDS sample buffer. GST pull-down samples were separated by SDS-PAGE and immune-blot. An antibody against HSP90 was used as previously reported (17). Ni2+ pull-down assay For the Ni2+ pull-down assay, 2.5 μM HSP90, XAP2, p23 and 1 mM ATP was added to 150 µl buffer D (25 mM HEPES-KOH pH 7.4/40 mM Imidazole/0.1% NP-40) and buffer E (25 mM HEPES-KOH pH 7.4/0.1% NP-40) upto 300 µl of total volume. The sample was incubated using a rotator with gentle rotation at 37 °C for 15 min. The incubated solution was loaded onto Ni2+-Sepharose resin equilibrated with buffer F (25 mM HEPES-KOH pH 7.4/20 mM Imidazole/0.1% NP-40). After incubation for 15 min at 4 °C with gentle rotation, centifuged at 4 °C, 5,000 rpm for 10 s to remove the supernatant. The beads were washed three times with buffer F and eluted by boiling at 100 °C for 5 min in SDS sample buffer. Ni2+ pull-down samples were separated by SDS-PAGE. Results Co-localization of AhR/HSP90 complex in the nucleus We have reported that AhR translocated from cytoplasm to nucleus with HSP90 under treatment with β-NF (17). In the current study, we confirmed of AhR nuclear translocation with chaperone complex in the presence of 3MC. As shown in Fig. 1A–C, we confirmed that the AhR-chaperone complex located in the nucleus in the presence of 3MC. Fig. 1 View largeDownload slide Co-localization of AhR and HSP90-chaperone complex. HeLa cells were treated with the vehicle or 3 µM 3MC for 2 h. Cells were incubated with anti-HSP90β and anti-AhR antibody (A), anti-HSP90 β and anti-p23 antibody (B), and anti-HSP90β and anti-XAP2 antibody (C). Blue staining indicatees DAPI staining of cell nuclei (A–C). Images were taken at 630× magnification. Fig. 1 View largeDownload slide Co-localization of AhR and HSP90-chaperone complex. HeLa cells were treated with the vehicle or 3 µM 3MC for 2 h. Cells were incubated with anti-HSP90β and anti-AhR antibody (A), anti-HSP90 β and anti-p23 antibody (B), and anti-HSP90β and anti-XAP2 antibody (C). Blue staining indicatees DAPI staining of cell nuclei (A–C). Images were taken at 630× magnification. Purification of GST-bHLH and DNA-binding ability The AhR activation mechanism is poorly understood in vitro. In the previous study, we have reported that the AhR-PAS domain is a HSP90 binding domain, and both AhR and HSP90 translocate to nuclear from cytoplasm (17). The AhR-bHLH domain has nuclear localization signal (NLS) and nuclear export signal (NES), so necessary for the transport to the nucleus. The AhR-bHLH domain is known to be as the HSP90 binding domain (13, 14). However, direct interactions between AhR-bHLH and HSP90 in vitro have not yet reported. In the present study, we focused on the AhR-bHLH domain and analyzed relations between AhR-bHLH and the molecular chaperone HSP90. GST-bHLH was expressed in E. coli for using in GST pull-down assay. A purification of GST-bHLH was carried out by GST affinity column chromatography. As shown in Fig. 2A, the purified bHLH, having about 33-kDa molecular mass, was a single protein band on SDS-PAGE. If bHLH has correct structure, it may be able to bind to XRE (Xenobiotic responsible element, so called dioxin responsible element). We analyzed the DNA-binding ability of bHLH using a XRE-Sepharose affinity column. Because of the 3′ end of XRE has OH- groups, we prepared the XRE affinity resin using an Epoxy-activated Sepharose 6B. Epoxy-activated Sepharose 6B is useful resin to fix the OH-, or NH2-groups. No protein bands were shown in mock-columns (Fig. 2B, lanes 1 and 3). Although almost of GST was not able to bind to XRE-Sepharose affinity resins (Fig. 2B, lane 2), certain amount of bHLH could bind to the affinity resin (Fig. 2B, lane 4). We also investigated the XRE binding ability of bHLH using gel mobility-shift assay (Fig. 2C). We could detect signals at higher end of the gels only in the presence of bHLH. No gel mobility-shift has been detected in the presence of GST. These results suggested that the purified bHLH possesses a DNA-binding ability. Fig. 2 View largeDownload slide Purification of GST-bHLH. (A) GST-bHLH was purified using a GST affinity column. Apply sample is suprnatant after sonication of GST-bHLH expressed in E. coli and centrifugation. The column was washed with 10 mM Tris-HCl pH 7.4. GST and GST-bHLH were eluted by Glutathione. Eluted proteins were analyzed by SDS-PAGE (11% gel). (B) The purified protein and GST (input) were incubated with to Mock resin or XRE-Sepharose affinity resin at 4 °C for 30 min, and the bound proteins were separated by SDS-PAGE (9% gel). Lanes 1, 3 and 2, 4 indicate Mock resins and XRE-Sepharose affinity resins, respectively. (C) The purified protein and GST (input) were incubated with CY3-XRE at 4 °C for 30 min, samples were separated by SDS-PAGE (4% gel). Free, GST, and GST-bHLH indicates CY3-XRE, CY3-XRE/GST, and CY3-XRE/GST-bHLH, respectively. Single- and double-closed triangles indicate free and protein bound CY3-XRE, respectively. Fig. 2 View largeDownload slide Purification of GST-bHLH. (A) GST-bHLH was purified using a GST affinity column. Apply sample is suprnatant after sonication of GST-bHLH expressed in E. coli and centrifugation. The column was washed with 10 mM Tris-HCl pH 7.4. GST and GST-bHLH were eluted by Glutathione. Eluted proteins were analyzed by SDS-PAGE (11% gel). (B) The purified protein and GST (input) were incubated with to Mock resin or XRE-Sepharose affinity resin at 4 °C for 30 min, and the bound proteins were separated by SDS-PAGE (9% gel). Lanes 1, 3 and 2, 4 indicate Mock resins and XRE-Sepharose affinity resins, respectively. (C) The purified protein and GST (input) were incubated with CY3-XRE at 4 °C for 30 min, samples were separated by SDS-PAGE (4% gel). Free, GST, and GST-bHLH indicates CY3-XRE, CY3-XRE/GST, and CY3-XRE/GST-bHLH, respectively. Single- and double-closed triangles indicate free and protein bound CY3-XRE, respectively. AhR-bHLH domain binds to HSP90, XAP2 and p23 We investigated that an association between bHLH and HSP90 by GST pull-down assay. As shown in Fig. 3A, HSP90 protein bands were detected in only GST-bHLH lanes, not in GST lanes. These results showed that the AhR-bHLH domain is also a HSP90 binding domain as same as the AhR-PAS domain. Its interaction was not changed in the presence or absence of ATP. We analyzed an effect of 17-DMAG on the interaction (Fig. 3B). The interaction between bHLH and HSP90 was not also affected by 17-DMAG in the presence or absence of ATP. Fig. 3 View largeDownload slide GST pull-down assay confirming the interaction of HSP90 with AhR-bHLH domain. (A) An association between AhR-bHLH and HSP90 was analyzed by GST pull-down assay in the absence or presence of 1 mM ATP. GST-bHLH or GST, HSP90, and ATP were incubated with GST resins. After washing, Glutathione specific-binding proteins were analyzed by SDS-PAGE (11% gel). Lanes 1–3 of gels were the inputs from purified GST (28 kDa), GST-bHLH (33 kDa), and HSP90 (90 kDa) as a control, respectively. (B) The purified GST, GST-bHLH, and HSP90 were incubated with GST resins in the absence or presence of 50 μM 17-DMAG. The elutants from the glutathione columns were analyzed by SDS-PAGE (11% gel). Pull-down assays were performed using purified GST or the GST-AhR-PAS domain and purified HSP90 in the absence (−) or presence (+) of ATP. Fig. 3 View largeDownload slide GST pull-down assay confirming the interaction of HSP90 with AhR-bHLH domain. (A) An association between AhR-bHLH and HSP90 was analyzed by GST pull-down assay in the absence or presence of 1 mM ATP. GST-bHLH or GST, HSP90, and ATP were incubated with GST resins. After washing, Glutathione specific-binding proteins were analyzed by SDS-PAGE (11% gel). Lanes 1–3 of gels were the inputs from purified GST (28 kDa), GST-bHLH (33 kDa), and HSP90 (90 kDa) as a control, respectively. (B) The purified GST, GST-bHLH, and HSP90 were incubated with GST resins in the absence or presence of 50 μM 17-DMAG. The elutants from the glutathione columns were analyzed by SDS-PAGE (11% gel). Pull-down assays were performed using purified GST or the GST-AhR-PAS domain and purified HSP90 in the absence (−) or presence (+) of ATP. We also analyzed the effects of co-chaperones p23 and XAP2 to the bHLH-HSP90 complex on SDS-PAGE. No proteins bound to GST (Fig. 4A). We could detect HSP90 and bHLH on SDS-PAGE, but XAP2 and p23 were difficult to detect, so confirmed in immune-blot using antibodies against HSP90, p23 and XAP2. The p23 and XAP2 protein bands were shown in GST-bHLH lanes (Fig. 4B). Thus, these results supports that AhR-bHLH interacts with HSP90, p23 and XAP2 in the cytoplasm. Furthermore, the complex was not affected by ATP. We investigated HSP90 co-chaperone complex using Ni2+ pull-down assay. As shown in Figs 5 and 6 × His-HSP90 binds to the Ni2+-Sepharose resin. We could detect HSP90, XAP2 and p23 protein bands on SDS-PAGE in the absence or presence of ATP. These results suggested that HSP90 makes a complex with XAP2 and p23 and AhR-bHLH binds to HSP90 complex via HSP90. Fig. 4 View largeDownload slide Conformational analysis of AhR, HSP90, and co-chaperone complex. An AhR-bHLH binding protein was identified by GST pull-down assay. In the assay, GST resins were incubated with HSP90, p23, XAP2, and GST or GST-bHLH, in addition, in the absence or presence of 1 mM ATP. The binding proteins were analyzed by SDS-PAGE (9% gel) (A) or immune-blot using anti-HSP90, anti-p23 and anti-XAP2 antibodies (B). Fig. 4 View largeDownload slide Conformational analysis of AhR, HSP90, and co-chaperone complex. An AhR-bHLH binding protein was identified by GST pull-down assay. In the assay, GST resins were incubated with HSP90, p23, XAP2, and GST or GST-bHLH, in addition, in the absence or presence of 1 mM ATP. The binding proteins were analyzed by SDS-PAGE (9% gel) (A) or immune-blot using anti-HSP90, anti-p23 and anti-XAP2 antibodies (B). Fig. 5 View largeDownload slide Conformational analysis of chaperone complex. The HSP90 and co-chaperones binding properties were identified by Ni2+ pull-down assay. In the assay, Ni2+-Sepharose resins were incubated with HSP90, p23, and XAP2 in the presence or absence of 1 mM ATP. The binding proteins were analyzed by SDS-PAGE (11% gel). Fig. 5 View largeDownload slide Conformational analysis of chaperone complex. The HSP90 and co-chaperones binding properties were identified by Ni2+ pull-down assay. In the assay, Ni2+-Sepharose resins were incubated with HSP90, p23, and XAP2 in the presence or absence of 1 mM ATP. The binding proteins were analyzed by SDS-PAGE (11% gel). Fig. 6 View largeDownload slide HSP90 and AhR domains. (A) Human HSP90 N-, M-, and C-domains or ΔN-, ΔM-, and ΔC-deletion mutants of HSP90 were constructed and purified under “Materials and Methods”. (B) The domain structure of human AhR. Fig. 6 View largeDownload slide HSP90 and AhR domains. (A) Human HSP90 N-, M-, and C-domains or ΔN-, ΔM-, and ΔC-deletion mutants of HSP90 were constructed and purified under “Materials and Methods”. (B) The domain structure of human AhR. bHLH binding domains of HSP90 HSP90 is composed from 3 domains. ATP binding domain (N domain), substrate binding domain (M domain), and dimerization domain (C domain) (20). We constructed and purified HSP90 domains and investigated the bHLH binding domain of HSP90 (Fig. 6A). The domain structure of human AhR was also shown in Fig. 6B. At first, we analyzed interactions between the GST-ΔAD binding domain of HSP90 using HSP90 domains (N-, M- and C-domain) as control. We could detect that AhR-PAS bound to the HSP90N domain (Fig. 7A). Neither HSP90M nor HSP90C domain bound to GST-ΔAD. Then, we investigated the interactions between bHLH and each HSP90 domain (N, M and C) using GST-pull down assay (Fig. 7B). No proteins bound to GST. On the contrary, HSP90 N-domain was pull-downed with bHLH. We could not detect other domains (HSP90 M- and HSP90 C-domain) on SDS-PAGE. ATP did not affect the interaction. We also confirmed the binding of the bHLH domain to HSP90 using HSP90 deletion mutants (HSP90-ΔC, ΔM and ΔN). As shown in Fig. 7C, no HSP90 deletion mutants were interacted with GST. Both HSP90-ΔC and -ΔM interacted with bHLH but not HSP90-ΔN (The same data were shown in Fig. 7B and C). These data suggested that AhR-bHLH bound to the HSP90 N-domain. Fig. 7 View largeDownload slide GST pull-down assay confirming the interaction of HSP90N-, M-, and C-domains and GST-ΔAD or GST-bHLH. (A) Purified GST, GST-ΔAD, HSP90 N-, M-, and C-domain were incubated with GST resins in the absence or presence of ATP. The elutants from the glutathione column were analyzed by SDS-PAGE (11% gel). Lanes 1–5 of gels were the inputs from purified HSP90 N-domain (38 kDa), HSP90 M-domain (40 kDa), HSP90 C-domain (16 kDa), GST (28 kDa), and GST-ΔAD (57 kDa) as a control, respectively. Pull-down assays were performed using purified GST (lanes 6–11) or the GST-AhR-PAS domain (lanes 12–17) and purified HSP90 in the absence (−) or presence (+) of ATP. (B) The purified GST, GST-bHLH, HSP90N-, M-, and C-domains were incubated with GST resins in the absence or presence of ATP. The elutants from the glutathione columns were analyzed by SDS-PAGE (11% gel). Lanes 1–5 of gels were the inputs from purified HSP90 N- domain (38 kDa), HSP90 M-domain (40 kDa), HSP90 C-domain (16 kDa), GST (28 kDa), and GST-bHLH (33 kDa) as a control, respectively. Pull-down assays were performed using purified GST or the GST-AhR-PAS domain and purified HSP90 in the absence (−) or presence (+) of ATP. (C) The purified GST, GST-bHLH, HSP90ΔN-, ΔM-, and ΔC-deletion mutants were incubated with GST resins in the absence or presence of ATP. The elutants from the glutathione columns were analyzed by SDS-PAGE (9% gel). Lanes 1-5 of gels were the inputs from purified HSP90ΔN (68 kDa), HSP90ΔC (60 kDa), HSP90ΔM (45 kDa), GST (28 kDa), and GST-bHLH (33 kDa) as a control, respectively. Pull-down assays were performed using purified GST or the GST-AhR-PAS domain and purified HSP90 in the absence −) or presence (+) of ATP. (D) Ni2+ pull-down assays were performed as described in the “Materials and Methods”. The purified HSP90N-, M-, C-domain, HSP90ΔN-, ΔC-deletion mutants, XAP2, and p23 were incubated with Ni2+-Sepharose resin in the absence or presence of ATP. Ni2+ pull-down samples were analyzed by SDS-PAGE (11% gel). Lanes 1–7 of gels were the inputs from purified HSP90N-domain (38 kDa), HSP90ΔC (60 kDa), HSP90 M-domain (40 kDa), HSP90ΔN (68 kDa), HSP90 C-domain (16 kDa), XAP2 (38 kDa), and p23 823 kDa) as a control, respectively. Ni-NTA pull-down assays were performed using purified HSP90N-domain, HSP90ΔC, HSP90M-domain, HSP90ΔN, and HSP90C-domain in the absence (−) or presence (+) of ATP. Fig. 7 View largeDownload slide GST pull-down assay confirming the interaction of HSP90N-, M-, and C-domains and GST-ΔAD or GST-bHLH. (A) Purified GST, GST-ΔAD, HSP90 N-, M-, and C-domain were incubated with GST resins in the absence or presence of ATP. The elutants from the glutathione column were analyzed by SDS-PAGE (11% gel). Lanes 1–5 of gels were the inputs from purified HSP90 N-domain (38 kDa), HSP90 M-domain (40 kDa), HSP90 C-domain (16 kDa), GST (28 kDa), and GST-ΔAD (57 kDa) as a control, respectively. Pull-down assays were performed using purified GST (lanes 6–11) or the GST-AhR-PAS domain (lanes 12–17) and purified HSP90 in the absence (−) or presence (+) of ATP. (B) The purified GST, GST-bHLH, HSP90N-, M-, and C-domains were incubated with GST resins in the absence or presence of ATP. The elutants from the glutathione columns were analyzed by SDS-PAGE (11% gel). Lanes 1–5 of gels were the inputs from purified HSP90 N- domain (38 kDa), HSP90 M-domain (40 kDa), HSP90 C-domain (16 kDa), GST (28 kDa), and GST-bHLH (33 kDa) as a control, respectively. Pull-down assays were performed using purified GST or the GST-AhR-PAS domain and purified HSP90 in the absence (−) or presence (+) of ATP. (C) The purified GST, GST-bHLH, HSP90ΔN-, ΔM-, and ΔC-deletion mutants were incubated with GST resins in the absence or presence of ATP. The elutants from the glutathione columns were analyzed by SDS-PAGE (9% gel). Lanes 1-5 of gels were the inputs from purified HSP90ΔN (68 kDa), HSP90ΔC (60 kDa), HSP90ΔM (45 kDa), GST (28 kDa), and GST-bHLH (33 kDa) as a control, respectively. Pull-down assays were performed using purified GST or the GST-AhR-PAS domain and purified HSP90 in the absence −) or presence (+) of ATP. (D) Ni2+ pull-down assays were performed as described in the “Materials and Methods”. The purified HSP90N-, M-, C-domain, HSP90ΔN-, ΔC-deletion mutants, XAP2, and p23 were incubated with Ni2+-Sepharose resin in the absence or presence of ATP. Ni2+ pull-down samples were analyzed by SDS-PAGE (11% gel). Lanes 1–7 of gels were the inputs from purified HSP90N-domain (38 kDa), HSP90ΔC (60 kDa), HSP90 M-domain (40 kDa), HSP90ΔN (68 kDa), HSP90 C-domain (16 kDa), XAP2 (38 kDa), and p23 823 kDa) as a control, respectively. Ni-NTA pull-down assays were performed using purified HSP90N-domain, HSP90ΔC, HSP90M-domain, HSP90ΔN, and HSP90C-domain in the absence (−) or presence (+) of ATP. Discussion The AhR activation mechanism has not yet been fully understood. AhR exists with HSP90, co-chaperone p23 and XAP2 in the cytoplasm. AhR has PAS and bHLH domains. The PAS domain is a ligand binding domain and a HSP90 binding domain. Recently, we have reported that the PAS domain binds to HSP90 directly (17). Because of the difficulty to purify the full length of AhR, we used some purified domains of AhR for experiments. We purified the PAS domain and checked the ligand-binding ability of purified the PAS domain using a β-naphthoflavone (β-NF) affinity resin. In this study, we investigated whether AhR-bHLH bound to HSP90, p23 and XAP2 or not. The AhR-bHLH domain has NLS, NES and the XRE binding domain. Moreover, the AhR-bHLH domain has been thought to be a HSP90 binding domain. First, we confirmed that purified AhR-bHLH protein bound to XRE. In the present study, we used XRE affinity resins. An oligonucleotide has OH- group in the 3′ end. We fixed the four tandem XRE to Epoxy Sepharose 6B columns. The affinity resin is able to fix the OH- or NH2- group of protein, ligand, and DNA. Until now, we have a number of reports in the drug-affinity (21–25). The purified bHLH could bind to XRE affinity resins, but not mock resins. GST did not bind to either XRE affinity resins or mock resins. We also analyzed the interaction between DNA and protein using gel mobility-shift assay. We used CY3-XRE at the methods. GST didn’t show the interaction. On the contrary, we could detect the gel mobility-shift between CY3-XRE and GST-bHLH. Thus, we confirmed the purification of functional bHLH. So far some of the detail of the interaction between AhR and HSP90 has been published. However, few data showed direct relations in molecular level. A nucleotide binding to the HSP90 N-domain induces a directionality and a conformational cycle. In the absence of ATP, HSP90 adopts an open conformation (V-shaped form). ATP induces conformational changes of HSP90 from open to closed form (26, 27). In the present study, we have demonstrated the direct interaction of bHLH, HSP90, XAP2 and p23. We have also determined bHLH is the binding domain of HSP90-N domain as same as AhR-PAS. ATP did not affect the interaction of bHLH, HSP90, XAP2 and p23. Interestingly, 17-DMAG did not affect on the interaction between bHLH and HSP90. We have recently reported that HSP90 was dissociated from AhR-PAS in the presence of 17-DMAG (17). The binding sites of bHLH and PAS to the HSP90 N-domain are slightly different each other. We speculate that the bHLH binding site of the HSP90 N-domain may be neighbor of the M-domain. On the contrary, the PAS binding site of the HSP90 N-domain may be end of the N-domain. ATP induces dramatically conformational changes of HSP90 from open to closed form. The conformational change of HSP90N end is bigger than that of HSP90N neighbor M domain. The differences of 17-DMAG to bHLH and PAS are thought to be due to such reasons. We propose the models of the AhR-HSP90 chaperone complex (Fig. 8). Fig. 8 View largeDownload slide Conformational change models of the AhR, HSP90, p23, XAP2 complex. The bHLH and PAS domains of AhR bind to HSP90 N-domain. Co-chaperones p23 and XAP2 bind to HSP90 N- and C-domains, respectively. When in the absence of ligand, the NLS of bHLH is hidden. When the ligands bind to AhR, the conformational changes will be occurred, then the NLS will be opened. Fig. 8 View largeDownload slide Conformational change models of the AhR, HSP90, p23, XAP2 complex. The bHLH and PAS domains of AhR bind to HSP90 N-domain. Co-chaperones p23 and XAP2 bind to HSP90 N- and C-domains, respectively. When in the absence of ligand, the NLS of bHLH is hidden. When the ligands bind to AhR, the conformational changes will be occurred, then the NLS will be opened. Based on the result, we inferred that HSP90 covers NLS when AhR is in the ligand-free state, and the conformational change of AhR complex lead to the exposure of NLS after AhR binds to ligand. For that reason, the AhR-bHLH domain interacts with HSP90. From the above, the AhR-bHLH domain is essential in the AhR activation mechanism similar to the PAS domain. Author Contributions HI in the research design; IK, MH, AH, NT, YN, HO, KF, YK, and TO conducted the experiment. IK, EG and HI wrote or contributed to the writing of the paper. Funding H.I. was supported by a Grant-in-Aid for Scientific Research (Exploratory Research: 16651056) from the Japanese Ministry of Education, Science, Sports and Culture. Conflict of Interest None declared. References 1 Kewley R.J., Whitelaw M.L., Chapman-Smith A. ( 2004) The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. Int. J. Biochem. Cell Biol . 36, 189– 204 Google Scholar CrossRef Search ADS PubMed  2 Sorg O. ( 2014) AhR signaling and dioxin toxicity. Toxicol. Lett.  230, 225– 233 Google Scholar CrossRef Search ADS PubMed  3 Mimura J., Fujii-Kuriyama Y. ( 2003) Functional role of AhR in the expression of toxic effects by TCDD. Biochim. et Biophys. Acta  1619, 263– 268 Google Scholar CrossRef Search ADS   4 Meyer B.K., Pray-Grant M.G., Vanden, Heuvel J.P., Perdew G.H. ( 1998) Hepatitis B virus X-associated protein 2 is a subunit of the unliganded aryl hydrocarbon receptor core complex and exhibits transcriptional enhancer activity. Mol. Cell. Biol . 18, 978– 988 Google Scholar CrossRef Search ADS PubMed  5 Nebert D.W., Gonzalez F. J. ( 1987) P450 genes: structure, evolution, and regulation. Annu. Rev. Biochem . 56, 945– 993 Google Scholar CrossRef Search ADS PubMed  6 Kazlauskas A., Poellinger L., Pongratz I. ( 1999) Evidence that the co-chaperone p23 regulates ligand responsiveness of the dioxin (aryl hydrocarbon) receptor. J. Biol. Chem . 274, 13519– 13524 Google Scholar CrossRef Search ADS PubMed  7 Karagöz G.E., Rüdiger S.G. ( 2015) Hsp90 interaction with clients. Trends Biochem Sci . 40, 117– 125 Google Scholar CrossRef Search ADS PubMed  8 Li J., Buchner J. ( 2013) Structure, function and regulation of the hsp90 machinery. Biomed. J . 36, 106– 117 Google Scholar CrossRef Search ADS PubMed  9 Erlejman A.G., Lagadari M., Toneatto J., Piwien-Pilipuk G., Galigniana M.D. ( 2014) Regulatory role of the 90-kDa-heat-shock protein (Hsp90) and associated factors on gene expression. Biochim Biophys Acta  1839, 71– 87 Google Scholar CrossRef Search ADS PubMed  10 Kobayashi A., Sogawa K., Fujii-Kuriyama Y. ( 1996) Cooperative interaction between AhR·Arnt and Sp1 for the drug-inducible expression of CYP1A1 gene. J. Biol. Chem . 271, 12310– 12316 Google Scholar CrossRef Search ADS PubMed  11 Beischlag T.V., Luis, Morales J., Hollingshead B.D., Perdew G.H. ( 2008) The aryl hydrocarbon receptor complex and the control of gene expression. Crit. Rev. Eukaryot. Gene Expr . 18, 207– 250 Google Scholar CrossRef Search ADS PubMed  12 Wu D., Potluri N., Kim Y., Rastinejad F. ( 2013) Structure and dimerization properties of the aryl hydrocarbon receptor PAS-A domain. Mol. Cell Biol . 33, 4346– 4356 Google Scholar CrossRef Search ADS PubMed  13 McGuire J., Coumailleau P., Whitelaw M.L., Gustafsson J.A., Poellinger L. ( 1996) The basic helix-loop-helix/PAS factor Sim is associated with hsp90. Implications for regulation by interaction with partner factors. J. Biol. Chem . 270, 31353– 31357 Google Scholar CrossRef Search ADS   14 Antonsson C., Arulampalam V., Whitelaw M.L., Pettersson S., Poellinger L. ( 1995) Constitutive function of the basic helix-loop-helix/PAS factor Arnt. Regulation of target promoters via the E box motif. J. Biol. Chem . 270, 13968– 13972 Google Scholar CrossRef Search ADS PubMed  15 Feng S., Cao Z., Wang X. ( 2013) Role of aryl hydrocarbon receptor in cancer. Biochim Biophys Acta  1836, 197– 210 Google Scholar PubMed  16 Kazlauskas A., Poellinger L., Pongratz I. ( 2000) The immunophilin-like protein XAP2 regulates ubiquitination and subcellular localization of the dioxin receptor. J. Biol. Chem . 275, 41317– 41324 Google Scholar CrossRef Search ADS PubMed  17 Tsuji N., Fukuda K., Nagata Y., Okada H., Haga A., Hatakeyama S., Yoshida S., Okamoto T., Hosaka M., Sekine K., Ohtaka K., Yamamoto S., Otaka M., Grave E., Itoh H. ( 2014) The activation mechanism of the aryl hydrocarbon receptor (AhR) by molecular chaperone HSP90. FEBS Open Bio . 4, 796– 803 Google Scholar CrossRef Search ADS PubMed  18 Fukunaga B.N., Probst M.R., Reisz-Porszasz S., Hankinson O. ( 1995) Identification of functional domain of the aryl hydrocarbon receptor. J. Biol. Chem . 270, 29270– 29278 Google Scholar CrossRef Search ADS PubMed  19 Lakhman S.S., Chen X., Gonzalez-Covarrubias V., Schuetz E.G., Blanco J.G. ( 2007) Functional characterization of the promoter of human carbonyl reductase 1 (CBR1). Role of XRE elements in mediating the induction of CBR1 by ligands of the aryl hydrocarbon receptor. Mol. Pharmacol . 72, 734– 743 Google Scholar CrossRef Search ADS PubMed  20 Ali M.M., Roe S.M., Vaughan C.K., Meyer P., Panaretou B., Piper P.W., Prodromou C., Pearl L.H. ( 2006) Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature  440, 1013– 1017 Google Scholar CrossRef Search ADS PubMed  21 Itoh H., Komatsuda A., Wakui H., Miura A.B., Tashima Y. ( 1999) Mammalian HSP60 is a major target for an immunosuppressant mizoribine. J. Biol. Chem . 274, 35147– 35151 Google Scholar CrossRef Search ADS PubMed  22 Itoh H., Ogura M., Komatsuda A., Wakui H., Miura A.B., Tashima Y. ( 1999) A novel chaperone-activity-reducing mechanism of the 90-kDa molecular chaperone HSP90. Biochem. J . 343, 697– 703 Google Scholar CrossRef Search ADS PubMed  23 Ishida R., Takaoka Y., Yamamoto S., Miyazaki T., Otaka M., Watanabe S., Komatsuda A., Wakui H., Sawada K., Kubota H., Itoh H. ( 2008) Cisplatin differently affects amino terminal and carboxyl terminal domains of HSP90. FEBS Lett . 582, 3879– 3883 Google Scholar CrossRef Search ADS PubMed  24 Miyazaki T., Sagawa R., Honma T., Noguchi S., Harada T., Komatsuda A., Ohtani H., Wakui H., Sawada K., Otaka M., Watanabe S., Jikei M., Ogawa N., Hamada F., Itoh H. ( 2004) 73-kDa molecular chaperone HSP73 is a direct target of antibiotic gentamicin. J. Biol. Chem . 279, 17295– 17300 Google Scholar CrossRef Search ADS PubMed  25 Yamamoto S., Nakano S., Owari K., Fuziwara K., Ogawa N., Otaka M., Tamaki K., Watanabe S., Komatsuda A., Wakui H., Sawada K., Kubota H., Itoh H. ( 2010) Gentamicin inhibits HSP70-assisted protein folding by interfering with substrate recognition. FEBS Lett . 584, 645– 651 Google Scholar CrossRef Search ADS PubMed  26 Saibil H. ( 2013) Chaperone machines for protein folding, unfolding and disaggregation. Nat. Rev. Mol. Cell Biol . 14, 630– 642 Google Scholar CrossRef Search ADS PubMed  27 Röhl A., Rohrberg J., Buchner J. ( 2013) The chaperone Hsp90: changing partners for demanding clients. Trends Biochem. Sci . 38, 253– 262 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations AhR aryl hydrocarbon receptor bHLH basic helix-loop-helix CYP1A1 cytochrome P450 1A1 17-DMAG 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin GST glutathione S-transferase HSP90 90-kDa of heat shock protein IPTG Isopropyl-1-thio-β-D-galactopyranoside 3MC 3-methylcholanthrene Arnt, AhR nuclear translocator NES nuclear export signal NLS nuclear localization signal PAS per-arnt-sim TCDD 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin XAP2 hepatitis B virus X-associated protein XRE xenobiotic responsible element © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Loading next page...
 
/lp/ou_press/the-regulation-mechanisms-of-ahr-by-molecular-chaperone-complex-CkIcr5AJ8Q
Publisher
Oxford University Press
Copyright
© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
ISSN
0021-924X
eISSN
1756-2651
D.O.I.
10.1093/jb/mvx074
Publisher site
See Article on Publisher Site

Abstract

Abstract The AhR, so called the dioxin receptor, is a member of the nuclear receptor superfamily. The ligand-free AhR forms a cytosolic protein complex with the molecular chaperone HSP90, co-chaperone p23, and XAP2 in the cytoplasm. Following ligand binding like 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), the AhR translocates into the nucleus. Although it has been reported that HSP90 regulates the translocation of the AhR to the nucleus, the precise activation mechanisms of the AhR have not yet been fully understood. AhR consists of the N-terminal bHLH domain containing NLS and NES, the middle PAS domain and the C-terminal transactivation domain. The PAS domain is familiar as a ligand and HSP90 binding domain. In this study, we focused on the bHLH domain that was thought to be a HSP90 binding domain. We investigated the binding properties of bHLH to HSP90. We analyzed the direct interaction of bHLH with HSP90, p23 and XAP2 using purified proteins. We found that not only the PAS domain but also the bHLH domain bound to HSP90. The bHLH domain forms complex with HSP90, p23 and XAP2. We also determined the bHLH binding domain was HSP90 N-domain. The bHLH domain makes a complex with HSP90, p23 and XAP2 via the HSP90 N-domain. Although the NLS is closed in the absence of a ligand, the structure of AhR will be changed in the presence of a ligand, which leads to NLS open, result in the nuclear translocation of AhR. AhR, aryl hydrocarbon receptor, co-chaperone, HSP90, molecular chaperone The aryl hydrocarbon receptor (AhR) is a member of the basic-helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family and has the function as a transcription factor (1, 2). AhR is the ligand-activated transcription regulator binding to environmental toxins with a high affinity such as 2, 3, 7, 8-tetrachlorodibenzeno-p-dioxin (TCDD) (3). In the absence of a ligand, the AhR exists with a dimer of the molecular chaperone heat shock protein 90 (HSP90), the HSP90-interacting protein p23 and hepatitis B virus X-associated protein 2 (XAP2) in the cytosol as a complex (4–6). Molecular chaperone HSP90 is abundant in eukaryotic cells and has the function of regulating more than 300 target substrates (7). HSP90 regulates AhR to maintain the AhR complex stably in ligand-free state in the cytoplasm (8, 9). In the presence of a ligand, the AhR translocates into the nucleus forming a heterodimer with the Aryl hydrocarbon receptor nuclear translocator (Arnt), and binds with xenobiotic responsible element (XRE) to act as a transcription factor inducing the toxicant metabolizing enzyme cytochrome P450 1A1 (CYP1A1) (10, 11). However, dioxin such as TCDD is accumulated in vivo without being metabolized because of the potent toxicity. The AhR is constituted by some functional domains. The PAS (per-arnt-sim) domain of the middle region has the PASA and PASB domains. The PASB domain includes a ligand binding site and the HSP90 binding sites (12). The bHLH (basic helix-loop-helix) domain near the N-terminal region has the DNA binding site, nuclear localization signal (NLS) and nuclear export signal (NES). The PAS domain has been thought to be a HSP90 binding domain (13, 14). Therefore, the bHLH domain is essential for nuclear translocation and interacts with XRE. The transcriptional activation domain is near the C-terminal region including glutamine rich site (1, 15). There are some reports about the association of AhR and HSP90 using in vitro transcription and translation system in rabbit reticulocyte lysate or wheat germ lysate (13, 14, 16). There are few reports about direct interaction of AhR complex due to the difficulty of the purification of AhR. We have recently reported the direct interaction between the AhR-PAS domain and HSP90 using purified proteins (17). HSP90 was dissociated from bHLH in the presence of 17-DMAG. After ligand binding, HSP90 and AhR translocate from cytoplasm to nucleus while maintaining complex. Although the AhR-bHLH domain is known as a HSP90 binding domain (18), this direct protein–protein interaction has not yet been confirmed using purified proteins. In the present study, we investigated the direct interaction of AhR and HSP90 and/or co-chaperones using purified bHLH, HSP90, XAP2, and p23. We determined the bHLH binding domain of HSP90 using purified HSP90 domains. We also investigated the influence of HSP90 inhibitor on the interactions. Methods and Materials Cell culture Cervical tumor-derived HeLa cells were obtained from ATCC. Cells were cultured in plastic dishes (Greiner, Germany) containing DMEM medium (Sigma-Aldrich Japan) supplemented with 5% fetal bovine serum (FBS) at 37 °C under 5% CO2 and 95% humidity. Materials 17-(Dimethylaminoethylamino)-17-demethoxygeldanamysin (17-DMAG) as the inhibitor of HSP90 was purchased from Invitrogen (San Diego, U.S.A.). Isopropyl-1-thio-β-D-galactopyranoside (IPTG) was purchased from Nakarai Tesque (Kyoto, Japan). 3-Methylcolanthrene (3MC) was purchased from Sigma-Aldrich Japan. An anti-HSP90β antibody (SPA-843) was purchased from Assay Designs (New York, U.S.A.), anti-AhR antibody (sc-5579) was purchased from Santa Cruz Biotechnology (Texas, U.S.A.), Alexa Fluor 488 conjugated anti-mouse antibody and Alexa Fluor 546 conjugated anti-rabbit antibody were purchased from Invitrogen. Immunofluorescence HeLa cells grown on a cover slip were treated with the vehicle or 3 µM 3MC for 2 h, fixed in ice-cold methanol at 4 °C for 15 min, washed three times with PBS, and incubated with 1% BSA in PBS at room temperature for 1 h. After washing with PBS, the primary antibody against HSP90β, AhR, p23, or XAP2 (diluted in 1% BSA/PBS) was mounted on a cover slip at 4 °C for 18 h. The cells were washed three times with PBS and incubated with an Alexa488- or Alexa546-conjugated secondary antibody for 3 h at room temperature. Finally, the cells were washed three times with PBS, incubated with DAPI (4′, 6-diamidino-2-phenylindole) for 30 min and mounted onto a slide glass with ProLong Gold antifade reagent (Invitrogen). Immunofluorescence images were obtained by the confocal laser microscopy (LSM780, Zeiss). Plasmid constructions PCR cycle was previously described. cDNA of bHLH was amplified by PCR using the forward primer 5′-GTCGACATGGCTGAAGGAATCAAGTCAA-3′ and reverse primer 5′-GCGGCCGCTCAATCAAAGAAGCTCTTGGCTCT-3′. The resulting PCR products were inserted into the Sal I/Not I sites of the pGEX-5X-3 vector. The constructs were confirmed by DNA sequencing. The cDNA of p23 was amplified by PCR using the forward primer 5′-CATATGCAGCCTGCTTCTGCAAAGTG-3′ and reverse primer 5′-GAATTCTTACTCCAGATCTGGCATTTTT-3′. The resulting PCR products were inserted into the Nde I/EcoR I sites of the pET21a vector. The constructs were confirmed by DNA sequencing. cDNA of XAP2 was amplified by PCR using the forward primer 5′-CATATGGCGGATATCATCGCAAG-3′ and reverse primer 5′-GAATTCTCAATGGGAGAAGATCCCC-3′. The resulting PCR products were inserted into the Nde I/EcoR I sites of the pET21a vector. The constructs were confirmed by DNA sequencing. HSP90, HSP90 N-, M-, C-, ΔN-, ΔM-andΔC-domains were amplified by PCR using the following primers: HSP90 and HSP90 N-domain N 5′-GGATCCATGCCTGAGGAAACCCAGACC-3′, HSP90 and HSP90 C-domain C 5′’-TCTAGATTAGTCTACTTCCATGCGTGA-3′, HSP90 N-domain C 5′-TCTAGATTCAGCCTCATCATCGCGTGA-3′, HSP90 C-domain N 5′-GGATCCGGTTACATGGCAG-3′, HSP90 M-domain N 5′-CATATGCTCAACAAAACAAAGCCCATC-3′, and HSP90 M-domain C 5′-CTCGAGTTCCAGGCCTTCTTTGGT-3′. The PCR products of HSP90, HSP90 N- and C-domains were digested with BamH I and Xba I restriction enzymes and cloned into the pCold I vector (TAKARA BIO, Inc. Japan). The PCR product of the HSP90 M-domain was digested with Nde I and Xho I restriction enzymes and cloned into the pET15b vector (Novagen, Inc. Japan). Recombinant protein expression and purification The bHLH was expressed in an Escherichia coli BL21 (DE3) arctic competent cell. The cells were grown at 37 °C, 250 rpm in LB BROTH medium supplemented with 100 μg/ml ampicillin until the OD600 reached 0.5. The cells were then induced by the addition of 0.1 mM IPTG, and the culture medium was incubated at 37 °C, 250 rpm for an additional 3 h. The cells were harvested by centrifugation at 4 °C, 13,000 rpm for 15 min, and cell pellets were suspended in 10 mM Tris-HCl pH 7.4. The cells were sonicated, centrifuged at 4 °C, 15,000 rpm for 15 min and the formed pellets were collected. The collected pellets were suspended in buffer (1 M Arginine, 10 mM Tris-HCl pH 7.4), then dialyzed with 10 mM Tris-HCl pH7.4, overnight to remove the Arginine. After dialysis, the lysates were cleared by centrifugation at 4 °C, 15,000 rpm for 15 min. The supernatant was applied to glutathione columns (Glutathione Sepharose 4B), washed with 10 mM Tris-HCl pH 7.4, and then eluted with elution buffer (20 mM Glutathione/10 mM Tris-HCl pH 7.4). Finally, the eluted proteins were concentrated by ultrafiltration. The p23 was expressed in an E. coli BL21(DE3) pLysS competent cell. The cells were grown at 37 °C, 250 rpm in LB BROTH medium supplemented with 100 μg/ml ampicillin and 30 μg/l chloramphenicol until the OD600 reached 0.5. The cells were then induced by the addition of 0.5 mM IPTG, and the culture medium was incubated at 37 °C, 250 rpm for an additional 3 h. The cells were harvested by centrifugation at 4 °C, 13,000 rpm for 15 min, and the cell pellets were suspended in 10 mM sodium acetate pH 6.0. The cells were sonicated, centrifuged at 4 °C, 15,000 rpm for 15 min, then the supernatants were collected. Proteins were applied to the DEAE column, and then washed with 10 mM sodium acetate buffer pH 6.0/0.17 M NaCl. After washing, the proteins were eluted with a linear gradient of 0.17–0.6 M NaCl in 10 mM sodium acetate buffer pH 6.0. The p23 fractions were dialyzed overnight with 10 mM Tris-HCl pH 7.4. After dialysis, the lysates were applied to the Q-sepharose column. Then, washed with 10 mM Tris-HCl pH 7.4/0.1 M NaCl and the proteins were eluted with linear gradient of 0.1–0.6 M NaCl in 10 mM Tris-HCl pH 7.4. The collected protein fractions were concentrated by ammonium sulphate fractionation and 60–90% fractions were collected. The protein fractions were applied to the sephacryl column and eluted with buffer (10 mM Tris-HCl pH 7.4/5% Glycerol/0.1 M NaCl). Finally, the collected fractions were concentrated by ultrafiltration. The XAP2 was expressed in an E. coli BL21 (DE3) arctic competent cell. The cells were grown at 37 °C, 250 rpm in LB BROTH medium supplemented with 100 μg/ml ampicillin until the OD600 reached 0.5. The culture medium was cooled for 30 min to 15 °C. The cells were then induced by the addition of 0.5 mM IPTG and incubated at 15 °C, 250 rpm for an additional 24 hr. The cells were harvested by centrifugation at 4 °C, 13,000 rpm for 15 min, and cell pellets were suspended in 10 mM Tris-HCl pH 7.4. The cells were sonicated for two cycles, centrifuged at 4 °C, 15,000 rpm for 15 min, then the supernatants were collected. Proteins were applied to the Heparin column, then washed with 10 mM Tris-HCl pH 7.4. After washing, the proteins were eluted with a linear gradient of 0-0.5 M NaCl in 10 mM Tris-HCl pH 7.4. The XAP2 fractions were dialyzed overnight with 10 mM Tris-HCl pH 7.4. After dialysis, collected proteins were applied to the Q-sepharose column, washed with 10 mM Tris-HCl pH 7.4. And then, proteins were eluted with a linear gradient of 0–0.6 M NaCl in 10 mM Tris-HCl pH 7.4. Finally, the eluted proteins were concentrated by ultrafiltration. The HSP90 N-, M-, C-, ΔN-, ΔM-, and ΔC-domains were expressed as a 6 × His fusion protein from the expression vector pCold I in the BL21 E. coli cells. The HSP90 M-domain was expressed as a 6 × His fusion protein from the expression vector pET15b in the arctic express (DE3) E. coli cells. The expression of the HSP90 N-, M-, C-, ΔN-, ΔM-, and ΔC-domains were induced by 0.5 mM IPTG. The cells were collected and each of cell extracts was applied to the Ni-NTA column, washed and eluted the same as the other chaperones. The AhR-ΔAD domain was amplified by PCR (iCycler, BioRad) using the forward primer 5′-GTCGACATGAACAGCAGCAGCC GCCAAC-3′ and reverse primer 5′-CTCGAGC TATTTTCGTAAATGCTCTGTTCC-3′. The resulting PCR products were inserted into the SalI/XhoI sites of the pGEX-5X-3 vector (Takara Bio, Japan). The constructs were confirmed by DNA sequencing (PRISM 3100, ABI). The GST-tagged AhR-ΔAD domain was expressed in an Escherichia coli BL21 (DE3) Arctic Competent Cell (Stratagene). The cells were grown at 30 °C in LB BROTH medium (Invitrogen) supplemented with 100μg/ml ampicillin for 3 hr, then cultured at 10 °C for 30 min. The cells were next induced by the addition of 0.5 mM IPTG and the cultures were incubated at 10 °C for an additional 24 hr. The cells were harvested by centrifugation at 20,000xg for 15 min at 4 °C, and the cell pellets were suspended in 10mM Tris-HCl, pH 7.4. The cells were sonicated, centrifuged at 20,000xg for 10 min at 4 °C and the formed pellets collected. The collected pellets were suspended in buffer (1M Arginine, 10 mM Tris-HCl, pH 7.4), then dialyzed with 10 mM Tris-HCl, pH 7.4, overnight to remove the Arginine. After dialysis, the lysates were cleared by centrifugation at 20,000 xg for 10 min at 4 °C. The supernatant was applied to glutathione columns (Glutathione Sepharose 4B, GE Healthcare Life Science), washed with 10 mM Tris-HCl, pH 7.4, and then eluted with elution buffer (20 mM Glutathione, 10 mM Tris-HCl, pH 7.4). The eluted proteins were concentrated by ultrafiltration. Antibody production An anti-p23 and anti-XAP2 antibody were produced by intramuscular injection into a rabbit of 1 mg of the purified each protein emulsified in complete Freund's adjuvant. Booster shots were given 3 times in the same manner as the original injection at 2-week intervals. The rabbit was bled 10 days after the last injection. The protocols for animal experimentation described in this paper were previously approved by the Animal Research Committee, Akita University School of Medicine; the “Guidelines for Animal Experimentation” of the University were completely adhered to in all subsequent animal experiments. XRE affinity chromatography and gel-shift assay The synthetic oligonucleotide which consist of four-tandem repeats of human XRE or CY3-XRE [10mer; (CY3) 5′-TTGCGTGCGG-3′] (19) were prepared (Fasmac Co., Ltd., Atsugi, Japan). XRE-Sepharose was prepared by coupling of XRE and Epoxy-activated Sepharose 6B (GE Healthcare Life Science) according to the manufacturer’s instructions. The purified GST-bHLH or GST were added to XRE-Sepharose or Mock (without XRE)-Sepharose column equilibrated with buffer (25 mM HEPES-KOH pH 7.4/5% Glycerol/0.1% NP-40/5 mM MgCl2) and incubated with gentle rotation using a rotator for 30 min at 4 °C. After washing with the same buffer three times, the bound proteins were separated by SDS-PAGE. Purified GST-bHLH (0.5 µM) and CY3-XRE (0.5 µM) were incubated with Binding Buffer (15 mM Tris-HCl pH 7.4, 75 mM NaCl, 1.5 mM EDTA, 1.5 mM DTT, 7.5% Glycerol, 0.5% NP-40) for 30 min at 4 °C. Gel-shift assay was performed using 4% gel and gel-shift mobility was detected using ChemiDoc XRS+ (BioRad). GST pull-down assay and 6 × His pull-down assay For the GST pull-down assay, 2.5 μM GST-bHLH, GST-ΔAD, or GST protein was added to a solution of 2.5 μM HSP90, p23, XAP2, 1 mM ATP and 150 μl buffer A (0.1 M KCl/10 mM MgCl2/20 mM Na2MoO4/0.6 M NaCl/5% Glycerol/0.1% NP-40 in 25 mM HEPES-KOH pH 7.4). The total volume of the sample was 300 μl by adding buffer B (5% Glycerol/0.1% NP-40 in 25 mM HEPES-KOH pH 7.4) and incubated using a rotator with gentle rotation at 37 °C for 15 min. The samples were loaded onto a GST resin equilibrated with buffer C (50 mM KCl/5 mM MgCl2/10 mM Na2MoO4/0.3 M NaCl/5% Glycerol/0.1% NP-40 in 25 mM HEPES-KOH pH 7.4) and incubated for 15 min at 4 °C with gentle rotation followed by centrifuged at 4 °C, 5,000 rpm for 10 s to remove the supernatant. The beads were washed three times with buffer C and eluted by boiling at 100 °C for 5 min in SDS sample buffer. GST pull-down samples were separated by SDS-PAGE and immune-blot. An antibody against HSP90 was used as previously reported (17). Ni2+ pull-down assay For the Ni2+ pull-down assay, 2.5 μM HSP90, XAP2, p23 and 1 mM ATP was added to 150 µl buffer D (25 mM HEPES-KOH pH 7.4/40 mM Imidazole/0.1% NP-40) and buffer E (25 mM HEPES-KOH pH 7.4/0.1% NP-40) upto 300 µl of total volume. The sample was incubated using a rotator with gentle rotation at 37 °C for 15 min. The incubated solution was loaded onto Ni2+-Sepharose resin equilibrated with buffer F (25 mM HEPES-KOH pH 7.4/20 mM Imidazole/0.1% NP-40). After incubation for 15 min at 4 °C with gentle rotation, centifuged at 4 °C, 5,000 rpm for 10 s to remove the supernatant. The beads were washed three times with buffer F and eluted by boiling at 100 °C for 5 min in SDS sample buffer. Ni2+ pull-down samples were separated by SDS-PAGE. Results Co-localization of AhR/HSP90 complex in the nucleus We have reported that AhR translocated from cytoplasm to nucleus with HSP90 under treatment with β-NF (17). In the current study, we confirmed of AhR nuclear translocation with chaperone complex in the presence of 3MC. As shown in Fig. 1A–C, we confirmed that the AhR-chaperone complex located in the nucleus in the presence of 3MC. Fig. 1 View largeDownload slide Co-localization of AhR and HSP90-chaperone complex. HeLa cells were treated with the vehicle or 3 µM 3MC for 2 h. Cells were incubated with anti-HSP90β and anti-AhR antibody (A), anti-HSP90 β and anti-p23 antibody (B), and anti-HSP90β and anti-XAP2 antibody (C). Blue staining indicatees DAPI staining of cell nuclei (A–C). Images were taken at 630× magnification. Fig. 1 View largeDownload slide Co-localization of AhR and HSP90-chaperone complex. HeLa cells were treated with the vehicle or 3 µM 3MC for 2 h. Cells were incubated with anti-HSP90β and anti-AhR antibody (A), anti-HSP90 β and anti-p23 antibody (B), and anti-HSP90β and anti-XAP2 antibody (C). Blue staining indicatees DAPI staining of cell nuclei (A–C). Images were taken at 630× magnification. Purification of GST-bHLH and DNA-binding ability The AhR activation mechanism is poorly understood in vitro. In the previous study, we have reported that the AhR-PAS domain is a HSP90 binding domain, and both AhR and HSP90 translocate to nuclear from cytoplasm (17). The AhR-bHLH domain has nuclear localization signal (NLS) and nuclear export signal (NES), so necessary for the transport to the nucleus. The AhR-bHLH domain is known to be as the HSP90 binding domain (13, 14). However, direct interactions between AhR-bHLH and HSP90 in vitro have not yet reported. In the present study, we focused on the AhR-bHLH domain and analyzed relations between AhR-bHLH and the molecular chaperone HSP90. GST-bHLH was expressed in E. coli for using in GST pull-down assay. A purification of GST-bHLH was carried out by GST affinity column chromatography. As shown in Fig. 2A, the purified bHLH, having about 33-kDa molecular mass, was a single protein band on SDS-PAGE. If bHLH has correct structure, it may be able to bind to XRE (Xenobiotic responsible element, so called dioxin responsible element). We analyzed the DNA-binding ability of bHLH using a XRE-Sepharose affinity column. Because of the 3′ end of XRE has OH- groups, we prepared the XRE affinity resin using an Epoxy-activated Sepharose 6B. Epoxy-activated Sepharose 6B is useful resin to fix the OH-, or NH2-groups. No protein bands were shown in mock-columns (Fig. 2B, lanes 1 and 3). Although almost of GST was not able to bind to XRE-Sepharose affinity resins (Fig. 2B, lane 2), certain amount of bHLH could bind to the affinity resin (Fig. 2B, lane 4). We also investigated the XRE binding ability of bHLH using gel mobility-shift assay (Fig. 2C). We could detect signals at higher end of the gels only in the presence of bHLH. No gel mobility-shift has been detected in the presence of GST. These results suggested that the purified bHLH possesses a DNA-binding ability. Fig. 2 View largeDownload slide Purification of GST-bHLH. (A) GST-bHLH was purified using a GST affinity column. Apply sample is suprnatant after sonication of GST-bHLH expressed in E. coli and centrifugation. The column was washed with 10 mM Tris-HCl pH 7.4. GST and GST-bHLH were eluted by Glutathione. Eluted proteins were analyzed by SDS-PAGE (11% gel). (B) The purified protein and GST (input) were incubated with to Mock resin or XRE-Sepharose affinity resin at 4 °C for 30 min, and the bound proteins were separated by SDS-PAGE (9% gel). Lanes 1, 3 and 2, 4 indicate Mock resins and XRE-Sepharose affinity resins, respectively. (C) The purified protein and GST (input) were incubated with CY3-XRE at 4 °C for 30 min, samples were separated by SDS-PAGE (4% gel). Free, GST, and GST-bHLH indicates CY3-XRE, CY3-XRE/GST, and CY3-XRE/GST-bHLH, respectively. Single- and double-closed triangles indicate free and protein bound CY3-XRE, respectively. Fig. 2 View largeDownload slide Purification of GST-bHLH. (A) GST-bHLH was purified using a GST affinity column. Apply sample is suprnatant after sonication of GST-bHLH expressed in E. coli and centrifugation. The column was washed with 10 mM Tris-HCl pH 7.4. GST and GST-bHLH were eluted by Glutathione. Eluted proteins were analyzed by SDS-PAGE (11% gel). (B) The purified protein and GST (input) were incubated with to Mock resin or XRE-Sepharose affinity resin at 4 °C for 30 min, and the bound proteins were separated by SDS-PAGE (9% gel). Lanes 1, 3 and 2, 4 indicate Mock resins and XRE-Sepharose affinity resins, respectively. (C) The purified protein and GST (input) were incubated with CY3-XRE at 4 °C for 30 min, samples were separated by SDS-PAGE (4% gel). Free, GST, and GST-bHLH indicates CY3-XRE, CY3-XRE/GST, and CY3-XRE/GST-bHLH, respectively. Single- and double-closed triangles indicate free and protein bound CY3-XRE, respectively. AhR-bHLH domain binds to HSP90, XAP2 and p23 We investigated that an association between bHLH and HSP90 by GST pull-down assay. As shown in Fig. 3A, HSP90 protein bands were detected in only GST-bHLH lanes, not in GST lanes. These results showed that the AhR-bHLH domain is also a HSP90 binding domain as same as the AhR-PAS domain. Its interaction was not changed in the presence or absence of ATP. We analyzed an effect of 17-DMAG on the interaction (Fig. 3B). The interaction between bHLH and HSP90 was not also affected by 17-DMAG in the presence or absence of ATP. Fig. 3 View largeDownload slide GST pull-down assay confirming the interaction of HSP90 with AhR-bHLH domain. (A) An association between AhR-bHLH and HSP90 was analyzed by GST pull-down assay in the absence or presence of 1 mM ATP. GST-bHLH or GST, HSP90, and ATP were incubated with GST resins. After washing, Glutathione specific-binding proteins were analyzed by SDS-PAGE (11% gel). Lanes 1–3 of gels were the inputs from purified GST (28 kDa), GST-bHLH (33 kDa), and HSP90 (90 kDa) as a control, respectively. (B) The purified GST, GST-bHLH, and HSP90 were incubated with GST resins in the absence or presence of 50 μM 17-DMAG. The elutants from the glutathione columns were analyzed by SDS-PAGE (11% gel). Pull-down assays were performed using purified GST or the GST-AhR-PAS domain and purified HSP90 in the absence (−) or presence (+) of ATP. Fig. 3 View largeDownload slide GST pull-down assay confirming the interaction of HSP90 with AhR-bHLH domain. (A) An association between AhR-bHLH and HSP90 was analyzed by GST pull-down assay in the absence or presence of 1 mM ATP. GST-bHLH or GST, HSP90, and ATP were incubated with GST resins. After washing, Glutathione specific-binding proteins were analyzed by SDS-PAGE (11% gel). Lanes 1–3 of gels were the inputs from purified GST (28 kDa), GST-bHLH (33 kDa), and HSP90 (90 kDa) as a control, respectively. (B) The purified GST, GST-bHLH, and HSP90 were incubated with GST resins in the absence or presence of 50 μM 17-DMAG. The elutants from the glutathione columns were analyzed by SDS-PAGE (11% gel). Pull-down assays were performed using purified GST or the GST-AhR-PAS domain and purified HSP90 in the absence (−) or presence (+) of ATP. We also analyzed the effects of co-chaperones p23 and XAP2 to the bHLH-HSP90 complex on SDS-PAGE. No proteins bound to GST (Fig. 4A). We could detect HSP90 and bHLH on SDS-PAGE, but XAP2 and p23 were difficult to detect, so confirmed in immune-blot using antibodies against HSP90, p23 and XAP2. The p23 and XAP2 protein bands were shown in GST-bHLH lanes (Fig. 4B). Thus, these results supports that AhR-bHLH interacts with HSP90, p23 and XAP2 in the cytoplasm. Furthermore, the complex was not affected by ATP. We investigated HSP90 co-chaperone complex using Ni2+ pull-down assay. As shown in Figs 5 and 6 × His-HSP90 binds to the Ni2+-Sepharose resin. We could detect HSP90, XAP2 and p23 protein bands on SDS-PAGE in the absence or presence of ATP. These results suggested that HSP90 makes a complex with XAP2 and p23 and AhR-bHLH binds to HSP90 complex via HSP90. Fig. 4 View largeDownload slide Conformational analysis of AhR, HSP90, and co-chaperone complex. An AhR-bHLH binding protein was identified by GST pull-down assay. In the assay, GST resins were incubated with HSP90, p23, XAP2, and GST or GST-bHLH, in addition, in the absence or presence of 1 mM ATP. The binding proteins were analyzed by SDS-PAGE (9% gel) (A) or immune-blot using anti-HSP90, anti-p23 and anti-XAP2 antibodies (B). Fig. 4 View largeDownload slide Conformational analysis of AhR, HSP90, and co-chaperone complex. An AhR-bHLH binding protein was identified by GST pull-down assay. In the assay, GST resins were incubated with HSP90, p23, XAP2, and GST or GST-bHLH, in addition, in the absence or presence of 1 mM ATP. The binding proteins were analyzed by SDS-PAGE (9% gel) (A) or immune-blot using anti-HSP90, anti-p23 and anti-XAP2 antibodies (B). Fig. 5 View largeDownload slide Conformational analysis of chaperone complex. The HSP90 and co-chaperones binding properties were identified by Ni2+ pull-down assay. In the assay, Ni2+-Sepharose resins were incubated with HSP90, p23, and XAP2 in the presence or absence of 1 mM ATP. The binding proteins were analyzed by SDS-PAGE (11% gel). Fig. 5 View largeDownload slide Conformational analysis of chaperone complex. The HSP90 and co-chaperones binding properties were identified by Ni2+ pull-down assay. In the assay, Ni2+-Sepharose resins were incubated with HSP90, p23, and XAP2 in the presence or absence of 1 mM ATP. The binding proteins were analyzed by SDS-PAGE (11% gel). Fig. 6 View largeDownload slide HSP90 and AhR domains. (A) Human HSP90 N-, M-, and C-domains or ΔN-, ΔM-, and ΔC-deletion mutants of HSP90 were constructed and purified under “Materials and Methods”. (B) The domain structure of human AhR. Fig. 6 View largeDownload slide HSP90 and AhR domains. (A) Human HSP90 N-, M-, and C-domains or ΔN-, ΔM-, and ΔC-deletion mutants of HSP90 were constructed and purified under “Materials and Methods”. (B) The domain structure of human AhR. bHLH binding domains of HSP90 HSP90 is composed from 3 domains. ATP binding domain (N domain), substrate binding domain (M domain), and dimerization domain (C domain) (20). We constructed and purified HSP90 domains and investigated the bHLH binding domain of HSP90 (Fig. 6A). The domain structure of human AhR was also shown in Fig. 6B. At first, we analyzed interactions between the GST-ΔAD binding domain of HSP90 using HSP90 domains (N-, M- and C-domain) as control. We could detect that AhR-PAS bound to the HSP90N domain (Fig. 7A). Neither HSP90M nor HSP90C domain bound to GST-ΔAD. Then, we investigated the interactions between bHLH and each HSP90 domain (N, M and C) using GST-pull down assay (Fig. 7B). No proteins bound to GST. On the contrary, HSP90 N-domain was pull-downed with bHLH. We could not detect other domains (HSP90 M- and HSP90 C-domain) on SDS-PAGE. ATP did not affect the interaction. We also confirmed the binding of the bHLH domain to HSP90 using HSP90 deletion mutants (HSP90-ΔC, ΔM and ΔN). As shown in Fig. 7C, no HSP90 deletion mutants were interacted with GST. Both HSP90-ΔC and -ΔM interacted with bHLH but not HSP90-ΔN (The same data were shown in Fig. 7B and C). These data suggested that AhR-bHLH bound to the HSP90 N-domain. Fig. 7 View largeDownload slide GST pull-down assay confirming the interaction of HSP90N-, M-, and C-domains and GST-ΔAD or GST-bHLH. (A) Purified GST, GST-ΔAD, HSP90 N-, M-, and C-domain were incubated with GST resins in the absence or presence of ATP. The elutants from the glutathione column were analyzed by SDS-PAGE (11% gel). Lanes 1–5 of gels were the inputs from purified HSP90 N-domain (38 kDa), HSP90 M-domain (40 kDa), HSP90 C-domain (16 kDa), GST (28 kDa), and GST-ΔAD (57 kDa) as a control, respectively. Pull-down assays were performed using purified GST (lanes 6–11) or the GST-AhR-PAS domain (lanes 12–17) and purified HSP90 in the absence (−) or presence (+) of ATP. (B) The purified GST, GST-bHLH, HSP90N-, M-, and C-domains were incubated with GST resins in the absence or presence of ATP. The elutants from the glutathione columns were analyzed by SDS-PAGE (11% gel). Lanes 1–5 of gels were the inputs from purified HSP90 N- domain (38 kDa), HSP90 M-domain (40 kDa), HSP90 C-domain (16 kDa), GST (28 kDa), and GST-bHLH (33 kDa) as a control, respectively. Pull-down assays were performed using purified GST or the GST-AhR-PAS domain and purified HSP90 in the absence (−) or presence (+) of ATP. (C) The purified GST, GST-bHLH, HSP90ΔN-, ΔM-, and ΔC-deletion mutants were incubated with GST resins in the absence or presence of ATP. The elutants from the glutathione columns were analyzed by SDS-PAGE (9% gel). Lanes 1-5 of gels were the inputs from purified HSP90ΔN (68 kDa), HSP90ΔC (60 kDa), HSP90ΔM (45 kDa), GST (28 kDa), and GST-bHLH (33 kDa) as a control, respectively. Pull-down assays were performed using purified GST or the GST-AhR-PAS domain and purified HSP90 in the absence −) or presence (+) of ATP. (D) Ni2+ pull-down assays were performed as described in the “Materials and Methods”. The purified HSP90N-, M-, C-domain, HSP90ΔN-, ΔC-deletion mutants, XAP2, and p23 were incubated with Ni2+-Sepharose resin in the absence or presence of ATP. Ni2+ pull-down samples were analyzed by SDS-PAGE (11% gel). Lanes 1–7 of gels were the inputs from purified HSP90N-domain (38 kDa), HSP90ΔC (60 kDa), HSP90 M-domain (40 kDa), HSP90ΔN (68 kDa), HSP90 C-domain (16 kDa), XAP2 (38 kDa), and p23 823 kDa) as a control, respectively. Ni-NTA pull-down assays were performed using purified HSP90N-domain, HSP90ΔC, HSP90M-domain, HSP90ΔN, and HSP90C-domain in the absence (−) or presence (+) of ATP. Fig. 7 View largeDownload slide GST pull-down assay confirming the interaction of HSP90N-, M-, and C-domains and GST-ΔAD or GST-bHLH. (A) Purified GST, GST-ΔAD, HSP90 N-, M-, and C-domain were incubated with GST resins in the absence or presence of ATP. The elutants from the glutathione column were analyzed by SDS-PAGE (11% gel). Lanes 1–5 of gels were the inputs from purified HSP90 N-domain (38 kDa), HSP90 M-domain (40 kDa), HSP90 C-domain (16 kDa), GST (28 kDa), and GST-ΔAD (57 kDa) as a control, respectively. Pull-down assays were performed using purified GST (lanes 6–11) or the GST-AhR-PAS domain (lanes 12–17) and purified HSP90 in the absence (−) or presence (+) of ATP. (B) The purified GST, GST-bHLH, HSP90N-, M-, and C-domains were incubated with GST resins in the absence or presence of ATP. The elutants from the glutathione columns were analyzed by SDS-PAGE (11% gel). Lanes 1–5 of gels were the inputs from purified HSP90 N- domain (38 kDa), HSP90 M-domain (40 kDa), HSP90 C-domain (16 kDa), GST (28 kDa), and GST-bHLH (33 kDa) as a control, respectively. Pull-down assays were performed using purified GST or the GST-AhR-PAS domain and purified HSP90 in the absence (−) or presence (+) of ATP. (C) The purified GST, GST-bHLH, HSP90ΔN-, ΔM-, and ΔC-deletion mutants were incubated with GST resins in the absence or presence of ATP. The elutants from the glutathione columns were analyzed by SDS-PAGE (9% gel). Lanes 1-5 of gels were the inputs from purified HSP90ΔN (68 kDa), HSP90ΔC (60 kDa), HSP90ΔM (45 kDa), GST (28 kDa), and GST-bHLH (33 kDa) as a control, respectively. Pull-down assays were performed using purified GST or the GST-AhR-PAS domain and purified HSP90 in the absence −) or presence (+) of ATP. (D) Ni2+ pull-down assays were performed as described in the “Materials and Methods”. The purified HSP90N-, M-, C-domain, HSP90ΔN-, ΔC-deletion mutants, XAP2, and p23 were incubated with Ni2+-Sepharose resin in the absence or presence of ATP. Ni2+ pull-down samples were analyzed by SDS-PAGE (11% gel). Lanes 1–7 of gels were the inputs from purified HSP90N-domain (38 kDa), HSP90ΔC (60 kDa), HSP90 M-domain (40 kDa), HSP90ΔN (68 kDa), HSP90 C-domain (16 kDa), XAP2 (38 kDa), and p23 823 kDa) as a control, respectively. Ni-NTA pull-down assays were performed using purified HSP90N-domain, HSP90ΔC, HSP90M-domain, HSP90ΔN, and HSP90C-domain in the absence (−) or presence (+) of ATP. Discussion The AhR activation mechanism has not yet been fully understood. AhR exists with HSP90, co-chaperone p23 and XAP2 in the cytoplasm. AhR has PAS and bHLH domains. The PAS domain is a ligand binding domain and a HSP90 binding domain. Recently, we have reported that the PAS domain binds to HSP90 directly (17). Because of the difficulty to purify the full length of AhR, we used some purified domains of AhR for experiments. We purified the PAS domain and checked the ligand-binding ability of purified the PAS domain using a β-naphthoflavone (β-NF) affinity resin. In this study, we investigated whether AhR-bHLH bound to HSP90, p23 and XAP2 or not. The AhR-bHLH domain has NLS, NES and the XRE binding domain. Moreover, the AhR-bHLH domain has been thought to be a HSP90 binding domain. First, we confirmed that purified AhR-bHLH protein bound to XRE. In the present study, we used XRE affinity resins. An oligonucleotide has OH- group in the 3′ end. We fixed the four tandem XRE to Epoxy Sepharose 6B columns. The affinity resin is able to fix the OH- or NH2- group of protein, ligand, and DNA. Until now, we have a number of reports in the drug-affinity (21–25). The purified bHLH could bind to XRE affinity resins, but not mock resins. GST did not bind to either XRE affinity resins or mock resins. We also analyzed the interaction between DNA and protein using gel mobility-shift assay. We used CY3-XRE at the methods. GST didn’t show the interaction. On the contrary, we could detect the gel mobility-shift between CY3-XRE and GST-bHLH. Thus, we confirmed the purification of functional bHLH. So far some of the detail of the interaction between AhR and HSP90 has been published. However, few data showed direct relations in molecular level. A nucleotide binding to the HSP90 N-domain induces a directionality and a conformational cycle. In the absence of ATP, HSP90 adopts an open conformation (V-shaped form). ATP induces conformational changes of HSP90 from open to closed form (26, 27). In the present study, we have demonstrated the direct interaction of bHLH, HSP90, XAP2 and p23. We have also determined bHLH is the binding domain of HSP90-N domain as same as AhR-PAS. ATP did not affect the interaction of bHLH, HSP90, XAP2 and p23. Interestingly, 17-DMAG did not affect on the interaction between bHLH and HSP90. We have recently reported that HSP90 was dissociated from AhR-PAS in the presence of 17-DMAG (17). The binding sites of bHLH and PAS to the HSP90 N-domain are slightly different each other. We speculate that the bHLH binding site of the HSP90 N-domain may be neighbor of the M-domain. On the contrary, the PAS binding site of the HSP90 N-domain may be end of the N-domain. ATP induces dramatically conformational changes of HSP90 from open to closed form. The conformational change of HSP90N end is bigger than that of HSP90N neighbor M domain. The differences of 17-DMAG to bHLH and PAS are thought to be due to such reasons. We propose the models of the AhR-HSP90 chaperone complex (Fig. 8). Fig. 8 View largeDownload slide Conformational change models of the AhR, HSP90, p23, XAP2 complex. The bHLH and PAS domains of AhR bind to HSP90 N-domain. Co-chaperones p23 and XAP2 bind to HSP90 N- and C-domains, respectively. When in the absence of ligand, the NLS of bHLH is hidden. When the ligands bind to AhR, the conformational changes will be occurred, then the NLS will be opened. Fig. 8 View largeDownload slide Conformational change models of the AhR, HSP90, p23, XAP2 complex. The bHLH and PAS domains of AhR bind to HSP90 N-domain. Co-chaperones p23 and XAP2 bind to HSP90 N- and C-domains, respectively. When in the absence of ligand, the NLS of bHLH is hidden. When the ligands bind to AhR, the conformational changes will be occurred, then the NLS will be opened. Based on the result, we inferred that HSP90 covers NLS when AhR is in the ligand-free state, and the conformational change of AhR complex lead to the exposure of NLS after AhR binds to ligand. For that reason, the AhR-bHLH domain interacts with HSP90. From the above, the AhR-bHLH domain is essential in the AhR activation mechanism similar to the PAS domain. Author Contributions HI in the research design; IK, MH, AH, NT, YN, HO, KF, YK, and TO conducted the experiment. IK, EG and HI wrote or contributed to the writing of the paper. Funding H.I. was supported by a Grant-in-Aid for Scientific Research (Exploratory Research: 16651056) from the Japanese Ministry of Education, Science, Sports and Culture. Conflict of Interest None declared. References 1 Kewley R.J., Whitelaw M.L., Chapman-Smith A. ( 2004) The mammalian basic helix-loop-helix/PAS family of transcriptional regulators. Int. J. Biochem. Cell Biol . 36, 189– 204 Google Scholar CrossRef Search ADS PubMed  2 Sorg O. ( 2014) AhR signaling and dioxin toxicity. Toxicol. Lett.  230, 225– 233 Google Scholar CrossRef Search ADS PubMed  3 Mimura J., Fujii-Kuriyama Y. ( 2003) Functional role of AhR in the expression of toxic effects by TCDD. Biochim. et Biophys. Acta  1619, 263– 268 Google Scholar CrossRef Search ADS   4 Meyer B.K., Pray-Grant M.G., Vanden, Heuvel J.P., Perdew G.H. ( 1998) Hepatitis B virus X-associated protein 2 is a subunit of the unliganded aryl hydrocarbon receptor core complex and exhibits transcriptional enhancer activity. Mol. Cell. Biol . 18, 978– 988 Google Scholar CrossRef Search ADS PubMed  5 Nebert D.W., Gonzalez F. J. ( 1987) P450 genes: structure, evolution, and regulation. Annu. Rev. Biochem . 56, 945– 993 Google Scholar CrossRef Search ADS PubMed  6 Kazlauskas A., Poellinger L., Pongratz I. ( 1999) Evidence that the co-chaperone p23 regulates ligand responsiveness of the dioxin (aryl hydrocarbon) receptor. J. Biol. Chem . 274, 13519– 13524 Google Scholar CrossRef Search ADS PubMed  7 Karagöz G.E., Rüdiger S.G. ( 2015) Hsp90 interaction with clients. Trends Biochem Sci . 40, 117– 125 Google Scholar CrossRef Search ADS PubMed  8 Li J., Buchner J. ( 2013) Structure, function and regulation of the hsp90 machinery. Biomed. J . 36, 106– 117 Google Scholar CrossRef Search ADS PubMed  9 Erlejman A.G., Lagadari M., Toneatto J., Piwien-Pilipuk G., Galigniana M.D. ( 2014) Regulatory role of the 90-kDa-heat-shock protein (Hsp90) and associated factors on gene expression. Biochim Biophys Acta  1839, 71– 87 Google Scholar CrossRef Search ADS PubMed  10 Kobayashi A., Sogawa K., Fujii-Kuriyama Y. ( 1996) Cooperative interaction between AhR·Arnt and Sp1 for the drug-inducible expression of CYP1A1 gene. J. Biol. Chem . 271, 12310– 12316 Google Scholar CrossRef Search ADS PubMed  11 Beischlag T.V., Luis, Morales J., Hollingshead B.D., Perdew G.H. ( 2008) The aryl hydrocarbon receptor complex and the control of gene expression. Crit. Rev. Eukaryot. Gene Expr . 18, 207– 250 Google Scholar CrossRef Search ADS PubMed  12 Wu D., Potluri N., Kim Y., Rastinejad F. ( 2013) Structure and dimerization properties of the aryl hydrocarbon receptor PAS-A domain. Mol. Cell Biol . 33, 4346– 4356 Google Scholar CrossRef Search ADS PubMed  13 McGuire J., Coumailleau P., Whitelaw M.L., Gustafsson J.A., Poellinger L. ( 1996) The basic helix-loop-helix/PAS factor Sim is associated with hsp90. Implications for regulation by interaction with partner factors. J. Biol. Chem . 270, 31353– 31357 Google Scholar CrossRef Search ADS   14 Antonsson C., Arulampalam V., Whitelaw M.L., Pettersson S., Poellinger L. ( 1995) Constitutive function of the basic helix-loop-helix/PAS factor Arnt. Regulation of target promoters via the E box motif. J. Biol. Chem . 270, 13968– 13972 Google Scholar CrossRef Search ADS PubMed  15 Feng S., Cao Z., Wang X. ( 2013) Role of aryl hydrocarbon receptor in cancer. Biochim Biophys Acta  1836, 197– 210 Google Scholar PubMed  16 Kazlauskas A., Poellinger L., Pongratz I. ( 2000) The immunophilin-like protein XAP2 regulates ubiquitination and subcellular localization of the dioxin receptor. J. Biol. Chem . 275, 41317– 41324 Google Scholar CrossRef Search ADS PubMed  17 Tsuji N., Fukuda K., Nagata Y., Okada H., Haga A., Hatakeyama S., Yoshida S., Okamoto T., Hosaka M., Sekine K., Ohtaka K., Yamamoto S., Otaka M., Grave E., Itoh H. ( 2014) The activation mechanism of the aryl hydrocarbon receptor (AhR) by molecular chaperone HSP90. FEBS Open Bio . 4, 796– 803 Google Scholar CrossRef Search ADS PubMed  18 Fukunaga B.N., Probst M.R., Reisz-Porszasz S., Hankinson O. ( 1995) Identification of functional domain of the aryl hydrocarbon receptor. J. Biol. Chem . 270, 29270– 29278 Google Scholar CrossRef Search ADS PubMed  19 Lakhman S.S., Chen X., Gonzalez-Covarrubias V., Schuetz E.G., Blanco J.G. ( 2007) Functional characterization of the promoter of human carbonyl reductase 1 (CBR1). Role of XRE elements in mediating the induction of CBR1 by ligands of the aryl hydrocarbon receptor. Mol. Pharmacol . 72, 734– 743 Google Scholar CrossRef Search ADS PubMed  20 Ali M.M., Roe S.M., Vaughan C.K., Meyer P., Panaretou B., Piper P.W., Prodromou C., Pearl L.H. ( 2006) Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature  440, 1013– 1017 Google Scholar CrossRef Search ADS PubMed  21 Itoh H., Komatsuda A., Wakui H., Miura A.B., Tashima Y. ( 1999) Mammalian HSP60 is a major target for an immunosuppressant mizoribine. J. Biol. Chem . 274, 35147– 35151 Google Scholar CrossRef Search ADS PubMed  22 Itoh H., Ogura M., Komatsuda A., Wakui H., Miura A.B., Tashima Y. ( 1999) A novel chaperone-activity-reducing mechanism of the 90-kDa molecular chaperone HSP90. Biochem. J . 343, 697– 703 Google Scholar CrossRef Search ADS PubMed  23 Ishida R., Takaoka Y., Yamamoto S., Miyazaki T., Otaka M., Watanabe S., Komatsuda A., Wakui H., Sawada K., Kubota H., Itoh H. ( 2008) Cisplatin differently affects amino terminal and carboxyl terminal domains of HSP90. FEBS Lett . 582, 3879– 3883 Google Scholar CrossRef Search ADS PubMed  24 Miyazaki T., Sagawa R., Honma T., Noguchi S., Harada T., Komatsuda A., Ohtani H., Wakui H., Sawada K., Otaka M., Watanabe S., Jikei M., Ogawa N., Hamada F., Itoh H. ( 2004) 73-kDa molecular chaperone HSP73 is a direct target of antibiotic gentamicin. J. Biol. Chem . 279, 17295– 17300 Google Scholar CrossRef Search ADS PubMed  25 Yamamoto S., Nakano S., Owari K., Fuziwara K., Ogawa N., Otaka M., Tamaki K., Watanabe S., Komatsuda A., Wakui H., Sawada K., Kubota H., Itoh H. ( 2010) Gentamicin inhibits HSP70-assisted protein folding by interfering with substrate recognition. FEBS Lett . 584, 645– 651 Google Scholar CrossRef Search ADS PubMed  26 Saibil H. ( 2013) Chaperone machines for protein folding, unfolding and disaggregation. Nat. Rev. Mol. Cell Biol . 14, 630– 642 Google Scholar CrossRef Search ADS PubMed  27 Röhl A., Rohrberg J., Buchner J. ( 2013) The chaperone Hsp90: changing partners for demanding clients. Trends Biochem. Sci . 38, 253– 262 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations AhR aryl hydrocarbon receptor bHLH basic helix-loop-helix CYP1A1 cytochrome P450 1A1 17-DMAG 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin GST glutathione S-transferase HSP90 90-kDa of heat shock protein IPTG Isopropyl-1-thio-β-D-galactopyranoside 3MC 3-methylcholanthrene Arnt, AhR nuclear translocator NES nuclear export signal NLS nuclear localization signal PAS per-arnt-sim TCDD 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin XAP2 hepatitis B virus X-associated protein XRE xenobiotic responsible element © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Journal

The Journal of BiochemistryOxford University Press

Published: Mar 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 12 million articles from more than
10,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Unlimited reading

Read as many articles as you need. Full articles with original layout, charts and figures. Read online, from anywhere.

Stay up to date

Keep up with your field with Personalized Recommendations and Follow Journals to get automatic updates.

Organize your research

It’s easy to organize your research with our built-in tools.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve Freelancer

DeepDyve Pro

Price
FREE
$49/month

$360/year
Save searches from Google Scholar, PubMed
Create lists to organize your research
Export lists, citations
Access to DeepDyve database
Abstract access only
Unlimited access to over
18 million full-text articles
Print
20 pages/month
PDF Discount
20% off