HSF1 pathway through the

Free Radical Biology and Medicine 104 (2017) 118–128

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Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed

Original article

1,4-Naphthoquinone activates the HSP90/HSF1 pathway through the Sarylation of HSP90 in A431 cells: Negative regulation of the redox signal transduction pathway by persulfides/polysulfides

MARK

Yumi Abikoa,b, Liang Shac, Yasuhiro Shinkaia,b,c, Takamitsu Unokia, Nho Cong Luongb, ⁎ Yukihiro Tsuchiyad, Yasuo Watanabed, Reiko Hirosea, Takaaki Akaikee, Yoshito Kumagaia,b,c, a

Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan Leading Graduate School Doctoral Program, Ph.D. Program in Human Biology, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan d Laboratory of Pharmacology, Showa Pharmaceutical University, Tokyo 194-8543, Japan e Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8575, Japan b c

A R T I C L E I N F O

A B S T R A C T

Keywords: 1,4-Naphthoquinone Electrophile Heat shock protein 90 Heat shock factor 1 Persulfides Polysulfides

The current consensus is that environmental electrophiles activate redox signal transduction pathways through covalent modification of sensor proteins with reactive thiol groups at low concentrations, while they cause cell damage at higher concentrations. We previously exposed human carcinoma A431 cells to the atmospheric electrophile 1,4-naphthoquinone (1,4-NQ) and found that heat shock protein 90 (HSP90), a negative regulator of heat shock factor 1 (HSF1), was a target of 1,4-NQ. In the study presented here, we determined whether 1,4-NQ activates HSF1. We also examined whether such redox signaling could be regulated by nucleophilic sulfur species. Exposure of A431 cells to 1,4-NQ covalently modified cellular HSP90, resulting in repression of the association between HSF1 with HSP90, thereby enhancing HSF1 translocation into the nuclei. Liquid chromatography-tandem mass spectrometry analysis with recombinant HSP90 revealed that the modifications site were Cys412 and Cys564. We found that HSF1 activation mediated by 1,4-NQ upregulated downstream genes, such as HSPA6. HSF1 knockdown accelerated 1,4-NQ-mediated cytotoxicity in the cells. While simultaneous treatment with reactive persulfide and polysulfide, Na2S2 and Na2S4, blocked 1,4-NQ-dependent protein modification and HSF1 activation in A431 cells, the knockdown of Cys persulfide producing enzymes cystathionine β-synthase (CBS) and/or cystathionine γ-lyase (CSE) enhanced these phenomena. 1,4-NQ-thiol adduct and 1,4-NQ-S-1,4-NQ adduct were produced during the enzymatic reaction of recombinant CSE in the presence of 1,4-NQ. The results suggest that activation of the HSP90–HSF1 signal transduction pathway mediated by 1,4-NQ protects cells against 1,4-NQ and that per/polysulfides can diminish the reactivity of 1,4-NQ by forming sulfur adducts.

1. Introduction Environmental electrophiles are believed to be harmful, causing cellular dysfunction and carcinoma. Endogenous electrophiles, such as 8-nitro-cyclic guanosine monophosphate and nitrated fatty acids, formed during oxidative processes and inflammation can modify thiol groups of sensor proteins. This activates some kinases and transcriptional factors, increasing antioxidant and anti-inflammation gene expression [1–6]. For example, the nuclear factor (erythroid-derived

2)-like 2 (Nrf2) pathway, epidermal growth factor receptor signaling, and heat shock factor 1 (HSF1) signaling are redox signaling pathways, which are negatively regulated by kelch-like ECH-associated protein 1 (Keap1), protein tyrosine phosphatase 1B, and heat shock protein 90 (HSP90), respectively [7–9]. We have previously reported that the environmental electrophile 1,2-naphthoquinone (1,2-NQ), which is found in diesel exhaust particles and airborne particles with aerodynamic diameters < 2.5 µm [10,11], causes cell damage at high concentrations but modifies protein tyrosine phosphatase 1B through

Abbreviations: CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; CysS-SH, cysteine persulfide; HSF1, heat shock factor 1; HSP, heat shock protein; Keap1, kelch-like ECHassociated protein 1; MeHg, methylmercury; Nrf2, nuclear factor (erythroid-derived 2)-like 2; 1,2-NQ, 1,2-naphthoquinone; 1,4-NQ, 1,4-naphthoquinone; PTEN, phosphatase and tensin homolog; RIPA, radio-immunoprecipitation assay ⁎ Corresponding author at: Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305–8575, Japan E-mail address: [email protected] (Y. Kumagai). http://dx.doi.org/10.1016/j.freeradbiomed.2016.12.047 Received 23 August 2016; Received in revised form 8 November 2016; Accepted 30 December 2016 Available online 31 December 2016 0891-5849/ © 2017 Elsevier Inc. All rights reserved.


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2.2. Animals, 1,4-NQ injection, and sample preparation for Western blot analysis

Cys121 at non-toxic concentrations [9]. This modification inhibits the catalytic activity of the enzyme, and causes epidermal growth factor receptor, which is associated with cell proliferation, to be activated [9]. We subsequently showed that the S-arylation of Keap1 by 1,2-NQ activates Nrf2, upregulating the downstream genes responsible for the detoxification and excretion of 1,2-NQ into extracellular space to be upregulated [12]. The activation of Nrf2 coupled with the covalent modification of Keap1 has also been found using tert-butyl-1,4-benzoquinone and methylmercury (MeHg) [13,14]. These findings strongly suggest that there are adaptive cellular responses to environmental electrophiles that occur through the activation of redox signal transduction pathways, and that these are initiated by the covalent modification of thiol groups on the sensor proteins. 1,4-Naphthoquinone (1,4-NQ), which is an environmental electrophile, is produced during the metabolic activation or photo-oxidation of naphthalene [10,15]. We have previously prepared a specific antibody against 1,4-NQ, and then found that HSP90 is a molecular target for the quinone in A431 cells [16]. Under basal conditions, HSP90 interacts with HSF1 to form a complex [17,18]. However, the oxidative and/or chemical modification of HSP90 allows HSF1 to become disassociated from the complex [18,19]. As a result, the transcriptional factor is translocated into the nucleus and upregulates heat shock proteins (HSPs) by binding to the heat shock elements [18,19]. We therefore hypothesized that the S-arylation of HSP90 by 1,4-NQ could activate HSF1. It has been believed for a long time that cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are enzymes that produce hydrogen sulfide. However, we recently found that cysteine persulfide (CysS-SH), which may have a higher reactivity against electrophiles, is the primary product of these enzymes when cystine (CysS-SCys) is used as a substrate and that CysS-SH readily undergoes S-transsulfuration, resulting in the spontaneous formation of glutathione persulfide (GSSH) and glutathione polysulfide (GS-SnH or GS-S-SG) [20]. Importantly, 1,4-NQ reacts with hydrogen sulfide anion to give a 1,4-NQ-thiol adduct (1,4-NQ-SH) and a 1,4-NQ-S-1,4-NQ adduct ((1,4-NQ)2S) [21]. Supporting this, we recently found that incubating MeHg (another environmental electrophile) with endogenous per/polysulfide species, such as GS-SH or GS-S-SG, causes bismethylmercury sulfide (MeHg)2S to be formed [22]. These observations suggest that per/polysulfide species may repress the 1,4-NQ-dependent activation of the redox signal transduction pathway by capturing 1,4-NQ. In the study described here, we investigated the activation of HSP90–HSF1 signaling mediated by 1,4-NQ and the contribution of per/polysulfides to the activated signaling pathway.

All of the animal protocols were approved by the University of Tsukuba Animal Care and Use Committee and were performed strictly adhering to the committee's guidelines for alleviating suffering. The C57BL/6J (Clea Japan, Tokyo, Japan) female mice used in the study were housed in an air-conditioned room on a 10 h (7 p.m. to 5 a.m.) dark, 14 h light cycle, and were allowed free access to a MF dry rodent diet (Oriental Yeast, Tokyo, Japan). Female mice 8 to 10 weeks old were injected orally with 1,4-NQ (0, 0.5, or 1 mg/kg) dissolved in corn oil. After 24 h, the mice were anesthetized and liver samples were collected. Aliquots of the mouse livers were homogenized in 2% sodium dodecyl sulfate (SDS) solution, and then centrifuged twice at 15,000g. The protein concentration was determined using the bicinchoninic acid assay, then SDS-polyacrylamide gel electrophoresis (PAGE) and western blot analyses were performed as described below.

2.3. Cell culture Human epidermoid carcinoma cell line A431 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutaMAX-I, and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2. The cells were starved overnight by incubating them with serum-free medium before being exposed to 1,4NQ.

2.4. Lysate preparation and nuclear extraction After being exposed to 1,4-NQ with or without Na2S2 or Na2S4, A431 cells were washed twice with ice-cold phosphate-buffered saline. A cell lysate was then prepared by boiling the cells in SDS sample buffer (50 mM Tris-HCl at pH 6.8, 2% SDS, and 10% glycerol) at 95 °C for 15 min or by sonicating the cells in radio-immunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 1% NP40, and 0.5% sodium deoxycholic acid) containing 1% protease inhibitor cocktail (Sigma). The cells lysed in RIPA buffer were centrifuged for 30 min at 20,000g. The nuclear extract was then prepared using an NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. Protein concentrations were determined using the bicinchoninic acid assay (Thermo Fisher Scientific).

2. Materials and methods 2.1. Materials

2.5. Detection of cellular HSP90 modified by 1,4-NQ

Avidin-agarose from egg white (A9207), dimethyl sulfoxide, Na2S4, and 1,4-NQ (98% purity determined by gas chromatography) were purchased from Sigma (St. Louis, MO, USA), Wako Pure Chemical Industries (Osaka, Japan), Alfa Aesar (Ward Hill, MA, USA), and Tokyo Chemical Industries (Tokyo, Japan), respectively. Biotin-PEAC5-maleimide and Na2S2 were obtained from Dojindo Laboratories (Kumamoto, Japan). Dynabeads M280-streptavidin and sheep anti-rabbit immunoglobulin G (IgG) were obtained from Invitrogen (Carlsbad, CA, USA). Anti-HSP70 antibody (ADI-SPA-810), geldanamycin-biotin, and recombinant human HSP90β (ADI-SPP-777) were obtained from Enzo Life Sciences (Farmingdale, NY, USA). Anti-HSP90 antibody (sc-7947), Anti-HSPA6 antibody (ab69408), and Anti-CBS antibody (M01) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Abcam (Cambridge, MA, USA), and Abnova (Taipei, Taiwan). Anti-HSF1 antibody (#4356), horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). All other reagents were of the highest purity available.

Cellular HSP90 was precipitated using geldanamycin-biotin and streptavidin-conjugated magnetic beads (Dynabeads M-280 Streptavidin; Thermo Fisher Scientific) following a modified procedure that has been described previously [23]. A431 cells were exposed to 1,4-NQ (10 µM) for 30 min, washed with ice-cold phosphate-buffered saline, then lysed using RIPA buffer. The mixture was centrifuged at 12,000g, then the protein concentration in the supernatant was determined using the bicinchoninic acid assay. An aliquot of the supernatant (1 mg/mL, 500 μL) was added geldanamycin-biotin (28 μg) and incubated while being rotated at 4 °C for 1 h. A 50 μL aliquot (slurry volume) of Dynabeads M-280 Streptavidin was washed twice with RIPA buffer, then added to the supernatant containing the capturing agent. The supernatant and magnetic beads were incubated together while being rotated at 4 °C for 2 h. The magnetic beads were then washed three times with RIPA buffer, then eluted with 50 μL of SDS-PAGE loading buffer containing 50 mM tris(2-carboxyethyl)phosphine. The eluted proteins were subjected to western blot analysis. 119


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Fig. 1. Chemical modification of cellular and recombinant HSP90 by 1,4-NQ. A: A431 cells were exposed to 1,4-NQ (10 μM) for 30 min, then cellular HSP90 was isolated using geldanamycin-biotin coupled with pull-down by streptavidin Dynabeads. The cell lysate preparations were then subjected to western blot analysis. B: Recombinant HSP90 (0.5 μg) was incubated with 1,4-NQ (0.5–8 μM) at 25 °C for 1 h. The reaction mixture was then subjected to immunoblot analysis with the antibodies indicated. C: Nucleophilic attack of protein thiol to 1,4-NQ, leading to 1,4-NQ-protein adduct formation. D: Results of the nanoUPLC-MSE analysis of 1,4-NQ-modified cysteine or lysine residues in human HSP90. Recombinant human HSP90 (2 μg) was incubated with 1,4-NQ (10 μM) at 25 °C for 30 min in a total volume of 10 μL of 50 mM Tris-HCl (pH 7.5). After the reaction, the HSP90 protein was digested using trypsin and analyzed by nanoUPLC-MSE. The corresponding MSE data are shown in Table 1.

2.6. Immunoprecipitation of HSF1

Table 1 MSE data of the 1,4-NQ-modified peptide in human HSP90. Position

Assignment

Calculated masses (Da)

Observed masses (Da)

Analyte modifiers

412–423

b10* b10-NH3* y12*

1291.55 1274.53 1552.69

1291.53 1274.50 1552.70

+1,4-NQ C (1) +1,4-NQ C (1) +1,4-NQ C (1)

560–565

y2*

406.14

406.13

*

y3

519.22

519.22

y4*

633.26

633.26

+1,4-NQ K (1) +1,4-NQ K (1) +1,4-NQ K (1) +1,4-NQ K (1) +1,4-NQ K (1) +1,4-NQ K (1) +1,4-NQ K (1)

y5*

762.31

762.30

*

615.25

615.27

y5-H2O*

744.30

744.31

y6*

909.38

909.37

y4-H2O

A431 cells were exposed to 1,4-NQ (5 or 20 μM) for 1 h, then a cell lysate was prepared using RIPA buffer as described above. The protein concentration was determined using the bicinchoninic acid assay. Antirabbit IgG conjugated magnetic beads (Dynabeads M-280 Sheep Antirabbit IgG; Thermo Fisher Scientific) were washed three times with Tris-buffered saline and Tween 20, then incubated with HSF1 antibodies at 4 °C for 2 h. The unbound antibodies were then removed, then the beads were resuspended in RIPA buffer, to which 400 μg of total lysate protein was added. The mixture was then incubated at 4 °C overnight. The beads were washed four times with lysis buffer, then the protein complexes were eluted by adding 30 μL of SDS-PAGE loading buffer. The eluted proteins were incubated at 95 °C for 5 min, then subjected to SDS-PAGE and western blot analysis.

C or C or C or C or C or

2.7. Western blot analysis

C or

The samples were normalized to have equal protein contents, then each sample was mixed with half its volume of SDS-PAGE loading buffer containing either 15 mM 2-mercaptoethanol or 50 mM tris(2-carboxyethyl)phosphine. Each mixture was then heated to 95 °C for 5 min, then applied to a SDS-polyacrylamide gel. The proteins were separated by SDSPAGE, then electro-transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA) at 2 mA/cm2 for 1 h. The

C or

Recombinant human HSP90 (2 μg) was incubated with 1,4-NQ (10 μM) at 25 °C for 30 min in 50 mM Tris-HCl (pH 7.5). After the reaction, the HSP90 protein was digested using trypsin and analyzed by nanoUPLC-MSE. The mass number of 156.02 was used for calculation of 1,4-NQ modification.

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Fig. 2. (A and B) Disruption of the interactions between HSF1 and HSP90 in A431 cells induced by exposure to 1,4-NQ and (C and D) nuclear translocation of HSF1 in A431 cells induced by 1,4-NQ. A: A431 cells were treated with dimethyl sulfoxide or 1,4-NQ for 1 h, then HSF1 was immunoprecipitated using specific antibodies. Western blot analysis using the antibodies indicated was then performed. B: The whole cell lysate was used as control. C: Cells were treated with phorbol-12-myristate-13-acetate (PMA) (1 μM), dimethyl sulfoxide (DMSO), or 1,4NQ (5 μM or 20 μM) for the time indicated, then the nuclei were extracted and the extracts subjected to immunoblot analysis using the antibodies indicated. D: The bands were quantified using ImageJ software. Each value is the mean ± the standard error for three independent experiments. * P < 0.05 compared with the vehicle.

Fig. 3. (A) Upregulation of HSPA6, HSP40, HSP90, and HSP105 mRNA in A431 cells by 1,4-NQ and (B) effects of the knockdown of HSF1 on increases in HSPA6 protein concentrations mediated by 1,4-NQ in A431 cells. A: Cells were exposed to different 1,4-NQ concentrations for 6 h. HSPA6, HSP40, HSP90, and HSP105 mRNA concentrations were determined by realtime PCR. All mRNA concentrations were normalized to the B2M mRNA concentration, and the concentrations are given as folds induced relative to the control. Each value is the mean ± the standard error for three independent experiments. * P < 0.05 and ** P < 0.01 compared with the vehicle. B: Cells were transfected with control or HSF1 siRNA for 72 h, then exposed to 1,4-NQ (20 μM) for the time indicated. The cell lysates were subjected to immunoblot analysis using the antibodies indicated. Each value is the mean ± the standard error for three independent experiments. ** P < 0.01 compared with the vehicle.

2.8. RNA interference

membranes were then blocked using 5% skim milk at 25 °C for 1 h, then incubated with primary antibodies at 4 °C overnight, and then incubated with secondary antibodies coupled to horseradish peroxidase at room temperature for 1 h. The proteins of interest were detected using an enhanced chemiluminescence system (Nacalai Tesque, Kyoto, Japan) using medical X-ray film (Konica Minolta Health Care, Tokyo, Japan) or a LAS 3000 imager (Fujifilm, Tokyo, Japan).

A431 cells were seeded at a density of 2.5×104 cells/cm2 and allowed to adhere for 24 h. Before transfection, the medium was removed and fresh medium without antibiotics was added. The transfection was performed using siRNAs (the HSF1 sense strand sequence 5′-GUGACCACUUGGAUGCUAUdTdT-3′, CBS sense strand 121


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Master Mix (Applied Biosystems), using 0.6 μg cDNA and 0.2 μM primers, and a 7500 Real Time PCR system (Applied Biosystems). The thermal cycling parameters were 50 °C for 2 min, 95 °C for 10 min, and 45 cycles of 95 °C for 15 s and 60 °C for 1 min. Melting curve analysis and agarose gel electrophoresis with ethidium bromide staining were performed to ensure that a single PCR product with the correct amplicon length was present. The HSPA6, HSP40, HSP90, HSP105, and beta-2-microglobulin (B2M) concentrations were determined using the relative standard curve method. Fold-changes in the genes were assessed after the intensity had been normalized to B2M. The human gene-specific primers used were as follows: HSPA6, forward, 5ʹAATGCAAGACAAGTGTCGGGAA-3ʹ; reverse, 5ʹ-CCCCCATAGAGCCT GGAGAA-3ʹ; HSP40, forward, 5ʹ-CCAGTCACCCACGACCTTC-3ʹ; reverse, 5ʹ-CCCTTCTTCACTTCGATGGTCA-3ʹ; HSP90, forward, 5ʹGCTTGACCAATGACTGGGAAG-3ʹ; reverse, 5ʹ-AGCTCCTCACAGTTAT CCATGA-3ʹ; HSP105, forward, 5ʹ-CCGGAAAGATGAACAGGTCAC-3ʹ; reverse, 5ʹ-GTGTAGCGCCTCCAACAATC-3ʹ; B2M, forward, 5ʹ-GGGTT TCATCCATCCGACATTG-3ʹ; reverse, 5ʹ-GTTCACACGGCAGGCATAC TCA-3ʹ.

Fig. 4. HSF1 knockdown increased the toxicity of 1,4-NQ to A431 cells. A: Cells were transfected with control or HSF1 siRNA for 72 h, then the lysate was subjected to immunoblot analysis using the antibodies indicated. B: After being transfected with siRNA for 72 h, cells were exposed to 1,4-NQ (10, 15, 20, 25, or 30 μM) for 24 h, then cell viability was measured using the 3-(4,5-dimethythiazol-2-yl)−2,5-triphenyl tetrazolium bromide assay. Each value is the mean ± the standard error for three independent experiments. *P < 0.05 and **P < 0.01 compared with the control siRNA.

2.10. Recombinant HSP90 modification and LC-MSE analysis

sequence 5′-GAACGAAAUCCCCAAUUCUdTdT-3′, and CSE sense strand sequence 5′-GAGCAGUUCCAUCUCCUAUdTdT-3′) and Lipofectamine RNAiMAX (Invitrogen) following the manufacturer's instructions. The cells were allowed to grow for 48 or 72 h before being exposed to 1,4NQ.

Wild type recombinant human HSP90 (0.5 μg) was incubated with 1,4-NQ at 25 °C for 1 h. The reaction mixture was then subjected to western blot analysis. A 2 μg aliquot of the same protein was incubated with 1,4-NQ (10 μM) at 25 °C for 30 min in a total volume of 10 μL of 50 mM Tris-HCl (pH 7.5). Samples of native and 1,4-NQ-modified HSP90 were incubated with 2 mM tris(2-carboxyethyl)phosphine at 25 °C for 10 min in a total volume of 20 μL of 50 mM ammonium bicarbonate solution. Each mixture was then alkylated by adding 5 μL of 30 mM 2-iodoacetamide in 50 mM ammonium bicarbonate solution and incubating the mixture at 25 °C for 20 min in the dark. The HSP90 samples were digested by adding 2.5 μL MS-grade modified trypsin (100 ng) and incubating the mixture at 37 °C overnight. NanoUPLC-

2.9. Real-time polymerase chain reaction After cells had been exposed to 1,4-NQ for 6 h, the total RNA was extracted using an RNeasy Mini kit (Qiagen, Valencia, CA, USA), and cDNA was synthesized from the mRNA using a high capacity RNA-tocDNA kit (Applied Biosystems, Foster, CA, USA). A real-time polymerase chain reaction (PCR) was performed using a Power SYBR Green PCR

Fig. 5. Participation of exogenous per/polysulfides in the covalent modification of cellular proteins and the nuclear translocation of HSF1 in A431 cells mediated by 1,4-NQ. A and B: 1,4NQ (500 µM) was incubated with (A) Na2S4 (0–2 mM) at 25 °C for 10 min or (B) Na2S4 (200 µM) at 25 °C for the time indicated. An aliquot of the reaction mixture was then analyzed by HPLC. 1,4-NQ was detected at 255 nm and the detected peak area was calculated. C: Cells were exposed to 1,4-NQ with or without Na2S4 (100 μM) or Na2S2 (100 μM) for 1 h, then the cell lysates were subjected to immunoblot analysis using 1,4-NQ antibodies. D: Cells were exposed to 20 µM of 1,4-NQ with or without Na2S4 (100 μM) or Na2S2 (100 μM) for 1 h, then the cell nuclei and cytoplasm were separated and subjected to immunoblot analysis using the antibodies indicated.

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Fig. 6. Participation of per/polysulfides in the 1,4-NQ-dependent S-arylation of cellular proteins and nuclear translocation of HSF1 in A431 cells. A and B: Cells were transfected with control siRNA, (A) CBS siRNA for 72 h, or (B) CSE siRNA for 48 h, then to 1,4-NQ (20 μM) for 1 h, then the cell lysates were subjected to immunoblot analysis using the antibodies indicated. C and D: Cells were transfected with control siRNA, (C) CBS siRNA for 72 h, or (D) CSE siRNA for 48 h, then to 1,4-NQ (20 μM) for 1 h, then the cell nuclei and cytoplasm were separated and subjected to immunoblot analysis using the antibodies indicated. Each value is the mean ± the standard error for three independent experiments. *P < 0.05 compared with the control siRNA.

MSE analysis was performed using a nanoAcquity UPLC system (Waters, Milford, MA, USA) equipped with a BEH130 nanoAcquity C18 column (100 mm long, 75 µm i.d., 1.7 µm particle size; Waters), which was kept at 35 °C. The analysis was performed in direct injection mode. Mobile phases A (0.1% formic acid) and B (acetonitrile containing 0.1% formic acid) were mixed using a gradient system, and the flow rate was 0.3 μL/ min. The mobile phase program started at 3% B for 1 min, then linearly increased over 74 min to 40% B, which was maintained for 4 min, then linearly increased over 1 min to 95% B, which was maintained for 5 min, then linearly decreased over 1 min to 3% B. The total run time (including conditioning the column at the initial conditions) was 100 min. The eluted peptides were transferred to the nano-electrospray source of a quadrupole time-of-flight mass spectrometer (a Synapt High Definition Mass Spectrometry system; Waters) through a Teflon capillary union and a precut PicoTip (Waters). The initial Synapt mass spectrometer parameters were a capillary voltage of 2.8 kV, a sampling cone voltage of 35 V, and a source temperature of 100 °C. A low (6 eV) or elevated (stepped from 15 to 30 eV) collision energy was used to generate either intact peptide precursor ions (low energy) or peptide product ions (elevated energy). The detector was operated in positive ion mode. The mass spectrometer performed survey scans from m/z 50 to m/z 1990. All analyses were performed using an independent reference, Glu-1-fibrinopeptide B (m/z 785.8426), which was infused through the NanoLockSpray ion source and sampled every 10 s and used as an external mass calibrant. Data were collected using MassLynx version 4.1 software (Waters). Protein Lynx Global Server Browser version 2.3 software (Waters) was used to identify the proteins. Biopharmlynx version 1.2 software (Waters) was used to perform baseline subtraction,

smoothing, deisotoping, de novo peptide sequence identification, and database searches. 2.11. Determination of 1,4-NQ by HPLC during its reaction with persulfides 1,4-NQ (0.5 mM) was incubated with Na2S4 (0, 0.5, 1, or 2 mM) at 25 °C for 10 min, and the mixture (10 μL) was analyzed by HPLC. CysS-SH was generated from cystine through enzymatic reactions with CBS or CSE, as has been described previously [20]. Briefly, CBS (0, 2, 4, or 8 µg) was incubated with 1 nmol of cystine in 20 mM HEPES (pH 7.5), 200 µM S-adenosylmethionine, and 1 mM pyridoxal phosphate buffer at 37 °C for 30 min. CSE (0, 0.5, 1, or 2 µg) was reacted with 1 nmol of cystine in 20 mM HEPES (pH 7.5) and 50 µM pyridoxal phosphate buffer at 37 °C for 30 min 1,4-NQ (1 mM, 10 μL) was added to 100 μL of a mixture containing CysS-SH, then the mixture was incubated at 25 °C for 30 min. The reaction was stopped by adding icecold acetonitrile (90 μL), then the mixture was centrifuged at 15,000g for 5 min. An aliquot of the supernatant (50 μL) was analyzed by HPLC. Each sample was monitored using an analytical HPLC system (YMCPack ODS-AM column (YMC, Kyoto, Japan), 250 mm long, 4.6 mm, i.d., 5 µm particle size; 30:70 v/v acetonitrile: water mobile phase; 1 mL/ min flow rate; detector set to 255 nm). 2.12. Determination of the products of the reaction between 1,4-NQ and CysS-SH by UPLC-MS/MS The products of the reaction between 1,4-NQ and CysS-SH were 123


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Fig. 7. Consumption of 1,4-NQ during the reaction between 1,4-NQ and cysteine persulfide and/or cysteine polysulfide. A: Cystine (0 or 100 nmol) was incubated with CBS (0 or 4 µg), pyridoxalphosphate (100 nmol), and S-adenosylmethionine (20 nmol) in 20 mM HEPES (pH 7.5) buffer at 37 °C for 30 min (in 100 μL total volume). Ten micro liter of 1,4-NQ (11 nmol) was added to the reaction mixture, and the mixture was incubated at 25 °C for 30 min. Ice-cold acetonitrile was then added to the mixture. The mixture was centrifuged at 15,000g, then an aliquot of the supernatant was analyzed by HPLC. B: Cystine (0 or 100 nmol) was incubated with CSE (0 or 1 µg) and pyridoxalphosphate (2 nmol) in 20 mM HEPES (pH 7.5) buffer at 37 °C for 30 min (total volume, 100 μL). Ten micro liter of 1,4-NQ (11 nmol) was added to the reaction mixture, and the mixture was incubated at 25 °C for 30 min. The samples were then prepared and analyzed as described above. C: Cystine (100 nmol) was incubated with CBS (0–8 µg), pyridoxalphosphate (100 nmol), and S-adenosylmethionine (20 nmol) in 20 mM HEPES (pH 7.5) buffer at 37 °C for 30 min (total volume, 100 μL). Ten micro liter of 1,4-NQ (11 nmol) was added to the reaction mixture, and the mixture was incubated at 25 °C for 30 min. The samples were then prepared and analyzed as described above. D: Cystine (100 nmol) was incubated with CSE (0–2 µg) and pyridoxalphosphate (2 nmol) in 20 mM HEPES (pH 7.5) buffer at 37 °C for 30 min (total volume, 100 μL). Ten micro liter of 1,4-NQ (11 nmol) was added to the reaction mixture, and the mixture was incubated at 25 °C for 30 min. The samples were then prepared and analyzed as described above. Each value is the mean ± the standard error for three independent experiments. *P < 0.05 and **P < 0.01 compared with the control.

2.13. Cellular viability

identified using an UPLC-MS/MS instrument (Waters) equipped with a BEH130 Acquity C18 column (50 mm long, 2.1 mm i.d., 1.7 µm particle size; Waters), which was kept at 35 °C. Mobile phases A (water containing 0.1% (v/v) formic acid) and B (acetonitrile containing 0.1% (v/v) formic acid) were mixed using a gradient system, and the flow rate was 0.3 μL/min. The mobile phase program started at 5% B for 2 min, then linearly increased over 8 min to 98% B, which was maintained for 5 min, then linearly decreased over 50 s to 5% B. The eluted compounds were transferred to the electrospray source of a Synapt High Definition Mass Spectrometry system (Waters). MassLynx software Ver. 4.1 was used to control the system and analyze the data produced. The electrospray source was used in positive ion mode with a capillary voltage of 2.8 kV and a sampling cone voltage of 20 V or in negative ion mode with a capillary voltage of 2.5 kV and a sampling cone voltage of 30 V. The low collision energy was 6 eV (to detect precursor ions) and the elevated collision energy was 10–40 eV (to generate product ions). All analyses were acquired using an independent reference, which was leucine-enkephalin (m/z 556.27 in positive ion mode, m/z 554.27 in negative ion mode).

The cellular toxicity of 1,4-NQ was estimated using the 3-(4,5dimethythiazol-2-yl)-2,5-triphenyl tetrazolium bromide assay, which has been described previously [24]. Briefly, A431 cells in the wells of 96-well plates were exposed to 1,4-NQ for 24 h, then the cells were treated with 5 mg/mL 3-(4,5-dimethythiazol-2-yl)-2,5-triphenyl tetrazolium bromide (added in 1/20 of the volume of liquid already in each well) for 1 h at 37 °C. The medium was removed, then dimethyl sulfoxide (100 μL/well) was added to dissolve the formazan. The absorbance of the solution at 540 nm was determined using an iMark microplate reader (Bio-Rad Laboratories). 2.14. Statistical analysis All data are expressed as the mean ± the standard error for at least three independent experiments. The results of statistical tests were considered to be significant at P < 0.05 or P < 0.01.

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Fig. 8. Products of the enzymatic reactions of CSE in the presence of 1,4-NQ. Cystine (100 nmol) was incubated with CSE (1 µg) and pyridoxalphosphate (5 nmol) in 20 mM HEPES (pH 7.5) buffer at 37 °C for 30 min 1,4-NQ (11 nmol) was added to the reaction mixture, and the mixture was incubated at 25 °C for 5 min, then ice-cold acetonitrile was added. The mixture was centrifuged at 15,000g, then an aliquot of the supernatant was analyzed by UPLC-MS/MS. The chromatograms in A, B, and C were acquired by monitoring m/z 278.0 in positive ion mode, m/z 189.0 in negative ion mode, and m/z 345.0 in negative ion mode, respectively. The dominant peaks found in chromatograms A, B, and C corresponded to the adducts 1,4-NQ-S-Cys, 1,4-NQ-SH, and (1,4-NQ)2S, respectively. The mass spectra (D, G, and J) and MS/MS spectra (E, H, and K) were obtained from peaks with UPLC retention times of 5.97 min (D, E, and F), 6.47 min (G, H, and I), and 8.34 (J, K, and L). The chemical structures of the adducts are shown (F, I, and L).

3. Results

glyceralaldehyde-3-phosphate dehydrogenase [27] and ubiquitin carboxylterminal hydrolase L1 [28] have indicated that molecular mass of 1,2-NQ covalently bound to these proteins was 156.02 in all cases (Fig. 1C), indicating that the 1,2-NQH2 easily auto -oxidized to 1,2-NQ on the protein adduction through C-S bond. In agreement with previous observations, we detected fragments modified by 1,4-NQ after incubation of recombinant HSP90 with 1,4-NQ, followed by trypsin digestion (Table 1), whereas little appreciable HSP90 fragments bound to 1,4-NQH2 were seen (data not shown). Ultra performance liquid chromatography (UPLC) tandem mass spectrometry (MSE) analysis showed that the modification sites were Cys412 and Cys564 (Fig. 1D), although the covalent modification of Lys565 by 1,4-NQ could not be excluded because N-arylation of Lys 565 may inhibit tryptic activity at Lys565 (see Table 1).

3.1. Modification of HSP90 by 1,4-NQ Modifications of HSP90 in A431 cells by 1,4-NQ were detected by precipitating the HSP90 using geldanamycin-biotin and streptavidinbeads. As shown in Fig. 1A, exposing the cells to 10 µM 1,4-NQ for 30 min caused the HSP90 to be modified. The modification of HSP90 by 1,4-NQ was evaluated by western blotting with the specific antibody for 1,4-NQ [16]. Recombinant human HSP90 protein was also modified by 1,4-NQ in a concentration-dependent manner (Fig. 1B). It is well understood that Michael acceptors such as NQs with α,βunsaturated carbonyl groups undergo 1,4-addition reaction by nucleophiles, resulting in adduct formations [25]. During reaction of 1,2-NQ, an isomer of 1,4-NQ, with protein thiol, we expected that product formed should be 1,2NQH2 (MW=158.02)-protein adduct as shown in Fig. 1C. Nevertheless, experiments with protein tyrosine phosphatase 1B [9], Keap1 [26],

3.2. Transactivation of HSF1 by 1,4-NQ to induce downstream genes Under basal conditions, HSP90 was co-precipitated with HSF1 using 125


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reactive persulfide sodium disulfide (Na2S2) and Na2S4 diminished covalent modification of cellular proteins by 1,4-NQ, but not by PMA, and quinone-mediated nuclear translocation of HSF1 (Figs. 5C, D, and S3). 3.4. Contributions of per/polysulfides-producing enzymes to the activation of HSP90–HSF1 signaling by 1,4-NQ While knockdown of CBS and CSE decreased the amounts of per/ polysulfide species in the cells [20], transfecting siRNA for these enzymes into A431 cells enhanced modification of cellular proteins by 1,4-NQ (Fig. 6A and B). Under these conditions, 1,4-NQ-mediated translocation of HSF1 into the nucleus was significantly enhanced (Fig. 6C and D). 3.5. Identification of sulfur adducts produced during CBS and CSE enzymatic reactions in the presence of 1,4-NQ Incubating 1,4-NQ (100 µM) with a mixture of Cys persulfidegenerating enzymes (CBS at 40 µg/mL (0.5 µM) or CSE at 10 µg/mL (250 nM)) in reaction buffers (1 mM cystine; 200 µM S-adenosylmethionine; 1 mM pyridoxalphosphate; 20 mM HEPES, pH 7.5 or 1 mM cystine; 50 µM pyridoxalphosphate; 20 mM HEPES, at pH 7.5), respectively, decreased the 1,4-NQ concentration. However, the 1,4-NQ did not decrease in the absence of the enzymes or the substrate (Fig. 7A and B). The 1,4-NQ was consumed in a concentration-dependent fashion by CBS and CSE (Fig. 7C and D). More per/polysulfides are produced from cystine by CSE than by CBS [20], so more 1,4-NQ was consumed by the reaction mixture containing CSE than by the reaction mixture containing CBS (Fig. 7). This suggested that 1,4-NQ reacts with cysteine persulfide and its related reactive persulfides/polysulfides. Consistent with this, UPLC-MS/MS analysis revealed that there were three reaction products. The products had retention times of 5.97 min (m/z 278 in positive ion mode), 6.47 min (m/z 189 in negative ion mode), and 8.34 min (m/z 345 in negative ion mode), which were identified as being 1,4-NQ-SH, 1,4-NQ-SCys, and (1,4-NQ)2S, respectively (Fig. 8).

Fig. 9. 1,4-NQ-mediated activation of HSP90–HSF1 signaling, which was negatively regulated by reactive persulfides and polysulfides with higher nucleophilicity. E, electrophile.

anti-HSF1 antibodies in A431 cells. Markedly less HSP90 precipitated with HSF1 when 1,4-NQ was present, whereas the expression level of HSP90 in the cells did not change (Fig. 2A), indicating that the interaction of HSP90 with HSF1 was disrupted by 1,4-NQ. Exposure of A431 cells to 1,4-NQ enhanced the translocation of HSF1 into the nucleus (Fig. 2B) and significantly induced downstream genes that are regulated by HSF1, such as HSPA6, HSP40, HSP90, and HSP105 in a concentration-dependent manner (Fig. 3A). HSPA6, which codes protein HSP70 [29], was strongly upregulated by 1,4-NQ. Although the exact mechanisms underlying the strong induction of HSPA6 by 1,4-NQ remain unclear, we speculate that such an induction is presumably due to the multiple HSEs in the promoter region of HSPA6 and also other factors such as AP1 [30]. The enhanced protein expression of HSPA6/ 70 was markedly blocked by the transfection of HSF1 siRNA (siHSF1) in A431 cells (Fig. 3B), confirming that the induction of HSPA6 mediated by 1,4-NQ was regulated by HSF1. Interestingly, the suppression of HSF1 by the siRNA significantly enhanced the cytotoxicity of 1,4-NQ in A431 cells (Fig. 4). Taking these results together, it seems likely that increased HSP levels mediated by the activation of HSF1 plays a role in an adaptive response to 1,4-NQ. In a separate experiment, we found that oral administration of 1,4-NQ to mice for 24 h induced HSP70 in the liver (Fig. S1). This suggests that the upregulation of HSP caused by 1,4-NQ is not a cell-specific event.

4. Discussion Our results indicated that the environmental electrophile 1,4-NQ is capable of activating HSP90–HSF1 signaling, resulting in upregulation of HSPs and that this activation of the redox signal transduction pathway was negatively regulated by persulfides/polysulfides with higher nucleophilicity. The activation of the HSPs/HSF1 signaling pathway by 4-hydroxynonenal and 6-methylsulfinylhexyl isothiocyanate has been found to be associated with the disruption of interactions between HSPs and HSF1 because of the HSPs being chemically modified [31,32]. Consistent with this, western blot analysis using anti-1,4-NQ antibody revealed that 1,4-NQ modified cellular and recombinant human HSP90 (Fig. 1A and B). UPLC-MS/MS analysis using recombinant HSP90 indicated that HSP90 was modified by 1,4-NQ at the Cys412 and Cys564 sites under conditions (Fig. 1D). Many studies of the oxidative- and chemical-modification of HSP90 have been published. For example, 6-methylsulfinylhexyl isothiocyanate has been found to modify Cys521 and 4-hydroxy-2-nonenal has been found to modify Cys572 in HSP90α, which corresponds to Cys564 in HSP90β [32,33]. In a preliminary study, we found that the electrophile cadmium can modify HSP90 through the same cysteine residue as modified by 1,4-NQ and activate HSF1 by inhibiting the association between HSF1 and HSP90 (Shinkai Y et al. [34]). We therefore speculate that Cys564 in HSP90 is easily covalently modified by a variety of electrophiles, potentially destabilizing the HSP90–HSF1 complex. Several pathways have been reported to activate HSF1 due to oxidation of Cys35 and Cys105 to form disulfide bonds in HSF1, the

3.3. Inhibition of 1,4-NQ-mediated HSP90–HSF1 signaling by per/ polysulfides The activation of HSP90–HSF1 by 1,4-NQ would be affected by addition of reactive persulfides or polysulfides to the cell medium if the persulfides and/or polysulfides can trap 1,4-NQ. To investigate this possibility, 1,4-NQ was incubated with a reactive polysulfide Na2S4 in vitro. As shown in Fig. S2A and B, 1,4-NQ was eluted from a high performance liquid chromatography (HPLC) column at a retention time of 5.4 min, and was monitored at a wavelength of 255 nm. The intensity of the peak decreased in the presence of Na2S4 (Fig. S2C and D). 1,4-NQ was also consumed in a concentration- and timedependent manner when it was incubated with Na2S4. The loss of 1,4NQ stopped at about 5 min (Fig. 5A and B). The limited decomposition of 1,4-NQ may have been caused by the decomposition of Na2S4 or by further reactions forming the ultimate product S8 through the oxidation of Na2S4. Treatment of the cells with 1,4-NQ in the presence of a 126


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Acknowledgment

phosphorylation of HSF1, the trimerization of HSF1, and the inhibition of HSP90 [7,35–38]. We did not determine the detail how HSF1 was activated, but 1,4-NQ was found to enhance the nuclear localization of HSF1 and induce HSPA6, HSP40, HSP90, and HSP105 messenger RNAs (Fig. 3A). HSP70, which is encoded by HSPA6, is a chaperon that inhibits cell damage, such as apoptosis and nucleolar fragmentation induced by hydrogen peroxide [7,39]. We suggest that HSP70, corresponding to HSPA6 induced by 1,4-NQ, may protect cells against 1,4NQ because the knockdown of HSF1, which upregulates HSPA6/HSP70, enhanced 1,4-NQ cytotoxicity (Fig. 4). Supporting this, we also found that HSP70 is certainly induced in the mouse livers exposed to 1,4-NQ in vivo (Fig. S1). Several lines of evidence indicate that HSF1 protects against oxidative stress induced by hydrogen peroxide and electrophilic stress induced by 4-hydroxynonenal [7,31,40–42]. 1,4-NQ extensively modifies cellular proteins (see Fig. 5C), so the activation of HSF1 may be essential for maintaining the proteostasis in counteracting 1,4-NQinduced electrophilic modification. Per/polysulfide species have higher nucleophilicities to react with electrophiles because of their low pKa values. For example, the pKa values of cysteine and hydrogen sulfide are 8.3 and 6.76, respectively, whereas the predicted pKa of CysS-SH is 4.34 and the estimated pKa of Na2S4 is 3.8 [43– 45]. The thiol groups of per/polysulfides with lower pKa values should be deprotonated to form thiolate anions at physiological pH values and thus they would be fairly reactive compared with glutathione (pKa 9.12). From the pKa values, we hypothesized that the knockdown of CBS/CSE may increase the 1,4-NQ-mediated activation of HSP90–HSF1 signaling due to reduction of the endogenous per/polysulfide levels, whereas addition of persulfide or polysulfide to a culture medium containing 1,4-NQ may suppress such redox signaling by capturing 1,4-NQ that would otherwise be involved in forming sulfur adducts. Experiments with CBS/CSE siRNA and treatments with Na2S2 and Na2S4 supported our hypothesis, as is shown in Figs. 5 and 6. Furthermore, in vitro analyses using recombinant CSE indicated that the enzymatic reaction products in the presence of 1,4-NQ were the 1,4-NQ-SCys adduct, 1,4-NQ-SH, and (1,4-NQ)2S (Figs. 7 and 8), whereas little of the 1,4-NQ-SSCys adduct was formed under the conditions used. This suggests that 1,4-NQ-SSCys may be an unstable adduct that undergoes nucleophilic attack by CysS-SH, eventually producing the 1,4NQ-SCys adduct. Overall, our findings led us to conclude that per/ polysulfides enzymatically produced by CBS and/or CSE negatively regulate the 1,4-NQ-mediated activation of HSP90–HSF1 signaling by capturing 1,4NQ, as is shown in Fig. 9. We have demonstrated that environmental electrophiles (e.g., cadmium, MeHg, 1,2-NQ, and 1,4-NQ) activate redox signal transduction pathways consisting of sensor proteins with reactive thiol groups (thiolate ions) and effector molecules (e.g., kinase and transcription factors), such as protein tyrosine phosphatase 1B–epidermal growth factor receptor signaling [9], the Keap1–Nrf2 pathway [12], and now HSP90–HSF1 signaling. In a separate study, we also found that 1,4-NQ covalently modifies the phosphatase and tensin homolog (PTEN) that is blocked by Na2S4, thereby activating Akt/CREB signaling associated with cell survival (Unoki T et al. unpublished observations). We recently showed that SH-SY5Y cells exposed to MeHg give a bell-shape PTEN/Akt/CREB signaling response. Lower concentrations of this environmental electrophile activate PTEN/Akt/CREB signaling through covalently modifying the PTEN, leading to upregulation of an antiapoptotic protein Bcl-2, whereas higher MeHg concentrations disrupt redox signaling through S-mercuration of CREB at Cys286, causing Bcl2 to be downregulated in SH-SY5Y cells [46]. However, the connection between PTEN pathway and HSP90 pathway is unclear. There is little doubt that exposing cultured cells and experimental animals to high concentrations of environmental electrophiles causes apoptotic cell death and toxicity to occur. However, we here describe a new concept: that exposure to exogenous electrophiles such as 1,4-NQ at non-toxic concentrations promotes adaptive responses to the electrophiles through the activation of redox signal transduction pathways by the S-arylation of the sensor proteins.

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