A Convenient Assay to Detect Protein Oxidation Caused by Redox-Active Quinones

Abstract

Redox-active quinones generate reactive oxygen species (ROS) through their redox cycling with electron donors. Hydrogen peroxide (H₂O₂) causes S-oxidation of proteins and is associated with activation of the redox signaling pathway and/or toxicity (Chem. Res. Toxicol., 30, 2017, Kumagai et al.). In the present study, we developed a convenient assay based on a combination of an enzyme-linked immunosorbent assay and a biotin-PEAC₅-maleimide assay and used it to determine protein S-oxidation by ROS during redox cycling of 9,10-phenanthrenequinone (9,10-PQ) and pyrroloquinoline quinone (PQQ). S-Oxidation of proteins in a mouse liver supernatant was detected during reaction of 9,10-PQ or PQQ with electron donors such as dithiothreitol or reduced nicotinamide adenine dinucleotide phosphate (NADPH), whereas cellular protein oxidation was not observed in the absence of electron donors. These results suggest that the developed assay is useful for the detection of S-oxidation of proteins.

INTRODUCTION

During our lifetime, we are exposed to various electron acceptors present in the environment and our diet. For example, 9,10-phenanthrenequinone (9,10-PQ, Fig. 1A) is found as a contaminant in diesel exhaust particles (DEP) and particulate matter 2.5,¹⁾ while pyrroloquinoline quinone (PQQ, Fig. 1A) is contained in various dietary sources.²⁾ Once these substances are incorporated into cells, they interact with biological electron donors to undergo redox cycling, thereby generating reactive oxygen species (ROS). We reported previously that 9,10-PQ undergoes one-electron reduction with dithiol compounds or reduced nicotinamide adenine dinucleotide phosphate (NADPH) to form its intermediate semiquinone radical (9,10-PQ^·−), which readily reacts with molecular oxygen (O₂) to yield superoxide and hydrogen peroxide (H₂O₂)³⁾ (Fig. 1B). 9,10-PQ^·− is also produced by a chemical disproportionation reaction between 9,10-PQ and 9,10-dihydroxyphenanthrene, a two-electron reductant of 9,10-PQ, that is catalyzed by NADPH-dependent reductases such as aldo-keto reductases or NADPH:quinone oxidoreductase 1 to produce excessive ROS in cells.³⁾ PQQ also acts as an efficient electron transfer catalyst from reducing agents such as NADPH to O₂ to yield ROS.⁴⁾

Fig. 1. Structures of Chemicals Used in This Study (A) and Schematic Procedure for the BPM-Labeling Assay to Detect Redox-Active Compounds (B, C)

(A) 9,10-PQ, 9,10-phenanthrenequinone; PQQ, pyrroloquinoline quinone; DDP, cis-9,10-dihydroxy-9,10-dihydrophenanthrene. (B) Redox cycling of redox-active compounds through one-electron reduction. NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; O₂, oxygen; O₂^·−, superoxide; H₂O₂, hydrogen peroxide; H⁺, hydrogen ion. (C) Schematic strategy of the BPM-labeling assay to detect redox-active compounds.

H₂O₂ generated from redox cycling of exogenous redox-active compounds causes S-oxidation of reactive thiols of proteins to yield sulfenic acid, sulfinic acid, and ultimately sulfonic acid, resulting in alterations of protein function.^3,5) While ROS at high concentrations cause cellular toxicity because of irreversible protein damage, low concentrations of ROS are found to activate redox signaling involved in homeostasis.^6,7) Therefore, detection of S-oxidation of cellular proteins and identification of redox-active compounds by xenobiotic chemicals are key steps to clarify the molecular mechanisms involved in the effects of the reactive substances on cellular homeostasis and redox signaling.

Previously, our collaboration with the Southern California Particle Center and Supersite resulted in the development of an assay to determine the redox activity of airborne particulate matter by measuring the consumption of dithiothreitol (DTT), which serves as an electron donor during redox cycling.⁸⁾ However, this assay could not be used to evaluate the oxidation of proteins by airborne particulate matter during redox cycling. Separately, we have established a biotin-PEAC₅-maleimide (BPM)-labeling method using Western blotting and an enzyme-linked immunosorbent assay (ELISA) to detect covalent S-modification of proteins by electrophiles.⁹⁾ As the maleimide cannot bind to modified thiols by electrophiles and ROS,¹⁰⁾ we hypothesize that the BPM-labeling method can be used to detect S-oxidation. In this research, we used 9,10-PQ and PQQ as the electron acceptors and applied our Redox-BPM-ELISA method to detect S-oxidation caused by ROS produced from the redox cycling of 9,10-PQ and PQQ with electron donors such as DTT and NADPH (Figs. 1B, C).

MATERIALS AND METHODS

Materials

9,10-PQ, NADPH, tris(2-carboxyethyl) phosphine hydrochloride (TCEP), phenanthrene, anthraquinone, BPM, and the horseradish peroxidase-linked anti-biotin antibody were from Sigma-Aldrich (St. Louis, MO, U.S.A.), Oriental Yeast (Tokyo, Japan), Hampton Research (Aliso Viejo, CA, U.S.A.), GL Sciences Inc. (Tokyo, Japan), Tokyo Chemical Industry (Tokyo, Japan), Dojindo (Kumamoto, Japan), and Cell Signaling Technology (Beverly, MA, U.S.A.), respectively. Bovine serum albumin (BSA) and DTT were from Nacalai Tesque (Kyoto, Japan). H₂O₂ and PQQ were purchased from Wako Pure Chemical Corporation (Osaka, Japan). cis-9, 10-Dihydroxy-9,10-dihydrophenanthrene (DDP) was synthesized as previously described.¹¹⁾

Cell Culture

HepG2 cells (RIKEN Cell Bank, Ibaraki, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Wako) supplemented with 10% fetal bovine serum, antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin), and 2 mM glutaMAX-I in a 5% CO₂ humidified atmosphere at 37 °C. The cells were seeded at a density of 8 × 10⁵ on 35 mm dishes for 24 h and then starved in serum-free DMEM for 24 h before treatment with the indicated compounds.

BPM-Labeling Assay Detected by Western Blotting

After treatment, the cells were washed three times with ice-cold phosphate-buffered saline (PBS) and collected by scraping in lysis buffer [50 mM Tris–HCl (pH 6.8), 0.5% deoxycholate (w/v), 1% NP-40 (w/v) and 150 mM NaCl] containing 100 µM BPM and 1% protease inhibitor cocktail (Sigma). The samples were then incubated on ice for 1 h and centrifuged at 15000 × g for 10 min at 4 °C to collect the supernatant. The protein concentration was determined using the bicinchoninic acid assay (Thermo Fisher Scientific, Roskilde, Denmark) according to the manufacturer’s instructions. Each sample was normalized to the same protein concentration before mixing with half the volume of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading buffer [6% SDS, 62.5 mM Tris–HCl (pH 6.8), 24% glycerol, 50 mM TCEP, and 0.015% bromophenol blue] and then was heated for 5 min at 95 °C. The cellular proteins were separated by SDS-PAGE. The gels were subjected to Coomassie brilliant blue staining or electro-transferred onto polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY, U.S.A.) at 2 mA/cm² for 1 h. The membranes were blocked with 5% skim milk in Tween 20-Tris buffered saline (TTBS) [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] for 1 h followed by incubation with a horseradish peroxidase-linked anti-biotin antibody at room temperature for 1 h. The immunoreactive proteins were detected with Amersham ECL Western blotting detection reagent (GE Healthcare, Buckinghamshire, U.K.). The results shown are representative of three independent experiments. The band intensity was measured using ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MD, U.S.A.).

Redox-BPM-Labeling ELISA

The BPM-labeling ELISA was modified based on the method described in our previous study.⁹⁾ In brief, mouse liver supernatant (200 µg/mL), which was prepared from the homogenized livers of adult C57BL/6J mice in three volumes of 3 M potassium chloride by centrifugation at 9000 × g for 10 min, or BSA (200 µg/mL) in 50 mM sodium carbonate-bicarbonate buffer (pH 9.5) and 1 mM TCEP (50 µL/well) was conjugated on Nunc-immunoplate F96 Maxisorp plates (Thermo Fisher Scientific) at 4 °C overnight. The coated proteins were reduced by subjecting them to a 30 min incubation with 10 mM TCEP in 50 mM Tris–HCl (pH 7.5) followed by washing with PBS three times. Then the proteins were reacted with electron acceptors in the presence and absence of DTT or NADPH in 50 mM Tris–HCl (pH 7.5) at 37 °C for 30 min. After washing three times with PBS, the free thiols on the proteins were labeled with 2 µM BPM in 50 mM Tris–HCl (pH 7.5) at 37 °C for 30 min. The unreacted BPM was removed by washing with PBS three times. The labeled proteins were incubated with streptavidin-peroxidase polymer diluted 1 : 5000 in TTBS at 37 °C for 30 min and then detected by the ABTS peroxidase substrate system (SeraCare Life Sciences, Milford, MA, U.S.A.) according to the manufacturer’s protocol.

Preparation of a Methanol Extract of DEP

DEP was prepared and extracted with methanol following the method described in the previous study.^12,13) The extract was dissolved in methanol/acetone (1 : 1, v/v) to a final concentration of 40 mg/mL and was kept under nitrogen gas at −80 °C before use.

Data Analysis

All data are presented as the mean ± standard error from at least three independent experiments. Statistical analysis was performed by two-way ANOVA followed by Tukey’s multiple-comparison test using GraphPad Prism version 8.4.3 software (San Diego, CA, U.S.A.).

RESULTS AND DISCUSSION

We first assessed the S-oxidation of cellular proteins using a BPM-labeling assay with Western blotting (Fig. 1C). Exposure of HepG2 cells to H₂O₂ or 9,10-PQ (Fig. 1A) decreased the blot intensities of the biotin signals in a concentration-dependent manner (Figs. 2A, B). DDP (Fig. 1A), an 9,10-PQ analog without redox activity, did not affect the signal (Fig. 2B). To detect the S-modification of proteins by ROS using the ELISA, proteins from the mouse liver supernatant or BSA-coated 96-well plates were prepared. Incubation of the proteins with H₂O₂ significantly suppressed the signal of the biotinylated proteins (Figs. 3A, B), indicating successful detection of S-oxidation. As shown in Figs. 3C and E, 9,10-PQ and PQQ (Fig. 1A) in the presence of DTT markedly decreased the biotin signal in a concentration-dependent manner using the mouse liver supernatant coated plates. The endogenous electron donor NADPH also triggered redox cycling of these quinones resulting in oxidation of the thiols (Figs. 3D, F). The non-redox-active compounds such as DDP, phenanthrene, and anthraquinone (Fig. 1A) did not consume thiols even in the presence of DTT and NADPH (Figs. 3G–L). Similar results were observed using the BSA-coated 96-well plates (data not shown). These results indicate that S-oxidation of cellular proteins caused by ROS derived from xenobiotic electron acceptors can be detected using the BPM-labeling assay with both Western blotting and ELISA (Fig. 1C). S-Oxidation of the mouse liver supernatant preparation during redox cycling of 9,10-PQ with DTT was greater than that of PQQ with DTT. However, the S-oxidation reaction caused by 9,10-PQ with NADPH was less than that of PQQ with NADPH (Figs. 3C–F). It seems likely that an electron acceptor was preferable to an electron donor to produce ROS, suggesting that a suitable electron acceptor and donor need to be chosen to increase the sensitivity of the assay.

Fig. 2. Western Blotting Analysis of S-Oxidation of Cellular Proteins by the BPM-Labeling Assay

HepG2 cells were exposed to (A) H₂O₂ (0 − 0.5 mM), (B) 9,10-phenanthrenequinone (9,10-PQ, 0 − 50 µM) or cis-9,10-dihydroxy-9,10-dihydrophenanthrene (DDP, 50 µM) for 30 min. The Redox-BPM-labeling assay was performed as described in Materials and Methods. * p < 0.01 compared with the control. CBB, Coomassie brilliant blue staining.

Fig. 3. S-Oxidation of Proteins by Redox-Active and -Inactive Compounds

(A, C–L) Mouse liver supernatant or (B) bovine serum albumin were incubated with (A, B) H₂O₂ (0 − 4 mM), (C, D) 9,10-phenanthrenequinone (9,10-PQ, 0 − 100 µM), (E, F) pyrroloquinoline quinone (PQQ, 0 − 800 µM), (G, H) cis-9,10-dihydroxy-9,10-dihydrophenanthrene (DDP, 0 − 100 µM), (I, J) anthraquinone (0 − 100 µM), and (K, L) phenanthrene (0 − 100 µM) in the absence and presence of (C, E, G, I, K) dithiothreitol (DTT, 0.5 mM) or (D, F, H, J, L) NADPH (2 mM) for 30 min at 37 °C. S-Oxidized proteins were detected by the Redox-BPM-labeling enzyme-linked immunosorbent assay as described in Materials and Methods. Data are the mean ± standard error of three determinations. * p < 0.05, ** p < 0.01 compared with the control. ^# p < 0.01 compared with the absence of DTT or NADPH.

Quinone-type DEP contaminants such as 9,10-PQ, 1,2-naphthoquinone (1,2-NQ), and 1,4-NQ¹⁾ interact with endogenous and exogenous electron donors and lead to the generation of ROS.^3,12) While our previous finding indicated that 9,10-PQ only has redox activity,³⁾ 1,2- and 1,4-NQ exhibit both redox activity and electrophilic properties that allow covalent S-modification even without an electron donor.⁵⁾ Consistent with these observations, S-modification of proteins from exposure to a methanol extract of DEP was detected using the BPM assay, whereas addition of DTT into the system tended to enhance the modification (Fig. 4). This result indicates that DEP-mediated S-modification of proteins in the absence of DTT is presumably attributable to electrophiles such as 1,2- or 1,4-NQ DEP contaminants, corresponding to our previous finding.¹⁴⁾ In the present study, we developed and characterized an easy assay for determination of S-oxidation of proteins. This assay may be useful to determine the presence of electron acceptors and donors in a variety of samples.

Fig. 4. Detection of S-Oxidation by Redox-Active Compounds in a Diesel Exhaust Particle Methanol Extract

(A) Mouse liver supernatant or (B) bovine serum albumin was exposed to a diesel exhaust particle (DEP) methanol extract (0 − 1 mg/mL) in the absence and presence of dithiothreitol (DTT, 4 mM) for 30 min at 37 °C. S-Oxidation of proteins was detected by the Redox-BPM-labeling enzyme-linked immunosorbent assay as described in Materials and Methods. Data are the mean ± standard error of three determinations. * p < 0.05, ** p < 0.01 compared with the control.

Acknowledgments

This work was supported in part by a Grant-in-Aid (#20K12180 to Y.A. and #18H05293 to Y.K.) for Scientific Research from the MEXT. We thank Renee Mosi, Ph.D.

Conflict of Interest

The authors declare no conflict of interest.

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