Epoxycyclohexenedione-type Compounds are a New Class of

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Epoxycyclohexenedione-type Compounds are a New Class of Inhibitors of the Bovine Mitochondrial ADP/ATP Carrier Ayaki Aoyama, Masatoshi Murai, Naoya Ichimaru, Shunsuke Aburaya, Wataru Aoki, and Hideto Miyoshi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01119 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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Biochemistry

Epoxycyclohexenedione-type Compounds are a New Class of Inhibitors of the Bovine Mitochondrial ADP/ATP Carrier

Ayaki Aoyama, Masatoshi Murai, Naoya Ichimaru, Shunsuke Aburaya, Wataru Aoki, and Hideto Miyoshi*

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

*Corresponding author: Hideto Miyoshi, Tel: (+81)-75-753-6119; E-mail: [email protected]

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ABBREVIATIONS AAC, ADP/ATP carrier; ADP, adenosine diphosphate; ATP, adenosine triphosphate; BKA, bongkrekic acid; CBB, Coomassie brilliant blue R250; CHCA, α-cyano-4-hydroxycinnamic acid; CNBr, cyanogen bromide; complex I, NADH-ubiquinone oxidoreductase; complex II, succinateubiquinone oxidoreductase; complex III, ubiquinol-cytochrome c oxidoreductase; ECHD, epoxycyclohexenedione; EDTA, ethylenediaminetetraacetic acid; HEPES, 2-[4-(2-hydroxyethyl)1-piperazinyl]ethanesulfonic acid; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; MOPS, 3-morpholinopropanesulfonic acid; MS, mass spectrometry; PAO, phenylarsine oxide; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SMP, submitochondrial particle; TAMRA, 6carboxy-N,N,N’,N’-tetramethylrhodamine; UQ0, ubiquinone-0.


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ABSTRACT Through the extensive screening of our chemical library, we found epoxycyclohexenedione (ECHD)-type compounds (AMM-59 and -120) as unique inhibitors of the bovine heart mitochondrial ADP/ATP carrier (AAC). The present study investigated the mechanism of the inhibition of AAC by ECHDs using submitochondrial particles (SMPs). Proteomic analyses of ECHD-bound AAC as well as biochemical characterization using different SH-reagents showed that ECHDs inhibit the function of AAC by covalently binding primarily to Cys57 and secondarily to Cys160. Interestingly, AAC remarkably aggregated in SMPs when incubated with high concentrations of ECHDs for a long period of time. This aggregation was observed under both oxidative and reductive conditions of the SDS-PAGE analysis of SMP proteins, indicating that aggregation is not caused by intermolecular S-S linkages. ECHDs are the first chemicals, to the best of our knowledge, to induce prominent structural alteration of AAC without forming intermolecular S-S linkages. When all solvent accessible cysteines (Cys57, Cys160, and Cys257) were previously modified by N-ethylmaleimide, the aggregation of AAC was completely suppressed. In contrast, when Cys57 or Cys160 is selectively modified by a SH-reagent, the covalent binding of ECHDs to a residual free residue of the two cysteines is sufficient to induce aggregation. The aggregation-inducing ability of another ECHD analogue (AMM-124), which has a shorter alkyl chain than AMM-59 and -120, was significantly less efficient than that of the two compounds. Based on these results, the mechanism underlying the aggregation of AAC induced by ECHDs was discussed.


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INTRODUCTION The mitochondrial machineries presiding over the synthesis of ATP via oxidative phosphorylation are promising targets for the synthetic development of agrochemicals and anthelmintic reagents. Many inhibitors of mitochondrial respiratory complexes (complexes I, II, and III) of harmful insects and fungi have been widely used in the agricultural field (1-3). Ohmura and coworkers reported that nafuredin and atpenins potently inhibit helminth (Ascaris suum) respiratory complexes I and II, respectively (4-6). In contrast to a wide variety of selective inhibitors of each of the respiratory enzymes, specific inhibitors of mitochondrial substrate carriers (mitochondrial carrier family proteins) are highly limited, except for the so-called SHreagents such as maleimide derivatives and organic mercurials. Only two classes of inhibitors (atractylosides and bongkrekic acids (BKAs)) have been identified for ADP/ATP carriers (AACs) so far, which are archetype members of the mitochondrial carrier family and one of the most abundant proteins of the inner mitochondrial membrane (7, 8). Recently, Todisco et al. (9) found two new inhibitors of AAC (suramin and chebulinic acid) through a docking-based virtual screening and in vitro Figure 1. Structures of compounds used in this study: AMM-59, AMM-120, AMM-124, compound transport assays. To the best of our knowledge, of spot A (in Figure 4), and TAMRA-N3. there is currently no specific inhibitor of phosphate carriers that also supplies ATP synthase with its substrate inorganic phosphate. The discovery of unique inhibitors, from the view point of chemical structures and/or action manners, of mitochondrial substrate carriers may contribute greatly not only to the acquisition of seed compounds for the development of new agrochemicals and anthelmintic reagents, but also to promising advances in basic research on mitochondrial carriers. The structural and functional properties of mitochondrial AACs have been extensively investigated using different techniques because of their high natural abundance and the availability of specific inhibitors (10-15); nevertheless, the mechanism underlying the transport of ADP/ATP remains largely elusive (16). Using our own and some commercially available chemical libraries, 4 ACS Paragon Plus Environment

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we initiated extensive screening three years ago aimed at discovering low-molecular-weight chemicals possessing novel molecular skeletons, which specifically inhibit the function of bovine heart mitochondrial AAC (AAC1, a major isoform). As an index of inhibitory effect, we chose the inhibition of [14C]ADP uptake by bovine heart submitochondrial particles (SMPs). This system is a representative assay that has been applied to the characterization of various inhibitors of AAC such as BKAs (17). During this screening (though we have not disclosed the results of screening yet), we found that epoxycyclohexenedione (ECHD)-type compounds (e.g. AMM-59, Figure 1), which are not similar in structure to AAC inhibitors hitherto known, are effective inhibitors of bovine AAC. In the present study, we investigated the action manner of AMM-59 using different SH-reagents for comparison and a synthetic derivative (AMM-120) that enables fluorescence detection of ECHDbound peptides for proteomic analyses. We found that they inhibit the function of AAC by covalently binding primarily to Cys57 and secondarily to Cys160, which are located on the 1st and 2nd matrix-side loops, respectively. Interestingly, when AAC in SMPs was incubated with high concentrations of ECHDs for a long period of time (> 4 h), the protein remarkably aggregated: the protein band of monomeric AAC completely disappeared from the SDS-PAGE gel that analyzed SMP proteins. Therefore, we also investigated the mechanism by which ECHDs induce the aggregation of AAC in SMPs. Although copper-O-phenanthroline (Cu(OP)2), which is an effective catalyst for the oxidation of SH groups, was previously reported to induce the dimerization (or higher polymerization) of AAC in SMPs by making an intermolecular S-S linkage (18), ECHDs was found to be the first chemicals, to the best of our knowledge, to induce such prominent structural alteration of AAC without forming intermolecular S-S linkages.


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EXPERIMENTAL PROCEDURES Materials Bongkrekic acid (BKA) and eosin-5-maleimide (EMA) were purchased from Sigma-Aldrich (St. Louis, MO). [14C]ADP was purchased from PerkinElmer (Waltham, MA). Protein standards (Precision Plus Protein Standards and Precision Plus Protein Dual Xtra Standards) for SDS-PAGE were from Bio-Rad (Hercules, CA). 4-HNE was obtained from Cayman Chemical (Ann Arbor, MI). Other reagents were all of analytical grade. Synthesis of AMM-59, AMM-120, and AMM124 The synthetic procedures for AMM-59, AMM-120, and AMM-124 are described in Supporting Information. All compounds were characterized by 1H- and 13C-NMR spectroscopy and mass spectrometry. Measurement of [14C]ADP uptake by bovine heart SMPs SMPs were prepared from isolated bovine heart mitochondria by the method of Matsuno-Yagi and Hatefi (19) and stored in buffer containing 250 mM sucrose and 10 mM Tris-HCl (pH 7.4) at -80 °C. Uptake of [14C]ADP by bovine SMPs was measured as reported previously (16). SMPs (1.2 mg of proteins/mL, 300 µL) were incubated with test compound at 37 ˚C for 20 min in buffer containing 125 mM KCl, 1 mM EDTA, 10 mM MOPS/KOH (pH 6.5), and oligomycin (5.0 µg/mL), the reaction was initiated by adding [14C]ADP (63 µM, 70 MBq/mmol) to the SMP suspension, and then further incubated on ice for 3 min. The reaction was terminated by the addition of BKA (10 µM), followed by immediate centrifugation (60,000 rpm at 4 ˚C for 20 min). Pellets were resuspended in the same buffer and centrifuged again, and the pellets obtained were solubilized in 2% (w/v) SDS and mixed with liquid scintillation cocktail (1 mL, Insta-Gel Plus, PerkinElmer). Radioactivity was assessed using a liquid scintillation counter (Wallac 1409 DSAS). The radioactivity in SMPs, which were incubated with an excess of BKA (10 µM, 10 min on ice) before the addition of [14C]ADP, was used to correct the nonspecifically bound [14C]ADP. Binding assay of mitochondrial proteins to immobilized phenylarsine oxide Phenylarsine oxide (PAO) was immobilized on agarose resin according to the procedures reported by Halestrap and colleagues (20-22). Briefly, HEPES was added to a final concentration of 50 mM to a solution of 4-aminophenylarsine oxide (5.0 mg, Toronto Research Chemicals, Toronto, Canada) in water (1 mL) containing aqueous HCl (pH 1.5), and this was followed by the addition of 1.0 M NaOH to pH 7.2. The mixture was reacted with Affi-Gel-10 (Bio-Rad, 5 mL) at room temperature for 15 h, and non-reacted groups were then blocked by ethanolamine (0.3 M, pH 8.0). Resin modified with PAO was washed several times with equilibration buffer (50 mM HEPES/NaOH, 150 mM Na2SO4, 1 mM EDTA, pH 7.2) containing 0.25% (w/v) Triton-X-100 and stored at 4 ˚C. Bovine SMPs (5.0 mg of proteins/mL, 50 µL) were incubated with various AAC-specific 6 ACS Paragon Plus Environment

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Biochemistry

ligands, such as BKA, PAO, UQ0, and ECHDs, in equilibration buffer (above) at room temperature for 20 min. The suspension was diluted with equilibration buffer (450 µL) containing 0.5% (w/v) Triton X-100 and the same concentration of ligands on ice for 1 h. Solubilized SMPs were centrifuged (60,000 rpm at 4 ˚C for 20 min), and the supernatant was incubated with the PAOimmobilized resin (50 µL) at room temperature for 30 min. The resin was washed five times with equilibration buffer containing 0.25% (w/v) Triton X-100, and the bound proteins were released in equilibration buffer containing 0.25% (w/v) Triton X-100 and 20 mM DTT at room temperature for 30 min. The specific capture/release procedures described above were monitored by Laemmli-type SDS-PAGE (23). Modification of bovine AAC by AMM-120 SMPs (2.0 mg of proteins/mL, 100–200 µL) were incubated with AMM-120 (1–10 µM) in buffer containing 250 mM sucrose, 1.0 mM MgCl2, and 50 mM KPi (pH 7.4) at 37 ˚C for 1–12 h. SMPs were then collected by centrifugation (60,000 rpm at 4 ˚C for 20 min) and denatured in 1% (w/v) SDS (25–50 µL). The AMM-120-bound proteins were conjugated with a fluorescent TAMRA-N3 tag (20 µM) via Cu+-catalyzed click chemistry using the Click-iT protein reaction buffer kit (Thermo Fisher Scientific, Waltham, MA) according to the same procedures described previously (24-26). Proteins were recovered by methanol/chloroform precipitation, followed by resolution on a 12.5% Laemmli-type SDS gel. The migration pattern of the TAMRA-conjugated proteins was visualized using a model FLA-5100 Bio-imaging analyzer (Fuji Film, Tokyo, Japan) with a 532 nm light source and LPG (575 nm) filter. Data processing and image quantification were performed using Multi Gauge software (Fuji Film). Modification of bovine AAC by EMA or NEM AAC in SMPs was modified by EMA or NEM according to the procedures reported by Majima et al. (27, 28). Regarding the modification by EMA, SMPs (20 mg of proteins/mL, 230 µL) were incubated with EMA (200 µM, 10 nmol/mg of protein) in buffer containing 250 mM sucrose and 10 mM Tris/HCl (pH 7.4) on ice for 1 min. Non-reacted (excess) EMA was quenched with 200 mM of DTT (10 µmol/mg of protein). Excess DTT was removed by centrifugation (60,000 rpm at 4 ˚C for 20 min, twice), and pellets were resuspended in the same buffer. SMP proteins were analyzed on a 12.5% Laemmli-type SDS gel and, subjected to fluorescent gel imaging and CBB staining. When the effects of AAC-specific ligands (AMM-59, ADP, BKA, and NEM) were examined (Figure 7), they were added and incubated at 37 ˚C for 1 h (in the case of AMM-59) or on ice for 10 min (in the case of ADP, BKA, and NEM) prior to the treatment with EMA. Regarding the modification of AAC by NEM, SMPs (20 mg of proteins/mL) were incubated with NEM (2.0 mM, 100 nmol/mg of protein) in a buffer containing 250 mM sucrose and 10 mM Tris/HCl (pH 7.4) on ice for 10 or 60 min, and non-reacted NEM was quenched with 200 mM of DTT. Excess DTT was removed by centrifugation (60,000 rpm at 4 ˚C for 20 min, twice), and pellets were resuspended in the same buffer. These SMP samples were used in the experiments 7 ACS Paragon Plus Environment

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shown in Figures 6B and 11C. Cross-linking of AAC by Cu(OP)2 The cross-linking of AAC by Cu(OP)2 was performed as described previously (18). SMPs (4.0 mg of proteins/mL) were suspended in buffer containing 250 mM sucrose and 10 mM Tris/HCl (pH 7.4), and incubated with AMM-59 (5–100 µM) at 37 ˚C for 1 h or NEM (0.4 mM) on ice for 10 min. SMP samples were then treated with 200 µM of Cu(OP)2 on ice for 10 min. The reaction was quenched by the addition of EDTA (5.0 mM) and NEM (5.0 mM), and the mixture was incubated on ice for 10 min. The SMPs were treated with Laemmli’s sample buffer (23) without 2-mercaptoethanol and analyzed on a 12.5% Laemmli-type SDS-PAGE, followed by CBB staining and a Western blot analysis. The SDS-PAGE analysis was also conducted under reductive conditions using Laemmli’s sample buffer containing 2-mercaptoethanol (0.35 M). Immunochemical analysis The mobility of AAC on the SDS gel was monitored by a mouse monoclonal anti-AAC1 antibody (MitoSciences, Eugene, OR). Proteins separated on the SDS gel were transferred onto a PVDF membrane according to the conditions described elsewhere (26). The membrane was blocked with 1% (w/v) gelatin in phosphate-buffered saline containing 0.5% (w/v) Tween 20 (Tween PBS) and then probed with the primary antibody (1:1000 dilution), followed by an incubation with the alkaline phosphatase-conjugated secondary antibody. The membrane was washed three times with Tween PBS and developed with NBT/BCIP colorimetric reagents (BioRad). Proteomic analysis To localize the region at which AMM-120 covalently bound, AAC was partially purified by hydroxyapatite chromatography (18, 26, 27). SMPs (400–800 µg of proteins/mL), which were incubated with 10 µM of AMM-120 (at 37 ˚C for 1 h), were solubilized in buffer containing 500 mM NaCl, 0.5 mM EDTA, 10 mM MOPS/NaOH (pH 7.4), and 5% (w/v) Triton X-100 on ice for 1 h, and insoluble materials were removed by centrifugation at 60,000 rpm for 20 min. The supernatant (300–400 µL) was loaded onto a small hydroxyapatite column (300–500 µL volume) equilibrated with buffer containing 100 mM NaCl, 0.05 mM EDTA, 10 mM MOPS/NaOH (pH 7.4), and 0.5% Triton X-100. The flow-through fraction (400-600 µL) was collected, precipitated with methanol/chloroform, and the resulting precipitate was subjected to conjugation with a fluorescent TAMRA–azido tag via click chemistry, as described above. The TAMRA-conjugated AAC was cleaved by 25 mM cyanogen bromide (CNBr) in 70% formic acid. Alternatively, AAC was digested with Lys-C in buffer containing 50 mM Tris/HCl and 0.1% SDS (pH 8.5). Digests were separated on a Schägger-type SDS gel (16.5% T, 6.0% C containing 6.0 M urea, ref. 29) In the mass spectrometric analysis of proteins, CBB-stained protein bands were in gel digested 8 ACS Paragon Plus Environment

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Biochemistry

with trypsin in buffer containing 25 mM NH4HCO3 at 37˚C overnight. Tryptic digests were desalted with ZipTip (Millipore) and spotted onto the target plate using CHCA as a matrix. A mass spectrometric analysis was conducted using a Bruker Autoflex III Smartbeam instrument (MALDI-TOF/TOF, Bruker Daltonics). The MS spectra obtained were analyzed according to the previous procedures (26). Identification of the cysteine that reacted with ECHD by mass spectrometry SMPs (3.5 mg of proteins/mL, total 1.6 mg) were incubated with AMM-120 (5 µM) in buffer containing 250 mM sucrose, 1.0 mM MgCl2, and 50 mM KPi (pH 7.4) at 37 ˚C for 3 h. AMM120-bound proteins were conjugated with cleavable biotin-SS-N3 (20 µM, Figure 1) via Cu+catalyzed click chemistry (24-26), followed by the recovery of proteins by methanol/chloroform precipitation. Biotinylated proteins were solubilized with 2% (w/v) SDS in a total volume of 60 µL, then diluted with Tris-buffered saline (1.0 mL) containing 1% (w/v) Triton X-100. Biotinylated proteins were enriched using streptavidin-agarose CL-4B (Sigma-Aldrich, St. Louis, MO) according to the procedures described previously (24-26). Specific capture/release procedures were monitored by SDS-PAGE (Figure 10), and the modified AAC was separated as a single band on the SDS gel, which was digested with trypsin. The identification and characterization of AAC were conducted using an LTQ Velos Orbitrap mass spectrometer equipped with Ultimate 3000 nano-LC (LC-MS, Thermo Scientific, Walthman) under the same experimental conditions as those reported previously (24, 25). Data were analyzed using Proteome Discoverer 2.1 (Thermo Scientific) with Mascot 2.3 (Matrix Science, London, U.K.). Regarding tryptic digestion, calbamidomethylation (Cys) was set as a static modification, while oxidation (Met) and the modification by AMM-120 ((C28H42N6O5S) at Cys (see Figure S1)) were set as dynamic modifications. The MS and MS/MS spectra were compared against SwissProt database, and they were filtered at a q value of ≤ 0.01 corresponding to a 1% false discovery rate.


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RESULTS ECHD-type compounds inhibit ADP uptake in bovine heart SMPs We initiated three years ago the comprehensive screening of chemicals, which inhibit the function of mitochondrial AAC. This screening was performed by directly measuring the uptake of adenine nucleotide by bovine heart SMPs: SMPs (1.2 mg of proteins/mL) were incubated with each test compound (10 µM) at 37˚C for 20 min, followed by the addition of 63 µM [14C]ADP on ice for 3 min, and transport was terminated by the addition of 10 µM BKA. During this screening, we found synthetic compounds possessing an ECHD skeleton to 14 inhibit [14C]ADP uptake; for example, AMM-59 Figure 2. Inhibition of [ C]ADP uptake by SMPs by AMM-59. Bovine SMPs (1.2 mg of (Figure 1) elicited distinct inhibitory effect. proteins/mL, 300 µL) were incubated with AMMIncorporated radioactivities decreased to 60 (± 59 or AMM-120 at 37˚C for 20 min according to the procedures described in Experimental Procedures, 12) and 22% (± 5) of the control in the presence and [14C]ADP (63 µM, 70 MBq/mmol) was then of 10 and 30 µM of AMM-59, respectively added to the suspension, followed by 14an incubation on ice for 3 min. The uptake of [ C]ADP was (Figure 2). We did not examine at higher terminated by the addition of BKA (10 µM), concentrations of AMM-59 because it induced followed by the quantification of radioactivity incorporated into SMPs. The nonspecifically aggregation of AAC, as described later. AMM- bound radioactivity was corrected by running the (10 µM, 10 min on 59 did not exert any inhibitory effect on incubation with an excess BKA ace) before the addition of [14C]ADP. The specific respiratory enzyme complexes (complexes I~IV), (BKA-sensitive) uptake of [14C]ADP by AAC was except complex I, at a concentration range that estimated to be approximately 430 pmol/mg of protein in the absence of AMM-59. Values were AMM-59 the averages of three independent measurements ± inhibited the [14C]ADP uptake. inhibited the complex I activity by 14% (± 5) at SD. the concentration of 25 nmol/mg of proteins, which is equivalent to 30 µM application in the [14C]ADP uptake experiment (Figure S2). Effect of ECHDs on the specific capture of AAC by immobilized PAO The vicinal thiol reagent phenylarsine oxide (PAO) was previously reported to cross-link to the cysteine residues Cys160 and Cys257 of bovine AAC, which are located on the 2nd and 3rd matrix-side loops, respectively (20). PAO-immobilized agarose resin, which was prepared by making use of this property, is useful for studying interactions between AAC and low-molecularweight chemicals (21, 22). Bovine SMPs (5.0 mg of proteins/mL) were solubilized in buffer containing 0.5% (w/v) Triton X-100, incubated with the PAO-immobilized resin, and bound proteins were released using buffer 10 ACS Paragon Plus Environment

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Biochemistry

containing 20 mM dithiothreitol (DTT), followed by 12.5% Laemmli-type SDSPAGE. As shown in Figure 3, a limited number of proteins were captured on the PAO-immobilized resin with a predominant CBB-stained band at ~30 kDa (a control lane in elute), which was identified as AAC by MALDI TOF-MS (12 peptides, 44% coverage) with a protein score of 82 (protein scores greater than 54 are significant (p < 0.05)). The specific binding of AAC to the PAO-immobilized resin was suppressed in the presence of free PAO or a short chain ubiquinone analogue UQ0 (Figure 3), as reported previously (22). AMM-59 also suppressed the binding of AAC to PAOimmobilized resin, suggesting that the inhibition of [14C]ADP uptake by this compound is attributable to the direct interaction of AMM-59 with AAC.

Figure 3. Effects of AMM-59 on the specific capture of AAC by immobilized PAO. SMPs (5.0 mg/mL, 50 µL) were incubated with PAO (100 µM), UQ0 (50 µM), or AMM-59 (20 µM) at room temperature for 20 min, followed by solubilization in buffer containing 0.5% (w/v) Triton X-100. Solubilized proteins were incubated with PAO-immobilized resin, and bound proteins were released by buffer containing 20 mM DTT according to the procedures described in Experimental Procedures. Specific capture and release procedures were monitored by 12.5% Laemmli-type SDS-PAGE (shown as unbound and elute). The ~30 kDa protein captured, indicated by the arrowhead, was identified as AAC using LC-MS/MS. Data are representative of three independent experiments.

Reactivity of the -SH group against AMM-59 Since ECHD-type compounds possess two electrophilic reaction points (an epoxy group and α,β-unsaturated carbonyl moiety), they may have a potential ability to react with some nucleophilic amino acids in mitochondrial AAC, such as cysteine (20, 27, 28). Taking this into consideration, the inhibition of the function of AAC by AMM-59 may be attributed to its covalent binding to the protein. To confirm this, we examined the reactivity of the -SH functional group against AMM-59 using ethanethiol (CH3CH2SH). AMM-59 (40 mM) and ethanethiol (200 mM) were incubated in dichloromethane (80 µL) at room temperature and no reaction was noted. However, when a catalytic amount of a weak base (diisopropylethylamine) was added to this mixture, the reaction occurred gradually with time, and, after a 30-min reaction, AMM-59 almost completely disappeared and two major products were detected using silica gel thin layer chromatography (spots A and B in Figure 4). We attempted to isolate these products using silica gel column chromatography. Although spot B compound was unstable under chromatographic conditions, we isolated spot A compound and analyzed its structure by 1H-NMR, 13C-NMR, and ESI-MS: 1H-NMR 400 MHz, CDCl3 δ 8.16 (d, 1H, J = 2.80 Hz), 7.75 (s, 1H), 7.10 (s, 1H), 6.75 (d, 1H, J = 2.88 Hz), 6.51 (s, 1H), 2.74 (q, 2H, J = 7.33 Hz), 2.43 (t, 2H, J = 7.40 Hz), 1.78-1.65 11 ACS Paragon Plus Environment

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(m, 2H), 1.41-1.21 (m, 14H), 1.23 (t, 3H, J = 7.34 Hz), 0.88 (t, 3H, J = 6.86 Hz); 13CNMR 100 MHz, CDCl3 δ 172.8, 150.0, 139.4, 126.0, 116.0, 109.0, 104.4, 38.4, 32.1, 30.8, 29.8, 29.7, 29.6, 29.5, 29.4, 25.9, 22.9, 15.1, 14.3; ESI-MS (m/z) 354 [M+H]+, 376 [M+Na]+. Based on the spectral data, we tentatively assigned its structure, as shown in Figure 1. This model reaction indicated that the -SH group indeed reacts with AMMFigure 4. Thin layer chromatography (TLC) of AMM59 in bulk medium. Since we were unable 59 reacted with ethanethiol (CH3CH2SH). AMM-59 (40 to assign all products, we cannot exclude the mM, 1.0 eq.) and ethanethiol (200 mM, 5.0 eq.) were incubated in dichloromethane in the presence of N,Npossibility that the -SH group also reacts diisopropylethylamine (21 mM, 0.5 eq.), and the reaction with the double bond of the α,β-unsaturated was allowed to continue at room temperature for 60 min. Aliquots of the reaction mixture at 5, 30, and 60 min were carbonyl moiety via Michael addition. The analyzed by TLC (Merk TLC plate Silica-gel 60F) using model reaction also indicated that potential 50% ethyl acetate/n-hexane as the mobile phase. Plates were sprayed with p-anisaldehyde, and separated spots products (or their intermediate compounds) were detected by heating to 300 ˚C for 2 min. are considerably unstable because the remarkable accumulation of polar decomposed substances was observed at the origin of thin layer chromatography (Figure 4). Synthesis of alkyne-tagged AMM-120 and its characterization Since AMM-59 was found to exhibit strong reactivity against the -SH group, our working hypothesis (namely, the inhibition of the function of AAC by AMM-59 is attributable to its covalent binding to the protein) gained practicality. To verify this, we needed to visualize the binding of AMM-59 to AAC in further experiments. Therefore, we synthesized AMM-120, which has a terminal alkyne in its hydrophobic tail (Figure 1). As we demonstrated previously (24, 25), the alkyne group allows the conjugation of molecular tags such as fluorophores and biotin via so-called Cu+-catalyzed click chemistry (azido-alkyne [3+2] cycloaddition) (30). We confirmed that AMM-120 also inhibits [14C]ADP uptake by SMPs and its inhibitory potency is almost identical to that of AMM-59 (Figure 2). Bovine SMPs (2.0 mg of proteins/mL) were incubated with various concentrations of AMM120 (1, 5, or 10 µM) at 37˚C for 12 h, solubilized with 1% (w/v) SDS, and then subjected to conjugation with a fluorescent TAMRA-N3 tag (Figure 1), followed by resolution on a 12.5% Laemmli-type SDS gel. A major fluorescent band was observed at ~30 kDa, the mobility shift of which was identical to that of AAC (Figure 5A, the lower panel). Fluorescent intensity increased as the concentrations of AMM-120 increased, and peaked within a ~2-h incubation. These results indicate that AMM-120 (also AMM-59) indeed covalently binds to AAC because if 12 ACS Paragon Plus Environment

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Biochemistry

the covalent bond is not formed between AMM-120 and AAC, TAMRA that was attached to AMM-120 via click chemistry is eliminated from the protein during the electrophoresis. AMM-120 also labeled the protein corresponding to a faint fluorescent band at ~40 kDa; the averaged fluorescence intensity of TAMRA bound to the protein, which was evaluated by image scanning, was 15% (± 3) of that observed for AAC. Although we were unable to identify this protein, it is likely that the binding affinity of AMM120 to the protein is substantially low because an excess AMM-59 could not efficiently suppress the labeling, as seen in Figure 5A (lower panel, the two lanes on the right side). The fluorescence intensities observed for other proteins were negligibly small (< 2%). To verify whether AMM-120 binds to the same position in AAC as that of AMM-59, we performed a competition test between AMM-120 and AMM-59; SMPs were incubated with AMM-120 (10 µM) for 12 h in the presence of AMM-59 (50 or 100 µM). Surprisingly, a monomeric AAC band disappeared from the SDS gel stained by CBB, whereas the behavior of other mitochondrial proteins almost did not change in the gel (Figure 5A, the upper panel). The fluorescence of TAMRA conjugated to AMM-120 Figure 5. Chemical modification of AAC in SMPs by AMM120. (A) SMPs (2.0 mg of proteins/mL) were incubated with various concentrations of AMM-120 at 37 ˚C for 12 h, denatured with 1% (w/v) SDS, and conjugated with fluorescent TAMRA-N3 (20 µM) via Cu +-catalyzed click chemistry. Proteins were resolved by a 12.5% Laemmli-type SDS gel and subjected to CBB staining (upper panel) or fluorescent gel imaging (lower panel). To verify the binding specificity of AMM-120 to AAC, SMP were also incubated in the presence of an excess of AMM-59 (50 or 100 µM). Approximately 50 µg of the proteins were loaded onto each lane. The position of AAC on the SDS gel is marked by an arrowhead. (B) SMPs (2.0 mg of proteins/mL) were incubated with AMM-59 or AMM-120 alone at 37 ˚C for 12 h and denatured with 1% (w/v) SDS. Proteins were resolved by a 12.5% Laemmli-type SDS gel and subjected to CBB staining. Approximately 50 µg of the proteins were loaded onto each lane. Data shown are the representative of three independent experiments.


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that covalently bound to AAC also completely disappeared (Figure 5A, the lower panel). Since aggregated proteins were observed at the point of origin, this result indicated that the incubation of SMPs with high concentrations of AMM-59 for a long period of time induces the aggregation of AAC. It should be noted that aggregation also occurred in the presence of high concentrations (>50 µM) of AMM-59 or AMM-120 alone, as shown in Figure 5B. The mechanism underlying the aggregation of AAC induced by ECHDs will be investigated later on; however, we characterized the covalent binding of low concentrations of ECHDs (< 10 µM) to AAC in the next section. Characterization of the covalent binding of ECHDs to AAC We characterized the covalent binding of ECHDs to AAC in bovine SMPs using AMM-120, which enables visualization by conjugation with TAMRA-N3. SMPs (2.0 mg of proteins/mL) were incubated with AMM-120 (10 µM) for 1 h in the presence of various compounds that bind to AAC, namely, phenylarsene oxide (PAO, 100 µM) that cross-links to Cys160 and Cys257 (20) and BKA (20 µM). We confirmed that AMM-120 inhibits AAC, but does not induce aggregation under the experimental conditions used (i.e. a short incubation period, 1 h). The covalent binding of AMM-120 was not interfered with PAO or BKA (Figure 6A), indicating that the binding site of ECHDs in AAC is not identical to those of PAO and BKA. Bovine AAC have three solvent-accessible cysteine residues: Cys57, Cys160, and Cys257, which are located on the 1st, 2nd, and 3rd matrixside loops, respectively (10). Of these, the cysteine reactive reagent N-ethylmaleimide (NEM) binds rapidly to Cys57, but slowly to Cys160 (or Cys257); Cys57 is predominantly modified when SMPs are incubated with NEM Figure 6. Characterization of the covalent binding of AMM-120 to AAC. (A) SMPs (2.0 mg of for a short incubation period (20, 27). Based on proteins/mL) were incubated with AMM-120 (10 for 1h in the presence of AAC-specific ligands the fluorescence intensity of TAMRA attached to µM) such as PAO (100 µM) and BKA (20 µM), followed AMM-120 (evaluated by image scanning), the by conjugation with TAMRA-N3 (20 µM) and SDSon a 2.5% Laemmli-type SDS gel. (B) covalent binding of AMM-120 (10 µM) to AAC PAGE SMPs were pre-incubated with 2.0 mM of NEM was suppressed by 81% (± 10) when we used (~100 nmol of NEM/mg of proteins) for 10 or 60 according to the procedures described in SMP samples pretreated with NEM (100 min Experimental Procedures. SMP samples modified nmol/mg of proteins) on ice for 10 or 60 min by NEM were further incubated with AMM-120 (10 for 1 h and conjugated with TAMRA-N3, (Figure 6B). When the incubation period of µM) followed by SDS-PAGE. Approximately 50 µg of NEM was short (10 min), the extent of the proteins were loaded onto each lane. The of AAC on the SDS gel is marked by an suppression somewhat varied in each experiment position arrowhead. Data shown are representative of three possibly because modification of the secondary independent experiments. 14 ACS Paragon Plus Environment

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Biochemistry

reactant (Cys160 or Cys257) by NEM is effected even by slight differences in incubation conditions. The residual labeling (~20%) after the NEM treatment for 60 min was observed possibly because Cys160 was not completely labeled by NEM even after 60 min incubation, as observed by Majima et al. (27). Together, it is conceivable that low concentrations of ECHDs predominantly bind to Cys57; however, we cannot exclude the possibility that they also bind to Cys160 and/or Cys257, albeit much less efficiently. Eosin-5-maleimide (EMA) is another cysteine reactive fluorescent reagent that predominantly binds to Cys160 in AAC of bovine heart SMPs (27, 28). SMPs (4.0 mg of proteins/mL) were incubated with various AAC-specific ligands (ADP, BKA, and 7. Effects of AMM-59 on specific modification NEM) including AMM-59 and then treated Figure of AAC by EMA. SMPs (4.0 mg of proteins/mL) were with 40 µM of EMA (10 nmol/mg of incubated with AMM-59 (5 or 10 µM, at 37 ˚C for 1 h), (5 or 10 mM, on ice for 10 min), BKA (10 or 20 µM, proteins) on ice for 10 min, followed by ADP at 37 ˚C for 1 h), or NEM (0.4 mM, on ice for 10 min), 12.5% Laemmli-type SDS-PAGE. We treated with fluorescent EMA (40 µM, 10 nmol/mg of on ice for 10 min, and then resolved on a 12.5% note that the experimental conditions proteins) Laemmli-type SDS gel, followed by fluorescent gel including the concentrations of ADP, BKA, imaging (upper panel) or CBB staining (lower panel). 50 µg of the proteins were loaded onto and NEM were set to be identical to those Approximately each lane. The position of AAC on the SDS gel is Data shown are used in ref. 27 to make comparison possible. marked by an arrowhead. representative of three independent experiments. Based on the fluorescence intensity of EMA evaluated by image scanning, ADP (5 and 10 mM) suppressed the binding of EMA to AAC by 94% (± 3) (Figure 7). BKA suppressed the binding by 25 (± 5) and 74% (± 8) at 10 and 20 µM, respectively. These suppressive effects are in agreement with the previous findings (28). In contrast, the pretreatment by NEM (100 nmol/mg of proteins, a 10-min incubation) suppressed the binding of EMA just by 16% (± 5) because NEM predominantly binds to Cys57 during short incubation period (20, 27). Although the pretreatment with 5.0 or 10 µM of AMM-59 suppressed the binding of EMA by 17 (± 5) and 34% (± 4), respectively, the extent of the suppression was much less than that by ADP (10 mM) or BKA (20 µM). These results strongly suggest that although a portion of AMM-59 binds to Cys160, this cysteine residue is not a predominant reactant of AMM-59 in the concentration range examined (< 10 µM), as described above. 15 ACS Paragon Plus Environment

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The cross-linking reagent copper-Ophenanthroline (Cu(OP)2) is a catalyst that causes the dimerization of AAC via the formation of an intermolecular S-S bridge between the Cys57 residues of each monomer (18). Therefore, we examined the effects of AMM-59 on the dimerization of AAC catalyzed by Cu(OP)2. SMPs (4.0 mg of proteins/mL) were incubated with different concentrations of AMM-59 (5–100 µM) at 37˚C for 1 h, treated with 200 µM Cu(OP)2 on ice for 10 min, and then resolved on a “non-reductive” 12.5% Laemmli-type SDS gel, followed by CBB staining and a Western blot analysis using an AAC1 antibody. We previously confirmed that no significant aggregation of AAC induced by AMM-59 alone was observed under the experimental conditions (i.e. 1 h incubation). As shown in Figure 8A, dimer and higher aggregates of AAC were observed without the pretreatment (a control lane). The polymerization was significantly suppressed with a concomitant increase in monomeric AAC by the treatment with NEM (100 nmol/mg of proteins, a 10-min incubation on ice), as reported previously (18). Dimerization and higher aggregation were gradually suppressed and the monomeric band intensity increased with an increase in the concentrations of AMM-59. Interestingly, the formation of a trimer was observed in the presence of high concentrations of AMM-59 (50 and 100 µM). While we currently have no clear explanation for this phenomenon, it is likely that since AMM-59 predominantly bound to Cys57 under the experimental conditions used, Cu(OP)2 catalyzed the formation of

Figure 8. Effects of AMM-59 on the dimerization of AAC catalyzed by Cu(OP)2. (A) SMPs (4.0 mg of proteins/mL) were incubated with various concentrations of AMM-59 (5–100 µM) at 37 ˚C for 1 h or NEM (0.40 mM) on ice for 10 min, then treated with 200 µM of Cu(OP)2 on ice for 10 min. Proteins were resolved by “non-reductive” 12.5% Laemmlitype SDS-PAGE using sample buffer without 2mercaptoethanol, followed by CBB staining and a Western blot analysis using a monoclonal AAC antibody. (B) The same protein samples were resolved in parallel by “reductive” 12.5% Laemmlitype SDS-PAGE using sample buffer containing 0.35 M 2-mercaptoethanol to cleave intermolecular S-S cross-linking. Approximately 50 µg of the proteins were loaded onto each lane. The position of AAC on the SDS gel is marked by an arrowhead. Data shown are representative of three independent experiments.


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Biochemistry

intermolecular S-S bridges via two residual free cysteines (Cys160 and Cys257) to provide the trimer. It is worth noting that when the SDS-PAGE analysis of the protein samples that were prepared in Figure 8A was conducted under “reductive” conditions containing 0.35 M 2-mercaptoethanol, the formation of dimer and higher aggregates of AAC was not detected because of the cleavage of intermolecular S-S linkages (Figure 8B), which is consistent with previous findings (18). Collectively, the results obtained using Cu(OP)2 also suggest that ECHDs bind predominantly to Cys57. Identification of the binding site of ECHDs in AAC The results described above strongly suggest that ECHDs covalently bind to cysteine residue(s)

Figure 9. Localization of the binding site of AMM-120 in AAC. (A) SMPs (2.0 mg of proteins/mL) were treated with AMM-120 (10 µM) for 1 h, solubilized with 5% (w/v) Triton X-100, and AAC was partially purified using hydroxyapatite chromatography, followed by conjugation with TAMRA-N3 (20 µM) via Cu+-catalyzed click chemistry. The TAMRA-attached AAC was digested with CNBr or Lys-C according to the procedures described in Experimental Procedures. Digests were separated on a Schägger-type SDS gel (16.5% T, 6% C containing 6.0 M urea), and subjected to fluorescent gel imaging or CBB staining. Data shown are representative of three independent experiments. (B) Schematic presentation of the digestion of AAC. The predicted cleavage sites are denoted with arrows and indicated by their residue numbers in the mature sequences of bovine AAC1 (SwissProt entry P02722).


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in AAC, predominantly to Cys57. To localize their binding site in AAC, SMPs (2.0 mg of proteins/mL) were incubated with 10 µM AMM-120 for 1 h, solubilized with 5% (w/v) Triton X100, and passed through a hydroxyapatite column, followed by conjugation with a fluorescent TAMRA-N3 tag via Cu+-catalyzed click chemistry. Hydroxyapatite chromatography provided AAC as an almost single band on a SDS gel (the control lane in Figure 9A), as reported previously (27, 31). The purified AMM-120-bound AAC was digested by CNBr or lysylendopeptidase (Lys-C), and the digests were separated on a 16.5% Schägger-type tricine gel. CNBr cleavage provided a strong fluorescent band in the ~20 kDa fragment (Figure 9A). MALDI-TOF MS analysis of the tryptic digests of this fragment revealed that it contains the internal sequences Tyr81–Lys92 and Gly172-Arg188 (m/z 1446.79 and 1927.11, respectively). Based on the theoretical cleavage sites by CNBr, the ~20 kDa fragment must be Ser2–Met201 (~21.9 kDa), which contains Cys57, Cys129, and Cys160 (Figure 9B). Lys-C digestion provided a strong fluorescent band, the apparent molecular mass of which was ~2 kDa, suggesting that the fragment is assigned to either or both of Gly53–Lys63 (~1.2 kDa) or Gly148–Lys163 (~1.7 kDa), containing Cys57 and Cys160, respectively (Figure 9B). The faint fluorescent bands at 3–5 kDa in the CNBr digests may be attributed to the binding of AMM-120 to Cys257, albeit to a lesser extent. To identify the cysteine that reacted with AMM-120, the SMP sample (3.5 mg of proteins/mL) treated with AMM-120 (5–10 µM) was conjugated with cleavable biotin-SS-N3 (20 µM, Figure 1) via Cu+-catalyzed click chemistry. The biotinylated proteins were specifically captured by immobilized streptavidin, followed by the “in gel” digestion of the enriched AAC with trypsin (Figure 10). Based on the product obtained from the model reaction using AMM-59 and ethanethiol (spot A in Figure 4), the digests Figure 10. Enrichment of the AAC modified by were extensively characterized using LC-MS/MS AMM-120. SMPs (3.5 mg of proteins/mL) were incubated with AMM-120 (5–10 µM) at 37 ˚C for 3 by setting a specific cysteine modification h, denatured with 1% (w/v) SDS, and then + [C28H42N6O5S, exact mass of 574.2937 (Figure conjugated with cleavable biotin-SS-N3 via Cu catalyzed click chemistry. The biotinylated S1)] as a variable protein modification. proteins were enriched using streptavidin-agarose, Although the enriched protein was identified as as described in Experimental Procedures. Specific capture/release procedures were monitored by SDSAAC with sequence coverage of 78% including PAGE (input, unbound, wash1, wash2, and elute). four cysteine residues (Figure S3), no modified The enriched AAC, indicated by an arrowhead, was digested with trypsin, and analyzed using an peptides were detected in the digests. The Orbitrap mass spectrometer. candidate peptides Gly53–Arg60 (containing 18 ACS Paragon Plus Environment

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Biochemistry

Cys57) and Glu153–Lys163 (containing Cys160) were identified as a standard carbamidomethylated form (m/z 931.50 and m/z 1239.60, respectively), not as the predicted adduct form. This may have been because the unstable modified product was cleaved under the analytical conditions used. There may be another reason why we failed to determine the modified tryptic digest in the enriched AAC. As we described in the above section (Figure 4), the reaction of AMM-59 with ethanethiol provided several products (mainly two products) in bulk medium. Among them, we were only able to isolate one product corresponding to spot A. Therefore, we cannot rule out the possibility that the reaction between AMM-120 and cysteine (predominantly Cys57) in AAC forms a different type of product, the structure of which has not yet been determined due to its instability under the experimental conditions used. The cysteine residue modified by AMM-59 was also assessed indirectly by mass spectrometry after the differential labeling of AAC with light (d0-) or heavy (d5-) NEM (32). SMPs were treated with d0- or d5-NEM in the absence or presence of AMM-59, respectively, followed by the isolation of AAC and digestion with trypsin. The combined tryptic digests were characterized based on the generated 5 Da mass shift. As a result, this trial was unsuccessful because the MS spectra of the tryptic digests containing Cys57 modified by d0- or d5-NEM were too weak to quantitatively evaluate the difference between the two groups (the absence or presence of AMM59). Mechanism of the aggregation of AAC induced by ECHDs We investigated the mechanism underlying the aggregation of AAC induced by ECHDs. SMPs (2.0 mg of proteins/mL) were incubated with different concentrations of AMM-59 (10–200 µM) at 37˚C for 4 h, and then analyzed by 12.5% Laemmli-type SDS-PAGE. We herein set the incubation period to be slightly short in order to make it easier to monitor the polymerization step including the formation of dimeric AAC. The CBB-stained band (~30 kDa) corresponding to monomeric AAC shifted slightly to the upper side, and the strength of the band decreased as the concentration of AMM-59 increased (Figure 11A). Traces of the dimeric state of AAC at ~60 kDa and higher polymerized AAC were observed in the Western blot analysis. The dimeric state of AAC was not detected by CBB staining because of the very small quantity present, indicating that the dimeric state rapidly progresses to further polymerized states. We also examined the time dependency of the aggregation of AAC induced by AMM-59 (100 µM). Aggregation progressed gradually with time and a monomeric AAC band significantly disappeared after an 8-h incubation (Figure 11B). Traces of dimeric AAC and higher polymerized AAC were also detectable by the Western blot analysis. It is important to emphasize that unlike the dimerization (and higher polymerization) of AAC catalyzed by Cu(OP)2 (Figure 8), the aggregation induced by AMM-59 was detected even when the SDS-PAGE analysis was conducted under reductive conditions: the experiments shown in Figures 11A and 11B were conducted under reductive conditions containing 0.35 M 2-mercaptoethanol. These results 19 ACS Paragon Plus Environment

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indicate that the aggregation of AAC does not involve intermolecular S-S bridges. Since it is conceivable that the covalent binding of ECHDs to cysteines in AAC triggers aggregation, we examined whether NEM, which suppressed the binding of AMM-120 to AAC (Figure 6), protects AAC from aggregation. When SMPs were pretreated with NEM (100 nmol/mg of proteins) on ice for 60 min (three solvent-accessible cysteines (Cys57, Cys160, and Cys257) are largely modified by NEM under these conditions, ref. 27), the aggregation induced by 100 µM of AMM-59 was significantly suppressed (by ~90%) (Figure 11C, middle panel). In contrast, when we used SMPs pretreated with NEM (100 nmol/mg of proteins) on ice for 10 min (Cys57 is predominantly modified under these conditions, ref. 22), the aggregation was still observed (Figure 11C, right panel). The whole pictures of SDS-PAGE gels of Figure 11C were presented in the Supporting Information (Figure S4). Similar results were obtained using AMM-120 (Figure S5). Figure 11. Characterization of the aggregation of AAC induced by AMM-59. (A) Concentration dependency of the aggregation of AAC. SMPs (2.0 mg of proteins/mL) were incubated with various concentrations of AMM-59 at 37 ˚C for 4 h, and resolved on a 12.5% Laemmli-type SDS gel containing 0.35 M 2-mercaptoethanol. AAC in each sample was analyzed by CBB staining or a Western blot analysis using an AAC antibody. (B) Time dependency of the aggregation of AAC. SMPs (2.0 mg of proteins/mL) were incubated with 100 µM of AMM-59 at 37 ˚C for various periods of time (1–20 h), followed by SDS-PAGE and a Western blot analyses. (C) Effects of the NEM treatment on the aggregation of AAC. SMPs (20 mg of proteins/mL) were previously incubated with 2.0 mM of NEM (100 nmol/mg of proteins) on ice for 10 or 60 min, according to the procedures described in the Experimental Procedures. SMP samples (2.0 mg of proteins/mL) were then incubated with AMM-59 (10 or 100 µM) at 37 ˚C for 10 h, followed by an analysis on a 12.5% Laemmli-type SDS gel. Approximately 50 µg of the proteins were loaded onto each lane. (D) Effects of the EMA treatment on the aggregation of AAC. SMPs (20 mg of proteins/mL) were previously incubated with 200 µM of EMA (10 nmol/mg of proteins) on ice for 1 min, according to the procedures described in Experimental Procedures. SMP samples (2.0 mg of proteins/mL) were incubated with AMM-59 (10 or 100 µM) at 37 ˚C for 10 h, followed by an analysis on a 12.5% Laemmli-type SDS gel. Approximately 50 µg of the proteins were loaded onto each lane. The position of AAC on the SDS gel is marked by an arrowhead. All data shown are representative of three independent experiments.


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Biochemistry

These results indicate that when Cys57 is previously masked by NEM, the covalent binding of AMM-59 solely to Cys160 may be sufficient to trigger the aggregation of AAC. Next, we examined the effects of the selective (predominant) modification of Cys160 by EMA on the aggregation of AAC. SMPs were pretreated with EMA (10 nmol/mg of proteins) on ice for 1 min, as shown in Figure 7. With this SMP sample, 100 µM of AMM-59 still induced aggregation (Figures 11D and S6), whereas a faint band of monomeric AAC was detected. These results indicate that the covalent binding of AMM-59 solely to Cys57 may be sufficient to induce aggregation. Taken together, when Cys57 or Cys160 is selectively masked by NEM or EMA, respectively, the covalent binding of AMM-59 to a residual free residue of the two cysteines is sufficient to induce the aggregation of AAC. We repeated the experiments shown in Figures 11A and 11B in the presence of sodium ascorbate (0.5 or 1.0 mM) or 2,6-di-tert-butyl-4-methylphenol (50 or 100 µM, often referred to as BHT). These radical scavengers were unable to suppress the aggregation of AAC (Figure S7). Therefore, the aggregation process does not appear to involve a radical intermediate(s). To investigate the effects of cations on the aggregation induced by AMM-59, we also repeated these experiments in the presence of EDTA (1.0 or 5.0 mM). The addition of EDTA did not affect the extent of aggregation (Figure S8).


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DISCUSSION We found ECHD-type compounds (AMM-59 and AMM-120) as specific inhibitors of bovine heart mitochondrial AAC (AAC1, a major isoform) through the extensive screening of lowmolecular-weight chemicals, whereas their inhibitory potencies are substantially weaker than those of atractylosides and BKAs, which exhibit strong inhibitory effects at the sub-micromolar level. To elucidate the inhibition mechanism of AAC by ECHDs, we performed proteomic analyses of AMM-120-bound AAC as well as biochemical characterization using different SH-reagents as reference compounds, such as NEM, EMA, and Cu(OP)2. Terada and colleagues extensively investigated the reactivities of three solvent-accessible cysteines in AAC (Cys57, Cys160, and Cys257) against these SH-reagents using bovine heart mitochondria and SMPs (18, 27, 28). It is worth noting that we were able to almost completely reproduce their experimental findings, as described in the text. The present results revealed that AMM-59 and AMM-120 inhibit the function of AAC by forming covalent bonds primarily with Cys57 and secondarily with Cys160, which are located in the 1st and 2nd matrix-side loops, respectively (10). However, we were unable to directly prove this using LC-MS/MS possibly because of the instability of the modified peptide. As far as this reactivity is concerned, ECHDs may be categorized, in a wide sense, into SH-reagents. Although an unknown ~40 kDa protein was faintly labeled by AMM-120, the extents of labeling of other proteins were negligibly small (< 2%) (Figure 5A). In light of this selectivity against AAC among many SMP proteins, which is attributed to their unique EDHD skeleton, they may not merely be ordinary SH-reagents, but rather a new class of inhibitors of AAC. The hydrophobic nature of ECHDs is also unique as AAC inhibitors because the inhibitors hitherto known (BKAs, atractylosides, suramin, and chebulinic acid) are very hydrophilic compounds possessing anionic functional groups at physiological pH conditions. Availability of a variety of inhibitors, which have largely different physicochemical properties, may be useful to characterize the functional and/or structural nature of AAC from different angles. Of particular interest is that when AAC was incubated with high concentrations of ECHDs for a long period of time, the protein remarkably aggregated in SMPs. Although there are various SH-reagents that covalently bind to cysteine residues in AAC, ECHDs are the first chemicals, to the best of our knowledge, that induce such prominent structural alterations of AAC without forming intermolecular S-S linkages. We note that the inhibition of AAC function by ECHDs via covalent binding and the aggregation of this protein induced by bound-ECHDs are not identical events; but, the two events may be successive and closely related. Further, it is currently unclear whether this unique phenomenon has any biological relevance to the function and/or dynamic conformational changes of AAC. However, the fact that even low-molecular-weight chemicals can induce the prominent structural alterations of AAC under mild experimental conditions may give a hint to consider the effects of various endogenous and exogenous chemicals on the protein, as discussed later taking 4-hydroxy-2-nonenal (4-HNE, a toxic decomposed product of polyunsaturated lipids) as an example. 22 ACS Paragon Plus Environment

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One of the important questions concerning the aggregation of AAC is which bond formation (ECHDs–Cys57, ECHDs–Cys160, or both) is a key event triggering aggregation. Since aggregated AACs is difficult to handle, we were unable to directly analyze the ECHDs that bound to it; therefore, we have to infer the answer to this question from the reactivities of ECHDs to the two cysteines. Although the reactivity of ECHDs against Cys57 is significantly higher than that against Cys160 (Figures 6 and 7), selective covalent binding to Cys57 must be impractical under the experimental conditions achieving the aggregation (i.e. a long incubation with high concentrations of ECHDs); substantial concomitant binding to Cys160 is unavoidable. On the other hand, the results shown in Figures 11C and 11D indicate that when Cys57 or Cys160 is predominantly masked by NEM or EMA, respectively, the covalent binding of AMM-59 to a residual free residue of the two cysteines (i.e. one molecule of bound AMM-59 per AAC) is sufficient to induce the aggregation of AAC. Taken together, it is highly conceivable that there are two AAC populations under actual experimental conditions achieving aggregation, which house one or two molecules of bound ECHD, and both may trigger the aggregation of AAC. Furthermore, concerning the mechanism of the aggregation of AAC induced by ECHDs, circumstantial evidence obtained herein suggest that the aggregation process does not involve a radical intermediate(s) as well as intermolecular S-S linkages (Figures 11A and 11B). Although Cu(OP)2 also induces the prominent dimerization (and higher polymerization) of AAC in SMPs, this phenomenon is attributed to the intermolecular S-S linkage catalyzed by this reagent (17). Some mitochondrial transmembrane proteins, such as the ND1 subunit of bovine mitochondrial respiratory complex I (33, 34), easily aggregate when they are heated because of their highly hydrophobic nature. Extensive efforts have been made to stabilize AAC because it is inherently unstable when purified in various 12. Effects of the alkyl side chain of ECHDs on detergents (8, 9, 35). Solubilized AAC Figure the aggregation of AAC. SMPs (2.0 mg of proteins/mL) using Triton X-100 is prone to aggregation were incubated with various concentrations of AMM-59, or AMM-124 at 37 ˚C for 12 h. (A) SMP depending on the delicate balance between AMM-120, proteins were denatured with 1% (w/v) SDS and with fluorescent TAMRA-N3 (20 µM) via hydrophobic and electrostatic interactions conjugated Cu+-catalyzed click chemistry. Proteins were resolved among the protein molecules (36). By by a 12.5% Laemmli-type SDS gel and subjected to taking these points into consideration, we fluorescent gel imaging, as conducted in Figure 7. (B) proteins were resolved on a 12.5% Laemmli-type made a working hypothesis that a critical SMP SDS gel, followed by CBB staining. Approximately 50 factor driving the aggregation of AAC is µg of the proteins were loaded onto each lane. The position of AAC on the SDS gel is marked by an hydrophobic stacking mediated by ECHDs arrowhead. SDS-PAGE data for control, AMM-59 (50 bound to Cys57 (and possibly to Cys160 in and 100 µM) and AMM-120 (50 and 100 µM) are the same as those used in Figure 5B. Data shown are part); namely, the insertion of a long representative of three independent experiments. 23 ACS Paragon Plus Environment

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hydrophobic alkyl chain(s) inside AAC may trigger the hydrophobic stacking. To confirm the likelihood of this, we synthesized another derivative AMM-124 (Figure 1), which has a shorter alkyl chain than AMM-120, and investigated its aggregation-inducing ability in SMPs. We speculated that the difference in hydrophobicities between AMM-120 (or AMM59) and AMM-124 is not a crucial factor for accessing Cys57 and Cys160 from bulk aqueous medium because both solvent-accessible cysteines face the outer side of SMP membrane (10). However, the binding affinity of AMM-124 to AAC, which was evaluated by the fluorescence intensity of TAMRA conjugated to bound AMM-124 or AMM-120, was significantly lower than that of AMM-120 (Figure 12A). Under these experimental conditions, 50 µM of AMM-59 and AMM-120 induced the complete aggregation of AAC, whereas much higher concentrations of AMM-124 were needed to induce aggregation because of its lower binding affinity (Figure 12B). Monomeric AAC was still determined even if AAC was incubated with 100 or 300 µM of AMM124. The whole pictures of SDS-PAGE gels of Figures 12A and 12B were presented in the Supporting Information (Figures S9 and S10). These results suggest that the alkyl chain moiety of ECHDs is located in the hydrophobic microenvironment of AAC and the tight hydrophobic interaction between ECHDs and AAC is energetically favorable in the bound state; consequently, this may be a key event triggering the aggregation of the protein. Further studies are needed to explore the dynamic process of aggregation in more detail. Looking at the present study in a different light, it suggests that hydrophobic chemicals possessing a strong electrophilic nature may have a possible ability to react with nucleophilic cysteine(s) in AAC and, subsequently, induce the polymerization of this protein. 4-Hydroxy-2nonenal (4-HNE) is the most abundant and toxic aldehyde generated by the reactive oxygen species-mediated peroxidation of polyunsaturated lipids. This hydrophobic compound has an electrophilic α,β-unsaturated carbonyl skeleton (37) and has been reported to covalently bind to AAC via Michael addition (38-40); however, the nucleophilic amino acid(s) was not identified in those studies. Since 4-HNE reaches fairly high local concentrations in mitochondria during oxidative stress (41, 42), we cannot rule out the Figure 13. Effects of 4-HNE on AAC in SMPs. SMPs (2.0 mg of proteins/mL) were incubated with possibility that 4-HNE bound to AAC also various concentrations of 4-HNE (10, 100, or 300 induces polymerization of this protein in µM) at 37 ˚C for 12 h, followed by separation on a 12.5% Laemmli-type SDS-PAGE. Proteins were mitochondria. To examine this possibility, we stained by CBB. Approximately 50 µg of the investigated the effects of 4-HNE on AAC. proteins were loaded onto each lane. The position of AAC on the SDS gel is marked by an arrowhead. SMPs (2.0 mg of proteins/mL) were incubated Data shown are representative of three independent 24

experiments.

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with different concentrations of 4-HNE (10, 100, and 300 µM) at 37˚C for 12 h, and then analyzed by 12.5% Laemmli-type SDS-PAGE. As shown in Figure 13, the monomeric AAC band (~30 kDa) shifted slightly to the upper side by the treatment with 100 and 300 µM of 4-HNE, as observed for AMM-59 (Figures 11A and 11B); however, the band did not disappear. These results indicate that while 4-HNE covalently binds to AAC, it does not induce the distinct polymerization of AAC. Therefore, hydrophobic chemicals that covalently bind to AAC do not necessarily induce the polymerization of AAC. The polymerization-inducing effect of ECHDs appears to be a unique characteristic of this type of chemical, which is closely related to some physicochemical properties of the cyclohexenedione skeleton. It may be mentioned that unique effects of 4-HNE on protein structure was reported by Kabuta et al. (43): Cys90 in ubiquitin-Cterminal hydrolase L1 (UCH-L1) is modified by 4-HNE, which leads to an increase in insolubility and formation of high molecular weight aggregates. In conclusion, we found ECHD-type compounds as specific inhibitors of bovine heart mitochondrial AAC. ECHDs inhibit the function of AAC by making a covalent bond primarily with Cys57. When AAC is incubated with high concentrations of ECHDs for a long period of time, this protein takes place remarkable aggregation in SMPs. A potentially critical factor driving the aggregation of AAC may be hydrophobic stacking mediated by ECHD bound to Cys57 (also Cys160 in part). Although various SH-reagents covalently bind to cysteine residues in AAC (including 4-HNE), ECHDs are the first chemicals, to the best of our knowledge, that induce the prominent structural alteration of the protein without forming intermolecular S-S linkages.

ACKNOWLEDGEMENTS We thank Drs. Fumihiko Sato and Kentaro Ifuku (Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University) and Dr. Mitsuyoshi Ueda (Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University) for allowing us access to their MALDI-TOF MS (Bruker Autoflex III Smartbeam) and LTQ Velos Orbitrap Mass Spectrometer, respectively, as well as for their helpful advice on the experiments. The experiments involving radioisotope techniques were performed at the Radioisotope Research Center, Kyoto University. Funding This study was supported by a Grant-in-Aid for Scientific Research (Grant 26292060 to H. M., Grant 15K07411 to M. M.) from the Japan Society for the Promotion of Science, and by Science and technology research promotion program for agriculture, forestry, fisheries and food industry (Grant 26021A). Notes The authors declare that they have no conflicts of interest with the contents of this article. 25 ACS Paragon Plus Environment

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SUPPORTING INFORMATION The synthetic procedures for AMM-59, AMM-120, and AMM-124, Figure S1 [predictable adducts formed by the modification by AMM-120, biotinylation (using biotin-SS-N3), and carbamidomethylation], Figure S2 (effects of AMM-59 on the activities of mitochondrial respiratory complexes, Figure S3 (characterization of the bovine AAC subunit by LC-MS), Figure S4 (whole pictures of SDS-PAGE gels corresponding to Figure 11C), Figure S5 (effects of the NEM treatment on the aggregation of AAC induced by AMM-120), Figure S6 (whole picture of SDS-PAGE gel corresponding to Figure 11D), Figure S7 (effects of radical scavengers on the aggregation of AAC induced by AMM-59), Figure S8 (effects of EDTA on the aggregation of AAC induced by AMM-59), Figure S9 (whole pictures of SDS-PAGE gels corresponding to Figure 12A) and Figure S10 (whole picture of SDS-PAGE gel corresponding to Figure 12B).


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REFERENCES 1. Earley, F., The biochemistry of oxidative phosphorylation: a multiplicity of targets for crop protection chemistry, in Krämer, W., Schirmer, U., Jeschke, P., and Witschel, M. (Eds.), Modern Crop Protection Compounds (2nd edition), Wiley-VCH, Verlag & Co., Weiheim, Germany 2012, pp. 559-584. 2. Sparks, T. C. and DeAmicis, C. V., Inhibitors of mitochondrial electron transport: acaricides and insecticides, in Krämer, W., Schirmer, U., Jeschke, P., and Witschel, M. (Eds.), Modern Crop Protection Compounds (2nd edition), Wiley-VCH, Verlag & Co., Weiheim, Germany 2012, pp. 1078-1108. 3. Murai, M. and Miyoshi, H. (2016) Current topics on inhibitors of respiratory complex I. Biochim. Biophys. Acta 1857, 884-891. 4. Ohmura, S., Miyadera, H., Ui, H., Shiomi, K., Yamaguchi, Y., Masuma, R., Nagamitsu, T., Takano, D., Sunazuka, T., Harder, A., Kölbl, H., Namikoshi, M., Miyoshi H., Sakamoto, K. and Kita, K. (2001) Anthelmintic compound, nafuredin, shows selective inhibition of complex I in helminth mitochondria. Proc. Natl. Acad. Sci. USA 98, 60-62. 5. Miyadera, H., Shiomi, K., Ui, H., Yamaguchi Y., Masuma, R., Tomoda, H., Miyoshi, H., Osanai, A., Kita, K. and Ohmura, S. (2003) Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase). Proc. Natl. Acad. Sci. USA 100, 473-477. 6. Shiomi, K., Ui, H., Suzuki, H., Hatano, H., Nagamitsu, T., Takano, D., Miyadera, H., Yamashita, T., Kita, K., Miyoshi, H., Harder, A., Tomoda, H., and Ōmura, S. (2005) A γ-Lactone form nafuredin, nafuredin-γ, also inhibits helminth complex I. J. Antibiot. 58, 50–55. 7. Klingenberg, M. (2008) The ADP and ATP transport in mitochondria and its carrier. Biochim. Biophys. Acta 1778, 1978-2021. 8. Crichton, P. G. Lee, Y., Ruprecht, J. J., Cerson, E., Thangaratnarajah, C., King, M. S., and Kunji, E. R. (2015) Trends in thermostability provide information on the nature of substrate, inhibitor, and lipid interactions with mitochondrial carriers. J. Biol. Chem. 290, 8206-8217. 9. Todisco, S., Di Noia, M. A., Onofrio, A., Parisi, G., Punzi, G., Redavid, G., De Grassi, A., and Pierri, C. L. (2016) Identification of new highly selective inhibitors of the human ADP/ATP carriers by molecular docking and in vitro transport assays. Biochem. Pharmacol. 100, 112-132. 10. Pebay-Peyroula, E., Dahout-Gonzalez, C., Kaun, R., Trezequet, V., Lauquin, G. J. M., and Brandolin, G. (2003) Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature 426, 39-44. 11. Ruprecht, J. J. and Kunji, E. R. (2006) Mitochondrial carriers in the cytoplasmic state have a common substrate binding site. Proc. Natl. Acad. Sci. USA 103, 2617-2622. 12. Robinson, A. J., Overy, C., and Kunji, E. R. (2008) The mechanism of transport by mitochondrial carriers based on analysis of symmetry. Proc. Natl. Acad. Sci. USA 105, 1776617771. 13. Dehez, F., Pebay-Peyroula, E., and Cjipot, C. (2008) Binding of ADP in the mitochondrial ADP/ATP carrier is driven by an electrostatic funnel. J. Am. Chem. Soc. 130, 12725-12733 27 ACS Paragon Plus Environment

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14. Wang, A. and Tajkhorshid, E. (2008) Electrostatic funneling of substrate in mitochondrial inner membrane carriers. Proc. Natl. Acad. Sci. USA 105, 9598-9603. 15. Monne, M., Miniero, D. V., Daddabbo, L., Robinson, A. J., Kunji, E. R., and Palmieri, F. (2012) Substrate specificity of the two mitochondrial ornithine carriers can be swapped by single mutation in substrate binding site. J. Biol. Chem. 287, 7925-7934. 16. Ruprecht, J. J., Hellawell, A. M., Harding, M., Crichton, P. G., McCoy, A. J., and Kunji, E. R. (2014) Structures of years mitochondrial ADP/ATP carriers support a domain-based alternatingaccess transport mechanism. Proc. Natl. Acad. Soc. USA 111, 426-434. 17. Lauquin, G. J., Villiers, C., Michejda, J. W., Hryniewiecka, L. V. and Vignais, P. V. (1977) Adenine nucleotide transport in sonic submitochondrial particles. Kinetic properties and binding of specific inhibitors. Biochim. Biophys. Acta. 460, 331-345. 18. Majima, E., Ikawa, K., Takeda, M., Hashimoto, M., Shinohara, Y., and Terada, H. (1995) Translocation of loops regulates transport activity of mitochondrial ADP/ATP carrier deduced from formation of a specific intermolecular disulfide bridge catalyzed by copper-Ophenanthroline. J. Biol. Chem. 270, 29548-29554. 19. Matsuno-Yagi, A., and Hatefi, Y. (1985) Studies on the mechanism of oxidative phosphorylation. J. Biol. Chem. 260, 14424–14427. 20. McStay, G. P., Clarke, S. J., and Halestrap, A. P. (2002) Role of critical thiol groups on the matrix surface of the adenine nucleotide translocase in the mechanism of the mitochondrial permeability transition pore. Biochem. J. 367, 541-548. 21. Halestrap, A. P., Woodfield, K. Y. and Connern, C. P. (1997) Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase. J. Biol. Chem. 272, 3346-3354. 22. Leung, A. W., Varanyuwatana, P., and Halestrap, A. P. (2008) The mitochondrial phosphate carrier interacts with cyclophilin D and may play a key role in the permeability transition. J. Biol. Chem. 283, 26312-26323. 23. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 24. Masuya, T., Murai, M., Ifuku, K., Morisaka, H. and Miyoshi, H. (2014) Site-specific chemical labeling of mitochondrial respiratory complex I through ligand-directed tosylate chemistry. Biochemistry 53, 2307-2317. 25. Ito, T., Murai, M., Morisaka, H. and Miyoshi, H. (2015) Identification of the binding position of amilorides in the quinone binding pocket of mitochondrial complex I. Biochemistry 54, 36773686. 26. Murai, M., Okuda, A., Yamamoto, T., Shinohara, Y. and Miyoshi, H. (2017) Synthetic ubiquinones specifically bind to mitochondrial voltage-dependent anion channel 1 (VDAC1) in Saccharomyces cerevisiae mitochondria. Biochemistry 56, 570-58. 27. Majima, E., Koike, H., Hong, Y.-M., Shinohara, Y., and Terada H. (1993) Characterization of 28 ACS Paragon Plus Environment

Page 28 of 31

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cysteine residues of mitochondrial ADP/ATP carrier with the SH-reagents eosin 5-maleimide and N-ethylmaleimide. J. Biol. Chem. 268, 22181-22187. 28. Majima, E., Shinohara, Y., Yamaguchi, N., Hong, Y. M. and Terada, H. (1994) Importance of loops of mitochondrial ADP/ATP carrier for its transport activity deduced from reactivities of its cysteine residues with the sulfhydryl reagent eosin-5-maleimide. Biochemistry 33, 9530– 9536. 29. Schägger, H. (2006) Tricine-SDS-PAGE. Nat. Protoc. 1, 16–21. 30. Wang, Q., Chan, T.R., Hilgraf, R., Fokin, V. V., Sharpless, K. B., and Finn, M. G. (2003) Bioconjugation by copper(I)-catalyzed azide-alkyne [3+2] cycloaddition. J. Am. Chem. Soc. 125, 3192-3193. 31. Yamamoto, T., Tamaki, H., Katsuda, C., Nakatani, K., Terauchi, S., Terada, H., & Shinohara, Y. (2013). Molecular basis of interactions between mitochondrial proteins and hydroxyapatite in the presence of Triton X-100, as revealed by proteomic and recombinant techniques. J. Chromatogr. A, 1301, 169-178. 32. Kahsai, A. W., Rajagopal, S., Sun, J., and Xiao, K. (2014) Monitoring protein conformational changes and dynamics using stable-isotope labeling and mass spectrometry (CDSiL-MS), Nat. Protoc. 9, 1301-1309. 33. Earley, F. G. P., Patel, S. D., Ragan, C. I., and Attardi, G. (1987) Photolabeling of a mitochondrially encoded subunit of NADH dehydrogenase with [3H]dihydrorotenone. FEBS Lett. 219, 108-113. 34. Murai, M., Ishihara, A., Nishioka, T., Yagi, T., and Miyoshi, H. (2007). The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I. Biochemistry, 46, 6409-6416. 35. Crichton, P. G., Harding, M., Ruprecht, J. J., Lee, Y., and Kunji, E. R. (2013) Lipid, detergent, and coomassie blue G-250 affect the migration of small membrane proteins in blue native gels: mitochondrial carriers migrate as monomers not dimers. J. Biol. Chem. 288, 22163-22173. 36. Drees, M. and Beyer, K. (1988) Interaction of phospholipids with the detergent-solubilized ADP/ATP carrier protein as studied by spin-label electron spin resonance. Biochemistry 27, 8584-8591. 37. Grimsrud, P. A., Xie, H., Griffin, T. J., and Bernlohr, D. A. (2008) Oxidative stress and covalent modification of protein with bioactive aldehydes. J. Biol. Chem. 283, 21837-21841. 38. Vieira, H. LA., Belzacq, A.-S., Haouzi, D., Bernassola, F., Cohen, I., Jacotot, E., Ferri, K. F., Hamel, C. E. I., Bartle, L. M., Melino, G., Brenner, C., Goldmacher, V., and Kroemer, G. (2001) The adenine nucleotide translocator: a target of nitric oxide, peroxynitrite, and 4hydroxynonenal. Oncogene 20, 4305-4316. 39. Echtay, K. S., Esteves, T. C., Pakay, J. L., Jekabsons, M. B., Lambert, A. J., Portero-‐Otín, M., Portero-Ótín, M., Pamplona, R., Vidal-Puig, A. J., Wang, S., Roebuch, S. J., and Brand, M. D. (2003) A signalling role for 4-‐hydroxy-‐2-‐nonenal in regulation of mitochondrial uncoupling. EMBO J. 22, 4103-4110. 29 ACS Paragon Plus Environment

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40. Choksi, K. B., Boylston, W. H., Rabek, J. P., Widger, W. R., and Papaconstantinou, J. (2004) Oxidatively damaged proteins of heart mitochondrial electron transport complexes. Biochim. Biophys. Acta, 1688, 95-101. 41. Lucas, D. T., and Szweda, L. I. (1998) Cardiac reperfusion injury: aging, lipid peroxidation, and mitochondrial dysfunction. Proc. Nat. Acad. Sci. U. S. A. 95, 510-514. 42. Winger, A. M., Taylor, N. L., Heazlewood, J. L., Day, D. A., and Millar, A. H. (2007) The cytotoxic lipid peroxidation product 4-hydroxy-2-nonenal covalently modifies a selective range of proteins linked to respiratory function in plant mitochondria. J. Biol. Chem. 282, 3743637447. 43. Kabuta, T., Setsuie, R., Mitsui, T., Kinugawa, A., Sakurai, M., Aoki, S., Uchida, K., and Wada, K. (2008) Aberrant molecular properties shared by familial Parkinson’s disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1, Hum. Mol. Genet. 17, 1482-1496.


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FOR TABLE OF CONTENTS USE ONLY

Epoxycyclohexenedione-type Compounds are a New Class of Inhibitors of the Bovine Mitochondrial ADP/ATP Carrier

Ayaki Aoyama, Masatoshi Murai, Naoya Ichimaru, Shunsuke Aburaya, Wataru Aoki, and Hideto Miyoshi*

O

H N

O

O O

Covalent binding to cysteines Mitochondrial ADP/ATP carrier (AAC)

Aggregation of AACs


Epoxycyclohexenedione-type Compounds are a New Class of

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