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Originally published In Press as doi:10.1074/jbc.M314148200 on April 9, 2004

J. Biol. Chem., Vol. 279, Issue 26, 27278-27285, June 25, 2004
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Immunochemical Identification of Coenzyme Q0-Dihydrolipoamide Adducts in the E2 Components of the {alpha}-Ketoglutarate and Pyruvate Dehydrogenase Complexes Partially Explains the Cellular Toxicity of Coenzyme Q0*

Michael J. MacDonald{ddagger}§, Rhonda D. Husain¶, Susanne Hoffmann-Benning¶, and Tracie R. Baker{ddagger}

From the {ddagger}Childrens Diabetes Center, University of Wisconsin Medical School, Madison, Wisconsin 53706 and Mass Spectrometry Facility, Michigan State University, East Lansing, Michigan 48824

Received for publication, December 24, 2003 , and in revised form, April 9, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Coenzyme Q0 (Q0), a strong electrophile, is toxic to insulin-producing cells. Q0 was incubated with rat and human pancreatic islets and INS-1 insulinoma cells, and its attachment to cellular proteins was studied with Western analysis using antiserum raised against the benzoquinone ring structure of ubiquinone (anti-Q). Q0 covalently bonded to two proteins, one of 50 kDa and another of 70 kDa. Both proteins were found to be mitochondrial in human and rat islet cells and in many rat organs. Mitochondria were incubated with Q0, and affinity-purified anti-Q was used to immunoprecipitate the 50-kDa protein. Amino acid sequencing identified it as dihydrolipoamide succinyltransferase, the E2 component of the {alpha}-ketoglutarate dehydrogenase complex (KDC). Western analysis also showed that Q bonds to the E2 components of the purified KDC and 0the pyruvate dehydrogenase complex (PDC). Dihydrolipoamide acetyltransferase, the E2 of the PDC, has a molecular mass of 70 kDa, and the 70-kDa protein was inferred to be this enzyme. Q0 was found to bond only to proteins containing dihydrolipoate, and in preparations of mitochondria, thiol reducing agents facilitated the attachment of Q0, but oxidizing agents prevented it, suggesting that Q0 bonds to thiols of dihydrolipoamide. Incubation of human or pig PDC with Q0 followed by matrix-assisted laser desorption ionization time-of-flight and liquid chromatography/electrospray ionization mass spectrometry analyses of chymotrypsin-digested peptides of PDC E2 confirmed that Q0 bonds to the dihydrolipoamide in these proteins. In mitochondria, coenzymes Q1 and Q2 did not bond to the 50-kDa protein but competed with the bonding of Q0 to this protein. The prevention by Q1 of characteristics the bonding of Q0 to KDC E2, as well as other of the Q0 effect, are reminiscent of the action of Q0 on the mitochondrial permeability transition pore described previously (Fontaine, E., Ichas, F., and Bernardi, P. (1998) J. Biol. Chem. 273, 25734–25740).


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ubiquinone analogs (1, 2) and other quinones (3–6) stimulate insulin release from pancreatic islets, but they also kill the insulin-producing cell. The insulin release is not simply due to insulin leaked from dying cells because numerous inhibitors of cellular respiration that kill the cell, such as rotenone, antimycin A, cyanide, and dinitrophenol, inhibit insulin release rather than stimulate it (7, 8). The injurious effects of these compounds may be due, in part, to redox cycling that collapses the proton gradient that maintains the electrical potential of the inner mitochondrial membrane. However, many quinones, because of their electrophilicity, can induce various patterns of oxidative damage to cells. Also, some ubiquinone analogs influence the mitochondrial permeability transition pore (PTP)1 (9–11). To learn more about the roles of quinones in these processes, we raised antisera to the ring structure of ubiquinone (anti-Q antibodies) and used them in Western analysis and for immunoprecipitation. This enabled us to identify several intracellular proteins to which Q0, an ubiquinone analog, covalently attached.

Numerous studies have implicated the PTP in apoptosis and cell death (12, 13), and Bernardi and co-workers (14, 15) observed that a ubiquinone binding site regulates the PTP. They found that Q0 was a potent inhibitor of calcium-dependent opening of the PTP and that Q0 was more potent than all other ubiquinone analogs including endogenous quinones (14). They additionally showed that electron flux through complex I of the respiratory chain is an important PTP regulator and that when complex I is turning over, Q0 becomes an even more potent inhibitor of the PTP (16), and they suggested that Q0 binds to sites previously occupied by endogenous quinones. Numerous ubiquinone analogs in which carbon 6 of the benzoquinone ring is occupied by various side chains were ineffective as PTP inhibitors, except for decylubiquinone, which was less potent than Q0. Bernardi and co-workers (14) also observed that Q1, which did not itself inhibit the PTP, specifically counteracted the inhibitory effect of Q0. The PTP is also regulated by the NAD/NADH, NADP/NADPH, and oxidized glutathione/reduced glutathione ratios, with the latter being the most important (17, 18). Oxidized ratios of pyridine nucleotides and oxidized glutathione/reduced glutathione and oxidized vicinal thiol groups favor pore opening (19, 20).

The current work shows that there are rough similarities between the effects of Q0 on the PTP (14–17) and its interactions with two mitochondrial proteins. The absence of a side chain at carbon 6 of the benzoquinone ring of Q0 enables it to be a potent electrophile that can react with cellular nucleophiles, such as peptide thiols and reduced glutathione, to form a strong carbon sulfur bond. The neighboring oxygen on carbon 1 permits resonance delocalization of the negative charge of the unsaturated bond at carbon 6, allowing a nucleophile to add to carbon 6 in a Michael reaction or a similar reaction.

We observed that Q0 bonds covalently to two proteins, one of about 50 kDa and another of about 70 kDa, in rat and human pancreatic islets and in INS-1 rat insulinoma cells as judged by Western analysis. The proteins were associated with the mitochondrial inner membrane of these tissues and of numerous rat organs. Although a large amount of Q0 was found in the two proteins when Q0 alone was added to mitochondria, prior incubation of mitochondria with a low concentration of dithiothreitol, which keeps thiols reduced, made both proteins more susceptible to Q0 bonding. In addition, agents such as oxidized dithiothreitol, N-ethylmaleimide, and t-butyl hydroperoxide that can oxidize thiols prevented the attachment of Q0. Q1 did not bond to the proteins, but it and Q2 inhibited bonding of Q0 to the 50-kDa protein, similar to the effect of Q1 preventing the action of Q0 on the PTP.

The 50-kDa protein was immunoprecipitated from mitochondria using affinity-purified anti-Q antibodies followed by amino acid sequencing of its trypsin-digested peptides and identified as the dihydrolipoamide succinyltransferase (E2) of the {alpha}-ketoglutarate dehydrogenase complex. At low micromolar concentrations, Q0 was found to bond to the E2 enzymes of purified {alpha}-ketoglutarate dehydrogenase and pyruvate dehydrogenase complexes. The E2 enzyme of the pyruvate dehydrogenase complex has a molecular mass of 70 kDa, and therefore it was inferred that the 70-kDa protein is the E2 (dihydrolipoamide acetyltransferase) of this complex. Both E2 enzymes are part of multienzyme complexes involved in metabolism of mitochondrial substrates that pass electrons to complex I of the respiratory chain. MALDI-TOF mass spectrometry and liquid chromatography/electrospray ionization mass spectrometry of the dihydrolipoamide acetyltransferase identified two protein fragments that contain lipoate as the targets of Q0. Q0 seems to bond most effectively and specifically to thiols of dihydrolipoamide because even at exceptionally high concentrations, it did not bond to the numerous cysteines in cellular proteins or to cysteines in the two purified E2 proteins. Thus, the results suggest that some of the toxic effects of Q0 in cells are due to its propensity to attack the dihydrolipoamides of the two acyltransferase enzymes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—{alpha}-2,3-Dimethoxy-5-methyl-6-(3'-carboxy-3'-methylpropyl)-1,4-benzoquinone (which we named Q acid) was generously provided by Dr. Masazumi Watanabe (Takeda Chemical Industries, Ltd., Tokyo, Japan). Protein A Trisacryl beads, KLH, and EDC were from Pierce. Q0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone), glutathione-agarose (catalog number G-0387), S-thiopropyl-Sepharose (catalog number T8387), additional KLH and EDC, and all other chemicals were obtained from Sigma in the highest purity available. Purified porcine heart {alpha}-ketoglutarate dehydrogenase and pyruvate dehydrogenase complexes were from Sigma. These two enzyme complexes purified from bovine heart, as well as recombinant human pyruvate dehydrogenase complex, were gifts of Thomas Roche. Pancreatic islets and other tissues were obtained from 300-g Sprague-Dawley rats as described previously (1, 21). Human pancreatic islets were from the Islet Isolation Core, Washington University School of Medicine (St. Louis, MO).

Antisera to Q Acid—Q acid was conjugated to KLH by mixing 0.5 ml of a 10 mM solution of Q acid in 0.9 M NaCl and 0.1 M MES buffer, pH 4.7, with 0.15 ml of 65 mM EDC. After 10 min, the activated Q acid was added to 0.2 ml of KLH (10 mg/ml), and the mixture was allowed to set at room temperature. After 2 h, the unconjugated Q acid was removed from the Q acid-KLH conjugate by gel filtration as described in the Pierce Immunogen EDC Conjugation Kit instructions. The conjugate was mixed with Freund's adjuvant, and 0.5–1.0 mg of hapten protein was injected into rabbits at 6-week intervals four times and again a fifth time after a year. Antisera were harvested 3 weeks after the third, fourth, and fifth injections of hapten.

Subcellular Fractionation—Organs were homogenized in 220 mM mannitol, 70 mM sucrose, and 5 mM potassium Hepes buffer, pH 7.5 (MSH), and centrifuged at 600 x g for 10 min to obtain a pellet of nuclei and cell debris. The resulting supernatant fraction was centrifuged at 5,500 x g (islets) or 15,000 x g (other tissues) for 10 min to obtain the mitochondrial pellet. Mitochondrial pellets from large tissues were resuspended in MSH and washed three times by centrifugation. Islet mitochondrial pellets were washed once. The post-mitochondrial supernatant fraction was centrifuged at 21,000 x g for 10 min to obtain a supernatant fraction consisting of cytosol plus endoplasmic reticulum (21, 22).

Incubation of Tissues with Q0—Intact islets were incubated in Krebs-Ringer bicarbonate buffer, pH 7.3. Homogenates of whole tissue and subcellular fractions were incubated with various concentrations of Q0 in MSH solution for 30 min or for 1 h at room temperature. When added, dithiothreitol or Q1, Q2, doxorubicin, genistein, 1,4-benzoquinone, aurovertin, or infrapeptin was present for 15 min before the addition of Q0 to mitochondria and also present during incubation with Q0. When added, N-ethylmaleimide, oxidized dithiothreitol (trans-4,5-dihydroxy-1,2-dithiane), or t-butylhydroperoxide was incubated with mitochondria for 1 h at room temperature and washed out by centrifugation of the mitochondria before Q0 was added. Compounds not soluble in aqueous buffers were dissolved in dimethyl sulfoxide and added to the aqueous mixture to give a final concentration of dimethyl sulfoxide of ≤1%.

Extraction of Proteins from Mitochondria—After mitochondria from various tissues were incubated with Q0, the mitochondria were centrifuged at 15,000 x g for 10 min, and the resulting pellet (about 600 µl) was suspended in 12 volumes of 20 mM sodium phosphate buffer, pH 7.5, containing 10 mM Triton X-100 and vortexed at room temperature for 1 h. The mixture was then centrifuged at 20,000 x g for 10 min, and the supernatant fraction containing the Q0-bonding proteins was saved for analysis (Figs. 5, 6, 7, 8). It was subsequently learned that a large amount, albeit less, of the 50-kDa protein could be extracted without Triton X-100. The Triton X-100 was omitted when the sample was used for immunoprecipitation.



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  		FIG. 5.
Bonding of Q0 to proteins in mitochondrial fractions from various organs. Mitochondria were incubated in the presence or absence of 1 mM Q0 for 1 h at room temperature, and immunoblot analysis with a late draw from antiserum 73613 was performed. Amounts of mitochondrial protein were as follows: adrenal, 40 µg; brain, 60 µg; kidney, 65 µg; liver, 120 µg; heart, 55 µg; lung, 20 µg; skeletal muscle, 53 µg; and spleen, 50 µg. Sizes of standard proteins are indicated in kDa at the left.

 



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  		FIG. 6.
Sulfhydryl reagents decrease the bonding of Q0 to the 50-kDa protein in rat kidney mitochondria. Rat kidney mitochondria (90 µg of protein) were incubated with sulfhydryl oxidizing agents for 1 h at room temperature, washed twice by centrifugation, and then incubated with 1 mM Q0 for 1 h at room temperature and subjected to immunoblot analysis. Blots were probed with an early draw anti-Q antibody from rabbit 73613. Lanes 1 and 2, t-butylhydropheroxide (Butyl), 0.1 and 1 mM; lanes 3 and 4, N-ethylmaleimide (NEM), 0.1 and 1 mM, lanes 5 and 6, oxidized dithiothreitol (oxDTT); lane 7, without an oxidizing agent (None).

 



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  		FIG. 7.
Competition of Q1 or Q2 with bonding of Q to proteins of rat kidney mitochondria. Rat kidney mitochondria (100 µl of 1 mg/ml mitochondrial protein) were incubated in the absence or presence of Q1 or Q2 (added in 10 µl of methanol) for 1 h, and then various concentrations of Q0 were added, and the incubation was continued for a second hour. Twenty µl of sample was applied to each well for PAGE followed by immunoblotting. The top and bottom panels each show a separate experiment. Samples in lanes 13 and 14 contained methanol equal to that in samples applied to lanes 5–12. The concentration of Q1 and Q2 was 1 mM except in mixtures shown in lanes 9, 11, and 12 of the bottom panel, where the concentration of Q2 was 0.5 mM. Blots were probed with antiserum from a late draw of anti-Q antiserum from rabbit 73613.

 



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  		FIG. 8.
Inhibition by benzoquinone of Q bonding to proteins of 50 and 70 kDa. Rat kidney mitochondria (70 µg of protein) were incubated in 100 µl of MSH solution in the absence or the presence of 0.1 or 1 mM 1,4-benzoquinone (Benz) for 1 h and then incubated in the absence or presence of 1 mM Q0 for a second hour. A sample (20 µl) was analyzed by Western analysis with antiserum from a late draw from rabbit 73613. The sample in lane 9 is the same as that in lane 2, except that this sample contained only methanol equal to the amount added with benzoquinone.

 
Immunoblotting—After incubation with Q0, pancreatic islets were washed and boiled in gel electrophoresis sample buffer (1.7% SDS, 5% glycerol, 62 mM Tris-chloride buffer, pH 6.8, and 100 mM dithiothreitol). Mitochondrial proteins were solubilized with 10 mM Triton X-100 in 20 mM sodium phosphate buffer, pH 7.5, and boiled in sample buffer. Proteins were separated by SDS-PAGE in 10% polyacrylamide gels and electrotransferred to nitrocellulose membranes that were probed with polyclonal antibodies to Q0. The antiserum from rabbit 73613 was used at a dilution of 1:6,000, and the antiserum from rabbit 73903 was used at a dilution of 1:500. Protein bands were visualized by chemiluminescence with the ECL Western blotting system (Amersham Biosciences).

Immunoprecipitation and Identification of the 50-kDa Q0 Bonding Protein—Affinity-purified antibodies were used to immunoprecipitate one of the proteins to which Q0 bonds. Q0 was immobilized on a solid support of 3 ml of either settled oxidized glutathione-agarose beads or S-thiopropyl-Sepharose beads. The beads were treated with 12 ml of 100 mM dithiothreitol for 1 h and washed five times with 12 ml of water. Q0 (40 mM, 12 ml) was added to the beads, and they were agitated gently for 18 h on a rocking platform. Beads were washed five times with water and twice with Tris-buffered saline (50 mM Tris-chloride, pH 7.5, and 150 mM NaCl). To affinity-purify the anti-Q antibodies, the beads were suspended in 4–8 ml of anti-Q serum from rabbit 73613 plus 4–7 ml of Tris-buffered saline and agitated gently for 18 h. Beads were washed five times with Tris-buffered saline and then sequentially treated for 15 min with 12 ml each of three different buffer solutions to elute the anti-Q antibodies: 100 mM glycine, pH 3.0; Tris-buffered saline; and 100 mM triethylamine, pH 11.5. Immediately after elution, the pH of each fraction was adjusted to 7.5 by adding 1 M Tris chloride to give a concentration of 0.2 M, and bovine serum albumin was added to give a concentration of 5 mg/ml. Western analysis indicated that the triethylamine eluate contained >90% of the anti-Q antibodies.

Affinity-purified anti-Q antibodies were covalently coupled to 2–3ml of protein A Trisacryl beads as described previously (23). Briefly, beads were washed three times with 10 volumes of 200 mM sodium borate buffer, pH 9.0, and incubated with all three of the elutant fractions of affinity-purified anti-Q antibodies. After 20 h, the beads were washed five times with 10 volumes of borate buffer and incubated in 10 volumes of 20 mM dimethylpimelimidate in the borate buffer. After 1 h, the beads were washed with borate buffer twice, and 10 volumes of 0.2 M ethanolanine, pH 8.7, were added to block any unreacted sites. After gentle agitation for 20 h, the beads were washed five times with 10 volumes of Tris-buffered saline and saved until use.

To immunoprecipitate the Q0-bonding protein, anti-Q-protein A Trisacryl beads were incubated for 18 h with 10–15 volumes of kidney mitochondrial supernatant fraction that had been extracted in 20 mM potassium phosphate buffer, pH 7.5. The extract was the supernatant fraction prepared by vortexing the mitochondria in 20 mM potassium phosphate buffer at room temperature for 1 h and removing the mitochondrial membranes by centrifuging the mixture for 10 min at 20,000 x g. The beads were washed five times with Tris-buffered saline and boiled in 1.5 volumes of SDS sample buffer in preparation for gel electrophoresis. After electrophoresis, the protein in the gel was stained with 0.1% Coomassie Blue R-250 in 10% acetic acid and 10% methanol for 15 min or electrotransferred to nitrocellulose for Western analysis. The Coomassie Blue R-250-stained band was excised from the gel and digested with trypsin, the internal peptides were separated by HPLC, and their amino acid sequences were determined with an Applied Biosystems Procise cLC Protein Sequencing System.

Mass Spectrometry of Peptides—Human and pig pyruvate dehydrogenase complexes were dialyzed into a solution of 20% glycerol, 1 mM EDTA, 50 µM dithiothreitol, and 25 mM potassium phosphate buffer, pH 7.5, and incubated with or without Q0, and the component proteins were separated by SDS-PAGE. The gel band was excised, cut into 1-mm3 pieces, alkylated with iodoacetamide, and then incubated for 22 h with 20 µg of chymotrypsin or chymotrypsin followed by trypsin in 50 mM ammonium bicarbonate buffer, pH 8.0. The digested peptides were extracted by sonication in 20 µl of 60% acetonitrile and 1% trifluoroacetic acid for 15 min, and then the supernatant fraction was concentrated in vacuo to 10 µl. For MALDI-TOF MS, 0.5 µl of the extract was mixed with 0.5 µl of saturated {alpha}-cyano-4-hydroxycinnamic acid matrix solution in 0.1% trifluoroacetic acid in 60% acetonitrile, air-dried, and introduced into the spectrometer. Spectra were obtained on a Voyager-DE STR MALDI-TOF mass spectrometer (PerSeptive Biosystems, Inc., Framingham, MA) in the linear mode. Time to mass calibration was achieved using bradykinin (MH+ at 1061.2 Da) and insulin MH+ at m/z 5734.59 Da. Average masses of all possible peptide fragments of the completely and incompletely digested proteins were calculated with the program Protein Prospector (http://prospector.ucsf.edu). Liquid chromatography/MS was performed on a Capillary liquid chromatography system (Waters Corp., Milford, MA) coupled to a LCQ DECA ion trap mass spectrometer (Thermofinnigan, San Jose, CA) equipped with a nanospray ionization source. An aliquot (6.4 µl) of the chymotryptic digest was trapped on a Michrom Cap Trap cartridge (Michrom BioResources, Inc., Auburn, CA) and flushed onto a 5 cm x 75 µm inner diameter Picofrit column packed with 5 µm of ProtoPep C18 material (New Objective, Woburn, MA). Peptides were eluted from the column with a gradient of 2–95% acetonitrile in 0.1% formic acid. Peptides were identified with the program Sequest.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Q Proteins—To study the toxic effects of Q0 on cells occurring at concentrations that also stimulate insulin release from pancreatic islets, the islets were incubated with 0–200 µM Q0. Intracellular Q0 and/or Q0 adhering to the outside of the cells was measured by absorbance at 263 nm (versus 274 nm for coenzyme Qs with long side chains) and by Craven's test. This indicated that the intracellular concentration of Q0 was no more than 45% of the concentration of Q0 added to the incubation medium. As judged by Western analysis with a late draw of anti-Q antiserum 73613, Q0 reacted with two proteins with molecular masses of about 50 and 70 kDa (Fig. 1). Q0 was also incubated with whole cell homogenates and subcellular fractions of rat and human pancreatic islets and the INS-1 rat insulinoma cell line. A solution containing Triton X-100 was used to extract proteins from the tissue fractions, and the extracts were used for Western analysis. In all three tissues, a protein of 50 kDa was the major reactant when a potent anti-Q antiserum from rabbit 73613 (from an early draw) was used at a dilution of 1:6,000 (Figs. 2, 3, 4). When a weaker anti-Q antiserum from a different rabbit (rabbit 73903) was used (at a dilution of 1:500) to probe the blots, a protein of about 70 kDa was the major reactant (Figs. 3 and 4). The initial different specificities of the anti-Q antisera from the two rabbits were puzzling but highly reproducible. It may indicate that the amino acid residues surrounding the Q0 bonding site were initially part of the epitope for these antisera. In any case, this initial apparent difference in specificity does not influence the arguments made later in the paper and does not influence our final conclusions. Antiserum from the weakly responding rabbit was not used in subsequent experiments because after the fourth and fifth immunizations, the strong antisera reacted with both the 50- and 70-kDa proteins.



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  		FIG. 1.
Bonding of Q0 to 50- and 70-kDa proteins in pancreatic islets. Rat pancreatic islets were incubated for 1 h at 37 °C with Krebs-Ringer bicarbonate buffer, pH 7.3, containing the concentrations of Q0 indicated at the bottom of each lane. Western analysis was performed with antisera from rabbit 73613. There was an equivalent of 25 islets (10 µg of protein) per lane. Protein size is indicated in kDa on the left. Visualization of the smallest band of about 30 kDa is not due to interaction of Q0 with the protein because it was present when preimmune serum was used for Western analysis and when islets were incubated with no Q0.

 



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  		FIG. 2.
Q0 bonding to protein in rat pancreatic islet subcellular fractions. Subcellular fractions of rat pancreatic islets were incubated in the absence (lanes 1, 3, 5, and 7) or presence of (lanes 2, 4, 6, and 8) of 1 mM Q0 for 1 h at room temperature. Proteins in the samples were analyzed by Western analysis with anti-Q antibody from an early draw from rabbit 73613. Lanes 1 and 2, whole cell homogenate (Homog) of pancreatic islets, 100 µg of protein; lanes 3 and 4, mitochondria (Mito), 50 µg of protein; lanes 5 and 6, nuclear and all debris fraction (Nuc), 150 µg of protein; lanes 7 and 8, cytosol (Cyto), 26 µg of protein. Sizes of standard marker proteins are indicated in kDa on the left.

 



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  		FIG. 3.
Bonding of Q0 to proteins in subcellular fractions of human pancreatic islets. Subcellular fractions of human islets were incubated with 1 mM Q0 in the absence or presence of 100 µM dithiothreitol for 1 h at room temperature and analyzed by immunoblotting. Fractions were nuclear and cell debris (Nuc; this fraction contains unbroken cells and mitochondria, which explains the presence of faint bonding of Q0 to proteins in this fraction), mitochondria (a 5,500 x g x 10 min post-nuclear pellet) (Mito), a 20,000 x g x 10 min post-mitochondrial pellet (20k pellet), and cytosol (Cyto) (20,000 x g x 10 min supernatant fraction (contains insulin granules and endoplasmic reticulum)). Blots were probed with anti-Q antisera from rabbit 73613 at a 1:6,000 dilution (top panel) and rabbit 73903 at a 1:500 dilution (bottom panel). Sizes of standard proteins in kDa are shown on the left.

 



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  		FIG. 4.
Q0 bonding to proteins in INS-1 subcellular fractions. Subcellular fractions of INS-1 cells were incubated in the absence (lanes 1, 3, 5, 7, and 9) or presence (lanes 2, 4, 6, 8, and 10 plus lane 3 of right panel) of 1 mM Q0 for 1 h at room temperature and analyzed by immunoblotting. Left panel, probed with antibody from rabbit 73613 at a 1:6,000 dilution. Right panel, probed with antibody from rabbit 73903 at a 1:500 dilution. Lanes 1 and 2, INS-1 whole cell homogenate, 150 µg of protein; lanes 3 and 4, mitochondria, 50 µg of protein; lanes 5 and 6, 20,000 x g x 10 min pellet of post-mitochondrial supernatant fraction, 10 µg of protein; lanes 7 and 8, nuclear and cell debris fraction, 50 µgof protein; lanes 9 and 10, cytosol (20,000 x g x 10 min supernatant fraction of post-mitochondrial supernatant fraction), 80 µg of protein. Sizes of standard proteins are indicated in kDa at the left.

 
Subcellular fractionation indicated that the Q0-bonding proteins are mitochondrial proteins (Fig. 2, lane 4; Fig. 3, lanes 4–6, top and bottom panels; Fig. 4, lane 4 of the left panel and lanes 3 and 4 of the right panel). The presence of a small amount of immunoreactive protein in the pellet of nuclei plus cell debris (Fig. 2, lane 6; Fig. 3, lane 3 of the top panel and lanes 1–3 of the bottom panel) is consistent with our previous observation that when pancreatic islets are fractionated, as much as 20% of total cellular mitochondrial inner membrane marker (mitochondrial glycerol phosphate dehydrogenase enzyme activity) is found in nuclei plus cell debris fraction in islets, most likely from unbroken cells (21). No immunoreactivity was seen in the cytosol fraction, which, because of the way it was deliberately prepared, also contains insulin granules and endoplasmic reticulum (Fig. 2, lane 8; Fig. 3, lanes 11 and 12, top and bottom panels; Fig. 4, lane 10, left and right panels). There was no immunoreactivity in cellular fractions not incubated with Q0 (Fig. 2, lanes 1, 3, 5, and 7; Fig. 3, lanes 1, 7, and 10, top and bottom panels; Fig. 4, lanes 1, 3, 5, 7, and 9 of the left panel and lanes 1, 5, 7, and 9 of the right panel).

It was initially thought that the 50- and 70-kDa Q0-bonding proteins were tightly associated with the inner mitochondrial membrane because after two cycles of freezing and thawing of mitochondria, substantial amounts of the proteins were still present in the pellet of mitochondrial membranes. The proteins could be released from the membranes with detergents, such as Triton X-100 (10 mM) or Nonidet P-40 (0.1%). Triton X-100 solubilized more of the proteins than Nonidet P-40. It was later discerned that a considerable amount (but less) of the immunoreactive 50-kDa protein could be dislodged from the mitochondrial membranes by vigorously vortexing the mitochondria in 20 mM potassium phosphate, pH 7.5, for 1 h without a detergent. Less of the 70-kDa protein than the 50-kDa protein could be solubilized from the mitochondria without detergents.

Tissue Distribution—The 50-kDa Q0-bonding protein was present in large amounts in mitochondria of all rat organs studied including heart, liver, kidney, adrenal gland, spleen, lung, skeletal muscle, and brain. High levels of the 70-kDa protein were detected in mitochondria of heart, moderate levels were detected in kidney and brain, and low levels were detected in adrenal gland and skeletal muscle when antiserum 73613 was used for Western analysis (Fig. 5). It is noteworthy that the tissues that contain high levels of the 70-kDa protein, such as heart, are also the tissues that are known to contain high levels of pyruvate dehydrogenase enzyme activity as discussed below. It was subsequently learned that the 70-kDa protein is a component of the pyruvate dehydrogenase complex.

Concentration Range of Q0 Bonding—The lowest concentration tested at which Q0 bonding to the 50-kDa protein of kidney mitochondria was visible by Western analysis was 10 µM (data not shown).

Effect of Oxidation/Reduction—Although considerable Q0 bonded to the two proteins when it was added by itself, adding 100 µM dithiothreitol to a tissue homogenate or mitochondria enhanced the reactivity of Q0 to the 50- and 70-kDa proteins. This suggests that bonding of Q0 was facilitated by increased availability of protein thiol groups (Fig. 3, lanes 3 and 6, top and bottom panels). The idea that Q0 bonds covalently to thiols was further supported by incubating mitochondria with agents that attack thiols, such as t-butyl hydroperoxide, N-ethylmaleimide, and oxidized dithiothreitol and removing the agents by washing the mitochondria before Q0 was added. These agents completely or partially inhibited the detection of Q0 in the proteins (Fig. 6), suggesting that these agents oxidized the protein's thiols and made them unsusceptible to attack by Q0.

Competition from Other Quinones—When Q1 or Q2 was incubated with kidney mitochondria, no protein was recognized by the antibody. This was expected because position 6 of these molecules is occupied by an isoprenoid chain, and thus these molecules are not electrophilic and cannot form a covalent bond at carbon 6. However, when Q1 and Q2 were co-incubated with Q0 and mitochondria, they diminished the bonding of Q0 to the 50-kDa protein, but not to the 70-kDa protein, in rat kidney mitochondria (Fig. 7). Other compounds that contain a quinone structure, doxorubicin and genestein, did not prevent the bonding of Q0 to the mitochondrial proteins (data not shown). However, 1,4-benzoquinone prevented the antibody from reacting with both the 50- and 70-kDa proteins (Fig. 8). Rotenone, a quinone inhibitor of NADH dehydrogenase found in the inner mitochondrial membrane, did not interfere with Q0 bonding to the proteins (data not shown).

Identity of the 50-kDa Q0 Protein—Kidney mitochondria were reacted with Q0 and extracted without detergent as described under "Experimental Procedures." Coomassie Blue staining of the immunoprecipitated and gel-electrophoresed Q0-incubated kidney mitochondrial extract revealed a single protein band of about 50 kDa. However, Western analysis, which is far more sensitive than Coomassie Blue staining, showed that a small amount of the 70-kDa protein was precipitated in addition to the 50-kDa protein. It is most likely that the 70-kDa protein was not well extracted from the mitochondria without detergent. Beads incubated with mitochondrial extraction buffer alone or mitochondrial extract previously incubated without Q0 or with 1,4-benzoquinone did not show any immunoprecipitated proteins that were visible by either Coomassie Blue staining or Western analysis.

The 50-kDa protein band was cut out of the gel and digested with trypsin, the resulting peptides were separated by HPLC, and the amino acid sequences of two of the peptides were determined. The sequences of the peptides (AAPEAPAAPPPPVAPVPTQMPPVP and GLVVPVIR) corresponded to residues 154–177 and 315–322 of dihydrolipoamide succinyltransferase (E2) (GenBankTM accession no. BAA14397 [GenBank] of the rat {alpha}-ketoglutarate dehydrogenase multienzyme complex (KDC). Because this enzyme contains dihydrolipoamide, and the E2 enzyme of the PDC has a molecular mass of 70 kDa and also contains dihydrolipoamide and is very similar to KDC E2, it was asked whether the 70-kDa immunoreactive protein was the E2 enzyme of the PDC. This enzyme contains two lipoate molecules. Western analysis of the purified porcine KDC and PDC (Fig. 9) or bovine enzyme complexes (data not shown) incubated with Q0 indicated that the antibody reacted with a 50-kDa protein of the KDC and a 70-kDa protein of the PDC. In addition, when a high concentration of PDC was used in immunoblotting, a second ~50-kDa protein of the PDC was detected (data not shown). This protein is likely the E3-binding protein of the PDC. It has a molecular mass of about 50 kDa and contains one lipoate molecule. This protein is much less abundant than the E2 enzyme in the PDC and would be expected to be visible at only high concentrations of the enzyme complex.



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  		FIG. 9.
Covalent bonding of Q0 to the E2 enzymes of {alpha}-ketoglutarate dehydrogenase and pyruvate dehydrogenase. Porcine {alpha}-ketoglutarate dehydrogenase multienzyme complex (KGDC) (0.5 µg of protein) and 0.9 µg of pyruvate dehydrogenase multienzyme complex (PDC) were incubated with various concentrations of Q for 1 h at room temperature and analyzed by Western analysis. The 50- and 70-kDa bands correspond to the E2 enzymes of {alpha}-ketoglutarate dehydrogenase multienzyme complex and PDC, respectively. The 70-kDa band in the preparation of {alpha}-ketoglutarate dehydrogenase multienzyme complex is due to its contamination with PDC as judged from our assays of enzyme activities and also as described by the vendor.

 
Mass Spectrometry Analysis—Results of mass spectrometry studies of pure human and partially pure pig PDC incubated with Q0 indicated that Q0 bonds only to the lipoyl domains of the dihydrolipoamide acetyltransferase (E2) component of PDC. The enzyme complexes were incubated with Q0 followed by SDS-PAGE, and the single peptide band that reacted with anti-Q0 antibody (the 70-kDa band) as judged from Western analysis was excised and digested with chymotrypsin and subjected to MALDI-TOF MS and electrospray ionization mass spectrometry analysis. Dihydrolipoic acid is known to be covalently attached to the epsilon amino group of the side chain of lysines at positions 99 and 226 of human PDC E2 (Swiss-Prot accession no. P10515 [GenBank] ). The MALDI-TOF mass spectrum showed two peaks with m/z values that corresponded to modification of the dihydrolipoates with Q0 as assigned with the aid of the Protein Prospector software program. Peaks with m/z values of 2604.30 and 2724.97 Da were observed in the spectrum of the Q0-treated sample that were not present in the spectrum of the sample that was not treated with Q0 (Fig. 10). These m/z values match the theoretical masses of chymotryptic petptide fragments 92–111 and 219–239 of the E2 component modified with Q0 (mass equal to peptide mass plus masses added by modifications by lipoate (189) and Q0 (182)). Electrospray ionization mass spectrometry analysis and a Sequest search of the chymotrypsin-digested 70-kDa protein band of Q0-treated human PDC followed by trypsin digestion confirmed that PDC E2 was the only major protein in the band and was consistent with the lysines at positions 99 and 226 being modified by lipoate plus Q0.



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  		FIG. 10.
MALDI-TOF MS spectra of human dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex incubated with or without Q0. The purified enzyme complex was incubated with or without Q0, and component proteins were separated with SDS-PAGE. The protein band containing the E2 component of PDC was excised, alkylated with iodoacetamide, digested with chymotrypsin, and analyzed by MALDI-TOF MS. Regions of the spectrum with peaks of m/z values consistent with Q0 bonded to peptide fragments containing lipoate (arrows), as well as the same regions from a sample of the E2 component from the enzyme complex not incubated with Q0, are shown. Other peaks in the spectra correlate with chymotryptic peptides arising from the digestion of human PDC E2.

 
The pig PDC E2 (GenBankTM accession no. BAB61720 [GenBank] is modified by lipoate attached to lysines at positions 132 and 259. The MALDI-TOF mass spectrum of the chymotrypsin-digested E2 peptide from the Q0-treated PDC showed a peak with an m/z of 5396.83 Da (Fig. 11). This mass corresponds to that of the consensus amino acid sequence of region 100–144 of the protein that is modified by lipoate plus Q0. No peak with this mass was observed in the spectrum of the sample not treated with Q0. Electrospray ionization mass spectrometry analysis of the chymotrypsin-digested pig E2 protein from the pig PDC sample that was not treated with Q0 followed by a Sequest search done against pig E2 of the PDC found two peptides with lysines modified by lipoate, one at position 132 and the other at position 259, when the search was done to include the relevant lysine modifications. The search ranked these peptides 1 and 2 with respect to peptides with modified lysines. A Sequest search of the electrospray ionization data from the digested pig PDC sample that was treated with Q0 found a peptide with the lysine at position 259 modified by lipoate plus Q0 and ranked this peptide number 1 when the search was carried out to include the relevant lysine modifications. The search indicated that the lipoate was also modified by carbamidomethylation arising from the iodacetamide reagent used to prevent dimerization of thiols during the proteolytic digestion of the proteins.



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  		FIG. 11.
MALDI-TOF MS spectrum of the pig dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex incubated with Q0. The arrow indicates a peak with an m/z value in the spectrum consistent with Q0 bonded to a peptide fragment containing lipoate. The same region of the spectrum from a sample of the protein not incubated with Q0 is shown for comparison. Methods were the same as those described in the Fig. 10 legend.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study, we incubated human and rodent pancreatic islets or mitochondria from various rat tissues with Q0 and used immunoblot analysis with antisera against the ring structure of ubiquinone to identify proteins containing covalently attached Q0. Two mitochondrial proteins, one of 50 kDa and another of 70 kDa, were detected. The stronger of the two antisera was affinity-purified, and the antibody was used to immunoprecipitate the 50-kDa protein from a kidney mitochondrial extract. By amino acid sequencing of several internal peptides from a tryptic digest of this protein, it was identified as dihydrolipoamide succinyltransferase, the E2 of the KDC.

At 1 µM or higher concentrations, Q0 was then shown to bond to a 50-kDa protein in purified porcine and bovine KDCs and to a 70-kDa protein in preparations of purified porcine and bovine PDCs (Fig. 9; data not shown). Dihydrolipoamide acetyltransferase, the E2 of the pyruvate dehydrogenase complex (PDC), has a molecular mass of 70 kDa, and the native 70-kDa Q0-bonding protein was inferred to be this protein. It is noteworthy that although the 50-kDa protein was easily seen in all tissues studied with Western analysis, the 70-kDa protein was more readily visualized in certain tissues, such as heart, kidney, and brain (Fig. 5) and islets (Fig. 1). This distribution roughly corresponds to the level of PDC enzyme activity among tissues of the body. The PDC complex also contains the E3-binding protein, which is another protein that contains lipoate and has a molecular mass of about 50 kDa. Q0 most likely bonds to this protein in addition to the E2 of KDC in intact mitochondria because two separate proteins with slightly different molecular masses of around 50 kDa were detected by Western analysis in extracts of rat kidney mitochondria that were fractionated with hydroxyapetite chromatography (data not shown).

Interestingly, unlike many other quinones or other agents that attack thiols, Q0 seems to specifically attack only dihydrolipoamide thiols, without attacking cysteine thiols of the same or other proteins. When Q0 was incubated at concentrations as low as 10 µM or as high as 10 mM with intact cells or subcellular fractions of various tissues, it bound only to the mitochondrial proteins later identified as ones known to possess lipoic acid. In addition, mass spectrometric analysis of purified PDC or KDC incubated with Q0 revealed covalent bonding of Q0 only to the dihydrolipoates of the E2 enzymes of these complexes and not to cysteines of these proteins. It seems unlikely that this is due solely to steric hindrance of the Q0 molecule preventing it from attacking cysteine sulfhydryls but allowing it to attack dihydrolipoamide sulfhydryls that extend from the peptide backbone on an 11-atom arm (formed by 4 lysine carbons plus the nitrogen of the amide bond and 6 carbons of lipoate) because even larger quinones with more complex structures, such as vitamin K analogs with bulky side groups (24, 25), attack cysteine thiols on many proteins. It is possible that, as an ubiquinone analog, the structure of Q0 enables it to specifically interact with the E2 components of these dehydrogenases when they are present in their native environment in the inner mitochondrial membrane. This idea is supported by the observation that Q1 and Q2 (which cannot covalently bond to thiols and therefore do not bond covalently to the 50- or 70-kDa proteins) inhibited Q0 bonding to the E2 of KDC in mitochondria (Fig. 7), but not in the purified enzyme complex (data not shown).

Q0 is often used in studies of mitochondria (14–19) and apoptosis (10, 11), and it also stimulates insulin release from isolated pancreatic islets (1, 2). Like several other similar quinone agents that stimulate insulin release (1–3), such as tyrosine kinase inhibitors (4–6), Q0 kills the insulin-producing cell. A quinone with a similar structure, 2,3-dimethoxy-1,4-naphthoquinone, causes Ca2+ overload and depletion of glutathione, ATP, and NAD in rat insulinoma cells, resulting in apoptosis (3). Some of these deleterious effects of quinones, such as causing Ca2+ overload, may be due to mitochondrial redox cycling, but quinones can also attack thiols, and this very likely accounts for the depletion of cellular glutathione. Interestingly, Q0 is reported to inhibit the opening of the mitochondrial PTP, and because this is the first step in apoptosis (12, 13), this should help keep the cell intact (14–19). However, it is noteworthy that Q0 differs from naturally occurring coenzyme Q molecules in that there is no group on carbon 6 except for a hydrogen. This makes Q0 a very reactive electrophile. The tyrosine kinase inhibitors and 2,3-dimethoxy-1,4-naphthoquinone also have carbons without side groups adjacent to carbonyl groups, also making them strong electrophiles. Some of the ability of Q0 to inhibit the mitochondrial permeability transition might be due to the fact that it can attack strongly nucleophilic thiol groups and that its ring structure is the same as that of ubiquinone.

Whether or not either of the two E2 enzymes is indirectly involved in the PTP will need to be resolved by future experiments. However, there are rough parallels between the effects of Q0 on the PTP and its bonding to these proteins. The effect of Q0 on the PTP involves complex I of the respiratory chain (16), and the reactions of KDC and PDC each contribute electrons to complex I. Q0 at low micromolar concentrations is a potent inhibitor of the PTP, and although its inhibiting action on the PTP is prevented by Q1, Q1 itself and other ubiquinone analogs have no effect on the PTP (14–19). Similarly, Q1 and Q2 do not bond to the E2 proteins, but they do prevent bonding of Q0 to the E2 protein of KDC. Thiol groups are involved in the PTP, and Q0 has a strong affinity for thiols. In this regard, it is of interest that aldehydes, such as 4-hydroxynonenal, have been shown to inhibit the PTP (26) and also to selectively inhibit PDC and KDC, most likely by attacking their dihydrolipoate groups (27, 28). Because 4-hydroxynonenal also inhibits glucose-induced insulin secretion from pancreatic islets (29), this supports the idea that the toxicity of Q0 may be related in part to covalent attack of the dihydrolipoamide sulfhydryls of the E2 components of PDC and KDC but that its ability to stimulate insulin release involves another mechanism, possibly by causing massive release of calcium from mitochondria (1, 2).

The results suggest that studies of mitochondrial physiology with Q0, because of its multiple effects, need to be interpreted carefully; more importantly, these results might have implications for human autoimmune disease. They further support the idea that unphysiologic post-translational modification of protein thiols can create an abnormal protein. This may initiate an immunogenic response against the protein (30), as in primary biliary cirrhosis, which is an autoimmune disease in which the major immune epitopes are the lipoyl domains of the E2 proteins of PDC and KDC (31, 32).


   FOOTNOTES
 
* This study was supported by National Institutes of Health Grant DK28348, the Oscar C. Rennebohm Foundation, and the Robert Wood Johnson Family Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Rm. 3459 Medical Science Center, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-1195; Fax: 608-262-9300; E-mail: mjmacdon{at}wisc.edu.

1 The abbreviations used are: PTP, permeability transition pore; EDC, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; KDC, {alpha}-ketoglutarate dehydrogenase complex; KLH, keyhole limpet hemocyanin; PDC, pyruvate dehydrogenase complex; Q acid, {alpha}-2,3-dimethoxy-5-methyl-6-(3'-carboxy-3'-methylpropyl)-1,4-benzoquinone; Q0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone; Q1, coenzyme Q1, 2,3, dimethoxy-5-methyl-6-[3-methyl-2-butenyl]-1,4-benzoquinone; Q2, coenzyme Q2, 2,3-dimethoxy-5-methyl-6-geranyl-1,4-benzoquinone; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; MES, 4-morpholineethanesulfonic acid; HPLC, high pressure liquid chromatography. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Ieva Reich, Fred Crane, Henry Lardy, Frank Ruzicka, and Rob Haworth for helpful discussion and Feng Lu, Heather Drought, Richard Raphael, Julian Buss, Michael Fallon, and Grzegorz Sabat for technical assistance. We thank Dr. Joseph Leykam at the Macromolecular Structure Facility of Michigan State University for amino acid sequence analysis and helpful advice.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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