Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M702385200 on October 18, 2007

J. Biol. Chem., Vol. 282, Issue 52, 37436-37447, December 28, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/52/37436    most recent
M702385200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Winger, A. M.
Right arrow Articles by Millar, A. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Winger, A. M.
Right arrow Articles by Millar, A. H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Cytotoxic Lipid Peroxidation Product 4-Hydroxy-2-nonenal Covalently Modifies a Selective Range of Proteins Linked to Respiratory Function in Plant Mitochondria*Formula

Alison M. Winger{ddagger}1, Nicolas L. Taylor{ddagger}2, Joshua L. Heazlewood{ddagger}2, David A. Day§, and A. Harvey Millar{ddagger}3

From the {ddagger}Australian Research Council Centre of Excellence in Plant Energy Biology, the University of Western Australia, 35 Stirling Highway, Crawley 6009, Western Australia and §Australian Research Council Centre of Excellence in Plant Energy Biology, School of Biological Sciences, University of Sydney, 2006 New South Wales, Australia

Received for publication, March 20, 2007 , and in revised form, October 17, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plants encounter a variety of environmental stresses that affect their cellular machinery and that they adapt to on a daily basis. Lipid peroxidation is one consequence, at the cellular level, of such stresses and yields cytotoxic lipid aldehydes, including 4-hydroxy-2-nonenal (HNE), that react with specific sites in proteins, leading to diverse changes in protein function and/or stability. We have assessed the sensitivity of plant mitochondrial proteins to HNE modification, using one-dimensional and two-dimensional denaturing PAGE and blue native-PAGE coupled to immunological detection and tandem mass spectrometry identification. A select range of proteins was modified by exogenous application of HNE to mitochondria isolated from Arabidopsis cell cultures. These included a number of proteins that directly interact with the ubiquinone pool, as well as a number of soluble matrix proteins. Mitochondria isolated from cell cultures following hydrogen peroxide, antimycin A, or menadione treatment had significantly reduced respiratory capacity and elevated levels of HNE adduction to specific subsets of proteins. Targets identified included the proteins affected by direct application of HNE but also some new proteins, including a number of matrix dehydrogenases, the inner membrane adenine nucleotide translocator, and the outer membrane voltage-dependent anion channel. Degradation products of some proteins were also found to be HNE adducted, suggesting a link between HNE adduction and protein turnover. Some of the major enzyme complexes that were HNE adducted did not show demonstrable changes in their maximal activity measured with artificial acceptors, but changes did occur in associations between respiratory chain complexes following stress treatments.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The polyunsaturated fatty acids of membrane phospholipids are highly susceptible to peroxidation by reactive oxygen species (ROS),4 and a self-propagating chain of free radical reactions can produce various aldehydes, alkenals, and hydroxyalkenals (1, 2). These aldehydes are cytotoxic, generally more stable than ROS, and can cause extensive damage to proteins and other cellular constituents. HNE is the most abundant and toxic aldehyde generated through ROS-mediated peroxidation of abundant lipids in plants such as linoleic acid (1, 3). HNE is a highly reactive electrophile, with the primary reactivity of the molecule lying at the unsaturated bond of the C-3 atom. HNE has been shown to form Michael adducts via this C-3 atom with the sulfhydryl group of Cys residues, the imidazole group of His residues, and the {epsilon}-amino group of Lys residues on a large number of proteins (3). Recently it has been proposed that HNE can also modify Arg residues of proteins (4). In addition to Michael adduct formation, Lys residues also form Schiff bases and pentylpyrrole adducts with HNE via the C-1 aldehyde group (5). HNE has also been shown to react via the C-3 position with the sulfhydryl groups of lipoic acid moieties on proteins (6). Modification of proteins by HNE has the potential to have serious detrimental effects in a cell because of modification of amino acids and the potential to form cross-links in proteins (1–5).

Investigations of the effects of HNE in mammalian mitochondria show that HNE can inhibit proteins involved in the respiratory process. Complex IV activity is inhibited by HNE in a dose-dependent manner in mammals (7, 8), predominantly by binding to subunit VIII of the complex (9). Cytochrome c (4) and the {alpha} subunit of the succinate dehydrogenase (complex II) (10, 11) have also been shown to be inhibited by HNE adduction in mammals. Complex I subunits have been shown to be adducted by HNE in heart mitochondria (12), but although complex I-linked respiration is inhibited by HNE in central nervous system mitochondria, direct assays of complex I using n-decylubiquinone failed to measure a loss of enzyme complex activity (10). Other mitochondrial localized proteins in mammals that are HNE targets include tricarboxylic acid cycle enzymes KGDC and PDC (6, 13, 14), the adenine nucleotide translocator (ANT) (15), heat shock protein 70 (16), and a protein-disulfide isomerase (17). It is interesting to note that not all modifications by HNE are detrimental or have a measurable effect on function. For example, increasing modification of KGDC by HNE in aged rat mitochondria leads to an increase in activity that correlated with a lowered Km value of the enzyme for {alpha}-ketoglutarate (14). Also, HNE has been shown to activate uncoupling protein function in plant and animal mitochondria by an undefined mechanism (18).

Plant mitochondria are known to respond to oxidative stress by the induction of respiratory bypasses of the phosphorylating respiratory chain (19), as well as the induction of antioxidant defenses such as the ascorbate/glutathione pathway (20) and a specific mitochondrial peroxiredoxin (21, 22). We have previously shown during oxidative stress in Arabidopsis (21) and environmental stress in pea (23) that, in addition to these defense responses, specific mitochondrial proteins are degraded under oxidative stress by an unknown pathway. In both cases this degradation was coincident with the loss of lipoic acid cofactors of decarboxylating dehydrogenases, which we had previously linked in potato mitochondria to modification by the lipid peroxidation product HNE (24). We have also shown in a detailed investigation of the impact of HNE on respiratory chain terminal oxidase function in plants that the stress-responsive nonphosphorylating alternative oxidase was much more sensitive to HNE inhibition than the cytochrome pathway (25). In that study, a combination of lipid peroxidation assays and the use of antibodies to HNE adducts revealed that hydroxyalkenals (HAEs) are produced in a dark-grown Arabidopsis cell culture model in response to H2O2, antimycin A, or menadione and that this led to an array of HNE-modified protein bands appearing in isolated mitochondrial samples (25). Here we have undertaken a more systematic analysis of the targets of HNE in plant mitochondria using antibodies to HNE and mass spectrometry analysis to identify the targets and to determine whether there are links between HNE adduction and mitochondrial response to, or damage by, oxidative stress.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Arabidopsis Cell Culture and Mitochondria Isolation—A heterotrophic Arabidopsis thaliana cell culture, established from callus of ecotype Lansberg erecta stem explants, was cultured in Murashige and Skoog basal salt mixture (Phyto-Technology Laboratories) supplemented with 3% (w/v) sucrose, 0.05% (w/v) naphthaleneacetic acid, and 0.005% (w/v) kinetin. Cell cultures (120 ml) were maintained in 100 µmol m–2 s–1 light at 22 °C with shaking (120 rpm). At 7 days, cells (20 ml) were subcultured into 100 ml of fresh media. Cell cultures used for experimental analysis in this work were maintained in the dark with shaking (120 rpm) following subculture. Mitochondria were purified from 7-day-old Arabidopsis cell cultures as described previously (26).

HNE Treatment of Isolated Mitochondria and Oxidative Stress Treatments of Cells—Cells from 7-day-old dark-grown Arabidopsis cell cultures were treated with 88 mM H2O2 (prepared in water by Asia Pacific Specialty Chemicals, Seven Hills, New South Wales, Australia), 25 µM antimycin A (prepared in ethanol), or 400 µM menadione (prepared in ethanol). Isolated mitochondria were incubated with indicated concentrations of HNE for 20 min at 25 °C with shaking (800 rpm). HNE treatment of mitochondria subsequently analyzed by oxygen electrode was carried out by incubation of HNE with 200–300 µgof mitochondrial protein in 250 µl of oxygen electrode reaction medium in the oxygen electrode chamber at 25 °C.

Measurement of Oxygen Consumption—All oxygen consumption measurements were performed using a Clark-type oxygen electrode (Hansatech, UK) attached to a personal computer. Calibration of the electrode was carried out by setting 100% to the current recorded in air-saturated water at 22 or 25 °C and by setting 0% to the current recorded after addition of sodium dithionite to the water to remove all oxygen in the electrode chamber. Various substrate, inhibitor, and effector standard additions are made as required. Arabidopsis cell culture (300 µl) was suspended in cell culture media to give a final volume of 1 ml. Total respiration of intact cells was measured at 22 °C following the addition of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (4 µM). Respiration through the alternative pathway or the cytochrome pathway in intact cells was measured following the additions of 1 mM KCN or 1 mM n-PG, respectively. Isolated mitochondria (200–300 µg of protein) were suspended in 1 ml of oxygen electrode reaction medium (0.3 M sucrose, 5 mM KH2PO4, 10 mM TES, 10 mM NaCl, 2 mM MgSO4, 0.1% (w/v) bovine serum albumin, pH 7.2) at 25 °C. Total isolated mitochondrial respiration was measured at 25 °C in the presence of various substrates as follows: for succinate-dependent respiration, succinate (10 mM) and ATP (0.5 mM); for malate plus glutamate-dependent respiration, malate (10 mM), glutamate (10 mM), thiamine pyrophosphate (0.2 mM), NAD (2 mM) and coenzyme A (12 µM). Respiration via the alternative pathway by isolated mitochondria was measured in the presence of the complex III inhibitor myxothiazol (2.5 µM), the reductant dithiothreitol (5 mM), and the AOX activator pyruvate (5 mM). Respiration via the cytochrome pathway was measured in the presence of the AOX inhibitor n-PG (0.5 mM).

Protein Electrophoresis—SDS-PAGE was performed according to Laemmli (27). Following electrophoresis gels were either stained with colloidal Coomassie or transferred to nitrocellulose membrane for Western blot analysis. IEF/SDS-PAGE was performed using immobilized pH gradient strips (Immobiline DryStrips, GE Healthcare) of pH range 3–10NL (nonlinear; 18 cm) or 4–7 (linear; 24 cm). Proteins were precipitated by addition of acetone to samples to a final concentration of 90% acetone. Samples were placed at –20 °C overnight to allow precipitation of proteins. Precipitates were centrifuged at 20,800 x g for 20 min and solubilized in either 350 µl (3–10NL) or 450 µl (4–7 linear) of IEF sample buffer (6 M urea, 2 M thiourea, 2% (w/v) CHAPS, 2% (w/v) IPG buffer pH 3–10NL or pH 4–7). 2 µl (3–10NL), or 2.6 µl (4–7 linear) of tributylphosphine (1:9 in IEF sample buffer) was added to each sample. Samples were then used to rehydrate either pH 3–10NL DryStrips, or pH 4–7 DryStrips overnight at room temperature. Isoelectric focusing was performed using a flatbed electrophoresis unit (Multiphor II, GE Healthcare) at 20 °C. Following isoelectric focusing, strips were removed and incubated for 50 min in IEF/SDS-PAGE transfer buffer (4 M urea, 20% (v/v) glycerol, 2% (w/v) SDS, trace bromphenol blue, 0.375 M Tris-HCl, pH 8.8). The second SDS-PAGE dimensions were run by placing the strips on top of 1-mm thick (25 x 18 cm) polyacrylamide gels (Ettan, Dalt 6, GE Healthcare) and following standard procedures. Blue native-PAGE was performed based on the method by Schägger and von Jagow (28). Mitochondrial samples (500 µg) were pelleted at 18,300 x g for 10 min at 4 °C. The pellet was resuspended in BN solubilization buffer with digitonin (150 mM acetate, 10% glycerol, 5% (w/v) digitonin, 30 mM HEPES, pH 7.4) at 10 µl per 100 µg of protein to solubilized the membranes. Samples were incubated on ice for 20 min and then centrifuged at 18,300 x g for 10 min to remove unsolubilized material. The supernatant was then transferred to a new tube containing 5% Serva Blue G at 1 µl per 20 µl of solubilization buffer. The first dimension separation was performed on 1.5-mm thick (16 x 11 cm) polyacrylamide gels using the Protean II system (Bio-Rad). The gel consisted of a stacking layer (0.25 M {epsilon}-aminocaproic acid, 4% (w/v) acrylamide:bisacrylamide (33:1), 0.05% (w/v) ammonium persulfate, 0.1% (v/v) TEMED, 25 mM BisTris-HCl, pH 7.0), and a gradient separating layer (0.25 M {epsilon}-aminocaproic acid, 4.5–16% (w/v) acrylamide:bisacrylamide (33:1), 0.05% (w/v) ammonium persulfate, 0.1% (v/v) TEMED, 25 mM Bis-Tris-HCl, pH 7.0). The gels were run with a cathode buffer (50 mM Tricine, 0.02% (w/v) Coomassie 250G, 15 mM BisTris-HCl, pH 7.0) in the upper chamber and an anode buffer in the lower chamber (50 mM BisTris-HCl, pH 7.0) of the gel tank. The gel was run initially at a constant voltage of 100 V for 45 min and thereafter with a constant current of 15 mA per gel, with voltage limited at 500 V, for 10 h. Gel runs containing samples used for in-gel assay analysis were paused after 3 h of electrophoresis, and the upper cathode buffer was exchanged for cathode buffer minus Coomassie to allow for a clearer background on the gel. Gels were stained with colloidal Coomassie, prepared for in-gel enzymatic analysis, or prepared for second dimension SDS-PAGE.

Enzyme Assays and In-gel Activity Staining—All assays were performed with mitochondrial samples ruptured by freeze/thaw thus allowing direct access of substrates to both sides of the inner membrane. Succinate dehydrogenase (complex II of the respiratory chain) was measured using a protocol modified from Singer et al. (29). 200 µg of protein was added to 1 ml of succinate dehydrogenase reaction mixture (60 µM 2,6-dichlorophenylindophenol, 0.03% (w/v) phenazine methosulfate, 1 mM KCN, 1 mM n-PG, 50 mM KH2PO4, pH 7.6). The reaction was started by the addition of 40 mM succinate to the mixture. Succinate dehydrogenase activity was measured as DCPIP reduction at 600 nm. Calculation of amount of DCPIP reduced was performed using {epsilon} = 15.7 mM–1 cm–1. In-gel measurement of complex I activity was performed based on the method described in Zerbetto et al. (30). Following blue native separation of mitochondrial protein, gels were washed four times for 5 min each with double distilled H2O and incubated in 50 ml per five lanes of NADH DH assay mixture (0.14 mM NADH, 1 mg/ml nitro blue tetrazolium, 0.1 M Tris-HCl, pH 7.4) until the stained complex was visible (10 min to 1 h). The reaction was stopped by rinsing the gel in water and transferring the gel to fixing solution (40% methanol, 10% acetic acid). NADH and deamino-NADH to FeCN [Fe(CN)3–6] was measured spectrophotometrically as NADH oxidation at 340 nm using {epsilon} = 6.22 mM–1 cm–1 (31). Pyruvate dehydrogenase complex and malate dehydrogenase were measured spectrophotometrically following NADH reduction and oxidation, respectively, at 340 nm using {epsilon} = 6.22 mM–1 cm–1 (24).

Western Blotting and Immunodetection—Proteins were transferred from polyacrylamide gels to HybondTM-C extra nitrocellulose blotting membrane according to the method of Towbin et al. (32) using a Hoefer SemiPhor (GE Healthcare) transfer system using standard procedures. Following Western blotting, the membrane was incubated in 1% blocking solution (Roche Applied Science), either for 1 h at room temperature on a rocker or overnight at 4 °C without rocking, to block nonspecific binding of the antibody. Following a brief rinse in TBS/Tween, the membrane was incubated with polyclonal HNE adduct antibodies (HNE11-S, Alpha Diagnostics International Inc.) at 1:2000 dilution overnight at 4 °C, with gentle rocking. The membrane was washed by three quick rinses, a 15-min and two 5-min washes with TBS/Tween, incubated with a 1:20,000 dilution of an anti-rabbit horseradish peroxidase-conjugated secondary antibody (GE Healthcare) in 1% Western blocking reagent for 1 h at room temperature with gentle shaking. The membrane was washed as before and detected using the BM chemiluminescence blotting substrate (POD) kit (Roche Applied Science). Images of the membranes were obtained using a LAS 1000 CCD camera and ImageGauge software (Fuji, Tokyo, Japan).

Peptide Extraction from Gels—Gel spots of interest were excised from colloidal Coomassie-stained diagonal PAGE or two-dimensional gels, placed into wells of 96-well PCR plates and frozen at –80 °C until analyzed. Gel spots were thawed and destained twice with de-stain solution (50% (v/v) acetonitrile, 10 mM NH4HCO3) for 45 min each time on an orbital shaker. Gel spots were then dried at 50 °C for 1 h and digested overnight with digestion solution (12.5 µg/ml trypsin, 10 mM NH4HCO3, 0.00125% (v/v) trifluoroacetic acid) at 37 °C. Peptides were extracted by incubation with 12 µl of 100% acetonitrile for 15 min on an orbital shaker. The supernatant from each sample was placed into a clean well of a new 96-well PCR plate. Each gel spot was incubated further with 10 µl of a solution containing 50% acetonitrile and 5% formic acid for 15 min with shaking. The supernatant was taken and placed with the previously extracted supernatant, and the previous 15-min incubation was repeated. Samples were dried down in a vacuum centrifuge and resuspended in 15 µl of resuspension solution (5% acetonitrile, 0.1% formic acid) immediately prior to mass spectrometry. Samples were analyzed using either an Applied Biosystems QSTAR Pulsar i LC/MS/MS system or an XCT Ultra (three-dimensional ion trap) from Agilent Technologies.

Protein Identification by Tandem Mass Spectrometry—Samples from HNE-treated isolated mitochondria (Fig. 2 and supplemental Table S1) were analyzed using an Agilent 1100 series capillary LC system and an Applied Biosystems QSTAR Pulsar i LC/MS/MS system equipped with the IonSpray source running Analyst QS software (version 1.1) with the instrument in positive ion mode. Each extracted peptide sample was loaded in turn with the Agilent 1100 series capillary LC system onto a 0.5 x 50 mm C18 (5 µm, 100 Å) reverse phase column (Higgins Analytical) with a C18 OPTI-GUARD guard column (Optimize Technologies) at 16 µl/min equilibrated with 5% acetonitrile and 0.1% formic acid. Peptides were eluted from the C18 reverse phase column into the QSTAR Pulsar i by a 7-min acetonitrile gradient (5–80%) at 16 µl/min under constant formic acid concentrations of 0.1%. During the period of ion detection, eluted peptides were analyzed by the mass spectrometer at 8 µl/min. The method used to analyze eluted ions employed the information-dependent acquisition capabilities of Analyst QS and the rolling collision energy feature for automated collision energy determination based on the ions m/z (Sciex/AB). The method employed a 1-s time of flight-MS scan, which automatically switched (using information-dependent acquisition) to a 2-s product ion scan (MS/MS) when a target ion reached an intensity of greater than 30 counts, and its charge state was identified as 2+, 3+, or 4+. Time of flight-MS scanning was undertaken in the m/z range of 400–1600 m/z using a Q2 transmission window of 380 atomic mass units (100%). Samples analyzed from oxidative stress-treated samples (Figs. 4 and 5; supplemental Tables S2 and S3) used an Agilent 1100 series capillary LC system and an Agilent Technologies XCT Ultra IonTrap equipped with an ESI source equipped with a low flow nebulizer in positive mode controlled by Chemstation (revision B.01.03, build 204, Agilent Technologies) and MSD Trap Control version 6.0 Build 38.15 software (Bruker Daltonik GmbH). Each extracted peptide sample was loaded in turn with the Agilent 1100 series capillary LC system onto a 0.5 x 50-mm C18 (5 µm, 100 Å; Varian) homemade reverse phase column at 10 µl/min equilibrated with 5% acetonitrile and 0.1% formic acid under a regulated temperature of 50 °C. Peptides were eluted from the C18 reverse phase column into the XCT Ultra by a 9-min acetonitrile gradient (5–60%) under constant formic acid concentrations of 0.1%. The method used for initial ion detection utilized a mass range of 200–1400 m/z with scan mode set to Standard (8100 m/z per s) and an ion charge control conditions set at 250,000 and three averages taken per scan. Smart mode parameter settings were employed using a target of 800 m/z, a compound stability factor of 90%, a trap drive level of 80%, and optimize set to normal. Ions were selected for MS/MS after reaching an intensity of 80,000 cps, and two precursor ions were selected from the initial MS scan. MS/MS conditions employed SmartFrag for ion fragmentation, a scan range of 70–2200 m/z using an average of three scans, the exclusion of singly charged ions option, and ion charge control conditions set to 200,000 in ultra scan mode (26,000 m/z per s).

Protein Identification Searches with Mascot—Data produced by both these methods were used to search the Mascot search engine version 2.1.04 (Matrix Biosciences) for protein identification. Search parameters at Mascot employed a peptide tolerance of ±1.2 Da and a MS/MS tolerance of ±0.6 Da, with the use of the variable modifications, oxidized methionine (oxidation M) and HNE (CHK). Searches were performed against the TAIR6 Arabidopsis data base (30,700 sequences; 12,656,682 residues, released from Arabidopsis website on July 9, 2006). Details of the peptide matches and the coverage of matched proteins by Mascot searches are in supplemental Tables S4–S6, respectively.


Figure 1
View larger version (79K):
[in this window]
[in a new window]

  		FIGURE 1.
Immunodetection of HNE-modified proteins separated by SDS-PAGE. Mitochondrial proteins (control (C) or HNE-treated (HNE)) were separated by one-dimensional electrophoresis. Proteins were either stained with colloidal Coomassie (A) or transferred to nitrocellulose membrane, probed with the polyclonal HNE adduct antibodies, and detected by chemiluminescence (B). Molecular mass markers are indicated on the left, and the size of major immunoreactive bands in the control gel on the right.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of HNE-modified and Modifiable Proteins in Plant Mitochondria—We initially aimed to systematically identify proteins in plant mitochondria that were selectively sensitive to modification by HNE both generated endogenously in response to stress treatments of cell cultures and supplied exogenously to isolated mitochondria. Commercial polyclonal antibodies raised in rabbit that react with HNE adducts on a variety of amino acid residues were used in Western blots of mitochondrial proteins (Fig. 1). Preliminary studies were undertaken to measure the changes in immunoreactivity of mitochondrial proteins following treatment of isolated mitochondria with 700 µM HNE for 30 min. A number of HNE-modified proteins reacted intensely with the antibodies in the treated samples in ~20-s exposures (data not shown). At longer exposure times of ~3 min, the major immunoreactive bands in the treated samples could be observed alongside lower level immunoreactivity of proteins in untreated mitochondria (Fig. 1). The pattern of immunoreactive proteins in the control and treated samples contrasted with the total protein pattern observed in the colloidal Coomassie-stained control lanes (Fig. 1). Predominant bands at about 70, 45–50, 40, and 22 kDa reacted with the HNE-protein adduct antibody in the control cells, whereas a much greater range of higher and lower molecular weight proteins became immunoreactive following HNE treatment.

As it was not feasible to identify the proteins reacting with the HNE antibody from one-dimensional gels, two-dimensional separation of proteins on the basis of pI and molecular mass was performed. Proteins from control and 700 µM HNE-treated mitochondria were separated by two-dimensional IEF/SDS-PAGE using pI 3–10 nonlinear first dimension IEF followed by standard SDS-PAGE. Proteins were then stained with colloidal Coomassie Blue (Fig. 2, A and B) or transferred to a nitrocellulose membrane and probed with polyclonal anti-HNE adduct antibodies (Fig. 2, C and D). Only a small proportion of the proteins detected by Coomassie staining showed immunoreactivity with the anti-HNE adduct antibodies, indicating that only a select group of mitochondrial proteins are modified by HNE. HNE treatment increased the number of immunoreactive proteins and also increased the intensity of protein spots apparent in control mitochondria (Fig. 2, C versus D). Western analysis of both the control and HNE-treated mitochondria was undertaken from four independent mitochondrial isolations and separate HNE treatments. Some variability of immunoreaction was observed between samples, especially in the control samples, where some protein spots were routinely found but others were only present occasionally. Protein spots that could be identified confidently by comparison of Western blots and total protein gels were excised, digested with trypsin, and analyzed by MS/MS (Fig. 2E and supplemental Table S1). The number of times these proteins were observed on Western blots is shown in the final two columns of supplemental Table S1.

From the 18 protein spots analyzed, 13 different proteins were identified. These proteins were ranked according to their degree of immunoreactivity and whether they were present in control or HNE-treated samples. Protein spots 1–7 represented the most immunoreactive protein bands that could be identified in the control samples. They were identified as PDC E1 β subunit, mercaptopyruvate sulfurtransferase, and the succinyl-CoA synthetase β subunit (supplemental Table S1), all matrix-located metabolic enzymes. In addition, the protein from a smaller spot (spot 8) was identified as a breakdown product of ATP synthase β subunit in the control samples. These proteins were found consistently in independent samples and appear to represent the background state of HNE-modified proteins in mitochondria isolated from this cell culture. Following incubation with added HNE, a number of new proteins were detected, whereas protein spots 1–8 became more prominent. The newly identified proteins (from spots 9 to 18) included subunits from protein complexes that deliver electrons to the ubiquinone pool (CI and electron transfer flavoprotein ubiquinone oxidoreductase) along with three more matrix metabolic enzymes, the Tu elongation factor, and chaperonin 10 (supplemental Table S1). These two-dimensional gel images of immunoreactive protein spots 1–18 (Fig. 2, C and D) are consistent with the dominant immunoreactive protein bands observed on one-dimensional gels at apparent molecular masses of ~70, 45–50, and 40 kDa (Fig. 1).


Figure 2
View larger version (51K):
[in this window]
[in a new window]

  		FIGURE 2.
Immunodetection of HNE-modified proteins separated by IEF/SDS two-dimensional electrophoresis (3–10NL). Mitochondrial proteins (control or HNE treated) were separated by two-dimensional electrophoresis, with the first dimension separation on 3–10NL, 18-cm ImmobilineTM Drystrips followed by SDS-PAGE second dimension. Proteins were either stained with colloidal Coomassie (A and B) or transferred to nitrocellulose membrane and probed with the polyclonal HNE adduct antibodies (C and D). Molecular mass and the pI gradient are indicated by axis numbers. The 18 protein spots located on colloidal Coomassie control gels and analyzed by mass spectrometry (see supplemental Table S1) are indicated in E but were analyzed from both control and HNE-treated gels. Asterisks on D indicate new immunoreactive protein spots present after HNE treatment.

 
Oxidative Stress Treatment of Intact Cells Leads to Changes in Whole Cell and Isolated Mitochondrial Respiration—To investigate whether HNE modified similar proteins in vivo, we used oxidative stress treatments previously developed to investigate primary metabolic function and mitochondrial dysfunction in Arabidopsis (21, 25, 33). These included treatment of cells with hydrogen peroxide, the respiratory inhibitor antimycin A, and the quinone analogue menadione. We have shown previously that these chemical treatments lead to enhanced levels of lipid peroxidation products in whole cells, measured either as malondialdehyde equivalents by the thiobarbituric acid-reactive substances assay or as HAE measured via a commercial HAE colorimetric detection method (25). Respiration of whole cells provided information on the viability of cell metabolism following oxidative stress and some general information about changes in respiratory pathways and respiratory flux. In the first few hours of treatment with these chemicals, the respiratory rates of whole cells were typically very low (25), but by 8 h post-treatment respiratory rates in all treatments had recovered, and only the H2O2 treatment had a respiration rate that was significantly lower than the control cells (Fig. 3A). Addition of n-PG, to block the alternative respiratory pathway only inhibited respiration significantly in the antimycin A-treated cells. In contrast, addition of KCN to block the cytochrome pathway inhibited control and menadione-treated cells by 60–70%, whereas H2O2 and antimycin A cell respiration were only inhibited by ~30%. This suggests a substantial increase in capacity of the alternative pathway in the H2O2 and antimycin A-treated cells. However, respiratory measurements in whole cells do not take into account increased or decreased substrate provision to mitochondria or changes in mitochondrial integrity that could affect the measurements. When mitochondria were isolated from these cells, a somewhat different profile of respiratory capacities was recorded (Fig. 3B). Using succinate as a substrate, respiratory rates were recorded in isolated mitochondria in the absence of inhibitors and also in the presence of n-PG to block the alternative or antimycin A to block the cytochrome pathway (Fig. 3B). The antimycin A-treated mitochondria retained their inhibition of cytochrome pathway activity, and the H2O2-treated samples had lower respiratory rates on a mitochondrial protein basis compared with controls (Fig. 3B). However, the menadione-treated cells yielded mitochondria with a significantly lower respiratory capacity (Fig. 3B) than was expected when compared with the whole cell respiratory assay results (Fig. 3A). This discrepancy cannot easily be explained. No other comparative measurements of inhibition of respiration by menadione in whole cells and isolated plant mitochondria have been reported, to our knowledge, that would allow this to be independently confirmed. The degree of inhibition following oxidative stresses in whole cells and isolated mitochondrial assays will likely depend upon which extramitochondrial rate-limiting steps in whole cell respiration are differential targets of oxidative stress by chemicals such as H2O2, menadione, and antimycin A.

The control respiratory rates (in the presence of ADP but the absence of inhibitors) were also recorded in mitochondrial samples using external NADH or malate plus glutamate as substrates (Fig. 3C). These data again showed lower respiratory rates by all mitochondria from treated samples. External NADH-dependent respiration was the most significantly inhibited by both direct HNE treatment and in vivo oxidative treatments, with all rates less than 35% of controls. Malate plus glutamate-dependent respiration was relatively less susceptible to inhibition, with rates in treated samples being 40–60% of controls and rates about 60% of controls after direct treatment with HNE.


Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 3.
Respiratory rates of whole cell culture samples (A) and mitochondria (B and C) isolated from cells treated with H2O2, antimycin A (AntiA), and menadione. A, for whole cell respiratory rates, white bars indicate total respiratory rate; gray bars indicate cytochrome pathway oxygen consumption rate in the presence of the alternative pathway inhibitor (n-PG); black bars indicate alternative pathway oxygen consumption rate in the presence of cytochrome pathway inhibitor (KCN). B, for succinate-dependent respiration by isolated mitochondria, white bars indicate total respiratory rate; gray bars indicate cytochrome pathway oxygen consumption rate in the presence of the alternative pathway inhibitor (n-PG); black bars indicate alternative pathway oxygen consumption rate in the presence of cytochrome pathway inhibitor (KCN), pyruvate, and dithiothreitol. C, for uninhibited respiratory rates by isolated mitochondria dependent on different substrates, white bars indicate respiratory rate dependent on external NADH; gray bars indicate respiratory rate dependent on malate plus glutamate. B and C, respiratory rates of isolated mitochondria directed treated with HNE (as indicated under "Experimental Procedures") are presented for comparison with the respiratory rates of mitochondria isolated from the cells treated with oxidizing agents.

 
A Selective Set of HNE-modified Proteins Were Identified in Mitochondria from the Oxidative Stress-treated Cell Cultures—Using an approach similar to that adopted in Fig. 2, proteins from mitochondria isolated from chemical-treated cell cultures at 8 h were separated by IEF/SDS-PAGE, followed by either staining for total protein or Western blotting and probed with the anti-HNE adduct antibodies. As the majority of immunoreactive protein spots from supplemental Table S1 were in the pI 5–7 range, a narrower range 4–7 linear gradient IEF was used to maximize separation of immunoreactive spots. Antibody reaction in control samples was compared on these gels with mitochondria from H2O2-, antimycin A-, and menadione-treated samples. We had previously used these antibodies to show that significant HNE modification of mitochondrial proteins occurs under these conditions in one-dimensional gel comparisons (25), but we did not identify the proteins concerned.

In control samples several immunoreactive protein spots were detected: protein spots 19, 21, 30, which were found on MS/MS analysis to be succinate dehydrogenase {alpha} subunit, ATP synthase β subunit, and pyruvate dehydrogenase E1 β subunit (supplemental Table S2). All three were more prominent in the treatment samples. This was broadly consistent with the identifications made following direct HNE treatment (Fig. 2 and supplemental Table S1). In addition, a range of new immunoreactive protein spots was present in the samples from oxidatively stressed cells (indicated by asterisks in Fig. 4). On MS/MS analysis, a number of these (protein spots 20 and 22–29) were found to be breakdown products of two proteins, namely succinate dehydrogenase {alpha} subunit and ATP synthase β subunit. A further protein spot (no. 31) represented an additional presentation of pyruvate dehydrogenase E1 β subunit with a slight acidic shift from the main protein spot for this protein (30). In addition 10 more proteins were identified, including elongation factor Tu, the 75-kDa subunit of complex I (also seen in supplemental Table S1 after HNE treatment), and a selection of other proteins over-represented in dehydrogenases and reductases (supplemental Table S2). Most of these new immunoreactive proteins were found in all three stress treatments but were most pronounced and most commonly found in the menadione treatments (supplemental Table S2).

Because several of the identified proteins are membrane-bound (supplemental Tables S1 and S2), blue native-PAGE separation of the electron transport chain complexes coupled to SDS-PAGE separation of complex subunits was employed to further analyze HNE modification of mitochondrial membrane protein samples (Fig. 5). Coomassie-stained gels of BN-PAGE/SDS-PAGE showed relatively similar abundance of oxidative phosphorylation complexes in the treatments, with typical banding patterns for complex I, complex V, complex III, and complex IV present. Immunoreaction with the anti-HNE-protein adduct antibodies in control samples identified protein spots representing the {alpha} and β subunits of ATP synthase (supplemental Table S3, spots 42 and 43) and a protein spot containing the E3 dihydrolipoamide dehydrogenase (supplemental Table S3, spot 44). Following H2O2 treatment, several new intensely immunoreactive protein spots were apparent, notably four dehydrogenases (for glutamate, formate, glyeraldehyde-3-phosphate, and succinate semialdehyde), two membrane proteins (voltage-dependent anion channel and ANT), manganese superoxide dismutase, and the {delta} subunit of ATP synthase. The same immunoreactive protein spots were also found to some degree in antimycin A and menadione treatments, but only ANT (spot 49) was found consistently (Fig. 5 and supplemental Table S3). Voltage-dependent anion channel, ANT, and ATP synthase β subunit are known to be HNE adduction sites in mammalian mitochondria (12, 15). Four subunits of respiratory complex I were also identified as immunoreactive protein bands in the BN-PAGE gels from treated samples (supplemental Table S3, protein spots 53–55).


Figure 4
View larger version (42K):
[in this window]
[in a new window]

  		FIGURE 4.
Immunodetection of HNE-modified mitochondrial proteins from H2O2-, antimycin A-, and menadione-treated cells, separated by IEF/SDS two-dimensional electrophoresis (4–7NL). Mitochondrial proteins (control, H2O2-, antimycin A-, and menadione-treated) were separated by two-dimensional electrophoresis, with the first dimension separation on 4–7NL, 18-cm ImmobilineTM Drystrips followed by SDS-PAGE second dimension. Proteins were transferred to nitrocellulose membrane and probed with the polyclonal HNE adduct antibodies (A–D). Molecular mass and the pI gradient are indicated by axis numbers. Asterisks on A–D indicate new immunoreactive protein spots present in each treatment that are numbered on E. The 23 protein spots located on colloidal Coomassie gels and analyzed by mass spectrometry (see supplemental Table S2) are indicated on the stained control gel in E but were analyzed from both control and treated samples.

 


Figure 5
View larger version (51K):
[in this window]
[in a new window]

  		FIGURE 5.
Immunodetection of HNE-modified mitochondrial proteins from H2O2-, antimycin A-, and menadione-treated cells, separated by BN-PAGE/SDS-PAGE two-dimensional electrophoresis. Mitochondrial proteins (control, H2O2-, antimycin A-, and menadione-treated) were separated by two-dimensional with the first dimension separation by blue native-PAGE, followed by an SDS-PAGE second dimension. Proteins were transferred to nitrocellulose membrane and probed with the polyclonal HNE adduct antibodies (A–D). Molecular mass are indicated on the y axis as numbers, and positions of major respiratory complexes are indicated on the x axis by roman numerals; the F1 subunit of complex V is noted separately. Asterisks on A–D indicate new immunoreactive protein spots present in each treatment that are numbered on E. The 16 protein spots located on colloidal Coomassie gels and analyzed by mass spectrometry (see supplemental Table S3) are indicated on the stained control gel in E but were analyzed from both control and treated samples.

 
Impact of HNE and Oxidative Stress Treatments on Enzyme Activities—We have performed a series of assays on enzymes that contain subunits modified by HNE (supplemental Tables S1–S3) to determine whether significant changes in maximal catalytic capacities can be measured. We have previously reported that complex II activity was not significantly inhibited by HNE addition in potato tuber mitochondria (24), and this was also confirmed here in Arabidopsis by succinate-dependent respiration rates after HNE addition in Fig. 3B. However, minimal inhibition of the catalytic capacity of complex II has been reported in mammalian mitochondria following HNE adduction (10, 11). Fig. 3B showed substantial decreases in succinate-dependent respiration by isolated mitochondria following oxidative stress treatments. To determine whether this effect was directly on complex II, we undertook enzyme assays of succinate-dependent DCPIP reduction in freeze/thawed extracts of mitochondrial samples isolated from Arabidopsis under the treatments tested in this study (Fig. 6A). Although there were some changes in mean values, there was no statistically significant change in complex II activity either by direct HNE addition or by the oxidative stress treatments.

Total NADH-dependent FeCN reduction in freeze/thawed extracts of mitochondria was not affected by HNE or the stress treatments (Fig. 6B). This was consistent with a lack of inhibition of NADH-FeCN activity by HNE previously reported in potato tuber mitochondrial extracts (24). However, as this artificial acceptor assay represents the sum of a range of enzyme activities in plant mitochondria (31, 34), it is not a good guide in plants to complex I activity alone. Deamino-NADH to FeCN is more selective for complex I (31, 34), so this assay was also performed, and no significant loss (p < 0.05) of maximal activity was recorded in freeze/thawed extracts either after direction addition of HNE to mitochondria or in freeze/thawed mitochondrial extracts isolated from the stress treatments (Fig. 6C). An alternative method to distinguish complex I and other NADH dehydrogenases is activity staining of complex I in BN-PAGE gels that separates the high molecular mass complex I from other dehydrogenases activities in plant mitochondria (30, 35). Activity staining of first dimension BN-PAGE gels for complex I function as NADH-nitro blue tetrazolium revealed that more of the activity staining was associated with a higher molecular mass CI·CIII supercomplex, and less with the classical CI band in H2O2- and antimycin A-treated samples (Fig. 6D).


Figure 6
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 6.
Assays of maximal activities of HNE-targeted mitochondrial enzymes in mitochondrial samples treated with HNE or isolated from H2O2-, antimycin A- (AA), and menadione (Mena)-treated cells. Assays are shown for succinate (Succ)-dependent DCPIP reduction (A), NADH oxidation linked to FeCN (B), deamino-NADH oxidation linked to FeCN (C), pyruvate dehydrogenase complex activity (E), and malate dehydrogenase activity (F). Assays were measured and averaged from three independent mitochondrial isolations from control and treated cells (+ S.E., n = 3). In-gel staining of complex I activity as NADH reduction of nitro blue tetrazolium is shown as a typical gel image in D highlighting the primary complex I band (CI), the CI–CIII supercomplex (CI–CIII), and the minor form of complex I band (CIb).

 
As malate plus glutamate-dependent respiration was significantly inhibited by HNE and the stress treatments (Fig. 3C), and pyruvate and malate dehydrogenases were HNE targets (supplemental Tables S1 and S2), we also measured PDC and malate dehydrogenase activities in freeze-thawed mitochondrial extracts (Fig. 6, E and F). Both showed small changes in mean maximal activities, but ultimately these could not be shown to be significantly lower in independent mitochondrial samples isolated from oxidatively stressed cell cultures. PDC was inhibited by direct treatment by HNE (p < 0.05) as shown previously in other plant species (24, 36). The variability in the measurements from independently isolated mitochondrial samples precluded the measurement of statistically significant decreases (p < 0.05) in the 15–20% range.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is apparent from this analysis that plant mitochondria contain proteins susceptible to HNE adduction. These are evident to some extent in mitochondria from untreated cell suspensions (presumably reflecting a base level of oxidative stress in these cells or oxidative damage during mitochondrial isolation) but are enhanced by HNE addition to isolated organelles or by oxidative stress in vivo. A series of studies focused on specific enzymes and electron transport chain complexes in mammalian mitochondria provide evidence for a broadly similar pattern as follows: modification of selective components of the electron transport chain (10–12) and of major tricarboxylic acid cycle dehydrogenases (6, 13). This study represents a more systematic analysis of mitochondrial targets in a single system. It also shows that protein modification by HNE can be relatively selective in the plant mitochondrial proteome and identifies the protein targets.

It is quite notable from our results that a significant proportion of the HNE adduction to mitochondrial membrane components focuses around the enzymes that deliver electrons to the ubiquinone pool (Figs. 2, 4, and 5 and supplemental Tables S1–S3). Several complex I subunits were identified as HNE targets, along with the FAD containing the {alpha} subunit of succinate dehydrogenase and the electron transfer flavoprotein ubiquinone oxidoreductase recently identified in Arabidopsis for its role in delivering electrons to UQ from the products of the branched chain amino acid degradation pathway (37). We have previously shown that AOX, which accepts electrons from UQH2, is another target of HNE and is inactivated by the aldehyde (25).

It seems logical that these UQ-interacting enzymes might be sites of HNE modification by oxidative stress in vivo as superoxide production by the electron transport chain generally arises from semiquinones in the membrane (38, 39). Thus, localized endogenous HNE production in the membrane and modification of nearby UQ-interacting proteins are feasible. Although this is a reasonable working hypothesis for oxidative stresses generated within cells (Figs. 4 and 5 and supplemental Tables S2 and S3), it does not readily explain such localized damage when HNE is exogenously supplied to mitochondria in vitro (Fig. 2 and supplemental Table S1). Rather, this suggests a link between the UQ pool, or at least proteins with a redox potential that allows them to interact with UQ, and susceptibility to modification by HNE. Extensive studies of oxidative stress-induced changes in plant gene expression also show that induction of transcripts for the alternative external and internal NADH:UQ oxidoreductases and the UQH2:O2 alternative oxidase are the major transcription changes among all the genes for mitochondrial proteins (40, 41).

Thus it appears that a range of events are occurring during oxidative stress focused on these nonphosphorylating bypass enzymes. First, their synthesis is being induced; second, they are being damaged; and third, their catalytic action might help lower the ROS production via the mitochondrial electron transport chain. The latter point is clear for AOX as it can help avoid over-reduction of the UQ pool (19). The NADH:UQ oxidoreductases could lower ROS production through avoiding reverse electron flow from succinate dehydrogenase to complex I when the UQ and/or matrix nucleotide redox poise is altered. We currently do not have any data to suggest that these processes are linked by feed-back or feed-forward mechanisms. However, we can propose an hypothesis that links the observed events. If modification by HNE leads to loss of activity, increased oxidative side reactions, or increased turnover of proteins in oxidative phosphorylation complexes, then coordinated changes in gene expression for nonphosphorylating bypasses can be interpreted as a convenient way of increasing or maintaining respiratory flux through the UQ pool by provision of a short, readily assembled electron transport chain. In this context, the inhibition of AOX activity by HNE may appear contradictory, but this could trigger a signal to stimulate gene expression resulting in increased compensatory AOX synthesis as well as NAD(P)H dehydrogenase synthesis and the turn on of other stress-related genes.

HNE, which is likely to be present or produced in the membrane, also appears to modify soluble enzymes of the matrix that are likely to be in close proximity to the surface of the mitochondrial inner membrane. Following the work of others on HNE modification of lipoic acid cofactors in PDC and KGDC (6, 13), we have previously shown that the loss of lipoic acid inactivates these enzymes in potato, pea, and Arabidopsis mitochondria (24, 36). Here we show that by directly monitoring HNE adduction of soluble proteins, two differences in perspective are gained on HNE targets in the mitochondrial matrix. First, even within the known susceptible protein complexes of PDC and KGDC, it is not only the E2 dihydrolipoamide acyltransferases, which contain bound lipoic acid, that are targets. PDC E1 β subunit and the shared E3 lipoamide dehydrogenase subunit are clearly major HNE adduction targets within these same protein complexes. Defining the precise molecular mechanism of PDC and KGDC damage during oxidative stress will thus require a thorough analysis of each step in the catalysis of these enzyme complexes and not simply a focus on lipoic acid modification. Second, the susceptibility of matrix dehydrogenases extends beyond these classical lipoic acid-containing proteins to other major dehydrogenase classes (supplemental Tables S1–S3). Glyceraldehyde-3-phosphate dehydrogenase (supplemental Table S3) is not a matrix enzyme but a glycolytic enzyme known to be associated with the outer mitochondrial membrane (42) and to accumulate on mitochondria during oxidative stress (21). It has recently been highlighted as an oxidative stress-sensitive protein in plants (43), and its HNE adduction could be involved in this phenomenon.

Interestingly, in a number of cases, proteins that are found to be HNE targets are also identified by the HNE adduct antibodies as smaller stable degradation products (supplemental Tables S1 and S2). We have found some of these products previously in a quantitative analysis of changes in abundance of proteins in mitochondria during the same stresses in cell culture (21). This could be interpreted in a number of ways: first, a link may exist between HNE adduction and protein degradation; second, HNE adduction may be blocking degradation, leading to accumulation of intermediates that would otherwise be rapidly degraded. A case for HNE adduction limiting protein degradation has been made in mammals (44, 45), but a case for specific proteolysis of oxidatively damaged proteins in mitochondria has also been made (46).

It is not clear to what degree HNE adduction leads directly to protein dysfunction. Although we have been able to observe inhibition of AOX activity by HNE (25), we have not observed significant inhibition of CI or CII by HNE adduction or oxidative stress (Fig. 6), but we do see significant losses in whole chain electron transport activity (Fig. 3B). It is also not possible to determine the degree of HNE adduction of a protein from these data using anti-HNE adduct antibodies. If less than 20% of a protein is adducted under these conditions, it is unlikely even if the activity assay undertaken could measure the functional effect (for example on Vmax, substrate, cofactor, or product-binding constants) that a statistically significant change would be recorded given technical and biological variability in activity measurements. However, in combination with other oxidation damage, inefficiency in a number of steps likely underlies the clearly measured losses in total respiratory rates observed both in vivo and in isolated mitochondria with a variety of substrates (Fig. 3). The degree of adduction could best be measured by defining the peptide that is modified and assessing the abundance of the modified and unmodified peptides by mass spectrometry. However, we have failed to identify any HNE-modified peptides in our mass spectral analysis, despite extensive searches of spectra for parent peptides +138 or +156 Da and searches in MS/MS for the 139.1 dehydrated, protonated HNE marker ion (data not shown). To our knowledge, there are very few cases where the sites of HNE adduction of mitochondrial proteins by in vivo modifications have been proven by mass spectrometry. Almost all reports of HNE modification sites in mitochondria are from HNE addition to purified proteins or peptides followed by immediate analysis by MS or MS/MS (4, 47–50). This inability to identify HNE peptides might be due to the reactivity of the aldehyde group of HNE, which has the potential to cross-link peptides or cyclize the peptide during sample handling prior to analysis (51).

Taking a different approach to this issue, we have compared the protein list generated in this report of HNE adducted proteins together with those generated in other reports in plant mitochondria looking at carbonyl group formation (52), tryptophan oxidation (53), and accessible thiols for thioredoxin binding (54) with proteins known to be degraded during oxidative stress in plant mitochondria (21, 23). Remarkably, of the 31 different proteins in this report, 24 have been listed in one of these categories (supplemental Table S7). It is even possible that the carbonyl group protein lists generated by Kristensen et al. (52) may be, at least in part, a direct analysis of HNE adducts. HNE adduction to amino acids by Michael addition from the C-3 unsaturation is the most common method of HNE adduction (3), but when this occurs the adducted HNE retains the C-1 aldehyde group, thus effectively introducing a carbonyl group into the protein. These HNE-Michael adducts can react with (2,4-dinitrophenyl)hydrazine via the carbonyl group on the HNE adduct (50, 51, 55).

Considering supplemental Table S7 as a whole, it appears that a common element in selective oxidative damage and degradation is the surface availability of thiols, which allows oxidative modification and generation of carbonyl groups, followed by protein degradation. The exact path of this process is not known, and the role of HNE modification as an inhibitor of enzyme activity, as a tag for degradation, and/or an inhibitor of protein turnover is likely to be complex and could be protein-specific. However, it is clear that HNE modification is an active process in plant mitochondria under oxidative stress and that tools to detect and localize this process could be used more broadly in plants to gain insights into the mechanism of oxidative stress and the link between lipid peroxidation and protein damage in plants.


   FOOTNOTES
 
* This work was supported in part by grants from the Australian Research Council through the Centres of Excellence Program (to A. H. M. and D. A. D.). 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S7. Back

1 Recipient of an Australian postgraduate award. Back

2 Supported as Australian Research Council postdoctoral fellow. Back

3 Supported as Australian Research Council professorial fellow. To whom correspondence should be addressed. Tel.: 61-8-6488-7245; Fax: 61-8-6488-4401; E-mail: hmillar{at}cyllene.uwa.edu.au.

4 The abbreviations used are: ROS, reactive oxygen species; ANT, adenine nucleotide translocator; AOX, alternative oxidase; DCPIP, dichlorophenylindophenol; FeCN, [Fe(CN)6]3–; HNE, 4-hydroxy-2-nonenal; IEF, isoelectric focusing; KGDC, {alpha}-ketoglutarate dehydrogenase complex; n-PG, n-propyl gallate; PDC, pyruvate dehydrogenase complex; TEMED, N',N',N',N'-tetramethylethylenediamine; UQ, ubiquinone; UQH2, reduced ubiquinone; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CI, complex I; HAE, hydroxyalkenal; MS/MS, tandem mass spectrometry; LC, liquid chromatography; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; BN, blue native; NL, nonlinear. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Schneider, C., Tallman, K. A., Porter, N. A., and Brash, A. R. (2001) J. Biol. Chem. 276, 20831–20838[Abstract/Free Full Text]
  2. Moller, I. M., Jensen, P. E., and Hansson, A. (2007) Annu. Rev. Plant Biol. 58, 459–481[CrossRef][Medline] [Order article via Infotrieve]
  3. Esterbauer, H., Schaur, R. J., and Zollner, H. (1991) Free Radic. Biol. Med. 11, 81–128[CrossRef][Medline] [Order article via Infotrieve]
  4. Isom, A. L., Barnes, S., Wilson, L., Kirk, M., Coward, L., and Darley-Usmar, V. (2004) J. Am. Soc. Mass Spectrom. 15, 1136–1147[CrossRef][Medline] [Order article via Infotrieve]
  5. Sayre, L. M., Arora, P. K., Iyer, R. S., and Salomon, R. G. (1993) Chem. Res. Toxicol. 6, 19–22[CrossRef][Medline] [Order article via Infotrieve]
  6. Humphries, K. M., and Szweda, L. I. (1998) Biochemistry 37, 15835–15841[CrossRef][Medline] [Order article via Infotrieve]
  7. Chen, J., Henderson, G. I., and Freeman, G. L. (2001) J. Mol. Cell. Cardiol. 33, 1919–1927[CrossRef][Medline] [Order article via Infotrieve]
  8. Chen, J., Schenker, S., Frosto, T. A., and Henderson, G. I. (1998) Biochim. Biophys. Acta 1380, 336–344[Medline] [Order article via Infotrieve]
  9. Musatov, A., Carroll, C. A., Liu, Y. C., Henderson, G. I., Weintraub, S. T., and Robinson, N. C. (2002) Biochemistry 41, 8212–8220[CrossRef][Medline] [Order article via Infotrieve]
  10. Picklo, M. J., Amarnath, V., McIntyre, J. O., Graham, D. G., and Montine, T. J. (1999) J. Neurochem. 72, 1617–1624[CrossRef][Medline] [Order article via Infotrieve]
  11. Lashin, O. M., Szweda, P. A., Szweda, L. I., and Romani, A. M. P. (2006) Free Radic. Biol. Med. 40, 886–896[CrossRef][Medline] [Order article via Infotrieve]
  12. Choksi, K. B., Boylston, W. H., Rabek, J. P., Widger, W. R., and Papaconstantinou, J. (2004) Biochim. Biophys. Acta 1688, 95–101[Medline] [Order article via Infotrieve]
  13. Humphries, K. M., Yoo, Y., and Szweda, L. I. (1998) Biochemistry 37, 552–557[CrossRef][Medline] [Order article via Infotrieve]
  14. Moreau, R., Heath, S. H., Doneanu, C. E., Lindsay, J. G., and Hagen, T. M. (2003) Antioxid. Redox. Signal. 5, 517–527[CrossRef][Medline] [Order article via Infotrieve]
  15. Vieira, H. L., Belzacq, A. S., Haouzi, D., Bernassola, F., Cohen, I., Jacotot, E., Ferri, K. F., El Hamel, C., Bartle, L. M., Melino, G., Brenner, C., Goldmacher, V., and Kroemer, G. (2001) Oncogene 20, 4305–4316[CrossRef][Medline] [Order article via Infotrieve]
  16. Perluigi, M., Fai Poon, H., Hensley, K., Pierce, W. M., Klein, J. B., Calabrese, V., De Marco, C., and Butterfield, D. A. (2005) Free Radic. Biol. Med. 38, 960–968[CrossRef][Medline] [Order article via Infotrieve]
  17. Carbone, D. L., Doorn, J. A., Kiebler, Z., and Petersen, D. R. (2005) Chem. Res. Toxicol. 18, 1324–1331[CrossRef][Medline] [Order article via Infotrieve]
  18. Smith, A. M., Ratcliffe, R. G., and Sweetlove, L. J. (2004) J. Biol. Chem. 279, 51944–51952[Abstract/Free Full Text]
  19. Vanlerberghe, G. C., and McIntosh, L. (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 703–734[CrossRef]
  20. Chew, O., Whelan, J., and Millar, A. H. (2003) J. Biol. Chem. 278, 46869–46877[Abstract/Free Full Text]
  21. Sweetlove, L. J., Heazlewood, J. L., Herald, V., Holtzapffel, R., Day, D. A., Leaver, C. J., and Millar, A. H. (2002) Plant J. 32, 891–904[CrossRef][Medline] [Order article via Infotrieve]
  22. Finkemeier, I., Goodman, M., Lamkemeyer, P., Kandlbinder, A., Sweetlove, L. J., and Dietz, K.-J. (2005) J. Biol. Chem. 280, 12168–12180[Abstract/Free Full Text]
  23. Taylor, N. L., Heazlewood, J. L., Day, D. A., and Millar, A. H. (2005) Mol. Cell. Proteomics 4, 1122–1133[Abstract/Free Full Text]
  24. Millar, A. H., and Leaver, C. J. (2000) FEBS Lett. 481, 117–121[CrossRef][Medline] [Order article via Infotrieve]
  25. Winger, A. M., Millar, A. H., and Day, D. A. (2005) Biochem. J. 387, 865–870[CrossRef][Medline] [Order article via Infotrieve]
  26. Millar, A. H., Liddell, A., and Leaver, C. J. (2001) Methods Cell Biol. 65, 53–74[Medline] [Order article via Infotrieve]
  27. Laemmli, U. K. (1970) Nature 227, 680–685[CrossRef][Medline] [Order article via Infotrieve]
  28. Schagger, H., and von Jagow, G. (1991) Anal. Biochem. 199, 223–231[CrossRef][Medline] [Order article via Infotrieve]
  29. Singer, T. P., Oestreicher, G., and Hogue, P. (1973) Plant Physiol. 52, 616–621[Abstract/Free Full Text]
  30. Zerbetto, E., Vergani, L., and Dabbeni-Sala, F. (1997) Electrophoresis 18, 2059–2064[CrossRef][Medline] [Order article via Infotrieve]
  31. Menz, R. I., Griffith, M., Day, D. A., and Wiskich, J. T. (1992) Eur. J. Biochem. 208, 481–485[Medline] [Order article via Infotrieve]
  32. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350–4354[Abstract/Free Full Text]
  33. Baxter, C. J., Redestig, H., Schauer, N., Repsilber, D., Patil, K. R., Nielsen, J., Selbig, J., Liu, J., Fernie, A. R., and Sweetlove, L. J. (2007) Plant Physiol. 143, 312–325[Abstract/Free Full Text]
  34. Rasmusson, A. G., and Moller, I. M. (1991) Physiol. Plant. 83, 357–365[CrossRef]
  35. Sabar, M., Balk, J., and Leaver, C. J. (2005) Plant J. 44, 893–901[CrossRef][Medline] [Order article via Infotrieve]
  36. Taylor, N. L., Day, D. A., and Millar, A. H. (2002) J. Biol. Chem. 277, 42663–42668[Abstract/Free Full Text]
  37. Ishizaki, K., Larson, T. R., Schauer, N., Fernie, A. R., Graham, I. A., and Leaver, C. J. (2005) Plant Cell 17, 2587–2600[Abstract/Free Full Text]
  38. Raha, S., and Robinson, B. H. (2000) Trends Biochem. Sci. 25, 502–508[CrossRef][Medline] [Order article via Infotrieve]
  39. Moller, I. M. (2001) Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 561–591[CrossRef][Medline] [Order article via Infotrieve]
  40. Clifton, R., Lister, R., Parker, K., Sappl, P., Elhafez, D., Millar, A., Day, D., and Whelan, J. (2005) Plant Mol. Biol. 58, 193–212[CrossRef][Medline] [Order article via Infotrieve]
  41. Clifton, R., Millar, A. H., and Whelan, J. (2006) Biochim. Biophys. Acta 1757, 730–741[Medline] [Order article via Infotrieve]
  42. Giege, P., Heazlewood, J. L., Roessner-Tunali, U., Millar, A. H., Fernie, A. R., Leaver, C. J., and Sweetlove, L. J. (2003) Plant Cell 15, 2140–2151[Abstract/Free Full Text]
  43. Hancock, J. T., Henson, D., Nyirenda, M., Desikan, R., Harrison, J., Lewis, M., Hughes, J., and Neill, S. J. (2005) Plant Physiol. Biochem. 43, 828–835[CrossRef][Medline] [Order article via Infotrieve]
  44. Friguet, B., and Szweda, L. I. (1997) FEBS Lett. 405, 21–25[CrossRef][Medline] [Order article via Infotrieve]
  45. Friguet, B., Szweda, L. I., and Stadtman, E. R. (1994) Arch. Biochem. Biophys. 311, 168–173[CrossRef][Medline] [Order article via Infotrieve]
  46. Marcillat, O., Zhang, Y., Lin, S. W., and Davies, K. J. (1988) Biochem. J. 254, 677–683[Medline] [Order article via Infotrieve]
  47. Aldini, G., Dalle-Donne, I., Vistoli, G., Maffei Facino, R., and Carini, M. (2005) J. Mass Spectrom. 40, 946–954[CrossRef][Medline] [Order article via Infotrieve]
  48. Carbone, D. L., Doorn, J. A., Kiebler, Z., Ickes, B. R., and Petersen, D. R. (2005) J. Pharmacol. Exp. Ther. 315, 8–15[Abstract/Free Full Text]
  49. Aldini, G., Gamberoni, L., Orioli, M., Beretta, G., Regazzoni, L., Facino, R. M., and Carini, M. (2006) J. Mass Spectrom. 41, 1149–1161[CrossRef][Medline] [Order article via Infotrieve]
  50. Carini, M., Aldini, G., and Facino, R. M. (2004) Mass Spectrom. Rev. 23, 281–305[CrossRef][Medline] [Order article via Infotrieve]
  51. Fenaille, F., Guy, P. A., and Tabet, J. C. (2003) J. Am. Soc. Mass Spectrom. 14, 215–226[CrossRef][Medline] [Order article via Infotrieve]
  52. Kristensen, B. K., Askerlund, P., Bykova, N. V., Egsgaard, H., and Moller, I. M. (2004) Phytochemistry 65, 1839–1851[CrossRef][Medline] [Order article via Infotrieve]
  53. Moller, I. M., and Kristensen, B. K. (2006) Free Radic. Biol. Med. 40, 430–435[CrossRef][Medline] [Order article via Infotrieve]
  54. Balmer, Y., Vensel, W. H., Tanaka, C. K., Hurkman, W. J., Gelhaye, E., Rouhier, N., Jacquot, J. P., Manieri, W., Schurmann, P., Droux, M., and Buchanan, B. B. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2642–2647[Abstract/Free Full Text]
  55. Deighton, N., Magill, W. J., Bremner, D. H., and Benson, E. E. (1997) Free Radic. Res. 27, 255–265[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Plant CellHome page
L. Oge, G. Bourdais, J. Bove, B. Collet, B. Godin, F. Granier, J.-P. Boutin, D. Job, M. Jullien, and P. Grappin
Protein Repair L-Isoaspartyl Methyltransferase1 Is Involved in Both Seed Longevity and Germination Vigor in Arabidopsis
PLANT CELL, November 1, 2008; 20(11): 3022 - 3037.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/52/37436    most recent
M702385200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Winger, A. M.
Right arrow Articles by Millar, A. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Winger, A. M.
Right arrow Articles by Millar, A. H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement