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Originally published In Press as doi:10.1074/jbc.M301990200 on June 18, 2003

J. Biol. Chem., Vol. 278, Issue 37, 34757-34763, September 12, 2003
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Rapid Suppression of Mitochondrial Permeability Transition by Methylglyoxal

ROLE OF REVERSIBLE ARGININE MODIFICATION*

Oliver Speer {ddagger} §, Sarune Morkunaite-Haimi {ddagger}, Julius Liobikas {ddagger} , Marina Franck {ddagger}, Linn Hensbo {ddagger}, Matts D. Linder {ddagger} ||, Paavo K. J. Kinnunen {ddagger}, Theo Wallimann § and Ove Eriksson {ddagger} **

From the {ddagger}Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine/Biochemistry, P. O. Box 63, Haartmaninkatu 8, FIN-00014 University of Helsinki, Helsinki, Finland and the §Swiss Federal Institute of Technology, ETH-Zürich, Institute of Cell Biology, ETH-Hönggerberg, CH-8093 Zürich, Switzerland

Received for publication, February 25, 2003 , and in revised form, May 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Methylglyoxal (MG) (pyruvaldehyde) is a reactive carbonyl compound produced in glycolysis. MG can form covalent adducts on proteins resulting in advanced glycation end products that may alter protein function. Here we report that MG covalently modifies the mitochondrial permeability transition pore (PTP), a high conductance channel involved in the signal transduction of cell death processes. Incubation of isolated mitochondria with MG for a short period of time (5 min), followed by removal of excess free MG, prevented both ganglioside GD3- and Ca2+-induced PTP opening and the ensuing membrane depolarization, swelling, and cytochrome c release. Under these conditions MG did not significantly interfere with mitochondrial substrate transport, respiration, or oxidative phosphorylation. The suppression of permeability transition was reversible following extended incubation in MG-free medium. Of the 29 physiological carbonyl and dicarbonyl compounds tested only MG and its analogue glyoxal were able to specifically alter the behavior of the PTP. Using a set of arginine-containing peptides, we found that the major MG-derived arginine adduct formed, following a short time exposure to MG, was the 5-hydro-5-methylimidazol-4-one derivative. These findings demonstrate that MG rapidly modifies the PTP covalently and stabilizes the PTP in the closed conformation. This is probably due to the formation of an imidazolone adduct on an arginine residue involved in the control of PTP conformation (Linder, M. D., Morkunaite-Haimi, S., Kinnunen, P. J. K., Bernardi, P., and Eriksson, O. (2002) J. Biol. Chem. 277, 937–942). We deduce that the permeability transition constitutes a potentially important physiological target of MG.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Methylglyoxal (MG)1 is a reactive dicarbonyl compound that is formed during glucose metabolism. MG binds covalently to proteins resulting in the formation of advanced glycation end products (AGEs), which are involved in several pathological processes, including cellular proliferative disorders (1) and diabetes mellitus (2). Increased formation of AGEs owing to hyperglycemia is a key factor in the development of diabetic complications such as microvascular disease and atherosclerosis (2). Inhibition of glycation reactions slows the progression of diabetic vascular disease manifestations (3, 4).

During glucose metabolism triose phosphates may undergo either spontaneous (5) or enzyme-facilitated decomposition to yield MG (6, 7). The active site of triose phosphate isomerase harbors a flexible loop surrounding enzyme-bound reaction intermediate. This loop flickers continuously between open and closed states leading to a small but constant leakage of the unstable intermediate enediolate phosphate (8) that immediately undergoes phosphate elimination to yield MG. Triose phosphate isomerase is a very efficient catalyst that is present at high concentration in tissues (9, 10), and therefore significant amounts of MG and MG-modified proteins may be produced (11).

At physiological concentrations MG primarily targets the arginine residues of proteins (12), resulting initially in the formation of reversible adducts. These adducts may consequently undergo a series of rearrangements that yield several possible end-products that contain either imidazolone- or pyrimidine-based ring systems (13–15). MG is known to target several proteins involved in the regulation of cell growth and differentiation (16–19), although the coupling between MG-induced alterations and subsequent cellular effects remains incompletely understood.

Mitochondria play an important role in programmed cell death by releasing proteins from the intermembrane compartment, where they are normally confined, to cytosol and nucleus (20). These proteins include cytochrome c and Smac/DIABLO, which activate caspases, endonuclease G that induces DNA fragmentation, and apoptosis-inducing factor, which promotes caspase-independent chromatin condensation and DNA fragmentation (21). The release of these pro-apoptotic proteins may be triggered by mitochondrial permeability transition (22). This event is induced by upstream apoptotic signals such as formation of the ganglioside GD3 (23–25) and mitochondrial Ca2+ uptake (22). Permeability transition is due to opening of the permeability transition pore (PTP) (22), which under normal cell life remains closed (26). In the open conformation the PTP permits free diffusion of solutes with a molecular mass of <1.5 kDa across the mitochondrial membrane. Permeability transition leads to mitochondrial depolarization and equalization of matrix and cytosolic ion and metabolite concentrations. Concomitant osmotic swelling of the mitochondrial matrix may lead to rupture of the outer membrane and release of proteins from the intermembrane compartment. According to the prevalent model, permeability transition pores are composed of the adenine nucleotide translocator, the voltage-dependent anion channel, and mitochondrial matrix cyclophilin (27, 28).

Ca2+ uptake does not elicit permeability transition in isolated mitochondria after treatment with the synthetic dicarbonyl compounds phenylglyoxal (PGO) or 2,3-butanedione (BAD) (29–32). These compounds react specifically with the guanidino group of arginine, indicating that modification of an arginine residue by PGO or BAD stabilizes the PTP in its closed conformation. Interestingly, modification with the PGO analogue OH-PGO results in an arginine adduct that induces the open conformation of the PTP (32). The profound effect of arginine modification demonstrates that structural rearrangements of an arginine residue are of key importance for the control of the PTP conformation, although the location of that arginine residue is still unclear. The possibility that physiological PTP regulators act via that site prompted us to investigate whether the natural dicarbonyl compound MG was capable of inducing changes in the function of the PTP.

In this study we have identified the mitochondrial PTP as a novel target of MG. Brief incubation of isolated rat liver mitochondria with MG led to complete suppression of both ganglioside GD3- and Ca2+-induced permeability transition. This also induced inhibition of cytochrome c release. Suppression of permeability transition by MG could be reversed by extended incubation in MG-free media. These findings demonstrate that MG induced a reversible covalent PTP modification, most likely an imidazolone derivative on an arginine residue involved in the control of the PTP conformation (29–32). The results of this study raise the possibility that MG reacts with the PTP under physiological conditions and that the altered PTP regulation perturbs the cell death program.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemical Modification of Mitochondria—Preparation of liver mitochondria from male Wistar rats was performed as described previously (30). Mitochondria (1 mg of protein/ml) were preincubated in modification medium containing 250 mM sucrose, 100 µM EGTA, 10 mM Hepes-KOH, pH 8.0, with or without MG for 5 min at 34 °C. The modification reaction was terminated by adjusting the pH to 6.8 with Hepes, cooling to 4 °C, and mitochondria were sedimented by centrifuging at 8000 x g for 5 min. Mitochondria were resuspended at a concentration of ~50 mg of protein/ml in 250 mM sucrose, 100 µM EGTA, and 10 mM Hepes-KOH, pH 7.4. Unless otherwise stated experiments were carried out at room temperature in assay medium containing 125 mM KCl, 5 mM succinate, 5 mM Pi-Tris, 2 mM Mg2+, 5 µM EGTA, 2 µM rotenone, 10 mM Hepes-Tris, pH 7.4. In one experimental series MG was substituted by PGO. Oxygen consumption was measured using a Clark electrode (Yellow Springs Instruments).

PTP Assays—Permeability transition was assayed by measuring membrane potential, Ca2+ transport and swelling. Medium [Ca2+] was measured using the fluorescent indicator dye Fluo-4FF (ex, 494 nm; em, 516 nm). Membrane potential was measured by TMRM fluorescence (ex, 550 nm; em, 575 nm), and swelling was monitored as a decrease in mitochondrial light scattering at 540 nm. Measurements were performed using a 96-well Tecan Spectrafluoplus plate reader or a PerkinElmer Life Sciences luminescence spectrometer LS50B. In swelling experiments, measured light scattering (I) was normalized by setting the initial scattering (I0) of the mitochondrial suspension to one unit. Permeability transition was quantified using the initial swelling rate following addition of Ca2+. The initial swelling rate for mitochondria preincubated in the absence of MG was set to 100% permeability transition. The initial swelling rate for mitochondria preincubated in the absence of MG but supplemented with 1 µM CsA was set to 0% permeability transition. GD3 was dissolved to 5 mM in water and sonicated in a water bath for 5 min before use. To test compounds containing the carbonyl group for effects on the PTP, mitochondria were incubated at 1 mg of protein/ml for 15 min at room temperature in modification medium supplemented with the compound of interest at the following concentrations: 10, 30, 100, and 300 nM, 1, 3, 10, 30, 100, and 330 µM, and 1, 3, and 10 mM. Mitochondria were diluted five times by addition of assay medium, and the effect of the compounds on the PTP was assessed following addition of Ca2+. Permeability transition was quantified as rate of swelling.

Cytochrome c Release—Release of cytochrome c was determined by immunoblotting. The mitochondrial suspension was removed from the photometer cuvette, cooled to +4 °C degrees and centrifugated for 3 min at 21,000 x g. The pellet and the supernatant were separated, and proteins were precipitated by addition of 10% trichloroacetic acid. Precipitated proteins were separated by SDS-PAGE using 12% gels and electrotransferred to a polyvinylidene fluoride membrane. Immunoblotting was performed using a monoclonal cytochrome c antibody (Zymed Laboratories) and visualized using the ECL system (Amersham Biosciences).

Mass Spectrometry—For MALDI-TOF mass spectrometry of MG-derived arginine adducts 100 µM of test peptide (NRVYIHPFHL, RVYVHPF, pEWPRQIPP, or YGGFMRF) was allowed to react with 2 mM MG in 10 mM Hepes-KOH, 100 µM EGTA, pH 8.0, at 34 °C. The reaction was stopped by adding 0.1% trifluoroacetic acid and cooling to 4 °C. The reaction mixture was desalted using a Zip Tip C18 silica bead microcolumn (Millipore). Peptides were eluted with acetonitrile/0.1% trifluoroacetic acid 1:2 and mixed with an equal volume of saturated {alpha}-HCA in the same solvent. Half a microliter of this solution was applied on the target. MALDI-TOF mass spectra were recorded on a Bruker Autoflex spectrometer using the linear detector in positive mode. Calibration of the machine was performed using the peaks of {alpha}-HCA and peptides of known masses. The rate constant value for formation of MG-derived arginine adducts was estimated from the relative peak intensity of native peptide and its MG derivatives after reaction for 1 min. For mass analysis of the GD3 ganglioside 0.5 nmol was mixed with 1 µl of 1 mM 2,4,6-trihydroxyacetophenone in acetonitrile/20 mM ammonium citrate (1:1) and applied on the target. Mass spectra were recorded using the linear detector in negative mode.

Electron Microscopy—Fixation of mitochondria was performed by adding 1% glutaraldehyde directly to the suspension. Embedding, sectioning, and staining were performed as described previously (30). Sections were viewed in a Jeol 1200 transmission electron microscope at a magnification of x10,000.

Chemicals—CsA was a gift from Novartis, OH-PGO was from Pierce, 3-deoxyglucosone was from Toronto Research Chemicals, TMRM and Fluo-4FF were from Molecular Probes, and ganglioside GD3 was from Calbiochem. Analysis of GD3 by MALDI-TOF demonstrated that the C-2 N-fatty acyl-sphingosine moiety was a C18 fatty acid in 9%, C19 in 15%, C20 in 25%, C21 in 31%, and C22 in 20%. Other chemicals were from Sigma. 1{Delta}-Pyrroline 5-carboxylate was regenerated from its 2,4-dinitrophenylhydrazone derivative as described previously (33).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We first studied the effect of MG on permeability transition induced either by the ganglioside GD3 or Ca2+. Mitochondria were preincubated for 5 min in the presence or absence of 2 mM MG followed by removal of free MG by centrifugation. These mitochondria were then incubated for 30 min in assay medium containing 25 µM GD3 or 10 µM Ca2+, whereupon mitochondria were processed for ultrastructural analysis by transmission electron microscopy and for analysis of cytochrome c release by immunoblotting. The results, shown in Fig. 1, demonstrated that both GD3 and Ca2+ caused extensive swelling and cytochrome c release in mitochondria preincubated in the absence of MG. Supplementing the medium with CsA, a selective high affinity inhibitor of permeability transition, prevented mitochondrial swelling and cytochrome c release, showing that both effects were due to permeability transition. Likewise, preincubation of mitochondria with MG completely prevented GD3- and Ca2+-induced swelling and cytochrome c release. These findings demonstrated that brief MG treatment effectively suppresses mitochondrial permeability transition.



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  		FIG. 1.
Prevention of mitochondrial swelling and cytochrome c release by MG. Mitochondria were preincubated for 5 min with or without 2 mM MG. Mitochondria were then sedimented by centrifugation and resuspended in assay medium at a concentration of 0.2 mg of protein/ml. The mitochondrial suspensions were supplemented with the following compounds: 25 µM GD3, 25 µM GD3 plus 1 µM CsA, 10 µM Ca2+, or 10 µM Ca2+ plus 1 µM CsA. The suspensions were incubated at room temperature for 30 min, whereupon mitochondria were sedimented by centrifugation. Aliquots of the supernatant and pellets were taken for SDS-PAGE and immunoblotting of cytochrome c. S and P indicate cytochrome c detected in the supernatant and pellet, respectively. Chemical fixation of mitochondria was performed using 1% glutaraldehyde followed by embedding, thin-sectioning, and staining. Sections were viewed at a magnification of x10,000. The scale bar is 1 µm.

 

The onset of permeability transition is dependent on Ca2+ uptake and production of {Delta}{psi} by substrate oxidation under the conditions used in Fig. 1. We verified that MG acted directly on the PTP, and not via these factors, investigating the effect of MG treatment on {Delta}{psi} and Ca2+ transport. The membrane potential-sensitive dye TMRM was used to measure {Delta}{psi}. Ca2+ uptake was assessed by measuring medium [Ca2+] using the membrane impermeable dye fluo-4FF, which becomes fluorescent upon Ca2+ binding. Mitochondrial swelling was monitored as a decrease in light scattering. First we studied the effect of GD3 on {Delta}{psi} and swelling (Fig. 2). Mitochondria preincubated in the presence or absence of MG were suspended in assay medium, which was supplemented with 5 mM succinate after 2 min. This addition led to a rapid accumulation of TMRM, regardless of whether MG had been present or not during preincubation, showing that MG did not interfere with the production of {Delta}{psi} by substrate oxidation (Fig. 2A). However, in mitochondria preincubated in the absence of MG the addition of 25 µM GD3 caused a release of TMRM, indicating a drop in {Delta}{psi} (trace a). Addition of GD3 also induced a decrease in light scattering indicating that these mitochondria underwent swelling (Fig. 2B, trace a). Supplementing the medium of these mitochondria with CsA (trace b) or using MG-treated mitochondria (trace c) prevented both the GD3-induced drop in {Delta}{psi} and swelling.



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  		FIG. 2.
Suppression of GD3-induced permeability transition by MG. Mitochondria were preincubated, centrifuged and resuspended as in Fig. 1. Succinate (5 mM) was added to the suspension after 2 min followed by GD3 ganglioside (25 µM) after 5 min. Panel A, depicts membrane potential measured using the fluorescent dye TMRM. Panel B, depicts swelling measured as light scattering at 540 nm. The initial intensity of the scattering was set to one unit. Traces a, b and d show mitochondria preincubated in the absence of MG and trace c shows MG-treated mitochondria. The suspension was supplemented with 1 µM CsA in trace b. In trace d GD3 was not added.

 

We then proceeded to study {Delta}{psi}, Ca2+ transport and swelling following Ca2+ addition (Fig. 3). Mitochondria were suspended in assay medium, and 10 µM Ca2+ was added after 2 min. The increase in medium [Ca2+] resulted in an increase in fluo-4FF fluorescence (panel C). Three minutes later mitochondria were energized by the addition of 5 mM succinate. In mitochondria preincubated without MG, this addition caused rapid swelling as indicated by the decrease in light scattering (panel B, trace a). Consistently, these mitochondria failed to produce {Delta}{psi} in the presence of Ca2+ as indicated by the lack of both TMRM and Ca2+ accumulation (panels A and C, trace a). However, supplementing the medium of these mitochondria with CsA prevented swelling, and the mitochondria were able both to maintain {Delta}{psi} (panel A, trace b) and take up Ca2+ (panel C, trace b). As expected, succinate addition to MG-treated mitochondria resulted in an immediate production of {Delta}{psi} (panel A, trace c), rapid Ca2+ uptake (panel C, trace c), and prevention of swelling. These results indicate that MG acts directly on the PTP and not on {Delta}{psi} production or Ca2+ uptake.



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  		FIG. 3.
Suppression of Ca2+-induced permeability transition by MG. Mitochondria were preincubated, centrifuged, and resuspended as in Fig. 1. Ca2+ (10 µM) was added after 2 min followed by succinate (5 mM) after 5 min. A, membrane potential as measured in Fig. 2. B, swelling as measured in Fig. 2. C, the Ca2+ concentration of the medium measured using the fluorescent dye Fluo-4FF. Traces a and b show mitochondria preincubated in the absence of MG, and trace c shows MG-treated mitochondria. The suspension was supplemented with 1 µM CsA in trace b.

 

This finding was further corroborated by measurements of respiration rate and {Delta}{psi} production by ATP hydrolysis (Fig. 4). The results indicated that mitochondria retained their maximal respiration rates in the presence of ADP or the protonophoric uncoupler FCCP following preincubation with up to 2 mM MG (left panel). Similarly, preincubation with 2 mM MG had no effect on {Delta}{psi} production by ATP hydrolysis as measured by the uptake of TMRM indicating that the function of the adenine nucleotide translocator and the ATPase was not affected by MG (right panel).



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  		FIG. 4.
Effect of MG on mitochondrial respiration and production of {Delta}{psi} by ATP hydrolysis. The left panel depicts mitochondrial oxygen consumption measured using a Clark electrode. Mitochondria were preincubated with the indicated concentrations of MG followed by centrifugation and resuspension at a concentration of 0.2 mg of protein/ml in assay medium. The suspension was added to the electrode chamber, and succinate (5 mM) was added immediately followed by ADP (200 µM) after 3 min and FCCP (1 µM) after 6 min. The oxygen consumption was calculated from the decrease in medium oxygen concentration following the addition of succinate, ADP, and FCCP, respectively. Values are expressed as mean ± S.E. obtained for three mitochondrial preparations. The right panel shows membrane potential as measured in Fig. 2. ATP (1 mM) was added to energize mitochondria after 3 min. Dotted trace, mitochondria treated with 2 mM MG. Solid trace, mitochondria preincubated in the absence of MG.

 

In these initial experiments we used a supraphysiological MG concentration. To investigate the physiological significance of our findings, it was necessary to determine the quantitative relationship between MG concentration and suppression of permeability transition. Therefore we preincubated mitochondria with varying concentrations of MG followed by permeability transition using Ca2+ as the triggering signal. Permeability transition was quantified using the initial swelling rates, which were plotted against the preincubation concentrations of MG (Fig. 5). The plot indicates that the apparent K50 for PTP inhibition is 600 µM and that MG had already a significant effect at 250 µM.



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  		FIG. 5.
Concentration dependence and reversibility of MG-induced suppression of permeability transition. Mitochondria were incubated with the indicated concentrations of MG followed by centrifugation and resuspension at a concentration of 0.2 mg of protein/ml in assay medium. Permeability transition was assayed exactly as in Fig. 3B. Swelling was quantified using the initial decrease in light scattering as described under "Experimental Procedures." Inset, mitochondria preincubated either with 2 mM MG (circles) or 2 mM PGO (triangles) were suspended in assay medium at a concentration of 0.2 mg of protein/ml at room temperature for the indicated time. Permeability transition was quantified as in the main figure. Values are expressed as mean ± S.E. obtained for three mitochondrial preparations.

 

Because MG reacts with proteins, resulting in both reversible and irreversible adducts, it was of interest to determine whether the effect of MG was reversible or not. To address this question we preincubated mitochondria with 2 mM MG or 2 mM PGO, which is known to form an irreversible adduct (30). Following the modification reaction and removal of free MG or PGO, mitochondria were incubated in assay medium at room temperature for up to 3 h whereupon permeability transition was measured by swelling. The results shown in Fig. 5 (inset) indicated that suppression of permeability transition by MG was transient and disappeared after 2 h at room temperature. As shown previously the effect of PGO was irreversible (30).

We then proceeded to investigate whether any physiological carbonyl compound other than MG was able to suppress permeability transition. We selected candidate carbonyl compounds from the main pathways of carbohydrate, amino acid, and fatty acid metabolism and some aliphatic aldehydes formed in the metabolism of alcohols. Mitochondria were incubated with each compound at concentrations of up to 10 mM in modification medium, whereupon permeability transition was assayed by swelling, using Ca2+ as the triggering signal. The results are presented in Fig. 6 and Table I. We first tested the {alpha}-oxoaldehydes glyoxal and 3-deoxyglucosone, both of which are implicated in the formation of AGEs. Data showed that, whereas glyoxal effectively suppressed permeability transition at an apparent K50 of 2 mM, 3-deoxyglucosone was completely without effect under the same conditions. CsA inhibited permeability transition in the submicromolar concentration range. For comparison we also used the highly toxic cross-linker glutaraldehyde, which was effective in the submillimolar concentration range. Other carbonyls were analyzed in a similar way, plots were constructed as in Fig. 6, and the results of these titrations are presented in Table I. The data indicate that none of the other compounds specifically altered the behavior of the PTP, despite the high chemical reactivity of several of them.



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  		FIG. 6.
Effect of compounds containing the carbonyl group on permeability transition. Mitochondria were preincubated for 15 min with the indicated concentrations of glutaraldehyde (triangles), glyoxal (circles), or CsA (squares) as control. Permeability transition was assayed and quantified as in Fig. 5.

 

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  		TABLE I
Effect of compounds containing the carbonyl group on permeability transition

Mitochondria were incubated for 15 min at room temperature with the listed compounds at concentrations up to 10 mM. Permeability transition was assayed and quantified as described in Fig. 5.

 

In general, formation of significant amounts of MG-derived arginine adducts on proteins requires hours or even days (12, 13, 34). In contrast, our findings showed that complete suppression of permeability transition by MG required a reaction time of only a few minutes. Therefore, it was of interest to clarify the nature of the MG adduct formed after the short incubation time used in this study. We analyzed the products formed in the reaction of 2 mM MG with the following arginine-containing peptides: NRVYIHPFHL (A), RVYVHPF (B), pEWPRQIPP (C), and YGGFMRF (D). The crude reaction mixture was subjected to desalting in C18 reversed-phase Zip tips followed by MALDI-TOF mass spectrometry analysis. After incubation with MG for 1 h, no adduct could be detected on peptides A and B, but prominent additional peaks indicating an increase in molecular mass of 54 could be detected on peptides C and D. Peptide D was selected for studying the time dependence of the reaction, because it was the most reactive. In the absence of MG the native peptide D gave rise to a single peak at m/z 877.4 (M+H+) (Fig. 7). After incubation for 1 min additional peaks appeared at m/z 931.7 corresponding to the 5-hydro-5-methylimidazol-4-one derivative, and at m/z 913.7 corresponding to the 5-methylimidazol-4-one derivative. Peaks corresponding to the expected molecular weight of pyrimidine- and tetrahydropyrimidine derivatives could not be detected. These findings demonstrate that the reaction between MG and peptidyl-arginine can proceed to completion within minutes and suggest that the predominating adduct formed under these conditions is the 5-hydro-5-methylimidazol-4-one derivative.



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  		FIG. 7.
Time dependence of the reaction between MG and the test peptide YGGFMRF. The peptide (100 µM) was incubated with 2 mM MG for the indicated time followed by desalting and analysis by MALDI-TOF mass spectrometry using the linear detector in positive mode. Peaks corresponding to the native peptide (m/z 877.4) are indicated with asterisks. Peaks of the peptide with 5-hydro-5-methylimidazol-4-one derivative (m/z 931.7) are marked with plus signs, and peaks of the 5-methylimidazol-4-one derivative (m/z 913.7) are marked with arrowheads.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have characterized a novel target of MG: the mitochondrial permeability transition pore. We have demonstrated that incubation of isolated mitochondria with MG in the millimolar concentration range for a short time was sufficient to completely suppress permeability transition. Under these conditions MG had no significant side effects on mitochondrial substrate transport, respiration, or oxidative phosphorylation demonstrating that MG acted selectively on the permeability transition. The suppression of permeability transition persisted following removal of free MG indicating that MG had formed a covalent ligand on the PTP or on an associated regulatory protein. Nevertheless, extended incubation in the absence of MG reversed the effect of MG on permeability transition. Because MG reacts almost exclusively with arginine residues under these conditions (12, 13), we suggest that MG targets the same arginine residue(s) as the synthetic reagents PGO, BAD, and OH-PGO (29–32).

In these experiments we have used the ganglioside GD3 or Ca2+ as signals to induce permeability transition. In several cell types the ganglioside GD3 is formed as a consequence of tumor necrosis factor-{alpha} or Fas receptor activation (35) and functions as an apoptosis mediator by transferring from the plasma membrane to mitochondria (36), where it induces permeability transition (23–25). Influx of Ca2+ through the plasma membrane leading to extensive mitochondrial Ca2+ accumulation and subsequent permeability transition is of key importance in neuronal death (37). In all likelihood, mobilization of either GD3 or Ca2+ during cell death processes is essential for induction of mitochondrial permeability transition. Because MG suppressed both GD3- and Ca2+-induced permeability transition, we conclude that the effect of MG is robust and not restricted to narrowly defined conditions.

To approach the question of whether MG can regulate the permeability transition in vivo we devised an experimental approach based on the predicted behavior of MG in the cell. In the intracellular milieu, production of MG is limited, and therefore several factors contribute to determine on which proteins MG-derived adducts are formed. The net production of MG is undoubtedly the most important factor influencing the total number of proteins modified by MG However, under conditions of limiting MG production the selection of individual targets is not a fortuitous event but is determined by the relative target reactivity, which in turn depends on the local chemical environments. If free and reversibly bound MG are in dynamic equilibrium (11), the extent of modification of different targets is expected to be proportional to their respective equilibrium constants. We therefore characterized the modification reaction regarding its concentration dependence, reversibility, and selectivity for the permeability transition. We also investigated whether physiological carbonyl compounds other than MG could modify the permeability transition.

Concentration dependence measurements showed that a significant suppression of permeability transition by MG could be observed already after 5 min of incubation at 250 µM. This concentration of free MG is in excess of that detected in living systems. However, in cells the MG production goes on more or less constantly leading to an equilibrium between free and protein-bound MG, which is shifted far in the direction toward the bound form. Consistent with this, the total concentration of MG in cells can be as high as 310 µM (11). Accordingly, MG-induced suppression of permeability transition was reversible, indicating that the MG adduct slowly decomposes and restores the PTP to its native state, suggesting that the level of modification of the PTP is proportional to the prevailing MG concentration. Furthermore, MG did not affect other mitochondrial functions suggesting that the arginine of interest is exceptionally reactive and hence that MG targets primarily the permeability transition.

A functional significance is also suggested by the observation that MG and its structural analogue glyoxal were the only physiological carbonyl compounds, among a large number of related compounds tested that selectively suppressed the permeability transition. Most notably, the reactive {alpha}-oxoaldehyde 3-deoxyglucosone, which is an important mediator of the formation of AGEs (38), completely lacked effect. Our results suggest that MG and glyoxal are the only physiological carbonyl compounds that form covalent ligands on the PTP. Because the reaction between MG and the PTP was fast, reversible, selective, and specific, this suggests the possibility that MG regulates the permeability transition in vivo.

The PTP can also be modified by the synthetic MG analogues PGO and OH-PGO (30–32). The resulting uncharged PGO adduct strongly suppresses permeability transition, whereas the negatively charged OH-PGO adduct promotes permeability transition (32). This led us to propose that the effect of arginine modification on the PTP conformation is due to the electrical charge of the resulting adduct. To test this hypothesis we investigated the reaction product of MG and a set of arginine-containing test peptides under the same conditions as were used for incubating mitochondria. The molecular mass of the detected compounds indicated that the major products formed during the first minutes of the reaction were imidazolone derivatives. This finding is consistent with our hypothesis that uncharged adducts stabilize the closed conformation of the PTP. The results also demonstrated that the reactivity of the arginine-peptides with MG varied largely, probably owing to the local chemical environment of the respective arginine residues. The rate constant value for the formation of the 5-hydro-5-methylimidazol-4-one derivatives from the most reactive test peptide and MG was 1.5 M–1 s–1. This value is 100–1000-fold greater than that previously reported for the formation of the same derivative from N-acetylarginine and MG (13). These findings demonstrate that modification of arginine residues by MG can proceed at an unprecedented speed.

The major intracellular precursors of MG are the glycolysis intermediates dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (5–8). The former compound takes part in the glycerolphosphate shuttle, in which cytosolic NADH reduces dihydroxyacetone phosphate to glycerol 3-phosphate, which is then reoxidized by glycerolphosphate dehydrogenase of the inner membrane. Owing to this design the local concentration of triose phosphates is presumably elevated in the vicinity of mitochondria. The PTP thus constitutes a nearby target for MG formed from triose phosphates. Consequently, we propose that MG may react with the PTP in living organisms, leading to MG-induced suppression of permeability transition and alterations of the regulation of the mitochondrial apoptosis pathway. This mechanism may be important under conditions of enhanced flux through glycolysis where the rate of MG production is increased over the normal level. The flux through glycolysis is increased not only during diabetic hyperglycemia (39) but also in many malignant tumors (40).

The findings of this and other recent studies (16–19) suggest that MG-induced modification of specific target proteins may play a role in cell signaling inducing specific alterations in cell behavior, as proposed previously (41). It is conceivable that these mechanisms operate at low MG concentration below the threshold of unspecific toxicity. The question of how specific MG-induced protein modifications are integrated in the regulation of cell signaling pathways should be the subject of future studies.

Addendum—During review of this paper, we became aware of a study on the effect of MG on mitochondrial respiration in cardiac cells (Roy, S. S., Biswas, S., Ray, M., and Ray, S. (2003) Biochem. J. 372, 661–669).


   FOOTNOTES
 
* This work was supported by grants from the University of Helsinki Research Funds, Sigrid Juselius Foundation, Finska Läkaresällskapet, and Perklén Memorial Foundation, by the Swiss Foundation for the Research on Muscle Diseases (to O. S. and T. W.), and by Swiss National Science Foundation NSF Grant 31-62024.00 (to T. W.). 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

On leave from the Institute for Biomedical Research, Kaunas University of Medicine, LT-3007 Kaunas, Lithuania. Back

|| Current address: Dept. of Molecular Medicine, National Public Health Institute, Helsinki, Finland. Back

** To whom correspondence should be addressed. Tel.: 358-9-191-25-405; Fax: 358-9-191-25-444; E-mail: ove.eriksson{at}helsinki.fi.

1 The abbreviations used are: MG, methylglyoxal; AGEs, advanced glycation end products; {alpha}-HCA, {alpha}-cyano-4-hydroxycinnamic acid; BAD, 2,3-butanedione; CsA, cyclosporin A; {Delta}{Psi}, mitochondrial transmembrane electrical potential difference; GD3, 1-O-[O-(N-acetyl-{alpha}-neuraminosyl)-(2->8)-O-(N-acetyl-{alpha}-neuraminosyl)-(2->3)-O-{beta}-D-galactopyranosyl-(1->4)-{beta}-D-glucopyranosyl]-ceramide; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; OH-PGO, p-hydroxyphenylglyoxal; PGO, phenylglyoxal; PTP, permeability transition pore; RFI, relative fluorescence intensity; TFA, trifluoroacetic acid; TMRM, tetramethylrhodamine methyl ester; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Paolo Bernardi for critical reading of the manuscript and Kaija Niva for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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