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 Ca 2؉ -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.

Methylglyoxal and synthetic glyoxal derivatives react covalently with arginine residue(s) on the mitochondrial permeability transition pore (PTP). In this study, we have investigated how the binding of a panel of synthetic phenylglyoxal derivatives influences the opening and closing of the PTP. Using both isolated mitochondria and mammalian cells, we demonstrate that the resulting arginine-phenylglyoxal adduct can lead to either suppression or induction of permeability transition, depending on the net charge and hydrogen bonding capacity of the adduct. We report that phenylglyoxal derivatives that possess a net negative charge and/or are capable of forming hydrogen bonds induced permeability transition. Derivatives that were overall electroneutral and cannot form hydrogen bonds suppressed permeability transition. When mammalian cells were incubated with low concentrations of negatively charged phenylglyoxal derivatives, the addition of oligomycin caused a depolarization of the mitochondrial membrane potential. This depolarization was completely blocked by cyclosporin A, a PTP opening inhibitor, indicating that the depolarization was due to PTP opening. Collectively, these findings highlight that the target arginine(s) is functionally linked with the opening/closing mechanism of the PTP and that the electric charge and hydrogen bonding of the resulting arginine adduct influences the conformation of the PTP. These results are consistent with a model where the target arginine plays a role as a voltage sensor.

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