Modification of Permeability Transition Pore Arginine(s) by Phenylglyoxal Derivatives in Isolated Mitochondria and Mammalian Cells

Abstract

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.

Antimelanoma Activity of Apoptogenic Carbonyl Scavengers

“…The apoptogenicity of carbonyl scavengers on human melanoma cell lines suggests that covalent trapping of RCS may be part of the apoptogenic mechanism of action observed with these agents. Recently, MG modulation of regulatory mechanisms of apoptosis, such as MPT pore opening and Hsp 27 oligomerization, have been demonstrated as part of an emerging cancer cell survival pathway that interferes with apoptosis by glycolytic formation of RCS (Speer et al, 2003;Johans et al, 2005;Wondrak et al, 2005). The possibility of a functional antagonism between carbonyl scavengers and RCS-mediated cell survival was further examined by inducing carbonyl scavenger-triggered apoptosis after external addition of the RCS phenylglyoxal (PG) as summarized in Fig.…”

“…MG modification of the MPT pore complex leads to suppression of pore opening and mitochondrial induction of apoptosis (Speer et al, 2003;Johans et al, 2005), and MG modification of Hsp 27 enhances its interaction with cytochrome c, preventing caspase activation (Bruey et al, 2000;Sakamoto et al, 2002). Evidence for covalent MG modification of an adenine nucleotide translocator-bound arginine residue leading to an unknown adduct in isolated mitochondria and live cells has been presented (Speer et al, 2003).…”

“…In addition to the established role of AGE-receptor for advanced glycation end product signaling in many human malignancies, increasing evidence supports the hypothesis that RCS, originating from increased tumor cell glycolysis and mitochondrial oxidative stress, are small molecule antiapoptotic effectors (Sakamoto et al, 2002;Speer et al, 2003;Johans et al, 2005). The ␣-dicarbonyl MG forms from glycolytic triose phosphates by spontaneous phosphate elimina-tion (Thornalley, 1995), and intracellular MG levels are elevated under conditions of increased glycolytic flux, such as hyperglycemia (Shinohara et al, 1998) and aerobic glycolysis associated with malignant transformation (Kawase et al, 1996).…”

“…MG is a potent glycating agent leading to the posttranslational modification of protein-arginine and lysine residues with formation of AGEs, such as arginine-pyrimidine (Shipanova et al, 1997), N-carboxyethyllysine (Ahmed et al, 1997), and the hydroimidazolone N ␦ -(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine (Thornalley, 2005). Recent evidence suggests that covalent modification of arginine residues by MG targets specific proteins involved in apoptosis, such as mitochondrial permeability transition (MPT) pore proteins (Johans et al, 2005) and heat shock protein 27 (Hsp 27) (Sakamoto et al, 2002). MG modification of MPT pore proteins interferes with pore opening, inhibiting mitochondrial swelling, loss of transmembrane potential, and subsequent release of proapoptotic factors such as cytochrome c and apoptosis-inducing factor in response to apoptotic stimuli such as high Ca 2ϩ and ganglioside GD3 (Speer et al, 2003;Johans et al, 2005).…”

“…Recent evidence suggests that covalent modification of arginine residues by MG targets specific proteins involved in apoptosis, such as mitochondrial permeability transition (MPT) pore proteins (Johans et al, 2005) and heat shock protein 27 (Hsp 27) (Sakamoto et al, 2002). MG modification of MPT pore proteins interferes with pore opening, inhibiting mitochondrial swelling, loss of transmembrane potential, and subsequent release of proapoptotic factors such as cytochrome c and apoptosis-inducing factor in response to apoptotic stimuli such as high Ca 2ϩ and ganglioside GD3 (Speer et al, 2003;Johans et al, 2005). In various human cancer cell lines, post-translational MG-mediated arginine-pyrimidine formation occurs at a single arginine residue of Hsp 27 with induction of Hsp 27 oligomerization essential for repression of cytochrome c-mediated apoptosome assembly (Bruey et al, 2000).…”