Protein S-glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake

• Published in: PloS one Vol. 13; no. 2; p. e0192801
• Main Authors: Gill, Robert M, O'Brien, Marisa, Young, Adrian, Gardiner, Danielle, Mailloux, Ryan J
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14.02.2018; Public Library of Science (PLoS)
• Subjects: Apoptosis; Binding sites; Biochemistry; Biology and Life Sciences; Carnitine; Cell regulation; Cellular proteins; Dehydrogenases; Disulfiram; Dithiothreitol; Electron transport chain; Fatty acids; Gene expression; Heart diseases; Hydrogen; Hydrogen ion concentration; Hydrogen peroxide; Incubation; Liver; Medicine and Health Sciences; Metabolism; Mitochondria; Muscle contraction; Muscles; Musculoskeletal system; NADH; Nicotinamide adenine dinucleotide; Oxidation; Oxidative stress; Oxygen; Physical Sciences; Properties; Protein S; Proteins; Pyruvic acid; Reactive oxygen species; Rodents; Signal transduction; Skeletal muscle; Substrate inhibition; Superoxide; Canada
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0192801

Protein S-glutathionylation is a reversible redox modification that regulates mitochondrial metabolism and reactive oxygen species (ROS) production in liver and cardiac tissue. However, whether or not it controls ROS release from skeletal muscle mitochondria has not been explored. In the present study, we examined if chemically-induced protein S-glutathionylation could alter superoxide (O2●-)/hydrogen peroxide (H2O2) release from isolated muscle mitochondria. Disulfiram, a powerful chemical S-glutathionylation catalyst, was used to S-glutathionylate mitochondrial proteins and ascertain if it can alter ROS production. It was found that O2●-/H2O2 release rates from permeabilized muscle mitochondria decreased with increasing doses of disulfiram (100-500 μM). This effect was highest in mitochondria oxidizing succinate or palmitoyl-carnitine, where a ~80-90% decrease in the rate of ROS release was observed. Similar effects were detected in intact mitochondria respiring under state 4 conditions. Incubation of disulfiram-treated mitochondria with DTT (2 mM) restored ROS release confirming that these effects were associated with protein S-glutathionylation. Disulfiram treatment also inhibited phosphorylating and proton leak-dependent respiration. Radiolabelled substrate uptake experiments demonstrated that disulfiram inhibited pyruvate import but had no effect on carnitine uptake. Immunoblot analysis of complex I revealed that it contained several protein S-glutathionylation targets including NDUSF1, a subunit required for NADH oxidation. Taken together, these results demonstrate that O2●-/H2O2 release from muscle mitochondria can be altered by protein S-glutathionylation. We attribute these changes to the protein S-glutathionylation complex I and inhibition of mitochondrial pyruvate carrier.