Respiratory substrates regulate S-nitrosylation of mitochondrial proteins through a thiol-dependent pathway.
S-Nitrosylation is a reversible post-translational modification on cysteinyl thiols that can modulate the function of redox-sensitive proteins. The S-nitrosylation of mitochondrial proteins has been shown to regulate various mitochondrial activities involved in energy-transducing systems and mitochondrion-driven apoptosis. In isolated rat brain mitochondria, we demonstrate that mitochondrial protein S-nitrosylation is regulated by respiratory substrates (glutamate/malate) through a thiol-dependent pathway. Mitochondrial proteins become susceptible to S-nitrosoglutathione (GSNO)-induced S-nitrosylation in mitochondria with an oxidized environment (low glutathione (GSH), NADH, and NADPH, and high GSSG, NAD(+), and NADP(+)) caused by isolation of mitochondria using a discontinuous Percoll gradient. Activation of mitochondrial respiration by respiratory substrates leads to increased NAD(P)H and GSH levels, which in turn reduces mitochondrial S-nitrosylated proteins. 1-Chloro-2,4-dinitrobenzene (CDNB), which depletes mitochondrial GSH and inhibits the thioredoxin-thioredoxin reductase system, prevented the denitrosylation of mitochondrial proteins caused by respiratory substrate treatment. Using biotin-switch coupled with LC-MS/MS, several mitochondrial proteins were identified as targets of S-nitrosylation including adenine nucleotide translocase (ANT) and voltage-dependent anion channel (VDAC), important components of the mitochondria permeability transition pore (MPTP), as well as ATP synthase. The S-nitrosylation of ATP synthase by GSNO was found to inhibit its activity. These findings emphasize the importance of respiratory substrates in regulating S-nitrosylation through a thiol-dependent (GSH and/or thioredoxin) pathway, with implications for mitochondrial bioenergetics and mitochondrion-driven apoptosis.
Department of Pharmacology & Pharmaceutical Sciences, School of Pharmacy, University of Southern California , Los Angeles, California 90089, United States.
Allen H K
AH
Chang
Harsh
H
Sancheti
Jerome
J
Garcia
Neil
N
Kaplowitz
Enrique
E
Cadenas
Derick
D
Han
D000818:Animals; D019069:Cell Respiration; D018698:Glutamic Acid / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D008297:Male; D024101:Mitochondrial Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D026422:S-Nitrosoglutathione / Q000378:metabolism*; D015398:Signal Transduction; D013438:Sulfhydryl Compounds / Q000378:metabolism
D008293:Malates; D024101:Mitochondrial Proteins; D013438:Sulfhydryl Compounds; D018698:Glutamic Acid; D026422:S-Nitrosoglutathione; C030298:malic acid