Protein glutathiolation by nitric oxide: an intracellular mechanism regulating redox protein modification.
This study was designed to examine whether NO regulates protein glutathiolation. Exposure to NO donors increased protein glutathiolation in COS-7 or rat aortic smooth muscle cells as detected by anti-protein glutathione (GSH) antibodies. This process was reversible and saturable. Stimulation with acetylcholine (ACh) increased protein glutathiolation in isolated rat aortic rings. This was prevented by inhibiting endothelial NO synthase (eNOS). In ACh-treated rings, proteins showing positive immunoreactivity with the anti-PSSG antibody (Ab) were identified by matrix assisted laser desorption-time-of-flight mass spectrometry to be actin, vimentin, and heat shock protein 70. Purified actin was more readily glutathiolated by S-nitrosoglutathione than by oxidized GSH as determined by electrospray-ionization mass spectrometry, and nitrosylated actin was glutathiolated by reduced GSH. Relative to wild-type (WT) mice, increased protein glutathiolation was observed in hearts of mice with cardiac-specific expression of inducible NO synthase (iNOS). Proteins immunoprecipitated from transgenic hearts revealed GSH-adducted peptides corresponding to adenine nucleotide translocator and the alpha-subunit of F1F0ATPase. These data suggest that exogenous NO or NO generated by eNOS or iNOS regulates protein adduction with GSH. This could be due to a direct reaction of proteins with S-nitrosoglutathione or denitrosylation of S-nitrosylated proteins by reduced GSH. Glutathiolation of cytoskeletal and mitochondrial proteins may be a significant feature of NO bioreactivity.
West
Matthew B
MB
Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky 40202, USA.
Hill
Bradford G
BG
Xuan
Yu-Ting
YT
Bhatnagar
Aruni
A
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D005978:Glutathione / Q000378:metabolism*; D066298:In Vitro Techniques; D051379:Mice; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D051381:Rats
D020030:Nitric Oxide Donors; D011506:Proteins; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496317:Nos2 protein, mouse; C496315:Nos3 protein, mouse; D005978:Glutathione; D000109:Acetylcholine