Protein glutathiolation by nitric oxide: an intracellular mechanism regulating redox protein modification

Abstract

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.

How widespread is stable protein S-nitrosylation as an end-effector of protein regulation?

“…However, if a thiol has been S-nitrosated by GSNO, the freed GSH may then attack the labile and thiol-reactive S-nitroso bond to generate an S-glutathiolated protein. This has been observed experimentally, whereby GSNO promoted S-glutathiolation of actin and Snitrosation was not detected [84]. This further illustrates how S-nitrosating chemical reactions can readily generate protein disulfides.…”

“…S-thiolation has been shown to be directly regulated by NO levels in vivo, with mice overexpressing cardiac-specific inducible NOS showing elevated protein S-glutathiolation [81]. This is likely explained by the elevated NO inducing S-nitrosated protein, which as explained above, then react with thiols, especially with abundant GSH, to form disulfides.…”

“…This is likely explained by the elevated NO inducing S-nitrosated protein, which as explained above, then react with thiols, especially with abundant GSH, to form disulfides. Additionally, acetylcholine increased cellular protein S-glutathiolation which was attenuated by inhibition of endothelial NOS [81]. As well as directly regulating protein function, S-thiolation is also thought to protect proteins from oxidative stress, possibly by abrogating cysteine hyperoxidation to the sulfinic or sulfonic acid states [82,83].…”

“…As well as directly regulating protein function, S-thiolation is also thought to protect proteins from oxidative stress, possibly by abrogating cysteine hyperoxidation to the sulfinic or sulfonic acid states [82,83]. Mechanistic studies suggest that during nitrosative stress protein S-glutathiolation is kinetically more likely via a protein S-nitrosothiol intermediate than an exchange reaction of the protein thiol with GSSG [84]. Despite protein S-thiolation having been recognised early on as a cellular response to nitrosative stress [85], the rates at specific protein S-nitrosothiols react with small thiols to promote S-thiolation have yet to be comprehensively determined.…”

“…Many proteins that are reported to be regulated by S-nitrosothiol formation in the cardiovascular system have also been shown, most often previously, to be regulated by disulfides, often at the same cysteine residue. For example, S-nitrosation and disulfide formation has been shown to induce the same functional change in hypoxia-inducible factor 1α-subunit [92,93], caspase-3 [60,94], Na + /K + ATPase [95,96] and actin [84,97]. A summary of proteins that form S-nitrosothiols as well as disulfides is provided in Table 1, some of which are discussed in more detail below.…”