Mechanism of protein decarbonylation

Abstract

Ligand/receptor stimulation of cells promotes protein carbonylation that is followed by the decarbonylation process, which might involve thiol-dependent reduction (C.M. Wong et al., Circ. Res. 102:301-318; 2008). This study further investigated the properties of this protein decarbonylation mechanism. We found that the thiol-mediated reduction of protein carbonyls is dependent on heat-labile biologic components. Cysteine and glutathione were efficient substrates for decarbonylation. Thiols decreased the protein carbonyl content, as detected by 2,4-dinitrophenylhydrazine, but not the levels of malondialdehyde or 4-hydroxynonenal protein adducts. Mass spectrometry identified proteins that undergo thiol-dependent decarbonylation, which include peroxiredoxins. Peroxiredoxin-2 and -6 were carbonylated and subsequently decarbonylated in response to the ligand/receptor stimulation of cells. siRNA knockdown of glutaredoxin inhibited the decarbonylation of peroxiredoxin. These results strengthen the concept that thiol-dependent decarbonylation defines the kinetics of protein carbonylation signaling.

Keywords: 2,4-dinitrochlorobenzene; 2,4-dinitrophenyl; 2,4-dinitrophenylhydrazine; BME; BSA; Carbonylation; DNCB; DNP; DNPH; DTT; Decarbonylation; ECL; Free radicals; GSH; Grx1; HRP; IEF; N-acetyl-l-cysteine; NAC; Oxidant signaling; Oxidative stress; PDGF; Peroxiredoxin; Protein oxidation; Prx; ROS; Reactive oxygen species; Redox signaling; SDS; SMC; Trx; bovine serum albumin; dithiothreitol; enhanced chemiluminescence; glutaredoxin-1; horseradish peroxidase; isoelectric focusing; l-glutathione; peroxiredoxin; platelet-derived growth factor; reactive oxygen species; smooth muscle cells; sodium dodecyl sulfate; thioredoxin; β-mercaptoethanol.