Abstract
Reactive oxygen species (ROS) have been recognized as second messengers, however, targeting mechanisms for ROS in cell signaling have not been defined. While ROS oxidizing protein cysteine thiols has been the most popular proposed mechanism, our laboratory proposed that ligand/receptor-mediated cell signaling involves protein carbonylation. Peroxiredoxin-6 (Prx6) is one protein that is carbonylated at 10 min after the platelet-derived growth factor (PDGF) stimulation of human pulmonary artery smooth muscle cells. In the present study, the SulfoBiotics Protein Redox State Monitoring Kit Plus (Dojindo Molecular Technologies) was used to test if cysteine residues of Prx6 are oxidized in response to the PDGF stimulation. Human Prx6 has a molecular weight of 25 kDa and contains two cysteine residues. The Dojindo system adds the 15 kDa Protein-SHifter if these cysteine residues are reduced in the cells. Results showed that, in untreated cells, the Prx6 molecule predominantly exhibited the 55 kDa band, indicating that both cysteine residues are reduced in the cells. Treatment of cells with 1 mM H 2O 2 caused the disappearance of the 55 kDa band and the appearance of a 40 kDa band, suggesting that the high concentration of H 2O 2 oxidized one of the two cysteine residues in the Prx6 molecule. By contrast, PDGF stimulation had no effects on the thiol status of the Prx6 molecule. We concluded that protein carbonylation is a more sensitive target of ROS during ligand/receptor-mediated cell signaling than sulfhydryl oxidation.
Highlights
Reactive oxygen species (ROS) have been shown to play important roles in cell signaling (Finkel, 2011; Suzuki et al, 1997)
Our laboratory has proposed that ligand/receptor-mediated cell signaling involves protein carbonylation (Wong et al, 2008; Wong et al, 2010), which occurs on four susceptible amino acid residues: proline, arginine, lysine, and threonine (Amici et al, 1989; Berlett & Stadtman, 1997)
Our results indicated that untreated human pulmonary artery smooth muscle cells predominantly contain the 55 kDa species, consistent with the Prx6 molecule, which has two Protein-SHifters incorporated, indicating that both cysteine residues occur in the reduced form in the cells (Figure 1A, lane 1)
Summary
Reactive oxygen species (ROS) have been shown to play important roles in cell signaling (Finkel, 2011; Suzuki et al, 1997). ROS targeting protein cysteine thiols has been the most popular proposed mechanism (D’Autreaux & Toledano, 2007; Forman et al, 2010; Moran et al, 2001; Rhee et al, 2000; Sen, 2000; Truong & Carroll, 2012; Veal et al, 2007), yet the occurrence of thiol oxidation requires levels of ROS that are much higher than what is expected to occur during cell signaling (Burgoyne et al, 2007). Our laboratory has proposed that ligand/receptor-mediated cell signaling involves protein carbonylation (Wong et al, 2008; Wong et al, 2010), which occurs on four susceptible amino acid residues: proline, arginine, lysine, and threonine (Amici et al, 1989; Berlett & Stadtman, 1997). In cultured cells, hydrogen peroxide (H O ) as low as 0.5 μM was found to promote protein carbonylation (Wong et al, 2008)
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