Cell signaling promoting protein carbonylation does not cause sulfhydryl oxidation: Implications to the mechanism of redox signaling

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.


Introduction
Reactive oxygen species (ROS) have been shown to play important roles in cell signaling (Finkel, 2011;Suzuki et al., 1997). In particular, the roles of ROS in cell growth signaling have been well documented (Rao & Berk, 1992;Sundaresan et al., 1995). For the mechanism of ROS signaling, the receptor activation producing ROS via NAD(P)H oxidase is a widely accepted concept (Griendling et al., 1994). However, molecular targeting mechanisms for ROS in cell signaling have been unclear. 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).
More recently, we identified proteins that are carbonylated in response to the platelet-derived growth factor (PDGF) stimulation. Among them, peroxiredoxin-6 (Prx6) was found to be carbonylated in response to a 10-min treatment of human pulmonary artery smooth muscle cells with PDGF (Wong et al., 2013). Peroxiredoxins have been shown to regulate cell signaling (Woo et al., 2010). The present study tested whether this signaling mechanism also promotes sulfhydryl oxidation within the Prx6 molecule. Briefly, cells were washed, proteins precipitated with trichloroacetic acid and "Protein-SHifters" were added to each sample. Samples were then loaded onto a sodium dodecyl sulfate polyacrylamide gel and electrophoresed. The gel was exposed to UV light to cut the "Protein-SHifters." The resultant non-reducing SDS polyacrylamide gel was electroblotted to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% milk for 30 min at room temperature and incubated with the anti-Prx6 antibody produced in rabbit (Sigma-Aldrich Chemical Company, St. Louis, MO, USA; Catalogue no. P0058; 1:1,000 dilution) at 4°C overnight. The membrane was then washed three times and incubated with goat anti-rabbit IgG-horseradish peroxidase conjugate (Bio-Rad; Catalogue no. 1706515; 1:3,000 dilution) for 45 min at room temperature. After washing three times, signals were obtained using an Enhanced Chemiluminescence System (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).

Results
The technology developed for SulfoBiotics Protein Redox State Monitoring Kit Plus, by Dojindo Molecular Technologies adds a 15 kDa Protein-SHifter on free sulfhydryl groups, allowing the visualization of the thiol status of a given protein by coupling with immunoblotting. The human Prx6 molecule with a molecular weight of 25 kDa has two cysteine residues. 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). Treatment of cells with PDGF (10 ng/ml) for 10 min, which promoted protein carbonylation of Prx6 (Wong et al., 2013), did not alter the thiol state of Prx6 ( Figure 1A, lane 1 and lane 2). The PDGF treatment for 30 min did not alter the thiol state of Prx6 either ( Figure 1A, lane 1 and lane 3). By contrast, treatment of H 2 O 2 at a high concentration (1 mM) eliminated the 55 kDa band and generated a 40 kDa band that is consistent with one sulfhydryl group being oxidized ( Figure 1A, lane 4). These results were reproduced at least five times. Dataset 1 (Suzuki et al., 2017) contains the uncropped version of Figure 1A and the uncropped repeats. The bar graph shows the data from five separate experiments with five separate cell treatments. Control experiments were performed to ensure that PDGF stimulated protein phosphorylation as well as carbonylation. Figure 1A and

Discussion
Unlike protein carbonylation of Prx6, which is promoted in response to PDGF-treatment of human pulmonary artery smooth muscle cells (Wong et al., 2013), PDGF stimulation of cells does not cause the oxidation of two cysteine residues within the human Prx6 molecule. By contrast, cysteine oxidation within the Prx6 molecule can be promoted by treating cells with mM concentrations of H 2 O 2 that are not likely to be generated in ligand/ receptor-mediated cell signaling. We conclude that protein carbonylation, but not sulfhydryl oxidation, is a likely ROS-targeting mechanism for growth factor stimulation and cell signaling.
Protein carbonylation is promoted by metal-catalyzed generation of hydroxyl radicals, which are known to promote oxidation indiscriminately. However, the caged and site-directed production of hydroxyl radicals via metals could confer specificity (Stadtman & Berlett, 1991;Wong et al., 2010).

Data availability
Dataset 1. The uncropped version of Figure 1A and the uncropped repeats.
DOI, 10.5256/f1000research.11296.d157362 (Suzuki et al., 2017 Author contributions YJS conceived the study and designed the experiments. CC, FA, LM, VR, and YJS carried out the research. YJS prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.

Competing interests
No competing interests were disclosed.

Grant information
This work was supported by the National Institutes of Health, National Heart, Lung, and Blood Institute and National Institute of Aging (Grants R01 HL72844 and R03 AG047824) to YJS. The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors describe the effects of PDGF and H2O2 treatment on the oxidation state of Prdx6 using a thiol probe, that when attached to free thiols increases the molecular weight of the protein by 15 kDa for each probe attached to the protein. The authors demonstrate that H2O2 treatment causes a change in the redox status of Prdx6 as compared to PDGF treatment. There are a number of issues that need to be resolved and validated by the authors before they can make some of the statements made within the manuscript.
It is essential that the authors fully describe the sample preparation before analysis as this could greatly affect the results and interpretations made. In Figure 1 the authors describe that Prdx6 when the "Protein-SHifter" is added the protein has a mol weight of 55 kDa in controls and the PDGF treatments and one free thiol with the H2O2 treatment, but in Fig1B they show the native state of Prdx6 forming an intra-disulphide, was a reducing agent used in the sample preparation to reduce this disulphide? Does the catalytic Cys47 of this 1-Cys peroxiredoxin form an intra-disulphide with Cys91? It would also be helpful if a non "Protein-SHifter" treated sample was included in the blot to demonstrate the native band at 25 kDa. From Fig1A it would appear that there is a much more intense band for Prdx6 in the H2O2 treated samples, is there a loading control that can be included for this blot?
Carbonylation usually refers to the introduction of an aldehyde or ketone group on an amino acid, I am not sure if this is what the authors are referring to in the title and throughout the manuscript. It is well known that Cys47 of Prdx6 forms a sulphinic (-SO2H) and/or sulphonic (-SO3H) acid. Indeed Prdx6 has been described as having quite a number of various modifications (Jeong, J et al, Proteomics, 2012) so the authors need to confirm the carbonylation or other modifications by mass spectrometry. It is clear that one of the Cys residues is not amenable to "Protein-SHifter" after H2O2 treatment, it would be helpful if they could identify which cysteine residue is susceptible to oxidation.

Are sufficient details of methods and analysis provided to allow replication by others? No
If applicable, is the statistical analysis and its interpretation appropriate? If applicable, is the statistical analysis and its interpretation appropriate? Yes