Evidence against Stable Protein S-Nitrosylation as a Widespread Mechanism of Post-translational Regulation

Abstract

S-nitrosation, commonly referred to as S-nitrosylation, is widely regarded as a ubiquitous, stable post-translational modification that directly regulates many proteins. Such a widespread role would appear to be incompatible with the inherent lability of the S-nitroso bond, especially its propensity to rapidly react with thiols to generate disulfide bonds. As anticipated, we observed robust and widespread protein S-nitrosation after exposing cells to nitrosocysteine or lipopolysaccharide. Proteins detected using the ascorbate-dependent biotin switch method are typically interpreted to be directly regulated by S-nitrosation. However, these S-nitrosated proteins are shown to predominantly comprise transient intermediates leading to disulfide bond formation. These disulfides are likely to be the dominant end effectors resulting from elevations in nitrosating cellular nitric oxide species. We propose that S-nitrosation primarily serves as a transient intermediate leading to disulfide formation. Overall, we conclude that the current widely held perception that stable S-nitrosation directly regulates the function of many proteins is significantly incorrect.

Keywords: S-nitrosation; S-nitrosylation; cysteine; disulfide; protein; redox; signaling; thiol.

Graphical abstract

Figure 1

Disulfide Formation Is Observed before an Increase in Detectable S-Nitrosothiols (A–C) SMCs treated with increasing doses of CysNO for 30 min were analyzed for protein S-nitrosothiol (PSNO) content using the biotin switch method. A dose-dependent increase in PSNO (A) was observed as well as a concurrent increase in PKAR1 (B) and PKG1α (C) disulfide dimer. (D) A significant increase in disulfide dimer formation in both proteins was observed before a significant change in detectable PSNO. (E and F) PSNO and PKG1α formation measured by immunoblotting over a time course of 30 min after SMCs were treated with 6 or 50 μM CysNO. (G) PSNO peaked at 10 and 5 min, respectively, before decreasing in abundance, whereas the PKG1α disulfide dimer increased with time. Data are represented as mean ± SEM; ^∗p < 0.05.

Figure 2

Functional Changes Regulated by Disulfides, Not S-Nitrosation (A) The oxidation state of PTEN was measured in SMCs treated with CysNO. (B) After exposure of CysNO, there was a decrease in reduced PTEN. Reduced PTEN was observed after reduction by DTT but not ascorbate. (C) PTEN’s enzymatic activity was inhibited after exposure to CysNO, as measured by in vitro phosphatase activity assay. Enzyme activity was restored after addition of DTT but not ascorbate. (D) Calpain-1’s enzymatic activity was inhibited in vitro after exposure to the nitrosating agent CysNO, the disulfide-inducing reagents GSSG, H₂O₂, or diamide, or the irreversible calpain inhibitor Z-LLY-FMK. Data are represented as mean ± SEM; ^∗p < 0.05.

Figure 3

An NO Donor Induces S-Thiolation (A) SMCs were exposed to CysNO for 30 min, and a dose-dependent increase in S-glutathiolated proteins was detected by immunoblotting. (B) A significant increase in S-glutathiolated proteins was observed at concentrations of CysNO above 4 μM. (C and D) A significant increase in S-cysteinated proteins was observed when SMCs were treated with biotin-CysNO (BioCysNO) compared with the biotin-cysteine (BioCys) control. (E) SMCs were treated with N-biotinyl glutathione ethyl ester (BioGEE), N-biotinyl S-nitrosoglutathione (GSNO) ethyl ester (BioGSNOEE), S-nitrosoglutathione ethyl ester (GSNOEE), or GSNO. N-biotinyl S-glutathiolated proteins and PKG1α were detected by immunoblotting. (F) BioGSNOEE induced significant S-glutathiolation compared with that induced by the non-nitrosated BioGEE control. BioGSNOEE or GSNOEE both also induced significant disulfide dimerization of PKG1α. Data are represented as mean ± SEM; ^∗p < 0.05.

Figure 4

Detection of S-Nitrosothiols Is Dependent on the Abundance of GSH (A and B) Ellman’s assay was performed to detect total and non-protein thiols in SMCs exposed to CysNO for 30 min. Although there was no change in total thiol concentration (A), a significant decrease in non-protein thiols was observed with increasing CysNO concentration (B). (C) Glutathione synthesis was inhibited in SMCs for 24 hr using buthionine sulfoximine (BSO) to deplete the cellular GSH concentration. SMCs exposed to CysNO were analyzed for PSNO content using the biotin switch method. (D) Depletion of GSH significantly increases detectable PSNO. (E) SMCs were exposed to CysNO, followed by treatment with increasing doses of GSH ethyl ester (GEE) to augment cellular GSH concentration. (F) A dose-dependent decrease in PSNO was detected after treatment with GEE, reaching significance at 5 mM GEE. (G) SMCs were pre-treated with or without the S-nitrosoglutathione reductase inhibitor N6022 for 1 hr prior to exposure to CysNO. (H) Exposure to N6022 significantly potentiated the accumulation CysNO-induced PSNO. Data are represented as mean ± SEM; ^∗p < 0.05.

Figure 5

The NO Donor Reduces SMCs’ Capacity to Recycle Disulfides (A) The activity of NADPH-dependent enzymes was measured using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay after SMCs were exposed to CysNO. Enzymes were significantly inhibited after exposure to 10 μM CysNO for 30 min. (B) When SMCs were allowed to recover for 1 hr after exposure to CysNO, changes in activity were abolished. (C) PSNOs were detected using the biotin switch method in SMCs that had no recovery or 1 hr recovery after CysNO treatment. SMCs that had no recovery period showed a dose-dependent increase in PSNO along with a concurrent increase in PKAR1 and PKG1α disulfide dimers. After a recovery period of 1 hr, there was no observable increase in PSNO, but a dose-dependent increase in PKAR1 and PKG1α disulfide dimers was apparent. (D) A recovery period of 1 hr abolished the detection of PSNO following treatment with CysNO. (E) A significant increases in PKAR1 and PKG1α disulfide dimers were observed in both SMCs with no recovery and after 1 hr recovery. (F) SMCs were treated with AF for 1 hr prior to exposure to CysNO, and then PSNO, PKG1α and α-actin were measured. (G) Treatment with AF prior to CysNO exposure significantly decreased observable PSNO. (H) AF treatment induced a significant increase in the PKG1α disulfide dimer that was exacerbated with exposure to CysNO. Data are represented as mean ± SEM; ^∗p < 0.05.

Figure 6

The Major Product of Exogenous or Endogenous Nitrosative Signaling Is Disulfide Formation (A) The double S-nitrosothiol/disulfide switch was used to compare disulfides with combined (PSNO + disulfides) protein oxidation in SMCs after treatment with CysNO for 30 min. A dose-dependent increase in total protein oxidation was observed upon treatment with CysNO. (B) A significant increases in both disulfides and PSNO + disulfides was observed from baseline. There was no statistical difference between the two treatment groups at any concentration of CysNO. (C) Global disulfides and disulfide formation in PKG1α and PKAR1 was measured in SMCs after 5 min treatment with 10 or 100 μM CysNO, SNAP, NCA, H₂O₂, or diamide. (D) Global disulfides were significantly increased after exposure to both doses of CysNO, H₂O₂, or diamide. (E) Significant disulfide formation in PKAR1 was induced by 10 μM NCA or 100 μM CysNO, H₂O₂, or diamide. Significant PKG1α dimerization occurred after exposure to 10 or 100 μM CysNO, NCA, H₂O₂, or diamide or 100 μM SNAP. (F) PSNO, PKG1α, and PKAR1 disulfide dimer formation was measured in SMCs over a 6-hr treatment with 500 μM DETANO. (G) Although no significant increase in PSNO was observed at any time point, a significant increase in PKAR1 disulfide formation was observed after 4 hr and PKG1α at 2 hr. Data are represented as mean ± SEM; ^∗p < 0.05.

Figure 7

Protein Thiols that Form S-Nitrosothiols Preferentially Form Disulfides (A) Using the Griess assay, an increase in nitrite was detected in SMCs treated with LPS for 18 hr, signifying elevated NO production. (B) Increased NO production correlated with a small but significant increase in PSNO. Increased PSNO was attenuated after treatment with the pan-specific NOS inhibitor L-NAME. Data are represented as mean ± SEM; ^∗p < 0.05. (C) PSNO and disulfides were labeled with iodoTMT sixplex reagents in SMCs treated with 10 μM CysNO, 1.5 μg/mL LPS, or under control conditions. Analysis by LC-MS/MS showed that the ratio of S-nitrosothiol to disulfide significantly decreased after SMCs were exposed to exogenous or endogenous nitrosative signaling. (D) The heatmap depicts the scaled and centered peptide peak area of iodoTMT-labeled PSNO or disulfide (PS-S) peptides under each condition. Accession numbers indicate the modification of an individual cysteine residue within a given protein. The color tracker represents the Z score for each cysteine modification and ranges from 0 (red), representing low-abundance modifications, to 2 (green), representing high-abundance modifications.