S-Nitrosylation of Paraxonase 1 (PON1) Elevates Its Hydrolytic and Antioxidant Activities

Figures

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  Figure 1. Mass spectra of rePON1 and rePON1-NO obtained by Q-TOF LC-MS. (A) Mass spectrum of rePON1 (1.25 μm) incubated in HEN buffer (50 mm HEPES buffer, 1 mM EDTA, 0.1 mm neocuproine, pH 7). (B,C) Mass spectrum of rePON1 (1.25 μm) after incubation with HSA-NO (10 μm) in HEN buffer for 1 h (B) and 3 h (C). Data are mean ± SEM of triplicate wells and are representative of three independent experiments. Statistical analysis by one-way ANOVA and GraphPad prism 8.0.1 software.

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  Figure 2. S-nitrosylation of rePON1 with HSA-NO as the donor increases the enzyme’s hydrolytic activities. RePON1 (1.25 µm) was incubated with different concentrations of HSA or HSA-NO (10, 20, 40 µm) for 1 and 3 h at 37 °C in HEN buffer (50 mm HEPES buffer, 1 mm EDTA, 0.1 mm neocuproine, pH 7). (A) Lactonase activity (5 µg/mL rePON1). (B) Esterase activity (5 µg/mL rePON1). (C) Paraoxonase activity (50 µg/mL rePON1). Each experiment was repeated separately three times with three replicates each time; a1 or b1 above a bar indicates p < 0.05 relative to rePON1 incubated with HSA for 1 and 3 h, respectively; a2 and b2 = p < 0.0001. Statistical analysis by one-way ANOVA and GraphPad prism 8.0.1.

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  Figure 3. Effect of rePON1-NO and HSA-NO on LDL oxidation induced by Cu2+. (A) RePON1 (5 and 10 μg/mL) and rePON1-NO (5 and 10 μg/mL) and (B) HSA (5 and 10 μg/mL) and HSA-NO (5 and 10 μg/mL) were incubated with LDL (0.05 mg protein/mL) and Cu2+ (10 μm). Absorbance at 234 nm was measured every 5 min for 2.5 h. Data are mean ± SEM of three representative independent experiments. Statistical analysis by one-way ANOVA and GraphPad prism 8.0.1 software.

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  Figure 4. FITC-rePON1-NO influx into macrophage cells compared to FITC-rePON1. RePON1 was labeled with FITC and incubated with GSNO for 3 h to obtain 50% rePON1 S-nitrosylation. Then, rePON1-NO was added to J774A.1 macrophages, incubated at 37 °C for 16 h and analyzed by FACS. (A) Histogram shows the shift in mean fluorescence intensity (MFI) in a representative experiment. Control (cells without treatment) in red, rePON1 without FITC as a negative control in black, rePON1 with FITC in blue, and rePON1-NO with FITC in purple. (B) MFI of a representative experiment. Results presented using NovoExpress and GraphPad by prism 8.0.1 software.