Peroxynitrite preferentially oxidizes the dithiol redox motifs of protein-disulfide isomerase

Abstract

Protein-disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs an array of cellular functions, such as cellular signaling and responses to cell-damaging events. PDI can become dysfunctional by post-translational modifications, including those promoted by biological oxidants, and its dysfunction has been associated with several diseases in which oxidative stress plays a role. Because the kinetics and products of the reaction of these oxidants with PDI remain incompletely characterized, we investigated the reaction of PDI with the biological oxidant peroxynitrite. First, by determining the rate constant of the oxidation of PDI's redox-active Cys residues (Cys⁵³ and Cys³⁹⁷) by hydrogen peroxide (k = 17.3 ± 1.3 m^-1 s^-1 at pH 7.4 and 25 °C), we established that the measured decay of the intrinsic PDI fluorescence is appropriate for kinetic studies. The reaction of these PDI residues with peroxynitrite was considerably faster (k = (6.9 ± 0.2) × 10⁴ m^-1 s^-1), and both Cys residues were kinetically indistinguishable. Limited proteolysis, kinetic simulations, and MS analyses confirmed that peroxynitrite preferentially oxidizes the redox-active Cys residues of PDI to the corresponding sulfenic acids, which reacted with the resolving thiols at the active sites to produce disulfides (i.e. Cys⁵³-Cys⁵⁶ and Cys³⁹⁷-Cys⁴⁰⁰). A fraction of peroxynitrite, however, decayed to radicals that hydroxylated and nitrated other active-site residues (Trp⁵², Trp³⁹⁶, and Tyr³⁹³). Excess peroxynitrite promoted further PDI oxidation, nitration, inactivation, and covalent oligomerization. We conclude that these PDI modifications may contribute to the pathogenic mechanism of several diseases associated with dysfunctional PDI.

Keywords: enzyme inactivation; hydrogen peroxide; kinetics; peroxynitrite; protein aggregation; protein disulfide isomerase; protein nitration; thiol oxidation.

Figure 1.

Kinetics of the oxidation of PDI by hydrogen peroxide. A, representative emission spectra (λ_excitation = 295 nm) of reduced PDI (5 μm) before (black trace) and 15 min after addition of 320 μm hydrogen peroxide (red trace) and after 15-min incubation of the latter oxidized sample with 500 μm DTT (blue trace). B, representative kinetics of reduced PDI (5 μm) oxidation by hydrogen peroxide (320 μm) and recovery by DTT addition (500 μm). The red trace corresponds to the fitting of the data to single-exponential decay. C, determination of the second-order rate constant of the reaction of PDI redox-active thiols with hydrogen peroxide. Pseudo-first-order rate constants (k_obs) were determined by mixing PDI (5 μm) with various concentrations of hydrogen peroxide (0.25–1.0 mm) as in B. All the incubations were performed in phosphate buffer (100 mm) containing DTPA (0.1 mm), final pH 7.4, at 25 °C. Error bars in C represent S.D. (n = 3 independent experiments). AU, arbitrary units.

Figure 2.

Peroxynitrite preferentially oxidizes the redox-active cysteines of PDI as revealed by PDI intrinsic fluorescence and limited proteolysis. A, representative kinetics of reduced PDI (5 μm) oxidation by peroxynitrite (PN) (2.5–10 μm). B, representative kinetics of reduced PDI (5 μm) oxidation by 10 μm peroxynitrite (black curve) or 20 μm peroxynitrite (blue curve) and reduction of the oxidized samples by DTT (500 μm). To monitor the variation of the fluorescence upon oxidized PDI reduction, the samples were oxidized at the bench (15 min) and mixed with DTT in the stopped-flow instrument. C, variation of the intrinsic fluorescence of PDI upon oxidation by hydrogen peroxide (500 μm) or by peroxynitrite (5, 10, and 20 μm) and re-reduction of oxidized PDI by DTT. The experiments were performed as in B. D, determination of the second-order rate constant of the reaction of PDI redox-active thiols with peroxynitrite. Initial rates were determined by experiments similar to those shown in A. E, representative gel of limited proteolysis of PDI (10 μm) promoted by trypsin before or after treatment with the specified concentrations of peroxynitrite or DTT (500 μm). The lanes marked with R correspond to 200 μm decomposed peroxynitrite (reverse addition). All the incubations were performed in phosphate buffer (100 mm) containing DTPA (0.1 mm), final pH 7.4, at 25 °C. Analyses were performed as described under “Experimental procedures.” Error bars in C and D represent S.D. (n = 3 independent experiments).

Figure 3.

Peroxynitrite preferentially oxidizes the redox-active cysteines of PDI but also decays to radicals. A, decay of 30 μm peroxynitrite monitored by its absorbance at 310 nm in the absence of PDI (red trace) and in the presence of 30 μm PDI (blue trace), the presence of 30 μm previously alkylated PDI (black trace), or the presence 30 μm previously oxidized PDI (dashed red trace). B, determination of the second-order rate constant of the reaction of PDI redox-active thiols with peroxynitrite. Initial rates were determined by experiments similar to those shown in A (blue trace) with the specified concentrations of PDI. C, decrease of the total thiol content of PDI (10 μm) upon addition of the specified concentrations of peroxynitrite. D, kinetic simulation of the reaction between the reactive thiols of PDI (10 μm) with peroxynitrite (20 μm) using Gepasi software (http://www.gepasi.org)⁶ and the equations shown in the text. To compare with the experimental values in C, all of the detectable PDI thiols were used in the plot of the simulation, although only the PDI reactive thiols are oxidized directly by peroxynitrite (Equation 2). All the incubations were performed in phosphate buffer (100 mm) containing DTPA (0.1 mm), final pH 7.4, at 25 °C Analyses were performed as described under “Experimental procedures.” Error bars in B and C represent S.D. (n = 3 independent experiments).

Figure 4.

Nano-ESI-Q-TOF MS/MS analysis of the alkylated and disulfide peptide ⁴³YLLVEFYAPWCGHCK⁵⁷ obtained from tryptic digests of reduced PDI (10 μm) untreated or treated with peroxynitrite (20 μm). A, MS/MS sequencing of the peak at m/z 648.3051, which corresponds to the alkylated peptide (monoisotopic mass, 1941.8906 Da) with a charge of 3, found in the tryptic digests of untreated PDI. R represents the carbamidomethyl group. B, MS/MS of the peak at m/z 609.6186, which corresponds to the oxidized peptide (monoisotopic mass, 1825.8320 Da) with a charge of 3, found in the tryptic digests of PDI treated with peroxynitrite. C, relative yields of carbamidomethyl and disulfide peptides of PDI untreated (control) or treated with 20 μm peroxynitrite (PN). The corresponding peptides of PDI domains a and a′ are shown at the left and right sides, respectively. Error bars represent S.D. (n = 3 independent experiments). The incubations and analyses were performed as described under “Experimental procedures” and in the text. XIC, extracted ion chromatogram; TIC, total ion chromatogram.

Figure 5.

Excess peroxynitrite promotes PDI oxidation, nitration, inactivation, and aggregation. A, representative SDS-PAGE analysis of PDI (10 μm) treated with the specified concentrations of peroxynitrite (PN). After 5-min incubation, the samples (8.4 μg of protein) were analyzed by SDS-PAGE and revealed with Coomassie Blue. B, representative Western blot of PDI (10 μm) treated with the specified concentrations of peroxynitrite as in A. After the incubations, the samples (8.4 μg of protein) were analyzed by SDS-PAGE and probed with an anti-nitrotyrosine antibody. C, the percentage of aggregated PDI after treatment with the specified concentrations of peroxynitrite. Gels similar to those shown in A were scanned, and the relative intensity of the bands corresponding to PDI (at ∼55 kDa) were quantified, and the percentage of covalent oligomerized PDI was taken as the remaining PDI/total PDI × 100. D, representative EPR spectra of DBNBS radical adducts produced by the decomposition of peroxynitrite (360 μm) in the presence of DBNBS (10 mm) and in the absence (upper trace) or presence of PDI (60 μm) (lower trace); the EPR parameter of the immobilized adduct is shown (2a_N = 56.9 G). E, PDI inactivation after treatment with the specified concentrations of peroxynitrite. After 5-min incubation, aliquots were removed, and PDI inactivation was monitored by the increase in the lag time required for insulin precipitation to attain an A_{540 nm} value of 0.1. All the incubations were performed in phosphate buffer (100 mm) containing DTPA (0.1 mm), final pH 7.4, at 25 °C Analyses were performed as described under “Experimental procedures.” Error bars in C and E represent S.D. (n = 3 independent experiments). ***, p < 0.001; *, p < 0.05.

Figure 6.

Nano-ESI-Q-TOF MS/MS analysis and estimation of the modified peptides obtained in the tryptic digests of PDI (10 μm) treated with peroxynitrite (200 μm). A, relative yields of the unmodified peptides present in untreated PDI (control) and PDI treated with 200 μm peroxynitrite (PN). The unmodified and modified peptides are specified; R and D in subscript represent the carbamidomethyl and the disulfide group, respectively. B, MS/MS sequencing of the peak at m/z 454.2268, which corresponds to the peptide ⁵⁸ALAPEY(NO₂)AK⁶⁵ (monoisotopic mass, 906.4447 Da) with a charge of 2. Error bars in A represent S.D. (n = 3 independent experiments). The incubations and analyses were performed as described under “Experimental procedures” and in the text. XIC, extracted ion chromatogram; TIC, total ion chromatogram.

Figure 7.

Schematic representation of the mechanism of the reaction between PDI and peroxynitrite (A) and of the PDI residues found to be hydroxylated and nitrated by excess oxidant (B). A, the PDI redox-active Cys residues are the preferential target of peroxynitrite, which also promotes PDI hydroxylation and nitration through the radicals produced from proton-catalyzed peroxynitrite decay. These radicals are produced in yields that depend on the relative concentrations of PDI and peroxynitrite. The figure shows the situation under the tested experimental conditions (10 μm PDI reacting with 20 or 200 μm peroxynitrite). Excess peroxynitrite over PDI reactive thiols decays mostly through the proton-catalyzed process to hydroxyl and nitrogen dioxide radicals, which in turn promote further PDI modifications. B, the aromatic residues found nitrated and/or hydroxylated by excess peroxynitrite over PDI reactive thiols are shown in red, whereas those unaltered are shown in blue. The structure of PDI with the redox-active Cys residues oxidized was adapted from Protein Data Bank code 4EL1. A solid ribbon structure of PDI domains a, b, b′, and a′ is shown. The residue numbering is of human PDI with the signal sequence.