Polysulfides link H2S to protein thiol oxidation

Abstract

Aims: Hydrogen sulfide (H2S) is suggested to act as a gaseous signaling molecule in a variety of physiological processes. Its molecular mechanism of action was proposed to involve protein S-sulfhydration, that is, conversion of cysteinyl thiolates (Cys-S(-)) to persulfides (Cys-S-S(-)). A central and unresolved question is how H2S-that is, a molecule with sulfur in its lowest possible oxidation state (-2)-can lead to oxidative thiol modifications.

Results: Using the lipid phosphatase PTEN as a model protein, we find that the "H2S donor" sodium hydrosulfide (NaHS) leads to very rapid reversible oxidation of the enzyme in vitro. We identify polysulfides formed in NaHS solutions as the oxidizing species, and present evidence that sulfane sulfur is added to the active site cysteine. Polysulfide-mediated oxidation of PTEN was induced by all "H2S donors" tested, including sodium sulfide (Na2S), gaseous H2S, and morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137). Moreover, we show that polysulfides formed in H2S solutions readily modify PTEN inside intact cells.

Innovation: Our results shed light on the previously unresolved question of how H2S leads to protein thiol oxidation, and suggest that polysulfides formed in solutions of H2S mediate this process.

Conclusion: This study suggests that the effects that have been attributed to H2S in previous reports may in fact have been mediated by polysulfides. It also supports the notion that sulfane sulfur rather than sulfide is the actual in vivo agent of H2S signaling.

FIG. 1.

PTEN activity can be monitored in real time using DiFMUP as substrate. (A) Schematic illustrating the real time PTEN activity assay used throughout this study. PTEN dephosphorylates the fluorogenic substrate DiFMUP, leading to an increase in the fluorescence signal, unless it is inhibited via oxidation to the disulfide-bonded state by oxidants such as H₂O₂. (B) Recombinant PTEN-WT or -C124S was left to react with DiFMUP in the presence of 5 mM DTT following the procedure described in Materials and Methods, and fluorescence emission was measured every minute. (C, D) The activity of PTEN-WT was measured under nonreducing conditions upon injection of buffer (untreated) or 100 μM of NEM (C), diamide or H₂O₂ (D) (indicated by first arrow). Ca. 25 min later, 10 mM DTT (or buffer) was added to half of the samples to demonstrate (ir)reversibility of the reaction (injection indicated by second arrow). Note that slight inactivation of untreated PTEN-WT was always observed, most of which was due to oxidation by atmospheric oxygen and could be reversed by addition of DTT. Curves represent means of duplicate (B) or triplicate (C, D) wells. (C, D) are representative of three independent experiments. DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate; DTT, dithiothreitol; H₂O₂, hydrogen peroxide; NEM, N-ethyl maleimide; PTEN, phosphatase and tensin homolog; WT, wild type. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

NaHS rapidly oxidizes the PTEN active site cysteine. (A) PTEN activity was measured under nonreducing conditions in response to buffer (untreated) or 5–50 μM NaHS (from Sigma; injection indicated by first arrow). Ca. 30 min later, samples received 20 mM DTT or buffer to demonstrate reversibility of the reaction (injection indicated by second arrow). (B) PTEN activity was measured under nonreducing conditions upon injection of buffer (untreated) or 50 μM GSSG, GSNO, ONOO⁻, ONOO⁻ vehicle (DMF), H₂O₂, or NaHS (from Sigma; injection indicated by arrow). (B′) As a measure of PTEN activity, the fluorescence intensity of the product at the time of oxidant addition was subtracted from the fluorescence signal at 60 min, and was normalized to the untreated control. Of all oxidants tested, NaHS inhibits the PTEN activity most efficiently. Bars denote means±SD of triplicate wells. All curves represent means of triplicate wells. (A) is representative of three, (B/B′) of two independent experiments. GSSG, glutathione disulfide; GSNO, S-nitrosoglutathione; ONOO⁻, peroxynitrite; NaHS, sodium hydrosulfide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

NaHS solutions facilitate oxidation of high pK_a thiols, but not disulfide bond reduction. (A) The fluorescence emission ratio of prereduced recombinant roGFP2 excited at 390 and 480 nm was determined continuously and used to calculate the degree of roGFP2 oxidation as described in Materials and Methods. Arrows indicate injection of 0.1–5 mM H₂O₂ (A) or NaHS (from Cayman; A′), followed by addition of 20 mM DTT to all samples ca. 40 min later to show reversibility. Data are representative of three independent experiments. (B) The activity of PTEN-WT was measured in the absence (B) or presence (B′) of 5 mM glutathione (GSH), which was added to wells as indicated. Subsequently, buffer (untreated) or 5–500 μM NaHS (from Cayman) were added (injection indicated by arrow). Quantification of the effect of NaHS on PTEN activity in the absence or presence of GSH was done as described for Figure 2B (B′′). Curves represent means, and bars denote mean±range, of duplicate wells. Data are representative of two independent experiments. (C) The degree of oxidation of preoxidized recombinant roGFP2 was determined in response to 0.01–1 mM DTT or NaHS (from Cayman) to test for their reducing effect (injection indicated by arrow). roGFP2, reduction-oxidation-sensitive GFP2. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

The oxidative properties of NaHS and Na₂S solutions are due to polysulfide formation. (A) UV/Vis spectra of NaHS (from Sigma or Cayman) and Na₂S (from Alfa Aesar) dissolved freshly to 150 mM in 200 mM Tris-HCl, pH 8.0, were recorded and compared to that of 0.83 mg/ml K₂S_x dissolved in 200 mM Tris-HCl, pH 9.2. Absorbance peaks at 300 and 372 nm (indicated by arrows) are characteristic of polysulfides. (B) The stock solution of K₂S_x used in (A) was subjected to cyanolysis as described in Materials and Methods to determine sulfane sulfur (S⁰) levels. Data are given for 13.2 mg/ml K₂S_x. Preincubation of K₂S_x with 200 mM DTT for 10 min abolished the signal due to polysulfide degradation. Bars denote mean±range of duplicate wells. (B′) Activity of PTEN-WT (380 nM) was recorded upon injection of dilutions of the same K₂S_x stock solution. S⁰ concentrations given in parentheses are calculated from the cyanolysis data in (B). Curves represent means of triplicate wells. (C) Sulfane sulfur (S⁰) levels of the same NaHS and Na₂S solutions as in (A) were determined by cyanolysis. Data are given for 120 mM stock solutions. Bars denote mean±range of duplicate wells. (C′) PTEN activity was measured upon addition of buffer (untreated) or 10 μM of these NaHS and Na₂S solutions (injection indicated by arrow). Curves represent means of triplicate samples. Quantification of the effect of these agents on PTEN activity (expressed as PTEN inhibition) was done as for Figure 2B and is shown in (C′′). Bars denote means±SD of triplicate wells. The experiment shown in (A–C′′) was repeated twice. (D) 5 mM of NaHS (Cayman) or Na₂S, or 0.55 mg/ml of K₂S_x were pretreated with (D′) or without (D) 50 mM cyanide (KCN) for 1 h before they were injected into a running PTEN activity assay to give a final concentration of 50 μM NaHS/Na₂S±500 μM KCN or 5.5 ng/ml K₂S_x±500 μM KCN. Curves represent means of triplicate samples. The same protective effect by KCN was observed for roGFP2 (not shown). K₂S_x, potassium (poly)sulfide; Na₂S, sodium sulfide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

Polysulfides also form in solutions of gaseous H₂S and GYY4137. (A) PTEN activity was recorded upon injection of 0.01–1 mM H₂S(aq) (indicated by arrow) prepared by passing H₂S gas through ultrapure water. (A′) The effect of these H₂S(aq) solutions on PTEN activity was quantified as described for Figure 2B. Bars denote means±SD of triplicate wells, and are representative of two independent experiments. (B) Activity of PTEN-WT was measured in response to 1 mM of the slow-releasing H₂S donor GYY4137 (GYY; B) or 50 μM H₂O₂ (B′) (injection indicated by second arrow), in the presence or absence of 45 mM KCN (injection indicated by first arrow). The KCN-only control sample is shown in (B′′). (B′′′) depicts the effects of GYY or H₂O₂ on PTEN activity in the absence of presence of KCN, quantified as for Figure 2B, but normalized to either the untreated sample or the KCN-only control. Bars denote means±SD of triplicate wells. All curves represent means of triplicate wells. GYY4137, morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate; H₂S, hydrogen sulfide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

Polysulfides inhibit PTEN by sulfane sulfur addition. (A) Recombinant PTEN-WT was prereduced with 20 mM DTT for 10 min, followed by buffer exchange into a nonreducing PTEN assay buffer and exposure to 100 μM H₂O₂ or NaHS (Cayman) for 15 min. The reaction was stopped with an excess of NEM to block free thiols for 15 min. Samples were then treated with or without 50 mM DTT for reduction and subjected to SDS-PAGE and immunoblotting for PTEN. Noncropped immunoblot images are shown in Supplementary Fig. S6. Data are representative of two independent experiments. (A′) depicts a representative PTEN activity assay showing complete inactivation of PTEN-WT within 15 min of exposure to 100 μM H₂O₂ or NaHS (Cayman). (B) PTEN-C71A (2.5 μM) was pretreated with buffer (untreated), 5 mM H₂O₂ or 5 mM NaHS for 15 min, then desalted, and subjected to the PTEN activity assay under nonreducing conditions (at a final protein concentration of 380 nM). DTT at 20 mM (or buffer) was added to half of the samples to demonstrate (ir)reversibility of the reaction (injection indicated by arrow). Data are representative of three independent experiments. (C) Prereduced PTEN-WT (10 μM) was exposed to 2 mM polysulfides (prepared by mixing hypochlorous acid and Na₂S solution) for 30 min, followed by buffer exchange and concentration of the protein. The sample was split in half and treated±1 mM DTT for 30 min to liberate protein-bound H₂S, which was detected using monobromobimane derivatization as described in Materials and Methods. Bars denote the released H₂S/PTEN ratio as means±SEM of four independent experiments. The Student's t-test yielded a two-tailed p-value of 0.006. (D) Activity of PTEN-C71A (380 nM) was measured under nonreducing conditions upon addition of 100 μM NaHS (or buffer), followed by injection of 400 μM H₂O₂ (or buffer), and then 10 mM DTT (or buffer), as indicated by arrows. The same results were observed for PTEN-C71S (not shown). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 7.

Polysulfides modify PTEN in intact cells. (A) HEK293T cells were exposed to the specified concentrations of H₂O₂, K₂S_x, NaHS (Cayman), Diallyl trisulfide (DATS), or DMSO for 15 min at 37°C, followed by NEM to block free thiols in intact cells. Cell lysates were subjected to nonreducing SDS-PAGE and immunoblotting to probe for PTEN. The NEM alkylation and immunoblot procedure was done for all panels of this figure. Cyanolysis of K₂S_x and NaHS stock solutions was performed immediately after dissolution to quantitate S⁰ levels, which were used to calculate S⁰ concentrations in the respective cell treatments. S⁰ levels for DATS treatments equal the applied DATS concentration. Data are representative of two (DATS/DMSO) or seven (H₂O₂, K₂S_x, NaHS) independent experiments. (B) HEK293T cells were exposed to the specified concentrations of H₂O₂, NaHS (Cayman), or Na₂S (Alfa Aesar or Sigma) for 45 min at 37°C. Data are representative of three independent experiments. (C) TCEP at 0.5 mM (or buffer) was added to HEK293T cells just before exposure to the specified concentrations of K₂S_x, H₂O₂, NaHS (Cayman), or Na₂S (Sigma) for 45 min. Similarly, 2000 U/well bovine liver catalase (or buffer) was injected before treatment with these stimuli for 45 min (D). The proportion of oxidized PTEN was quantified by densitometry and is given below immunoblots (C, D). (C, D) are representative of two independent experiments. TCEP, tris(2-carboxyethyl)phosphine.