A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems

Abstract

Hydrogen sulfide signaling involves persulfide formation at specific protein Cys residues. However, overcoming current methodological challenges in persulfide detection and elucidation of Cys regeneration mechanisms from persulfides are prerequisites for constructing a bona fide signaling model. We here establish a novel, highly specific protein persulfide detection protocol, ProPerDP, with which we quantify 1.52 ± 0.6 and 11.6 ± 6.9 μg/mg protein steady-state protein persulfide concentrations in human embryonic kidney 293 (HEK293) cells and mouse liver, respectively. Upon treatment with polysulfides, HEK293 and A549 cells exhibited increased protein persulfidation. Deletion of the sulfide-producing cystathionine-γ-lyase or cystathionine-β-synthase enzymes in yeast diminished protein persulfide levels, thereby corroborating their involvement in protein persulfidation processes. We here establish that thioredoxin (Trx) and glutathione (GSH) systems can independently catalyze reductions of inorganic polysulfides and protein persulfides. Increased endogenous persulfide levels and protein persulfidation following polysulfide treatment in thioredoxin reductase-1 (TrxR1) or thioredoxin-related protein of 14 kDa (TRP14) knockdown HEK293 cells indicated that these enzymes constitute a potent regeneration system of Cys residues from persulfides in a cellular context. Furthermore, TrxR1-deficient cells were less viable upon treatment with toxic amounts of polysulfides compared to control cells. Emphasizing the dominant role of cytosolic disulfide reduction systems in maintaining sulfane sulfur homeostasis in vivo, protein persulfide levels were markedly elevated in mouse livers where hepatocytes lack both TrxR1 and glutathione reductase (TR/GR-null). The different persulfide patterns observed in wild-type, GR-null, and TR/GR-null livers suggest distinct roles for the Trx and GSH systems in regulating subsets of protein persulfides and thereby fine-tuning sulfide signaling pathways.

Keywords: Glutathione system; Life sciences; ProPerDP method; Proteins; Thioredoxins; biochemistry; cell signaling; hydrogen sulfide; persulfide.

Fig. 1. Protein Persulfide Detection Protocol.

Thiol and persulfide functional groups are alkylated by IAB to form the corresponding thioether and dialkyl disulfide derivatives, respectively (-R denotes the electrophile moiety of the alkylating agent). Oxidized Cys residues of proteins in the original sample (Sample 1) will not be derivatized by IAB. Affinity purification of alkylated proteins is achieved by pulldown with streptavidin-coated magnetic beads, leaving proteins only containing oxidized Cys residues in the supernatant (Sample 2). Resuspension of purified beads in a reducing buffer selectively cleaves the original persulfides off as thiols, which can be analyzed after separation with recovery from the beads, thus allowing the determination of protein persulfides (Sample 3). Note that some sulfenic acid (-SOH) or nitrosothiol (-SNO) derivatives might become alkylated in Sample 1, but these reactions give thioethers such as free thiols and hence do not appear as false positives in Sample 3. Native Cys residues with free thiols in the original sample will not be released from the beads by reduction but can be recovered by boiling of the beads in SDS (Sample 4). The diamond symbol (◊) denotes the biotin tag, and the pictogram in the inset refers to the biotin-streptavidin binding interaction. The order and nomenclature of the samples are maintained for all gels and blots in this study, thereby referred to as S1 to S4, respectively.

Fig. 2. Detection of protein persulfide formation on HSA.

S1 to S4 refer to sampling according to Fig. 1. (A) IAB alkylated HSA-SSH samples are efficiently pulled down by streptavidin-coated magnetic beads (compare S1 to S2). Lanes S3 and S4 represent HSA-SSH that was reduced off the beads by TCEP and HSA thiol that was released during boiling, respectively. No bands were detected in S3 when HSA-SSH was reduced by TCEP before alkylation. Gels are representative of n = 9 experiments. (A′) Incubation of HSA-SSH with 5 mM sulfide for 30 min before alkylation reduces some of the HSA-SSH by shifting the equilibrium of reaction 1 (see text). (B and B′) HSA-SSH formation was observed in HS_x⁻-treated plasma samples (B), but no endogenous HSA-SSH was detectable in untreated plasma (B′) by Colloidal Coomassie Blue staining. Gels are representative of n = 3 experiments. (C and C′) Concentration-dependent (C) HS_x⁻ or (C′) H₂S treatment induced HSA-SSH formation in plasma detected by immunoblot analyses against HSA. Blots only show S3. Plasma protein (100 ng) and pure HSA (20 ng) were applied as loading controls. Blots are representative of n = 3 experiments. The observed mobility shifts on each gels are due to the reduction of structural disulfide bonds in HSA during the final reduction step. Mass spectrometry confirmed that the shifted bands indeed represent HSA.

Fig. 3. Catalytic reduction of HS_x⁻ and BSA-SSH by the Trx system.

(A) Kinetic traces show catalytic reduction of increasing concentrations of HS_x⁻ at 100 nM TrxR1 and 250 μM NADPH following the consumption of NADPH (at 340 nm). (B) Initial rates of NADPH consumption by TrxR1 using HS_x⁻ as substrate show linear dependence on the HS_x⁻ concentration up to 1 mM. (C) Addition of 50 μM sodium selenite (at the indicated time point by the arrow) to similar reaction mixtures as in (A) resulted in increased NADPH consumption rates (also see fig. S2). (D) The GCSG mutant of TrxR1 is inactive in a similar activity assay as in (A), indicating the need for catalysis of the Sec residue (see the activities of further mutants in fig. S3, A to C). (E) TrxR1 concentration–corrected initial rates were increased in the presence of 5 μM Trx1 and 2 μM TRP14 compared to TrxR1 alone with linear HS_x⁻ concentration dependencies. (F and G) Addition of HS_x⁻ had no inhibitory potential on TrxR1-coupled (F) insulin-reducing Trx1 activities or (G) cystine-reducing TRP14 activities. (H) Kinetic traces show catalytic reduction of 170 μM BSA-SSH by 50 nM TrxR1 at 250 μM NADPH, which are further accelerated by 5 μM Trx1 or 2 μM TRP14.

Fig. 4. Protein persulfide detection in cells.

S1 to S4 refer to sampling according to Fig. 1. (A) Detection of persulfides in HS_x⁻-treated and untreated A549 cell lysates (Coomassie staining). (B) Persulfide detection in HS_x⁻-treated intact A549 cells, where the alkylating agent is washed away before the cell lysis step, as visualized by Coomassie staining. The indicated bands in S3 were subsequently subjected to tryptic digestion and mass spectrometry analyses. (C) List of identified persulfidated proteins from the bands indicated on (B). (D) Endogenous persulfidation can only be visualized by silver staining. Gels are representative of n = 3 experiments. (E) More protein persulfides were detected in wild type (WT) compared to cys4Δ and cys3Δ yeast strains representing the corresponding CBS and CSE deleted variants, respectively. For sample preparation and the relevant genotypes, see Materials and Methods.

Fig. 5. TrxR1 and TRP14 counteract intracellular protein persulfide accumulation.

S3 refers to sampling according to Fig. 1. (A) Western blot showing the knockdown of TRP14 and TrxR1 in stably transfected HEK293 cells, compared to control cells. GAPDH is applied as a loading control. (B) Representative silver-stained gel (of n = 4 experiments) shows that more protein persulfides are detected in TrxR1 and TRP14 knockdown HEK293 cells than in the control (control cells have been transfected with a plasmid that transcribes a scramble shRNA) under normal growth conditions. (C) Increases in protein persulfide levels in TrxR1 and TRP14 knockdown HEK293 cells compared to control reached statistical significance (*P < 0.05 using the paired t test); 100% in the control cells corresponds to 1.52 ± 0.55 μg/mg total protein. Error bars represent SDs of n = 4 experiments. (D) Two hours of treatment with 200 μM polysulfide induced higher levels of intracellular protein persulfides in intact TrxR1 and TRP14 knockdown cells compared to control cells. (E) A modified SRB cytotoxicity assay revealed that control cells (•) are significantly more viable upon polysulfide exposure than TrxR1 knockdown (○) cells (***P < 0.0001 at 0.5 and 1 mM polysulfide concentration). Data points and error bars represent the average and SD, respectively, of n = 4 independent experiments of triplicate measurements.

Fig. 6. Catalytic reduction of HS_x⁻ and BSA-SSH by the glutaredoxin system.

(A and B) Kinetic traces show catalytic reduction of increasing concentrations of HS_x⁻ at 6 μg/ml GR, 250 μM NADPH, and 1 mM GSH in the absence (A) or presence (B) of 1 μM Grx1 following the consumption of NADPH (at 340 nm). (C) Initial rates of NADPH consumption by the GSH system using HS_x⁻ as a substrate in the enzyme systems described in (A) and (B), represented by “•” and “○” symbols, respectively, show linear dependence on the HS_x⁻ concentration up to 1 mM. (D) Kinetic traces show catalytic reduction of 170 μM BSA-SSH at 6 μg/ml GR, 250 μM NADPH, and 1 mM GSH in the absence or presence of 1 μM Grx1.

Fig. 7. Trx and GSH systems orchestrate protein persulfide levels in vivo.

(A) Representative silver-stained gel demonstrates the detected differences in protein persulfide pools of liver samples from mice engineered to lack GR (GR-null) or TrxR1 and GR (TR/GR-null) in hepatocytes compared to that of an age-matched healthy control (WT). S3 refers to sampling according to Fig. 1. (B) Increased levels of protein persulfides were detected by ProPerDP (see Materials and Methods) in GR-null and TR/GR-null mouse liver samples compared to WT, reaching statistical significance (*P < 0.05 using nonpaired t test) for TR/GR-null; 100% in the WT animals corresponds to 11.6 ± 6.9 μg/mg total protein (n = 7 different animals). Error bars represent SDs of n = 8 experiments with different animals for TR/GR-null and n = 5 for GR-null.

Fig. 8. Potential caveats of the ProPerDP method.

(A) In proteins with more than one free Cys that exhibit different persulfidation properties, because of a nonpersulfidated Cys residue, the protein can stay immobilized on the streptavidin beads despite containing a persulfide, leading to false-negative signals. (B) Intermolecular protein disulfide bonds with nonpersulfidated extra Cys residues on one of the polypeptide chains might appear as false-positive signals in the persulfide proteome. Upon reduction, P₂ proteins are cleaved off the beads and could erroneously be present in the persulfide proteome fractions (Sample 3 in Fig. 1). (C) A potential way to overcome the abovementioned caveats is to digest the alkylated proteins before the pulldown step because it is highly unlikely that both the free and persulfidated Cys [for (A)] or the disulfide and the free Cys moieties [for (B)] will end up in the same peptide using this method. With this approach, the alkylated peptide persulfides could be detected by mass spectrometry after they are cleaved off the beads by the reducing agent. This method improvement is under development in our laboratory.