Identification and Quantification of Glutathionylated Cysteines under Ischemic Stress

Abstract

Ischemia reperfusion injury contributes to adverse cardiovascular diseases in part by producing a burst of reactive oxygen species that induce oxidations of many muscular proteins. Glutathionylation is one of the major protein cysteine oxidations that often serve as molecular mechanisms behind the pathophysiology associated with ischemic stress. Despite the biological significance of glutathionylation in ischemia reperfusion, identification of specific glutathionylated cysteines under ischemic stress has been limited. In this report, we have analyzed glutathionylation under oxygen-glucose deprivation (OGD) or repletion of nutrients after OGD (OGD/R) by using a clickable glutathione approach that specifically detects glutathionylated proteins. Our data find that palmitate availability induces a global level of glutathionylation and decreases cell viability during OGD/R. We have then applied a clickable glutathione-based proteomic quantification strategy, which enabled the identification and quantification of 249 glutathionylated cysteines in response to palmitate during OGD/R in the HL-1 cardiomyocyte cell line. The subsequent bioinformatic analysis found 18 glutathionylated cysteines whose genetic variants are associated with muscular disorders. Overall, our data report glutathionylated cysteines under ischemic stress that may contribute to adverse outcomes or muscular disorders.

Keywords: cardiomyocytes; clickable glutathione; glutathionylation; ischemic stress; proteomics.

Figure 1.. Clickable glutathione approach for detection of glutathionylation.

Glutathione synthetase mutant (GS M4) uses azido-Ala to synthesize azido-glutathione (γGlu-Cys-azido-Ala, N₃-GSH). After incubation of azido-Ala to HL-1 cells expressing GS M4, azido-glutathione forms S-glutathionylation, which can be detected after the click reaction.

Figure 2.. Global protein glutathionylation is significantly induced upon glucose deprivation (GD) but relatively weak in hypoxia or oxygen-glucose deprivation (OGD).

(A-C) Analysis of protein glutathionylation under GD, hypoxia, or OGD. HL-1 cells expressing GS M4 were incubated with azido-Ala. Cells were then subjected to decreasing concentrations of glucose (0–25 mM) under normoxia (A), glucose deprivation in comparison to hypoxia and OGD (B), or glucose deprivation in different percentages of oxygen (1, 5, 21%) (C). After the stimulus, cells were lysed and subjected to click reaction with Cy5-alkyne and analyzed for fluorescence (glutathionylation level) and Coomassie stains (protein loading control). Data are representative of at least 3 independent experiments. (D-G) Analysis of redox environment under GD, hypoxia, or OGD. HL-1 cells were independently analyzed, without using the clickable glutathione approach, for ROS (D), a ratio of NADP⁺ over NADPH (E), a ratio of oxidized glutathione (GSSG) over reduced glutathione (GSH) (F), and viability (G) after subjecting cells in GD, hypoxia, or OGD for 18 h. Data represent the mean ± SD, n = 3 independent experiments. Difference is significant by one-way ANOVA followed by Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.. Global protein glutathionylation increases upon the addition of palmitate during repletion of glucose and oxygen after OGD (OGD/R).

(A-C) In-gel fluorescence detection of global levels of glutathionylation during repletion of glucose or oxygen after OGD (A), repletion of palmitate after OGD (B), or co-addition of fatty acid oxidation inhibitors during OGD/R (C). HL-1/GS M4 cells incubated with azido-Ala were subjected to the indicated conditions after OGD (1% O₂ and no glucose) in a hypoxic chamber, or co-treated with Etomoxir (Et), Ranolazine (Ra), or 4-pentenoic acid (PA) in the presence of palmitate (1 mM) and BSA (170 μM). Cells were then lysed and analyzed by in-gel fluorescence (glutathionylation level) or Coomassie stains (protein loading control) after click reaction with Cy5-alkyne. Data are representative of at least 3 independent experiments. (D-G) Analysis of redox environment during OGD/R. HL-1 cells were analyzed for ROS (D), a ratio of NADP⁺ over NADPH (E), a ratio of oxidized glutathione (GSSG) over reduced glutathione (GSH) (F), and viability (G) after repletion of glucose, oxygen, or palmitate for 2 h, following OGD for 8 h. Data represent the mean ± SD, n = 3 independent experiments. Difference is significant by one-way ANOVA followed by Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.. Identification and quantification of glutathionylated cysteines upon addition of palmitate.

(A) Isotopically labeled clickable glutathione approach to identify and quantify levels of glutathionylation. After incubation of light or heavy azido-Ala, two cohorts of HL-1 cells expressing GS M4 were subjected to OGD for 8 h (step 1), followed by repletion of glucose and oxygen (OG) without or with palmitate for 2 h (step 2). Lysates collected were combined and processed for click reactions with biotin-DADPS-alkyne, pull-down with streptavidin beads, on-bead tryptic digestion, and elution of glutathionylated peptides by acidic cleavage of DADPS linker, followed by LC-MS/MS to determine an MS1-peak area ratio (R_H/L) of heavy- to light-labeled peptides. (B) Two experimental conditions [E1: OGD, followed by repletion of oxygen, glucose, and palmitate (OGF) for both heavy and light isotopes. E2: OGD, followed by OGF for heavy isotope or repletion of oxygen and glucose only (OG) for light isotope] and the number of quantified glutathionylated peptides. (C) Plots of R_H/L values and the coefficient of variation (CV) of glutathionylated peptides. Box plots are shown with the median value (line), box (25–75%), and whiskers (10–90%). (D) R_H/L values of individual glutathionylated peptides. Distribution of R_H/L values (top). Examples of identified proteins with their cysteine residues are shown by the arrow and yellow color. Graphical display of MS1-peaks showing relative quantification of heavy (blue)- to light (red)-labeled peptides (bottom).

Figure 5.. Bioinformatic analysis of glutathionylated proteins upon addition of palmitate.

(A) The major biological process associated with the identified glutathionylated proteins. Each biological process is shown with a plot of R_H/L values and selected clusters in STRING analysis. (B) Analysis of glutathionylated proteins in association with sarcomere, mitochondria, and cardiomyopathy. Among identified proteins, sarcomere-associated proteins (left) and mitochondrial proteins (middle) were identified from the DAVID GO database. Glutathionylated proteins associated with cardiomyopathy (right) were identified from cardiomyopathy disease query in STRING analysis. The identified proteins are shown after STRING and cluster analysis with plots of R_H/L values. The names of clusters were assigned based on proteins in individual clusters after DAVID GO analysis. Glutathionylated proteins with low R_H/L values (below 50% in the group) and high R_H/L values [over 50% in the group and NL (no light but heavy label found)] are shown with white and pink colors in circles, respectively.

Figure 6.. Desmin and BAG3 are glutathionylated upon addition of palmitate during OGD/R.

(A) Glutathionylation of desmin C332. (B) Glutathionylation of BAG3 C179. HL-1 cells expressing GS M4 were used without transfection. Alternatively, HEK293/GS M4 cells were transfected with wild-type or cysteine mutants of desmin or BAG3. Cells were then subjected to OGD for 8 h, followed by the addition of oxygen, glucose, and palmitate with BSA (OGD/OGF) or oxygen-glucose only (OGD/OG) for 2 h. Glutathionylated proteins were processed for click reaction with biotin-alkyne and pull-downs with streptavidin beads and analyzed by western blotting with individual antibodies for desmin, BAG3, and FLAG. Data are representative of 2 independent experiments. A symbol (►) indicates a non-specific band. Two bands in the FLAG-BAG3 blot appear to result from two BAG3 forms.