Global profiling of distinct cysteine redox forms reveals wide-ranging redox regulation in C. elegans

Abstract

Post-translational changes in the redox state of cysteine residues can rapidly and reversibly alter protein functions, thereby modulating biological processes. The nematode C. elegans is an ideal model organism for studying cysteine-mediated redox signaling at a network level. Here we present a comprehensive, quantitative, and site-specific profile of the intrinsic reactivity of the cysteinome in wild-type C. elegans. We also describe a global characterization of the C. elegans redoxome in which we measured changes in three major cysteine redox forms after H₂O₂ treatment. Our data revealed redox-sensitive events in translation, growth signaling, and stress response pathways, and identified redox-regulated cysteines that are important for signaling through the p38 MAP kinase (MAPK) pathway. Our in-depth proteomic dataset provides a molecular basis for understanding redox signaling in vivo, and will serve as a valuable and rich resource for the field of redox biology.

Fig. 1. Labeling of cysteine redox forms with chemoselective probes.

IPM is an iodoacetamide-based alkyne probe for cysteinyl thiol (–SH); BTD is a benzothiazine-based alkyne probe for cysteine sulfenic acid (–SOH); DiaAlk is a diazene-based alkyne probe for cysteine sulfinic acid (–SO₂H). The alkyne group can be conjugated to azide-bearing tags via click chemistry for detection and enrichment. In mammalian cells, –SO₂H within peroxiredoxins can be reduced by a sulfiredoxin. In C. elegans no known sulfiredoxin is present, and –SO₂H is presumably irreversible. Oxidation and reduction processes are depicted in red and blue arrows, respectively.

Fig. 2. Mapping hyperreactive cysteines in C. elegans.

a Schematic diagram of our quantitative chemoproteomic workflow for site-specific quantification of the intrinsic reactivity of cysteines in the C. elegans proteome. Lysates of C. elegans harvested under the same condition were labeled with either 10 or 100 µM IPM, respectively, and digested by trypsin. The resulting IPM-modified peptides were conjugated to light (10 µM, in red) or heavy (100 µM, in blue) azido biotin reagents with a photocleavable linker (Az-UV-biotin) via CuAAC, also known as click chemistry. The light and heavy-labeled samples were then mixed equally in amount and subjected to streptavidin-based enrichment. After several washing steps, the modified peptides were selectively eluted from beads under 365 nm wavelength of UV light for LC-MS/MS-based proteomic analysis. b Bar chart showing the numbers of quantified cysteine sites in two different studies, with common sites in black and different sites in red. c Scatter plot showing the R_100:10 values measured for cysteines quantified in both studies, and those with similar R_100:10 values (less than 1.5-fold difference) in both studies are colored in red. d Correlation of R_100:10 values with functional annotations from the UniProt database, where active sites, disulfide bonds, or metal-binding sites are shown in red, and all other quantified cysteines are in black. A moving average line of functional annotated sites is shown in a dashed blue line. e–g Representative extracted ion chromatograms (XICs) showing changes in IPM-labeled peptides from UBA-1 (e, active site is shown in red), GSTO-1 (f), and GPDs (g). The profiles for light- and heavy-labeled peptides are shown in red and blue, respectively. The average R_100:10 values calculated from biological triplicates are displayed below each XIC.

Fig. 3. Defining the oxidation-sensitive C. elegans redoxome.

a Schematic diagram of our quantitative chemoproteomic workflow for profiling cysteine redox forms in C. elegans upon peroxide treatment. Worms treated with (red) or without (blue) H₂O₂ were labeled in vitro with the chemoselective probes IPM (for –SH), BTD (for –SOH), or DiaAlk (for –SO₂H), in parallel (Fig. 1). The probe-tagged proteomes were processed into tryptic peptides, followed by reactions with light or heavy Az-UV-biotin reagents via CuAAC. The light and heavy-labeled samples were then mixed equally in amount, cleaned with SCX, and enriched on streptavidin beads. Labeled peptides were then photoreleased and subjected to LC-MS/MS-based proteomic analysis for identification and quantification of individual cysteine residues. b Distribution of the Log-transformed R_T/C values for cysteine redox forms. c Heatmap showing dynamic changes in redox forms for cysteine sites identified by all three probes. d, e Representative XICs showing changes in probe-labeled peptides from Y41D4A.5 (C120) (d) and GPD-3 (C158) (e). Profiles for light- and heavy-labeled peptides are shown in red and blue, respectively. The average R_T/C values calculated from two biological replicates are displayed below each XIC. f Structure of GPD-3 ortholog showing the distance between C158 and C162. g A Pie chart showing the distribution of the number of dynamically-changed cysteine redox forms per protein in the C. elegans redoxome. h A selected group of known redox-reactive metabolic enzymes identified in this study. Functionality information is retrieved from the UniProt knowledge base.

Fig. 4. Cysteine-mediated redox regulation is involved in various biological processes and pathways.

a A pie chart showing the distribution of the 1537 proteins that exhibit at least one redox-sensitive cysteine with respect to their functions in different organelles. Subcellular localizations for C. elegans proteins are retrieved from the UniProt knowledge base. b, c Biological processes and pathways enriched in the C. elegans redoxome, indicated by GO (b, blue) and KEGG (c, red) analysis. d, e Representative XICs showing changes in IPM-labeled peptides from LET-363/mTOR (d) and DAF-16/FOXO (e). The profiles for light- and heavy-labeled peptides are shown in red and blue, respectively. The average R_T/C values calculated from two biological replicates are displayed below individual XICs. f Fluorescent images and quantification g showing nuclear accumulation of DAF-16::GFP after 5 h of t-BOOH treatment (mean ± SEM, n = 3 experiments, at least 100 worms per condition). **P = 0.0100; Two-tailed Student’s t-test. Scale bar = 100 µm. h A schematic diagram showing that various stress stimuli promote eIF2a phosphorylation by GCN-2. i Representative XICs showing changes in IPM-labeled peptides from GCN-1. j Representative western blots and quantification k showing that eIF2a phosphorylation in response to 30 min t-BOOH exposure is impaired in gcn-1(−) animals (mean, n = 2 experiments). Lysates were loaded onto two different gels for detection with different antibodies, and blots were processed in parallel.

Fig. 5. Oxidant-sensitive cysteines are essential for p38 activation.

a The p38 MAPK pathway in C. elegans. Cysteines in pink were identified as redox-sensitive in our analysis, while cysteines in white were not responsive to H₂O₂ treatment. b Representative XICs showing changes in IPM-labeled peptides from SEK-1 (C213) and PMK-1 (C173). The profiles for light- and heavy-labeled peptide are shown in red and blue, respectively. The average R_T/C values calculated from two biological replicates are displayed below each XIC. c C213 (in red) in SEK-1 and C173 (in red) in PMK-1 are evolutionarily conserved and located close to the magnesium-binding DFG motifs (in blue). d, e Representative western blots in wild-type, SEK-1, and PMK-1 mutant animals carrying individual cysteine-to-serine mutations, and quantification showing that the two reactive cysteines are each important for t-BOOH-induced p38 phosphorylation (mean ± SEM, n = 3 experiments in (d); mean, n = 2 experiments in (e).) *P = 0.0318; ns, not significant (P = 0.3443); One-way ANOVA with Bonferroni post-test. Lysates were loaded onto two different gels for detection with different antibodies, and blots were processed in parallel. f Quantification of mRNA levels of the SKN-1 target gst-10 by qRT-PCR in wild-type, the PMK-1 C173S mutant (pmk-1(syb1415)), and the SEK-1 C213S mutant (sek-1(syb1398)) animals with or without 30 min t-BOOH treatment (mean, n = 2 experiments). g Survival curves of wild-type animals, and pmk-1(−)(km25), sek-1(−)(km4), PMK-1 C173S (syb1415), and SEK-1 C213S (syb1398) mutants in the presence of Pseudomonas aeruginosa PA14. n = 2 experiments with 280–388 worms per condition. Survival summary data are provided in Supplementary Table 1.