Research ArticleCell Biology

Spatial and temporal alterations in protein structure by EGF regulate cryptic cysteine oxidation

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Science Signaling  21 Jan 2020:
Vol. 13, Issue 615, eaay7315
DOI: 10.1126/scisignal.aay7315
  • Fig. 1 The OxRAC workflow to globally profile cysteine oxidation and overview of results.

    (A) Serum-starved A431 cells were left untreated (0 min) or stimulated with EGF (100 ng/ml) for the times indicated before lysis. (B) OxRAC workflow schematic in which free cysteine residues are trapped with NEM, and oxidized thiols are enriched by thiopropyl sepharose resin and trypsin digested on-resin. The oxidized cysteine residues remain bound during washing, then are eluted by reduction, and labeled with iodoacetamide (IAC) to differentiate oxidized (IAC-labeled) from nonoxidized (NEM-labeled) cysteine residues. Peptides are analyzed by data-dependent acquisition (DDA) to identify peptides and data-independent acquisition (DIA) mass spectrometry for quantification purposes based on high-resolution MS2 scans. (C) DIA-MS2 scans of the pyruvate kinase (PKM) peptide CCSGAIIVLTK. The site defining y10 fragment ion (red line) between the two labeled cysteine residues confirms peak identity. (D and E) Time-dependent changes in the relative oxidation of PKM Cys423 and Cys424 (D) and of cysteine residues in procollagen-lysine 2-oxoglutarate 5-dioxygenase 1 (PLOD1), catenin delta-1 (CTNND1), and dihydrolipoyl dehydrogenase (DLD) in response to EGF stimulation. *P < 0.05, **P < 0.01, based on one-way ANOVA with Dunnett’s post hoc test. Error bars are SEM for n = 3 independent biological replicates. Fold change is log2 transformed. (F) Enumeration of the functional annotation, disulfide bond types, and number of cysteine residue per peptide in the dataset.

  • Fig. 2 EGF-dependent regulation of cysteine redox networks cluster into three distinct temporal profiles associated with unique subcellular locations and biological processes.

    (A) Average log2 fold change of all peptides compared to baseline (n = 3 independent biological replicates). Lines indicate normal distributions, and the red line indicates the fold change used for normalization. (B) Heatmap of all statistically significantly oxidized cysteine-containing peptides (Q < 0.05, ANOVA corrected by Benjamini-Hochberg) clustered by K-means (1 − Pearson correlation, K = 3) of relative oxidation levels. (C) Fuzzy c-means clustering of significantly oxidized peptides and selected Gene Ontology (GO) and Reactome annotations. P values are from Panther. Fold change is log2 transformed. tRNA, transfer RNA.

  • Fig. 3 Cysteine residues in all major organelles are oxidized by EGF, but location influences the temporal dynamics.

    (A) Membrane orientation of 182 modified sites on the cytoplasmic, extracellular, or luminal side presented as percentage significant over time in response to EGF. Significance per time point is based on Q < 0.05, ANOVA corrected by Benjamini-Hochberg. n = 3 independent biological replicates. (B) Differential response over time of the extracellular and cytoplasmic side of the transmembrane protein Plexin-2B. (C) The percentage of cysteine residues in 942 sentinel proteins detected by OxRAC and annotated to a single cellular compartment that have at least one cysteine residue significantly oxidized by EGF (ANOVA corrected by the Benjamini-Hochberg method). (D) Estimated percent oxidation of all cysteine residues in sentinel proteins. (E) Selected examples of differentially oxidized peptides containing two cysteine residues. A disulfide bond in EGFR between Cys248 and Cys251 becomes reduced upon endocytosis in response to EGF. Examples of two previously unknown functional cysteine residues in TXNDC5, which are potentially disulfide linked. 1Ox and 2Ox indicate singly or doubly oxidized forms of the peptide, respectively. (F) Linear regression (r, Pearson) of the fold change over time of the singly oxidized (1Ox) forms compared to the variation (SD) between the two singly oxidized sites. Sites annotated as disulfide linked, active, or metal binding are indicated. Includes differentially oxidized sites identified in peptides spanning two cysteine residues. N/A, not applicable.

  • Fig. 4 Synchronized redox regulation of cysteine residues throughout canonical EGF signaling pathways at 15 and 30 min.

    Select enriched canonical pathways from IPA downstream of EGFR are pictured. All genes with a statistically significantly regulated cysteine residue are colored green. P < 0.05 based one-way ANOVA with Dunnett’s post hoc test, n = 3 independent biological replicates. Proteins detected but not significantly oxidized by EGF are filled in gray, and those undetected, but important for continuity of a pathway, were left unfilled. EGF redox dynamics over 60 min for significantly changing peptides in each pathway are represented in heatmaps.

  • Fig. 5 Protein domains redox regulated by EGF stimulation.

    (A) Enrichment of protein domains detected in the entire dataset “all identified” or statistically significantly oxidized by EGF, “EGF response,” as compared to all domains in the human proteome. P values determined by two-tailed Fisher’s exact test. (B) Domain organization of PRDX1 and locations and function of its cysteine residues. The relative oxidation of the resolving, peroxidatic, and noncatalytic cysteine residues in PRDXs over time in response to EGF stimulation. *P < 0.05, **P < 0.01, based on one-way ANOVA with Dunnett’s post hoc test. Error bars are SEM for n = 3 independent biological replicates. (C) Functional annotation of all cysteine residues detected compared to those significantly regulated in response to EGF. (D) Average ratio of all peptides assigned to the most enriched domains compared to their overall significance (q), overlaid with functional annotation when available. Closed circles, enriched sites in response to EGF; open circles, not enriched in response to EGF. Adjusted P values (q) were calculated from ANOVA results by applying the Benjamini-Hochberg method to correct for multiple comparisons.

  • Fig. 6 EGF-dependent redox regulation of buried cysteine residues in 14-3-3, small GTPase proteins, and ERK2.

    (A) Density plot of relative solvent accessibility (RSA) that compares cysteine residues significantly and not regulated by EGF. (B) RSA for all cysteine residues in proteins identified compared to all cysteine residues specifically quantified and those significantly regulated. Sites of >0.25 are considered solvent accessible. ***P < 0.001 by one-way ANOVA. For the box-whisker plot, center line is median, limits are upper and lower quartiles, and whiskers are 5 and 95 percentiles. (C) RSA prediction for sulfenated cysteine residues in (24). (D) Amino acid sequence conservation of the cysteine residues identified in 14-3-3 proteins. Structure of dimerized 14-3-3 sigma (PDB ID: 4DAU) with helix 4 shown in purple and locations of the remaining oxidized cysteine residues shown in black. Purple arrows point to the conserved cysteine residue in helix 4, represented as a sphere. RSA is indicated for each cysteine site or the average across 14-3-3 family members. (E) Time-dependent changes in the oxidation of 14-3-3 cysteine residue. **P < 0.01, based one-way ANOVA with Dunnett’s post hoc test. Error bars are SEM for n = 3 independent biological replicates. (F) Unmodified and phosphorylated (Thr183 and Tyr185) ERK2 crystal structures (PDB: 1ERK and 2ERK, respectively) with Cys65 highlighted in red and solvent as a cyan sphere. (G) Crystal structures of the GTP binding pocket in CDC42 (PDB ID: 5CJP) and RAC1 (PDB ID: 5O33 GTP analog). Cys157 is highlighted in red. Yellow structure is the bound GTP nucleotide analog. Cyan spheres indicate solvent molecules. (H) Representative crystal structure of KRAS (left; PDB: 5VQ2) in which Cys80 (red) is solvent inaccessible and a representative structure from molecular dynamics (MD) simulations of KRAS (right) in which Cys80 is solvent accessible. Amino acid side chains within 7 Å of Cys80 are shown as spheres, and Cys80 is shown in red as a ball and stick.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/615/eaay7315/DC1

    Fig. S1. In situ DYn-2 treatment alters EGFR-dependent ERK phosphorylation.

    Fig. S2. OxRAC method validation and controls.

    Fig. S3. Redox regulation of protein kinases and phosphatases.

    Fig. S4. EGF-dependent redox regulation of AAA ATPases and small GTPases.

    Fig. S5. Biochemical properties of the identified oxidized cysteine residues.

    Data file S1. Table of all cysteine residues detected, their LC-MS information and intensities, and redox regulation.

    Data file S2. PANTHER and Reactome annotations for peptides (and associated genes) assigned to each temporal cluster.

    Data file S3. IPA canonical pathways enriched at each time point.

    Data file S4. Representation of Pfam domain and family annotations within the OxRAC dataset.

  • The PDF file includes:

    • Fig. S1. In situ DYn-2 treatment alters EGFR-dependent ERK phosphorylation.
    • Fig. S2. OxRAC method validation and controls.
    • Fig. S3. Redox regulation of protein kinases and phosphatases.
    • Fig. S4. EGF-dependent redox regulation of AAA ATPases and small GTPases.
    • Fig. S5. Biochemical properties of the identified oxidized cysteine residues.
    • Legends for data files S1 to S4

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Table of all cysteine residues detected, their LC-MS information and intensities, and redox regulation.
    • Data file S2 (Microsoft Excel format). PANTHER and Reactome annotations for peptides (and associated genes) assigned to each temporal cluster.
    • Data file S3 (Microsoft Excel format). IPA canonical pathways enriched at each time point.
    • Data file S4 (Microsoft Excel format). Representation of Pfam domain and family annotations within the OxRAC dataset.

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