Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites

Abstract

Hydrogen peroxide (H₂O₂) is an important messenger molecule for diverse cellular processes. H₂O₂ oxidizes proteinaceous cysteinyl thiols to sulfenic acid, also known as S-sulfenylation, thereby affecting the protein conformation and functionality. Although many proteins have been identified as S-sulfenylation targets in plants, site-specific mapping and quantification remain largely unexplored. By means of a peptide-centric chemoproteomics approach, we mapped 1,537 S-sulfenylated sites on more than 1,000 proteins in Arabidopsis thaliana cells. Proteins involved in RNA homeostasis and metabolism were identified as hotspots for S-sulfenylation. Moreover, S-sulfenylation frequently occurred on cysteines located at catalytic sites of enzymes or on cysteines involved in metal binding, hinting at a direct mode of action for redox regulation. Comparison of human and Arabidopsis S-sulfenylation datasets provided 155 conserved S-sulfenylated cysteines, including Cys181 of the Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE4 (AtMAPK4) that corresponds to Cys161 in the human MAPK1, which has been identified previously as being S-sulfenylated. We show that, by replacing Cys181 of recombinant AtMAPK4 by a redox-insensitive serine residue, the kinase activity decreased, indicating the importance of this noncatalytic cysteine for the kinase mechanism. Altogether, we quantitatively mapped the S-sulfenylated cysteines in Arabidopsis cells under H₂O₂ stress and thereby generated a comprehensive view on the S-sulfenylation landscape that will facilitate downstream plant redox studies.

Keywords: Arabidopsis; S-sulfenylation; chemoproteomics; posttranslational modification; redox regulation.

Fig. 1.

Quantitative Cys-SOH identification in Arabidopsis. (A) Workflow for in vivo S-sulfenylation labeling and analysis in Arabidopsis cell cultures. The BTD-based probe was sketched as an arrow (chemical warhead) followed by 3 lines indicating the alkyne handle. (B) Mapping of the 1,537 identified Cys-SOHs sites on 1,394 (Araport11) proteins, of which 200 proteins were identified to be S-sulfenylated previously (–19) (Right) and 1,194 were discovered as protein targets (Left) (Dataset S1). (C) Ratios (R_H2O2/Control, y axis) plotted for 463 Cys-SOH sites quantified in at least 2 out of 3 replicates (x axis). Ratios of at least 1.5-fold increases (red, 92 Cys-SOHs) or decreases (green, 3 Cys-SOHs) are shown. (Inset) For the catalytic Cys of MsrB3/7/8/9 (encircled in bold), the extracted-ion chromatogram is displayed (H₂O₂/light in red, and control/heavy in blue).

Fig. 2.

Subcellular localization distribution and functional enrichment of S-sulfenylation events. (A) Subcellular distribution of 809 S-sulfenylated proteins defined as high-confidence markers (HCMs) by the Multiple Marker Abundance Profiling tool (28, 29). Organelles are colored proportionally to the S-sulfenylated HCMs. (B) Gene set enrichment of S-sulfenylated proteins for KEGG pathways (blue) and custom protein classes (SI Appendix, Materials and Methods) (Top). Fold enrichment (y axis) is plotted as a function of statistical significance (minus log Q value, x axis). Node size corresponds to the number of S-sulfenylated proteins. Bar chart outlining the number of Cys-SOHs for the enriched KEGG pathways and protein classes (Bottom). Induced Cys-SOHs (R_H2O2/Ctrl ≥ 1.5) are plotted, and their respective proportion is displayed. (C) Cys-SOH domain enrichment (SI Appendix, Materials and Methods). Fold enrichment (y axis) is plotted as a function of statistical significance (minus log Q value, x axis). Node size is scaled to the number of Cys-SOHs. ER, endoplasmic reticulum; PM, plasma membrane.

Fig. 3.

Evolutionary comparison of Arabidopsis and human Cys-SOH sites. (A) Overrepresentation motifs of Cys-SOH sequence windows (−6 to +6 residues) of human (20) and Arabidopsis BTD-labeled sites constructed by iceLogo (39). Basic residues Lys, Arg, and His are in blue, and Cys is in red. (B) Overview of Arabidopsis Cys-SOH alignment scenarios and their occurrence. (C) Examples of Arabidopsis (green)–human (blue) protein sequence alignments with exact alignments of S-sulfenylation events (red, with protein position indicated). Ratios (R_H2O2/Ctrl) were derived from this study (Dataset S1), with the number of biological replicates indicated between parentheses, and as reported for human RKO cells treated with H₂O₂ (23).

Fig. 4.

Sulfenylation of Cys181 of AtMAPK4 that is crucial for the kinase activity. (A) S-sulfenylation of the H₂O₂-sensitive Cys in the MAP kinase signature (PS01351 PROSITE pattern) in Arabidopsis (Cys176 in AtMAPK3; Cys181 in AtMAPK4; Cys201 in AtMAPK6; and Cys178 in AtMAPK11) and human (Cys161 in HsMAPK1). (B) Dimedone blot showing AtMAPK4 S-sulfenylation under 1 mM H₂O₂ for 1 h. (C). Annotated spectrum match of ‘DLKPSNLLLNANC₁₈₁DLK’ with Cys181 modified by dimedone (+138.0 Da). (D) Kinase activity of AtMAPK4 and its C181S variant as measured on phosphorimage displaying myelic basic protein (MBP) phosphorylation with [³²P]ATP. (E) Kinase activity of autophosphorylated (P_i) and nonautophosphorylated AtMAPK4 and its C181S variant as measured by quantitative Cerenkov counting after 45 min of incubation with MBP.