DYn-2 Based Identification of Arabidopsis Sulfenomes

Abstract

Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H2O2) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H2O2 dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H2O2 treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana.

Fig. 1.

Schematic views of the molecular mechanism of the DYn-2 probe and the strategy to identify DYn-2 trapped sulfenylated proteins. A, DYn-2 specifically detects sulfenic acid modifications, but no other thiol modifications. B, Biotinylation of the DYn-2 tagged proteins by click reaction. C, Once DYn-2 tagged proteins are biotinylated, a streptavidin-HRP (Strep-HRP) blot visualizes sulfenylation, or alternatively, after enrichment on avidin beads, proteins are identified by mass spectrometry analysis.

Fig. 2.

The DYn-2 chemical probe competes with the YAP1C genetic probe. YAP1C/YAP1A overexpressing cell cultures were treated with 0 or 20 mm H₂O₂ in the presence or absence of DYn-2 for 1 h. Proteins were extracted in the catalase-supplemented extraction buffer, and YAP1C complexes (marked with an arrow) were visualized with the PAP antibody complex. YAP1C complex formation was reduced in the presence of DYn-2 in both nontreated, and H₂O₂ treated YAP1C-GS overexpressing Arabidopsis cells. Treatment of protein samples with 50 mm TCEP led to the reduction of the YAP1C complexes, indicating the disulfide nature of the complexes.

Fig. 3.

DYn-2 detects time- and dose-dependent changes of H₂O₂ mediated sulfenylation in Arabidopsis. A, Cell cultures were treated with 0, 0.5, 1, 2, 5, 10, or 20 mm H₂O₂ for 1 h in the presence of 500 μm DYn-2 probe. After the click reaction, the H₂O₂ dose-dependent sulfenylation was visualized on a Strep-HRP developed Western blot. B, Cell cultures were treated with 0, 1, or 20 mm H₂O₂ for 15, 30, 60, and 120 min in the presence of 500 μm DYn-2. After the click reaction, the time-dependent sulfenylation was visualized on a Strep-HRP developed Western blot.

Fig. 4.

Analysis of the sulfenome identified in Arabidopsis under H₂O₂ stress. A, Enrichment of DYn-2 tagged proteins. Cell cultures were treated with 0 or 10 mm H₂O₂ for 30 min in the presence of 500 μm DYn-2 probe. After extraction, the DYn-2 tagged proteins were biotinylated and enriched using avidin beads. L: lysates, L(p): Lysates after precipitation, E: eluted proteins, S: Supernatant, the nonbound part of the lysate. On a Strep-HRP developed Western blot, an increased signal was observed under stress conditions even after enrichment of the DYn-2 tagged proteins on avidin beads. B, After subtraction of the background datasets of nontreated samples, 226 proteins were identified from three independent experiments as the H₂O₂ mediated DYn-2 sulfenome. C, The number of the identified proteins predicted to be present in the different subcellular compartments. D, Percentage of the candidates previously identified as having redox-active cysteines. E, The previously reported 103 proteins contain sulfenic acids (SOH), disulfides (S-S), S-glutathionylated (SSG), and S-nitrosylated proteins (SNO). F, The 123 cytoplasmic sulfenylated proteins identified by DYn-2 contain 16 proteins in common with the YAP1C cytoplasmic sulfenome.