Abstract
In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known as S-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identified S-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond. However, the identity of the S-sulfenylated amino acid residues within a protein remained enigmatic. By using the same transgenic YAP1C probe, we present here a technological advancement to identify in situ sulfenylated cysteine sites in Arabidopsis thaliana cells under control condition and oxidative stress. Briefly, the total extract of transgenic YAP1C A. thaliana cells was initially purified on IgG-Sepharose beads, followed by a tryptic digest. Then, the mixed disulfide-linked peptides were further enriched at the peptide level on an anti-YAP1C-derived peptide (C598SEIWDR) antibody. Subsequent mass spectrometry analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating sulfenylated cysteines in over 1,000 proteins. Approximately 55% of these YAP1C-linked cysteines had previously been reported as redox-sensitive cysteines (S-sulfenylation, S-nitrosylation, and reversibly oxidized cysteines). The presented methodology provides a noninvasive approach to identify sulfenylated cysteines in any species that can be genetically modified.
Highlights
Biotic and abiotic stresses increase the production of reactive oxygen species (ROS) in plants
This genetic construct was fused with an N-terminal tandem affinity purification (TAP) tag, containing two IgG-binding domains of protein G and a streptavidin-binding peptide (SBP), separated by the Human Rhinovirus (HRV) 3C protease cleavage site
We had identified S-sulfenylated Arabidopsis proteins in vivo by means of a transgenically expressed YAP1C probe fused to an affinity purification tag (Waszczak et al, 2014; De Smet et al, 2019)
Summary
Biotic and abiotic stresses increase the production of reactive oxygen species (ROS) in plants. In Arabidopsis, H2O2-dependent S-sulfenylation of BRASSINAZOLE-RESISTANT 1 (BZR1) promotes its interaction with transcriptional factors (TFs) and drives gene expression (Tian et al, 2018), whereas, S-sulfenylation of BRASSINOSTEROID INSENSITIVE 2 (BIN2) enhances the formation of phosphorylated BRI1-EMSSUPPRESSOR 1 (BES1), which cannot transport and bind TFs in nuclei, thereby decreasing brassinosteroid (BR) signaling outputs (Song et al, 2019). Another example is the impact on the levels of the auxin biosynthetic precursor tryptophan by S-sulfenylation of a tryptophan synthetase β subunit 1 (Yuan et al, 2017). The identification of sulfenylated cysteine sites is a crucial step to advance our understanding of redox-regulated processes
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