Identification of Sulfenylated Cysteines in Arabidopsis thaliana Proteins Using a Disulfide-Linked Peptide Reporter

Abstract

In proteins, hydrogen peroxide (H 2 O 2) 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 (C 598 SEIWDR) 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.

Hydrogen sulfide signaling in plant adaptations to adverse conditions: molecular mechanisms

“…There is a lot of evidence that there is an overlap between ROS and H 2 S; therefore, together, protein cysteine oxidation and persulfidation may represent a mechanism for the modulation of signaling processes induced by developmental or environmental stress events. In recent years, many proteins have been described as sulfenylation targets in Arabidopsis in several works, revealing >2000 targets for this modification ( De Smet et al , 2019 ; Huang et al , 2019 ; Wei et al , 2020 ). In a comparison performed between the sulfenylated and the previously identified persulfidated proteins, >6000 targets ( Aroca et al , 2015 , 2017 a ; Laureano-Marín et al ., 2020 ; Jurado-Flores et al , 2021 ) revealed that 82% of the sulfenylated proteome described in Arabidopsis also undergo persulfidation ( Fig.…”

Measuring ROS and redox markers in plant cells

“…102 As well as this chemical probe, a non-invasive SOHtrapping biosensor, the plant-optimized and affinity-tagged yeast AP-1 (YAP1C), has been exploited to capture and identify sulfenylated proteins in Arabidopsis, including characterisation of the sulfenome at a subcellular level. 103,104 These probes are currently revealing the extent to which overall cysteine oxidation is impacted by ROS stress; it will be interesting to determine whether these modifications initiate specific signalling events, e.g. by modulating the stability of proteins with N-terminal cysteine residues via the Cys/Arg branch of the N-degron pathway.…”

The Modus Operandi of Hydrogen Sulfide(H2S)-Dependent Protein Persulfidation in Higher Plants

“…GSH also interacts with NO to form S -nitrosoglutathtione (GSNO) which is regarded as a NO reservoir [ 58 ]. S -sulfenylation, which results from the oxidation of the thiol group to Cys sulfenic acid, is mediated by H 2 O 2 [ 59 , 60 ]. S -acylation is a reversible oxiPTM involving the addition of a fatty acid, such as palmitate or stearate, to specific cysteines through thioester bonds [ 61 , 62 ].…”