Identification of sulfenylated cysteines inArabidopsis thalianaproteins using a disulfide-linked peptide reporter

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 remained enigmatic. Here, we present a technological advancement to identify in situ sulfenylated cysteines directly by means of the transgenic Yap1 probe. In Arabidopsis thaliana cells, after an initial affinity purification and a tryptic digestion, we further enriched the mixed disulfide-linked peptides with an antibody targeting the YAP1C-derived peptide (C598SEIWDR) that entails the redox-active cysteine. Subsequent mass spectrometry analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating sulfenylated cysteines in over 1,000proteins. 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.

Redox regulation of enzymes involved in sulfate assimilation and in the synthesis of sulfur-containing amino acids and glutathione in plants

“…While higher plant ATPS usually have a single cysteine residue in the C-terminal region (Cys435 in AtATPS1) which is absent in algal sequences, similar redox PTMs have been reported for A. thaliana ATPS. AtATPS1 was found to be prone to sulfenylation and nitrosylation (Hu et al, 2015;Wei et al, 2020) and AtATPS2 to glutathionylation (Dixon et al, 2005). Moreover, ATPS1, 2, and 4 have been reported as persulfidated (Jurado-Flores et al, 2021).…”

“…Five cysteine residues are conserved in SIR sequences and four of them serve as Fe-S cluster ligands. In fact, an additional partially conserved Cys468 is targeted by sulfenylation (Wei et al, 2020). As for most other enzymes of the pathway, SIR may be persulfidated, but neither the cysteine affected nor the effect on activity have been explored (Figure 2 and Supplementary Table 2).…”

“…Because these cysteines have very different numbering depending on the isoform, we refer to CysA for the cysteine that is present only in the N-terminal part of organellar OAS-TL (OAS B and OAS C), whereas CysB and CysC represent the two cysteine residues that are conserved in all Arabidopsis OAS-TL. In OAS A1 and OAS A2, only CysC (Cys42) may be oxidized both by nitrosylation and sulfenylation (Figure 2 and Supplementary Table 2; Hu et al, 2015;Wei et al, 2020). In OAS B, CysB (Cys98), and CysC (Cys112) could be sulfenylated (Huang et al, 2019;Wei et al, 2020).…”

“…In OAS A1 and OAS A2, only CysC (Cys42) may be oxidized both by nitrosylation and sulfenylation (Figure 2 and Supplementary Table 2; Hu et al, 2015;Wei et al, 2020). In OAS B, CysB (Cys98), and CysC (Cys112) could be sulfenylated (Huang et al, 2019;Wei et al, 2020). In OAS C, the three cysteines may be subject to redox PTM.…”

“…In OAS C, the three cysteines may be subject to redox PTM. CysA (Cys99) and CysB (Cys136) can be reversibly oxidized but the modification is unknown (Liu et al, 2014;Nietzel et al, 2020) whereas CysC (Cys150) could be sulfenylated (Wei et al, 2020). The fact that CysC is sulfenylated in all OAS together with the observed persulfidation of three OAS isoforms may suggest a two-step mechanism in which sulfenylation precedes persulfidation, i.e., when sulfenic acids react with H 2 S.…”