Cysteine-based redox regulation and signaling in plants

Abstract

Living organisms are subjected to oxidative stress conditions which are characterized by the production of reactive oxygen, nitrogen, and sulfur species. In plants as in other organisms, many of these compounds have a dual function as they damage different types of macromolecules but they also likely fulfil an important role as secondary messengers. Owing to the reactivity of their thiol groups, some protein cysteine residues are particularly prone to oxidation by these molecules. In the past years, besides their recognized catalytic and regulatory functions, the modification of cysteine thiol group was increasingly viewed as either protective or redox signaling mechanisms. The most physiologically relevant reversible redox post-translational modifications (PTMs) are disulfide bonds, sulfenic acids, S-glutathione adducts, S-nitrosothiols and to a lesser extent S-sulfenyl-amides, thiosulfinates and S-persulfides. These redox PTMs are mostly controlled by two oxidoreductase families, thioredoxins and glutaredoxins. This review focuses on recent advances highlighting the variety and physiological roles of these PTMs and the proteomic strategies used for their detection.

Keywords: cysteine; disulfide bond; glutathionylation; nitrosylation; redox regulation; sulfenic acid; thiolate.

FIGURE 1

Principal oxidative modifications of cysteinyl residues and their reduction pathways. Reactive cysteine residues mostly exist in thiolate forms at physiological pH and can form a sulfenic acid (SOH) by reacting with H₂O₂. This sulfenic acid is an intermediate for most other redox PTMs forming (i) intra- or intermolecular disulfide bonds, (ii) glutathione adducts in the presence of GSH, (iii) sulfenyl-amides, (iv) persulfides upon reaction with hydrogen sulfide, (v) thiosulfinates by reacting with another sulfenic acid, and (vi) sulfinic (SO₂H) and sulfonic (SO₃H) acids by further reacting with H₂O₂. Another possible glutathionylation pathway could rely on the reaction between a thiolate and nitrosoglutathione (GSNO). Interestingly, the latter compound will also promote S-nitrosylation reactions similarly, to some other derived forms of NO not represented here (see the text). Most of these redox PTMs are reversible and the enzymatic reduction of these different oxidation forms is essentially achieved by glutaredoxins (mainly glutathionylated proteins but also some disulfides) and thioredoxins (disulfides and possibly persulfides, nitrosothiols and some glutathionylated proteins). In addition to specifically reducing sulfinic acids formed on peroxiredoxins, sulfiredoxin (Srx) may also catalyze Prx deglutathionylation (Findlay et al., 2006; Park et al., 2009). It is not yet demonstrated that plant Trxs have a denitrosylase activity. The reduction pathway of thiosulfinates, sulfenyl-amides is unclear but glutathione and subsequently Grxs might be involved.

FIGURE 2

Indirect chemical detection of redox PTMs by proteomics. Besides the wish for detecting basal redox PTMs, experiments are usually designed to assess in vivo redox changes after applying an oxidative stress treatment. Most current approaches to detect redox PTMs rely on the same three-step strategy. The first step consists of blocking free thiol groups with alkylating agents such as N-ethylmaleimide (NEM), iodoacetamide (IAM) and its isotopically light ¹²C- derivative or methyl methanethiosulfonate (MMTS) (1). The second step consists of the reduction of reversibly oxidized cysteine residues (2). General reductants as dithiothreitol (DTT) or Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) are used to identify all types of oxidized cysteines. In contrast, selective chemical agents (ascorbate, arsenite, dimedone) or enzymes (glutaredoxins, thioredoxins) are used for the reduction of specific redox PTMs. Finally, the third step corresponds to the labeling of liberated thiol groups by reaction with biotinylated-, fluorescent- or isotopically heavy ¹³C-derivatives of alkylating reagents mentioned previously, keeping in mind that fluorescent reagents are rather devoted to the detection of modified proteins, whereas biotinylated reagents are devoted to protein enrichment for subsequent mass spectrometry analyses (3). Readers interested by the qualitative ICAT approach can obtain more details in a recent review (Leonard and Carroll, 2011). Interestingly, because MMTS does not seem to reduce persulfide groups, Mustafa et al. (2009) demonstrated that these persulfide groups could be detected without the reduction step if MMTS is used for the first alkylation step (4).