Sulfenic Acid Modification Research Articles

Chemical Dissection of an Essential Redox Switch in Yeast

Saccharomyces cerevisiae responds to elevated levels of hydrogen peroxide in its environment via a redox relay system comprising the thiol peroxidase Gpx3 and transcription factor Yap1. In this signaling pathway, a central unresolved question is whether cysteine sulfenic acid modification of Gpx3 is required for Yap1 activation in cells. Here we report that cell-permeable chemical probes, which are selective for sulfenic acid, inhibit peroxide-dependent nuclear accumulation of Yap1, trap the Gpx3 sulfenic acid intermediate, and block formation of the Yap1-Gpx3 intermolecular disulfide directly in cells. In addition, we present electrostatic calculations that show cysteine oxidation is accompanied by significant changes in charge distribution, which might facilitate essential conformational rearrangements in Gpx3 during catalysis and intermolecular disulfide formation with Yap1.

Facile synthesis and biological evaluation of a cell-permeable probe to detect redox-regulated proteins

We have developed an improved synthesis for the cell-permeable, sulfenic acid probe DAz-1. Using DAz-1, we detect sulfenic acid modifications in the cell-cycle regulatory phosphatase Cdc25A. In addition, we show that DAz-1 has superior potency in cells compared to a biotinylated derivative. Collectively, these findings set the stage for the development of activity-based inhibitors of Cdc25 cell-cycle phosphatases, which are sensitive to the redox state of the active-site cysteine and demonstrate the advantage of bioorthogonal conjugation methods to detect protein sulfenic acids in cells.

The Requirement of Reversible Cysteine Sulfenic Acid Formation for T Cell Activation and Function

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in mediating cellular responses. We have examined the importance of reversible cysteine sulfenic acid formation in naive CD8(+) T cell activation and proliferation. We observed that, within minutes of T cell activation, naive CD8(+) T cells increased ROI levels in a manner dependent upon Ag concentration. Increased ROI resulted in elevated levels of cysteine sulfenic acid in the total proteome. Analysis of specific proteins revealed that the protein tyrosine phosphatases SHP-1 and SHP-2, as well as actin, underwent increased sulfenic acid modification following stimulation. To examine the contribution of reversible cysteine sulfenic acid formation to T cell activation, increasing concentrations of 5,5-dimethyl-1,3-cyclohexanedione (dimedone), which covalently binds to cysteine sulfenic acid, were added to cultures. Subsequent experiments demonstrated that the reversible formation of cysteine sulfenic acid was critical for ERK1/2 phosphorylation, calcium flux, cell growth, and proliferation of naive CD8(+) and CD4(+) T cells. We also found that TNF-alpha production by effector and memory CD8(+) T cells was more sensitive to the inhibition of reversible cysteine sulfenic acid formation than IFN-gamma. Together, these results demonstrate that reversible cysteine sulfenic acid formation is an important regulatory mechanism by which CD8(+) T cells are able to modulate signaling, proliferation, and function.

Fluorescent and Affinity-Based Tools To Detect Cysteine Sulfenic Acid Formation in Proteins

Cysteine sulfenic acid formation in proteins results from the oxidative modification of susceptible cysteine residues by hydrogen peroxide, alkyl hydroperoxides, and peroxynitrite. This species represents a biologically significant modification occurring during oxidant signaling or oxidative stress, and it can modulate protein function. Most methods to identify such oxidatively modified proteins rely on monitoring the loss of one or more thiol group(s) or on selective labeling of nascent thiol groups following reduction of oxidized proteins. Our previous work reported the direct labeling of these chemically distinct modifications with a dimedone analogue, 1,3-cyclohexadione, to which a linker and functional group (an alcohol) had been added; further addition of a fluorescent isatoic acid or methoxycoumarin reporter allowed detection of the incorporated tag by fluorescence techniques ( Poole, L. B., Zeng, B. B., Knaggs, S. A., Yakubu, M., and King, S. B. ( 2005) Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjugate Chem . 16, 1624-1628 ). We have now expanded our arsenal of tagging reagents to include two fluorescein-, two rhodamine-, and three biotin-conjugated probes based on the original approach. The new tools provide readily detectable fluorescent and affinity probes to identify sulfenic acid modifications in proteins and have been used in subsequent mass spectrometric analyses to confirm covalent attachment of the conjugates and directly determine the site of modification.

Molecular Mechanism of Oxidative Stress Perception by the Orp1 Protein

In this study we investigated the molecular mechanism by which the Orp1 (Gpx3) protein in Saccharomyces cerevisiae senses and reacts with hydrogen peroxide. Upon exposure to H(2)O(2) Orp1(Cys36) forms a disulfide-bonded complex with the C-terminal domain of the Yap1 protein (Yap1-cCRD). We used 4-nitrobenzo-2-oxa-1,3-diazole to identify a cysteine sulfenic acid (Cys-SOH) modification that forms on Cys(36) of Orp1(Cys36) upon exposure to H(2)O(2). Under similar conditions, neither Cys(82) of Orp1(Cys82) nor Cys(598) of Yap1 forms Cys-SOH. A homology-based molecular model of Orp1 suggests that the structure of the active site of Orp1 is similar to that found in mammalian selenocysteine glutathione peroxidases. Proposed active site residues Gln(70) and Trp(125) form a catalytic triad with Cys(36) in the Orp1 molecular model. The remainder of the active site pocket is formed by Phe(38), Asn(126), and Phe(127), which are evolutionarily conserved residues. We made Q70A and W125A mutants and tested the ability of these mutants to form Cys-SOH in response to H(2)O(2). Both mutants were unable to form Cys-SOH and did not form a H(2)O(2)-inducible disulfide-bonded complex with Yap1-cCRD. The pK(a) of Cys(36) was determined to be 5.1, which is 3.2 pH units lower than that of a free cysteine (8.3). In contrast, Orp1 Cys(82) (the resolving cysteine) has a pK(a) value of 8.3. The pK(a) of Cys(36) in the Q70A and W125A mutants is also 8.3, demonstrating the importance of these residues in modulating the nucleophilic character of Cys(36). Finally, we show that S. cerevisiae strains with ORP1 Q70A and W125A mutations are less tolerant to H(2)O(2) than those containing wild-type ORP1. The results of our study suggest that attempts to identify novel redox-regulated proteins and signal transduction pathways should focus on characterization of low pK(a) cysteines.

Synthesis of Chemical Probes to Map Sulfenic Acid Modifications on Proteins

Cysteine sulfenic acids in proteins can be identified by their ability to form adducts with dimedone, but this reagent imparts no spectral or affinity tag for subsequent analyses of such tagged proteins. Given its similar reactivity toward cysteine sulfenic acids, 1,3-cyclohexadione was synthetically modified to an alcohol derivative and linked to fluorophores based on isatoic acid and 7-methoxycoumarin. The resulting compounds retain full reactivity and specificity toward cysteine sulfenic acids in proteins, allowing for incorporation of the fluorescent label into the protein and "tagging" it based on its sulfenic acid redox state. Control experiments using dimedone further show the specificity of the reaction of 1,3-diones with protein sulfenic acids in aqueous media. These new compounds provide the basis for an improved method for the detection of protein sulfenic acids.