Protein-thiol oxidation, from single proteins to proteome-wide analyses.

Protein-thiol oxidation subserves multiple biological functions, from enzymatic catalysis to protein oxidative folding, protein trafficking, reactive oxygen (ROS) and nitrogen (RNS) species sensing and signaling and, more generally, protein redox regulation. Protein-thiol oxidation may also constitute a sequel of ROS and RNS toxicity. Accurate and robust methods aimed at monitoring the in vivo redox state of cysteine residues are thus warranted. To this aim, we have developed biochemical approaches that rely on trapping cysteine residues in their in vivo redox state using acidic conditions, followed by the differential labeling of reduced versus oxidized cysteine residues by thiol-specific reagents. These methods have been instrumental in the discovery of eukaryotic peroxide receptors and new ROS-scavenging enzymes and in identifying the repertoire of cytoplasmic oxidized protein thiols. Proteome-wide approaches also contributed to establish the functions of the thioredoxin and glutathione pathways in eukaryotic cytoplasmic thiol-redox control.

The system biology of thiol redox system in Escherichia coli and yeast: differential functions in oxidative stress, iron metabolism and DNA synthesis.

By its ability to engage in a variety of redox reactions and coordinating metals, cysteine serves as a key residue in mediating enzymatic catalysis, protein oxidative folding and trafficking, and redox signaling. The thiol redox system, which consists of the glutathione and thioredoxin pathways, uses the cysteine residue to catalyze thiol-disulfide exchange reactions, thereby controlling the redox state of cytoplasmic cysteine residues and regulating the biological functions it subserves. Here, we consider the thiol redox systems of Escherichia coli and Saccharomyces cerevisiae, emphasizing the role of genetic approaches in the understanding of the cellular functions of these systems. We show that although prokaryotic and eukaryotic systems have a similar architecture, they profoundly differ in their overall cellular functions.

The Saccharomyces cerevisiae proteome of oxidized protein thiols: contrasted functions for the thioredoxin and glutathione pathways.

Protein thiol oxidation subserves important biological functions and constitutes a sequel of reactive oxygen species toxicity. We developed two distinct thiol-labeling approaches to identify oxidized cytoplasmic protein thiols in Saccharomyces cerevisiae. Inone approach, we used N-(6-(biotinamido)hexyl)-3'-(2'-pyridyldithio)-propionamide to purify oxidized protein thiols, and in the other, we used N-[(14)C]ethylmaleimide to quantify this oxidation. Both approaches showed a large number of the same proteins with oxidized thiols ( approximately 200), 64 of which were identified by mass spectrometry. We show that, irrespective of its mechanism, protein thiol oxidation is dependent upon molecular O(2). We also show that H(2)O(2) does not cause de novo protein thiol oxidation, but rather increases the oxidation state of a select group of proteins. Furthermore, our study reveals contrasted differences in the oxidized proteome of cells upon inactivation of the thioredoxin or GSH pathway suggestive of very distinct thiol redox control functions, assigning an exclusive role for thioredoxin in H(2)O(2) metabolism and the presumed thiol redox buffer function for GSH. Taken together, these results suggest the high selectivity of cytoplasmic protein thiol oxidation.

Two redox centers within Yap1 for H2O2 and thiol-reactive chemicals signaling.

The Yap1 transcription factor regulates yeast responses to H2O2 and to several unrelated chemicals and metals. Activation by H2O2 involves Yap1 Cys303-Cys598 intra-molecular disulfide bond formation directed by the H2O2 sensor Orp1/Gpx3. We show here that the electrophile N-ethylmaleimide activates Yap1 by covalent modification of Yap1 C-terminal Cys598, Cys620, and Cys629, in an Orp1 and Yap1-oxidation-independent way, thus establishing an alternate and distinct mode of Yap1 activation. We also show that menadione, a superoxide anion generator and a highly reactive electrophile, operates both modes of Yap1 activation. Further, the Yap1 C-terminal domain reactivity towards other electrophiles (4-hydroxynonenal, iodoacetamide) and metals (cadmium, selenium) suggests a common mechanism for sensing thiol reactive chemicals, involving thiol chemical modification. We propose that Yap1 has two distinct molecular redox centers, one triggered by ROS (hydroperoxides and the superoxide anion) and the other by chemicals with thiol reactivity (electrophiles and divalent heavy metals cations). These data indicate that yeast cells cannot sense these compounds through the same molecular devices, albeit they are all electrophilic.