The Saccharomyces cerevisiae Proteome of Oxidized Protein Thiols

Abstract

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. In one 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 (ϳ200), 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.The amino acid cysteine subserves important biological functions due to the unique redox properties of the sulfur atom of its thiol side chain (1). By engaging in a wide variety of redox reactions and coordinating metals, cysteine serves as a key residue in enzyme catalysis, protein oxidative folding (2-4) and trafficking (5), and redox signaling and regulation (6 -8). However, these unique redox properties also make the cysteine residue vulnerable to reaction with a wide spectrum of non-physiological electrophiles, especially the reactive oxygen and nitrogen species, potentially leading to unwanted redox modifications and protein loss of function (9).The cysteine residue exists in vivo in the fully reduced free thiol form (-SH or -S Ϫ ) and in different oxidation forms: the thiyl radical (-S ⅐ ); the disulfide bond (Cys-S-S-Cys); the sulfenic (-SOH), sulfinic (SO 2 H), and sulfonic (-SO 3 H) acid forms; and the S-nitrosylated form (-S-NO) (1, 7, 10). The cysteine thiyl radical and cysteine sulfenic acid are very unstable because of their highly reactive nature and thus cannot be easily identified biochemically. In contrast, the cysteine sulfinic and sulfonic acids are irreversible forms of protein oxidation, although the cysteine sulfinic acid that forms in the peroxide-reducing enzyme peroxiredoxin is enzymatically retroreduced by sulfiredoxin (11, 12). Disulfide bonds are relatively stable, reversing to the reduced state by thioldisulfide exchange with kinetics depending on the protein context (10). Their occurrence is thought to be restricted to specific subcellular compartments. In the endoplasmic reticulum, disulfide bond formation drives the correct folding of secreted proteins and is catalyzed by a FAD-containing sulfhydryl oxidase (Ero1) and protein-disulfide isomerase ...

Quantifying changes in the thiol redox proteome upon oxidative stress in vivo

“…4) and concluded from these results that E. coli cells are exposed to a considerable amount of ROS during aerobic growth. This observation agrees with earlier reports in both E. coli (10) and yeast cells (22) and provides an excellent explanation for the essential roles that the thioredoxin and glutaredoxin systems play in maintaining the reducing character of pro-and eukaryotic cytoplasm (23).…”

“…This mechanism might contribute to the Pasteur effect in bacteria, which describes the influence of oxygen on the glucose metabolism (27). Similar observations have recently also been made in aerobically growing yeast cells, where a large proportion of glycolytic enzymes including GapA were shown to be oxidatively modified in vivo (22).…”

“…It is via reversible thiol modifications that transcriptional (e.g., OxyR, Yap1) and posttranslational (e.g., Hsp33, Prx-2) responses are triggered and that physiological processes (e.g., GapA) start to change during oxidative stress conditions (9). To identify target proteins that use redoxsensitive thiol groups to modulate their protein activity, the monitoring of relative changes in the overall thiol oxidation state (10,11,13,15,22) or the selective identification of proteins with distinct thiol modifications (12, 37) is an invaluable approach. Previously established methods, however, were unable to directly quantify the extent of thiol-modified protein and identify the cysteines affected.…”

Swapping of the N‐terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti‐aging features to the cell

“…In another systematic study focused onto the oxidation of protein thiols in yeast, the oxidized proteome of unstressed cells was identified [44]. In this survey, among 200 mostly cytoplasmatic proteins, cysteines were reported to be oxidized at a various extent.…”

Cellular Defenses against Superoxide and Hydrogen Peroxide

“…In bacteria and yeast, where Gpx is a minor scavenger or absent altogether, the evidence of protein thiol oxidation by physiological doses of H 2 O 2 is not strong. High H 2 O 2 doses have typically been employed in proteomics studies that detect oxidized proteins (121,122). One wonders, then, whether glutaredoxins and thioredoxins are actually needed only when H 2 O 2 stress is of such long duration as to compensate for the sluggish reactivity of protein thiols; whether they repair a small cohort of extremely reactive thiolate enzymes that have so far escaped detection; whether thiolate oxidations are primarily driven by reactions with oxygen or hydroxyl radicals, rather than H 2 O 2 ; or whether disulfide stress arises in natural environments from a different types of stressor, mimicked by thiol agents such as diamide.…”