Protein Thiol Modifications Visualized In Vivo

Abstract

Thiol-disulfide interconversions play a crucial role in the chemistry of biological systems. They participate in the major systems that control the cellular redox potential and prevent oxidative damage. In addition, thiol-disulfide exchange reactions serve as molecular switches in a growing number of redox-regulated proteins. We developed a differential thiol-trapping technique combined with two-dimensional gel analysis, which in combination with genetic studies, allowed us to obtain a snapshot of the in vivo thiol status of cellular proteins. We determined the redox potential of protein thiols in vivo, identified and dissected the in vivo substrate proteins of the major cellular thiol-disulfide oxidoreductases, and discovered proteins that undergo thiol modifications during oxidative stress. Under normal growth conditions most cytosolic proteins had reduced cysteines, confirming existing dogmas. Among the few partly oxidized cytosolic proteins that we detected were proteins that are known to form disulfide bond intermediates transiently during their catalytic cycle (e.g., dihydrolipoyl transacetylase and lipoamide dehydrogenase). Most proteins with highly oxidized thiols were periplasmic proteins and were found to be in vivo substrates of the disulfide-bond-forming protein DsbA. We discovered a substantial number of redox-sensitive cytoplasmic proteins, whose thiol groups were significantly oxidized in strains lacking thioredoxin A. These included detoxifying enzymes as well as many metabolic enzymes with active-site cysteines that were not known to be substrates for thioredoxin. H2O2-induced oxidative stress resulted in the specific oxidation of thiols of proteins involved in detoxification of H2O2 and of enzymes of cofactor and amino acid biosynthesis pathways such as thiolperoxidase, GTP-cyclohydrolase I, and the cobalamin-independent methionine synthase MetE. Remarkably, a number of these proteins were previously or are now shown to be redox regulated.

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

“…To quantitatively describe the extent of oxidative thiol modifications in proteins, we combined a mass spectrometric global approach using isotope coded affinity tag (ICAT) technology (14-16) with our previously established differential thiol trapping technique (10). Both methods rely on the rapid and irreversible modification of accessible cysteines in proteins with the thiol-trapping reagent iodoacetamide (IAM).…”

“…The recent interest in redox-regulated proteins fueled by the increasing recognition of their important physiological roles however, led to the development of several global thiol trapping techniques (10)(11)(12)(13). These methods, albeit capable of detecting proteins with redox-sensitive cysteines in lysates and intact cells, often lack the ability to directly identify the proteins or cysteines involved, and more importantly, to quantify the extent of oxidative thiol modifications.…”

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…The subsequent addition of reductant coupled to the use of a thiol-specific labeling agent such as biotinylated IAA or biotinylated NEM will then identify the oxidized Cys (Figure 3, top panel) [47]. This methodology, also referred to as "the thiol trapping technique" [220] has been successfully applied to cells in culture and demonstrated reversible oxidation and inactivation of tyrosine phosphatases in response to stimulation with growth factor receptors [29,116,117,219]. A similar strategy has been developed to detect Snitrosylated proteins, widely known as the "biotin switch method" (Figure 3, middle panel) [56,221].…”

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.…”