Quantifying the global cellular thiol–disulfide status

Abstract

It is widely accepted that the redox status of protein thiols is of central importance to protein structure and folding and that glutathione is an important low-molecular-mass redox regulator. However, the total cellular pools of thiols and disulfides and their relative abundance have never been determined. In this study, we have assembled a global picture of the cellular thiol-disulfide status in cultured mammalian cells. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated protein (PSSG) in all cellular protein, including membrane proteins. These data were combined with quantification of reduced and oxidized glutathione in the same cells. Of the total protein cysteines, 6% and 9.6% are engaged in disulfide bond formation in HEK and HeLa cells, respectively. Furthermore, the steady-state level of PSSG is <0.1% of the total protein cysteines in both cell types. However, when cells are exposed to a sublethal dose of the thiol-specific oxidant diamide, PSSG levels increase to >15% of all protein cysteine. Glutathione is typically characterized as the ''cellular redox buffer''; nevertheless, our data show that protein thiols represent a larger active redox pool than glutathione. Accordingly, protein thiols are likely to be directly involved in the cellular defense against oxidative stress.cysteine ͉ glutathione ͉ protein B ecause of the thiol group (SH), cysteine is the most chemically reactive natural amino acid found in cells. SH is mainly found in proteins (PSH) and in low-molecular-mass metabolites such as the highly abundant glutathione (GSH). Two SH groups can be oxidized to form a disulfide bond, and, in the cell, disulfides are mainly found in proteins (PSSP), in glutathione disulfide (GSSG), or as mixed disulfides between protein and glutathione (PSSG). In addition, SH groups can be reversibly oxidized by reactive oxygen species to nitrosothiols or sulfenic acids. Higher oxidation states, such as sulfinic and sulfonic acids, are generally considered to be irreversible.Disulfides are important for protein structure and folding, and formation of disulfide bonds can regulate protein function (1). Furthermore, glutathionylation, here used to denote the formation of PSSG, has been implicated as a mechanism for protection against irreversible protein oxidation during oxidative stress (2).The cytosolic glutathione pool is highly reducing (3) and consequently, disulfide bond formation is infrequent and typically only takes place transiently during catalysis of redox processes. In contrast, disulfide bond formation is favored in other compartments of the cell, such as the endoplasmic reticulum (ER) and the intermembrane space of mitochondria (4). The overall distribution of thiols and disulfides in different cellular pools defines the global thiol-disulfide redox status of the cell. Maintenance of thioldisulfide redox homeostasis in different subcellular compartments is important, and failure to do so can be detrimental to the structure, stability and activity of variou...

Cocaine-Induced Adaptations in Cellular Redox Balance Contributes to Enduring Behavioral Plasticity

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