Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis

Lind, Christina; Gerdes, Robert; Hamnell, Ylva; Schuppe-Koistinen, Ina; Löwenhielm, Helena Brockenhuus von; Holmgren, Arne; Cotgreave, Ian A.

doi:10.1016/s0003-9861(02)00468-x

Cited by 304 publications

(243 citation statements)

References 35 publications

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“…Based on this scheme, the OxICAT method detects all reversibly oxidized cysteines, whereas higher oxidation states, such as sulfinic and sulfonic acids, will not be detected. However, substitution of the nonspecific thiol reductant TCEP with more specific reductants such as glutaredoxin or ascorbate will allow the use of OxICAT to specifically detect glutathionylations or nitrosylations, respectively (17,18).…”

Section: Resultsmentioning

confidence: 99%

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

Leichert

Gehrke

Gudiseva

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

481

542

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Antimicrobial levels of reactive oxygen species (ROS) are produced by the mammalian host defense to kill invading bacteria and limit bacterial colonization. One main in vivo target of ROS is the thiol group of proteins. We have developed a quantitative thiol trapping technique termed OxICAT to identify physiologically important target proteins of hydrogen peroxide (H 2O2) and hypochlorite (NaOCl) stress in vivo. OxICAT allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. It also identifies the affected proteins and defines their redox-sensitive cysteine(s). Using this technique, we identified a group of Escherichia coli proteins with significantly (30 -90%) oxidatively modified thiol groups, which appear to be specifically sensitive to either H 2O2 or NaOCl stress. These results indicate that individual oxidants target distinct proteins in vivo. Conditionally essential E. coli genes encode one-third of redoxsensitive proteins, a finding that might explain the bacteriostatic effect of oxidative stress treatment. We identified a select group of redox-regulated proteins, which protect E. coli against oxidative stress conditions. These experiments illustrate that OxICAT, which can be used in a variety of different cell types and organisms, is a powerful tool to identify, quantify, and monitor oxidative thiol modifications in vivo.chaperone ͉ proteomics ͉ thiol modification

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Section: Resultsmentioning

confidence: 99%

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

Leichert

Gehrke

Gudiseva

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

481

542

View full text Add to dashboard Cite

show abstract

“…This implies that the vast majority of PSSG should be found in secretory compartments. Previous methods for detection of PSSG require manipulation of the cell system by either preventing protein synthesis (23)(24)(25)(26) or by loading cells with biotin-labeled glutathione (27,28), which could affect the steady-state levels of PSSG. We have quantified the absolute basal levels of glutathionylation directly in acid quenched cells.…”

Section: Discussionmentioning

confidence: 99%

Quantifying the global cellular thiol–disulfide status

Hansen

Roth²,

Winther³

2009

Proc. Natl. Acad. Sci. U.S.A.

375

307

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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...

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“…In this endeavor, catalytically active Grx is used to reduce S-glutathionylated derivatives to the sylfhydryl group, followed by labeling of the newly reduced cysteine with a specific tag ( Figure 3, bottom panel). This approach has been used in proteomic studies and identified a number of proteins targeted by S-glutathionylation, in addition to identification of specific sites of S-glutathionylation [154,227]. Related approaches to detect protein S-glutathionylation include the use of an antibody directed against GSH, GSH affinity purification, glutathione-S-transferase overlay, or the use of cellular labeling strategies with tagged and cell permeable glutathione, such as glutathione ethyl ester which becomes incorporated into proteins following an oxidative event and therefore is useful to detect proteins that are targets for Sglutathionylation [110,[228][229][230].…”

Section: Methodologies and To Detect Reversible Cysteine Oxidations Imentioning

confidence: 99%