A fluorometric method to quantify protein glutathionylation using glutathione derivatization with 2,3-naphthalenedicarboxaldehyde

Menon, Deepthi; Board, Philip G.

doi:10.1016/j.ab.2012.10.009

Cited by 23 publications

(20 citation statements)

References 31 publications

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“…To get a first insight if trypanosomes undergo protein S -thiolation at all, BS T. brucei were treated with oxidative stressors and total protein S -thiolation was measured after labeling the released thiols with 2,3-naphthalene dicarboxaldehyde (NDA) ( 44 ).…”

Section: Resultsmentioning

confidence: 99%

Stress-Induced ProteinS-Glutathionylation andS-Trypanothionylation in African Trypanosomes—A Quantitative Redox Proteome and Thiol Analysis

Ulrich

Finkenzeller

Merker

et al. 2017

Antioxidants & Redox Signaling

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Aims: Trypanosomatids have a unique trypanothione-based thiol redox metabolism. The parasite-specific dithiol is synthesized from glutathione and spermidine, with glutathionylspermidine as intermediate catalyzed by trypanothione synthetase. In this study, we address the oxidative stress response of African trypanosomes with special focus on putative protein S-thiolation.Results: Challenging bloodstream Trypanosoma brucei with diamide, H2O2 or hypochlorite results in distinct levels of reversible overall protein S-thiolation. Quantitative proteome analyses reveal 84 proteins oxidized in diamide-stressed parasites. Fourteen of them, including several essential thiol redox proteins and chaperones, are also enriched when glutathione/glutaredoxin serves as a reducing system indicating S-thiolation. In parasites exposed to H2O2, other sets of proteins are modified. Only three proteins are S-thiolated under all stress conditions studied in accordance with a highly specific response. H2O2 causes primarily the formation of free disulfides. In contrast, in diamide-treated cells, glutathione, glutathionylspermidine, and trypanothione are almost completely protein bound. Remarkably, the total level of trypanothione is decreased, whereas those of glutathione and glutathionylspermidine are increased, indicating partial hydrolysis of protein-bound trypanothione. Depletion of trypanothione synthetase exclusively induces protein S-glutathionylation. Total mass analyses of a recombinant peroxidase treated with T(SH)2 and either diamide or hydrogen peroxide verify protein S-trypanothionylation as stable modification.Innovation: Our data reveal for the first time that trypanosomes employ protein S-thiolation when exposed to exogenous and endogenous oxidative stresses and trypanothione, despite its dithiol character, forms protein-mixed disulfides.Conclusion: The stress-specific responses shown here emphasize protein S-trypanothionylation and S-glutathionylation as reversible protection mechanism in these parasites. Antioxid. Redox Signal. 27, 517–533.

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Section: Resultsmentioning

confidence: 99%

Stress-Induced ProteinS-Glutathionylation andS-Trypanothionylation in African Trypanosomes—A Quantitative Redox Proteome and Thiol Analysis

Ulrich

Finkenzeller

Merker

et al. 2017

Antioxidants & Redox Signaling

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“…Total Protein Glutathionylation Assay-Protein glutathionylation was determined by a previously described method (44) Cells were lysed in a non-reducing buffer (0.5 M Tris, 300 mM NaCl, 1% Nonidet P-40, 50 mM N-ethylmaleimide, 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride), and the protein concentration was determined using a BCA protein estimation kit according to manufacturer's instructions (Pierce). Cellular proteins (100 g) were precipitated in ice-cold acetone at Ϫ20°C for Ͼ2h.…”

Section: Methodsmentioning

confidence: 99%

“…These results suggest that under these conditions GSTO1-1 acts primarily as a deglutathionylating enzyme. (54,55), it has previously been shown to strongly induce glutathionylation in cultured cells (44,56). In this study we examined the time course of intracellular glutathionylation after treatment with 1 mM GSNO and compared T47-D cells transfected with either an empty pCDNA3 plasmid vector, cells transfected with pCDNA3-GSTO1C32A, or cells transfected with pCDNA3-GSTO1.…”

Section: Deglutathionylation Of Actin By Gsto1-1 Influences Its Physimentioning

confidence: 99%

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

Menon

Board

2013

Journal of Biological Chemistry

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122

132

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Background: Glutathionylation is a major post-translational modification that regulates protein function. Results: Human glutathione transferase Omega 1 (GSTO1-1) can catalyze the deglutathionylation of protein thiols in vitro and in cell culture. Conclusion: GSTO1-1, but not GSTO2-2, catalyzes the deglutathionylation of actin and other proteins under physiological conditions. Significance: Genetic variation in GSTO1-1-catalyzed deglutathionylation may influence the function of multiple proteins in signaling pathways.

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“…This method can also be used to monitor antioxidant therapy, as demonstrated recently in children with mitochondrial encephalomyopathies [40]. Using 2,3-naphthalenedicaboxaldehyde as the fluorescence probe and direct fluorescence measurement, Menon and Board [41] quantitatively assayed the total content of S -glutathionylated proteins in several mouse tissues treated with the anti-cancer drug, doxorubicin, and in a lymphoblastoid cell line before and after a pro-oxidant stimulus. Their method allows a rapid and simple quantification of changes in the level of protein glutathionylation in response to oxidative stress and drug treatment.…”

Section: Tools and Strategies To Monitor Protein S-glutathionylationmentioning

confidence: 99%

Protein Glutathionylation in Cardiovascular Diseases

Pastore

Piemonte

2013

IJMS

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The perturbation of thiol-disulfide homeostasis is an important consequence of many diseases, with redox signals implicated in several physio-pathological processes. A prevalent form of cysteine modification is the reversible formation of protein mixed disulfides with glutathione (S-glutathionylation). The abundance of glutathione in cells and the ready conversion of sulfenic acids to S-glutathione mixed disulfides supports the reversible protein S-glutathionylation as a common feature of redox signal transduction, able to regulate the activities of several redox sensitive proteins. In particular, protein S-glutathionylation is emerging as a critical signaling mechanism in cardiovascular diseases, because it regulates numerous physiological processes involved in cardiovascular homeostasis, including myocyte contraction, oxidative phosphorylation, protein synthesis, vasodilation, glycolytic metabolism and response to insulin. Thus, perturbations in protein glutathionylation status may contribute to the etiology of many cardiovascular diseases, such as myocardial infarction, cardiac hypertrophy and atherosclerosis. Various reports show the importance of oxidative cysteine modifications in modulating cardiovascular function. In this review, we illustrate tools and strategies to monitor protein S-glutathionylation and describe the proteins so far identified as glutathionylated in myocardial contraction, hypertrophy and inflammation.

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A fluorometric method to quantify protein glutathionylation using glutathione derivatization with 2,3-naphthalenedicarboxaldehyde

Cited by 23 publications

References 31 publications

Stress-Induced ProteinS-Glutathionylation andS-Trypanothionylation in African Trypanosomes—A Quantitative Redox Proteome and Thiol Analysis

Stress-Induced ProteinS-Glutathionylation andS-Trypanothionylation in African Trypanosomes—A Quantitative Redox Proteome and Thiol Analysis

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

Protein Glutathionylation in Cardiovascular Diseases

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