The effect of oxidant and the non-oxidant alteration of cellular thiol concentration on the formation of protein mixed-disulfides in HEK 293 cells.

Cellular molecules possess various mechanisms in responding to oxidant stress. In terms of protein responses, protein S-glutathionylation is a unique post-translational modification of protein reactive cysteines forming disulfides with glutathione molecules. This modification has been proposed to pl...

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Autores principales:	Jasen Lee Gilge, Michael Fisher, Yuh-Cherng Chai
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2008
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b16eac69d54e4fbf9eb3347fafc7204a
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spelling	oai:doaj.org-article:b16eac69d54e4fbf9eb3347fafc7204a2021-11-25T06:18:02ZThe effect of oxidant and the non-oxidant alteration of cellular thiol concentration on the formation of protein mixed-disulfides in HEK 293 cells.1932-620310.1371/journal.pone.0004015https://doaj.org/article/b16eac69d54e4fbf9eb3347fafc7204a2008-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19107210/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Cellular molecules possess various mechanisms in responding to oxidant stress. In terms of protein responses, protein S-glutathionylation is a unique post-translational modification of protein reactive cysteines forming disulfides with glutathione molecules. This modification has been proposed to play roles in antioxidant, regulatory and signaling in cells under oxidant stress. Recently, the increased level of protein S-glutathionylation has been linked with the development of diseases. In this report, specific S-glutathionylated proteins were demonstrated in human embryonic kidney 293 cells treated with two different oxidative reagents: diamide and hydrogen peroxide. Diamide is a chemical oxidizing agent whereas hydrogen peroxide is a physiological oxidant. Under the experimental conditions, these two oxidants decreased glutathione concentration without toxicity. S-glutathionylated proteins were detected by immunoblotting and glutathione concentrations were determined by high performance liquid chromatography. We further show the effect of alteration of the cellular thiol pool on the amount of protein S-glutathionylation in oxidant-treated cells. Cellular thiol concentrations were altered either by a specific way using buthionine sulfoximine, a specific inhibitor of glutathione biosynthesis or by a non-specific way, incubating cells in cystine-methionine deficient media. Cells only treated with either buthionine sulfoximine or cystine-methionine deficient media did not induce protein S-glutathionylation, even though both conditions decreased 65% of cellular glutathione. Moreover, the amount of protein S-glutathionylation under both conditions in the presence of oxidants was not altered when compared to the amount observed in regular media with oxidants present. Protein S-glutathionylation is a dynamic reaction which depends on the rate of adding and removing glutathione. Phenylarsine oxide, which specifically forms a covalent adduct with vicinal thiols, was used to determine the possible role of vicinal thiols in the amount of glutathionylation. Our data shows phenylarsine oxide did not change glutathione concentrations, but it did enhance the amount of glutathionylation in oxidant-treated cells.Jasen Lee GilgeMichael FisherYuh-Cherng ChaiPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 3, Iss 12, p e4015 (2008)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jasen Lee Gilge Michael Fisher Yuh-Cherng Chai The effect of oxidant and the non-oxidant alteration of cellular thiol concentration on the formation of protein mixed-disulfides in HEK 293 cells.
description	Cellular molecules possess various mechanisms in responding to oxidant stress. In terms of protein responses, protein S-glutathionylation is a unique post-translational modification of protein reactive cysteines forming disulfides with glutathione molecules. This modification has been proposed to play roles in antioxidant, regulatory and signaling in cells under oxidant stress. Recently, the increased level of protein S-glutathionylation has been linked with the development of diseases. In this report, specific S-glutathionylated proteins were demonstrated in human embryonic kidney 293 cells treated with two different oxidative reagents: diamide and hydrogen peroxide. Diamide is a chemical oxidizing agent whereas hydrogen peroxide is a physiological oxidant. Under the experimental conditions, these two oxidants decreased glutathione concentration without toxicity. S-glutathionylated proteins were detected by immunoblotting and glutathione concentrations were determined by high performance liquid chromatography. We further show the effect of alteration of the cellular thiol pool on the amount of protein S-glutathionylation in oxidant-treated cells. Cellular thiol concentrations were altered either by a specific way using buthionine sulfoximine, a specific inhibitor of glutathione biosynthesis or by a non-specific way, incubating cells in cystine-methionine deficient media. Cells only treated with either buthionine sulfoximine or cystine-methionine deficient media did not induce protein S-glutathionylation, even though both conditions decreased 65% of cellular glutathione. Moreover, the amount of protein S-glutathionylation under both conditions in the presence of oxidants was not altered when compared to the amount observed in regular media with oxidants present. Protein S-glutathionylation is a dynamic reaction which depends on the rate of adding and removing glutathione. Phenylarsine oxide, which specifically forms a covalent adduct with vicinal thiols, was used to determine the possible role of vicinal thiols in the amount of glutathionylation. Our data shows phenylarsine oxide did not change glutathione concentrations, but it did enhance the amount of glutathionylation in oxidant-treated cells.
format	article
author	Jasen Lee Gilge Michael Fisher Yuh-Cherng Chai
author_facet	Jasen Lee Gilge Michael Fisher Yuh-Cherng Chai
author_sort	Jasen Lee Gilge
title	The effect of oxidant and the non-oxidant alteration of cellular thiol concentration on the formation of protein mixed-disulfides in HEK 293 cells.
title_short	The effect of oxidant and the non-oxidant alteration of cellular thiol concentration on the formation of protein mixed-disulfides in HEK 293 cells.
title_full	The effect of oxidant and the non-oxidant alteration of cellular thiol concentration on the formation of protein mixed-disulfides in HEK 293 cells.
title_fullStr	The effect of oxidant and the non-oxidant alteration of cellular thiol concentration on the formation of protein mixed-disulfides in HEK 293 cells.
title_full_unstemmed	The effect of oxidant and the non-oxidant alteration of cellular thiol concentration on the formation of protein mixed-disulfides in HEK 293 cells.
title_sort	effect of oxidant and the non-oxidant alteration of cellular thiol concentration on the formation of protein mixed-disulfides in hek 293 cells.
publisher	Public Library of Science (PLoS)
publishDate	2008
url	https://doaj.org/article/b16eac69d54e4fbf9eb3347fafc7204a
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The effect of oxidant and the non-oxidant alteration of cellular thiol concentration on the formation of protein mixed-disulfides in HEK 293 cells.

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