Analysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates

Esha Sircar; Detcho A. Stoyanovsky; Timothy R. Billiar; Arne Holmgren; Rajib Sengupta

doi:10.1016/j.niox.2021.10.008

Analysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates

Esha Sircar, Detcho A. Stoyanovsky, Timothy R. Billiar, Arne Holmgren, Rajib Sengupta^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

10 Scopus citations

Abstract

The intracellular concentration of reduced glutathione (GSH) lies in the range of 1–10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1-SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).

Original language	English
Pages (from-to)	26-30
Number of pages	5
Journal	Nitric Oxide - Biology and Chemistry
Volume	118
DOIs	https://doi.org/10.1016/j.niox.2021.10.008
State	Published - 1 Jan 2022
Externally published	Yes

Keywords

Denitrosylation
Glutaredoxin
Glutathione
Nitric oxide
S-nitrosylation
Thioredoxin

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10.1016/j.niox.2021.10.008

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title = "Analysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates",

abstract = "The intracellular concentration of reduced glutathione (GSH) lies in the range of 1–10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1-SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).",

keywords = "Denitrosylation, Glutaredoxin, Glutathione, Nitric oxide, S-nitrosylation, Thioredoxin",

author = "Esha Sircar and Stoyanovsky, {Detcho A.} and Billiar, {Timothy R.} and Arne Holmgren and Rajib Sengupta",

note = "Publisher Copyright: {\textcopyright} 2021 Elsevier Inc.",

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doi = "10.1016/j.niox.2021.10.008",

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AU - Sircar, Esha

AU - Stoyanovsky, Detcho A.

AU - Billiar, Timothy R.

AU - Holmgren, Arne

AU - Sengupta, Rajib

PY - 2022/1/1

Y1 - 2022/1/1

N2 - The intracellular concentration of reduced glutathione (GSH) lies in the range of 1–10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1-SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).

AB - The intracellular concentration of reduced glutathione (GSH) lies in the range of 1–10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1-SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).

KW - Denitrosylation

KW - Glutaredoxin

KW - Glutathione

KW - Nitric oxide

KW - S-nitrosylation

KW - Thioredoxin

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U2 - 10.1016/j.niox.2021.10.008

DO - 10.1016/j.niox.2021.10.008

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AN - SCOPUS:85118563501

SN - 1089-8603

VL - 118

SP - 26

EP - 30

JO - Nitric Oxide - Biology and Chemistry

JF - Nitric Oxide - Biology and Chemistry

ER -

Analysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates

Abstract

Keywords

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