Identification of proteins containing redox-sensitive thiols after PRDX1, PRDX3 and GCLC silencing and/or glucose oxidase treatment in Hepa 1-6 cells.

Abstract

The oxidation and reduction of cysteine thiols are thought to be a major mechanism for redox regulation. The aim of this study was to identify proteins with reactive thiols and determine their oxidation profiles under oxidative stress induced by simultaneous silencing of antioxidant defences (peroxiredoxin-1, peroxiredoxin-3, and the catalytic subunit of the glutamate-cysteine ligase), and/or treatment with glucose oxidase (GO). Using an approach that combined the labelling of reversibly oxidised cysteines, 2-DE protein separation and MS analysis, we identified 26 proteins with cysteines prone to reversible oxidation belonging to different functional classes. Among these proteins are those that have not been previously recognised as reversible oxidation targets, including cytoplasmic aspartate aminotransferase, proteasome subunit alpha type-6, heterogeneous nuclear ribonucleoproteins isoA2/B1 and A/B, and histidine triad nucleotide-binding protein 1. We provide the first evidence of reversible oxidation for specific cysteines, including Cys112 and Cys146 in glutamate dehydrogenase 1, Cys17 in actins, Cys5 in protein disulfide-isomerase A3, and Cys267 in the heat shock cognate 71 kDa protein. Silencing induced lower oxidative stress than GO treatment. Nevertheless, we detected some proteins particularly sensitive to oxidation by silencing. We hypothesised that these proteins may play a role in regulatory mechanisms by redox stress.

• Authors: Carlos A Fuentes-Almagro, María-José Prieto-Álamo, Carmen Pueyo, Juan Jurado
• Journal: Journal of proteomics (J Proteomics) Vol. 77 Pg. 262-79 (Dec 21 2012) ISSN: 1876-7737 [Electronic] Netherlands
• PMID: 22975676 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2012 Elsevier B.V. All rights reserved.

Chemical References

• Sulfhydryl Compounds
• Glucose Oxidase
• PRDX1 protein, human
• PRDX3 protein, human
• Peroxiredoxin III
• Peroxiredoxins
• Glutamate-Cysteine Ligase

Topics

• Catalytic Domain
• Cell Line
• Gene Expression Regulation (drug effects, genetics)
• Gene Silencing
• Glucose Oxidase (pharmacology)
• Glutamate-Cysteine Ligase
• Humans
• Oxidation-Reduction (drug effects)
• Peroxiredoxin III
• Peroxiredoxins
• Sulfhydryl Compounds (metabolism)