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

Deepthi Menon,Philip G Board

doi:10.1074/jbc.m113.487785

Deepthi Menon, Philip G Board

Open Access

https://doi.org/10.1074/jbc.m113.487785

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Abstract

The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.

Highlights

Glutathionylation is a major post-translational modification that regulates protein function
Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways
A comparison of the structures of GSTO1, GSTO2, CLIC2, and glutaredoxin shown in Fig. 6 reveals several conserved features including their thioredoxin-like domains and a glutathione binding site where glutathione can form a disulfide bond with a conserved active site cysteine residue positioned near the amino-terminal end of helix 1

Summary

Background

Glutathionylation is a major post-translational modification that regulates protein function. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways. The active site of GSTO1 is positioned in a wide crevice that can potentially accommodate large substrates such as proteins [14] Since their first description, the Omega class GSTs have been investigated in relation to a number of biologically significant pathways and clinical disorders including drug resistance [21], Alzheimer disease [22,23,24], Parkinson disease [25], vascular dementia and stroke [26, 27], and amyotrophic lateral sclerosis [28], the action of anti-inflammatory drugs [29], the disposition

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EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION