Limits...
γ-Glutamylcysteine detoxifies reactive oxygen species by acting as glutathione peroxidase-1 cofactor.

Quintana-Cabrera R, Fernandez-Fernandez S, Bobo-Jimenez V, Escobar J, Sastre J, Almeida A, Bolaños JP - Nat Commun (2012)

Bottom Line: Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases.In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment.Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Institute of Neurosciences of Castile and Leon, University of Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain.

ABSTRACT
Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases. Through its ability to down modulate reactive oxygen species, glutathione is considered an essential thiol-antioxidant derivative, yet under certain circumstances it is dispensable for cell growth and redox control. Here we show, by directing the biosynthesis of γ-glutamylcysteine-the immediate glutathione precursor-to mitochondria, that it efficiently detoxifies hydrogen peroxide and superoxide anion, regardless of cellular glutathione concentrations. Knocking down glutathione peroxidase-1 drastically increases superoxide anion in cells synthesizing mitochondrial γ-glutamylcysteine. In vitro, γ-glutamylcysteine is as efficient as glutathione in disposing of hydrogen peroxide by glutathione peroxidase-1. In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment. Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor.

Show MeSH

Related in: MedlinePlus

γ-Glutamylcysteine is a GPx1 cofactor detoxifying mitochondrial H2O2.(a) Expression of an shRNA against GCL (shGCL) in HEK293T cells efficiently knockdowns wild-type mitoGCL, but not a mutant form of mitoGCL (mitoGCL (mut)) that is refractory to shGCL. (b) Treatment of cells with rotenone (1 μM; 4 h) increased mitochondrial O2·–, as revealed by MitoSox fluorescence; this was further enhanced by GCL silencing (shGCL), and rescued by mitoGCL (mut) expression; GCL silencing in the absence of rotenone did not significantly modify mitochondrial O2·– in HEK293T cells (c) Western blots showing the efficacy of siRNA duplexes, used at 100 nM for 3 days in HEK293T cells, against glutathione synthetase (GSS), glutathione peroxidase-1 (GPx1), glutathione reductase (GSR) and superoxide dismutase-2 (SOD2); control siRNA (siControl) was an siRNA against luciferase. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as loading control. (d) Rotenone (1 μM; 4 h) increased mitochondrial O2·–, as judged by MitoSox fluorescence, in mitoGCL-expressing cells; this effect was abolished by GSS silencing and enhanced by GPx1 knockdown; silencing GSS did not rescue the increase in O2·– caused by GPx1 knockdown; GSR silencing did not alter, and SOD2 silencing enhanced rotenone-induced ROS. (e) γ-Glutamylcysteine (γGC) or GSH dose-dependently disposed H2O2in vitro, but the presence of GPx1 (0.5 U ml−1) in the incubation medium was necessary for H2O2 disposal, as judged by the lack of effect of 500 μM γGC or GSH in the absence of GPx1 (w/o GPx1). (f) H2O2 disposal by GPx1 was not potentiated by the presence of GSR (0.5 U ml−1) in the incubation medium when γGC (200 μM) was used (left panel); however, the presence of GSH (200 μM) potentiated GPx1-mediated H2O2 disposal (right panel). *P<0.05; #P<0.05 versus siControl (mitoGCL; analysis of variance; n=3 independent experiments); in vitro assays (e and f) were performed in sextuplicate. All data are expressed as mean±S.E.M.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3316877&req=5

f2: γ-Glutamylcysteine is a GPx1 cofactor detoxifying mitochondrial H2O2.(a) Expression of an shRNA against GCL (shGCL) in HEK293T cells efficiently knockdowns wild-type mitoGCL, but not a mutant form of mitoGCL (mitoGCL (mut)) that is refractory to shGCL. (b) Treatment of cells with rotenone (1 μM; 4 h) increased mitochondrial O2·–, as revealed by MitoSox fluorescence; this was further enhanced by GCL silencing (shGCL), and rescued by mitoGCL (mut) expression; GCL silencing in the absence of rotenone did not significantly modify mitochondrial O2·– in HEK293T cells (c) Western blots showing the efficacy of siRNA duplexes, used at 100 nM for 3 days in HEK293T cells, against glutathione synthetase (GSS), glutathione peroxidase-1 (GPx1), glutathione reductase (GSR) and superoxide dismutase-2 (SOD2); control siRNA (siControl) was an siRNA against luciferase. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as loading control. (d) Rotenone (1 μM; 4 h) increased mitochondrial O2·–, as judged by MitoSox fluorescence, in mitoGCL-expressing cells; this effect was abolished by GSS silencing and enhanced by GPx1 knockdown; silencing GSS did not rescue the increase in O2·– caused by GPx1 knockdown; GSR silencing did not alter, and SOD2 silencing enhanced rotenone-induced ROS. (e) γ-Glutamylcysteine (γGC) or GSH dose-dependently disposed H2O2in vitro, but the presence of GPx1 (0.5 U ml−1) in the incubation medium was necessary for H2O2 disposal, as judged by the lack of effect of 500 μM γGC or GSH in the absence of GPx1 (w/o GPx1). (f) H2O2 disposal by GPx1 was not potentiated by the presence of GSR (0.5 U ml−1) in the incubation medium when γGC (200 μM) was used (left panel); however, the presence of GSH (200 μM) potentiated GPx1-mediated H2O2 disposal (right panel). *P<0.05; #P<0.05 versus siControl (mitoGCL; analysis of variance; n=3 independent experiments); in vitro assays (e and f) were performed in sextuplicate. All data are expressed as mean±S.E.M.

Mentions: To decipher how γ-glutamylcysteine detoxified ROS but otherwise avoiding the influence of cytosolic GCL, we expressed a silent mutant mitoGCL form (mitoGCL (mut)) refractory to the action of a small hairpin RNA against GCL (shGCL)5, in HEK293T cells (Fig. 2a). Knocking down GCL markedly reduced total cellular GSH in both mitoGCL and mitoGCL (mut) transfected cells (Supplementary Fig. S1e). GCL knockdown was insufficient to trigger a significant increase in mitochondrial O2·–, but this was strongly potentiated by rotenone and rescued by mitoGCL (mut; Fig. 2b). Thus, in unstressed HEK293T cells, γ-glutamylcysteine may not contribute to the basal O2·– regulation that can occur in primary neurons, in which we previously reported a significant increase in O2·– by shGCL13. It therefore appears that the impact of γ-glutamylcysteine as a physiological redox regulator differs among cell types. Next, we knocked down GSS (Fig. 2c), which resulted in endogenous γ-glutamylcysteine accumulation and GSH decrease (Supplementary Table S1). We found that GSS knockdown abolished the increase in rotenone-induced O2·–, both in the absence (Supplementary Fig. S1f) and in the presence of mitoGCL (Fig. 2d).


γ-Glutamylcysteine detoxifies reactive oxygen species by acting as glutathione peroxidase-1 cofactor.

Quintana-Cabrera R, Fernandez-Fernandez S, Bobo-Jimenez V, Escobar J, Sastre J, Almeida A, Bolaños JP - Nat Commun (2012)

γ-Glutamylcysteine is a GPx1 cofactor detoxifying mitochondrial H2O2.(a) Expression of an shRNA against GCL (shGCL) in HEK293T cells efficiently knockdowns wild-type mitoGCL, but not a mutant form of mitoGCL (mitoGCL (mut)) that is refractory to shGCL. (b) Treatment of cells with rotenone (1 μM; 4 h) increased mitochondrial O2·–, as revealed by MitoSox fluorescence; this was further enhanced by GCL silencing (shGCL), and rescued by mitoGCL (mut) expression; GCL silencing in the absence of rotenone did not significantly modify mitochondrial O2·– in HEK293T cells (c) Western blots showing the efficacy of siRNA duplexes, used at 100 nM for 3 days in HEK293T cells, against glutathione synthetase (GSS), glutathione peroxidase-1 (GPx1), glutathione reductase (GSR) and superoxide dismutase-2 (SOD2); control siRNA (siControl) was an siRNA against luciferase. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as loading control. (d) Rotenone (1 μM; 4 h) increased mitochondrial O2·–, as judged by MitoSox fluorescence, in mitoGCL-expressing cells; this effect was abolished by GSS silencing and enhanced by GPx1 knockdown; silencing GSS did not rescue the increase in O2·– caused by GPx1 knockdown; GSR silencing did not alter, and SOD2 silencing enhanced rotenone-induced ROS. (e) γ-Glutamylcysteine (γGC) or GSH dose-dependently disposed H2O2in vitro, but the presence of GPx1 (0.5 U ml−1) in the incubation medium was necessary for H2O2 disposal, as judged by the lack of effect of 500 μM γGC or GSH in the absence of GPx1 (w/o GPx1). (f) H2O2 disposal by GPx1 was not potentiated by the presence of GSR (0.5 U ml−1) in the incubation medium when γGC (200 μM) was used (left panel); however, the presence of GSH (200 μM) potentiated GPx1-mediated H2O2 disposal (right panel). *P<0.05; #P<0.05 versus siControl (mitoGCL; analysis of variance; n=3 independent experiments); in vitro assays (e and f) were performed in sextuplicate. All data are expressed as mean±S.E.M.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3316877&req=5

f2: γ-Glutamylcysteine is a GPx1 cofactor detoxifying mitochondrial H2O2.(a) Expression of an shRNA against GCL (shGCL) in HEK293T cells efficiently knockdowns wild-type mitoGCL, but not a mutant form of mitoGCL (mitoGCL (mut)) that is refractory to shGCL. (b) Treatment of cells with rotenone (1 μM; 4 h) increased mitochondrial O2·–, as revealed by MitoSox fluorescence; this was further enhanced by GCL silencing (shGCL), and rescued by mitoGCL (mut) expression; GCL silencing in the absence of rotenone did not significantly modify mitochondrial O2·– in HEK293T cells (c) Western blots showing the efficacy of siRNA duplexes, used at 100 nM for 3 days in HEK293T cells, against glutathione synthetase (GSS), glutathione peroxidase-1 (GPx1), glutathione reductase (GSR) and superoxide dismutase-2 (SOD2); control siRNA (siControl) was an siRNA against luciferase. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as loading control. (d) Rotenone (1 μM; 4 h) increased mitochondrial O2·–, as judged by MitoSox fluorescence, in mitoGCL-expressing cells; this effect was abolished by GSS silencing and enhanced by GPx1 knockdown; silencing GSS did not rescue the increase in O2·– caused by GPx1 knockdown; GSR silencing did not alter, and SOD2 silencing enhanced rotenone-induced ROS. (e) γ-Glutamylcysteine (γGC) or GSH dose-dependently disposed H2O2in vitro, but the presence of GPx1 (0.5 U ml−1) in the incubation medium was necessary for H2O2 disposal, as judged by the lack of effect of 500 μM γGC or GSH in the absence of GPx1 (w/o GPx1). (f) H2O2 disposal by GPx1 was not potentiated by the presence of GSR (0.5 U ml−1) in the incubation medium when γGC (200 μM) was used (left panel); however, the presence of GSH (200 μM) potentiated GPx1-mediated H2O2 disposal (right panel). *P<0.05; #P<0.05 versus siControl (mitoGCL; analysis of variance; n=3 independent experiments); in vitro assays (e and f) were performed in sextuplicate. All data are expressed as mean±S.E.M.
Mentions: To decipher how γ-glutamylcysteine detoxified ROS but otherwise avoiding the influence of cytosolic GCL, we expressed a silent mutant mitoGCL form (mitoGCL (mut)) refractory to the action of a small hairpin RNA against GCL (shGCL)5, in HEK293T cells (Fig. 2a). Knocking down GCL markedly reduced total cellular GSH in both mitoGCL and mitoGCL (mut) transfected cells (Supplementary Fig. S1e). GCL knockdown was insufficient to trigger a significant increase in mitochondrial O2·–, but this was strongly potentiated by rotenone and rescued by mitoGCL (mut; Fig. 2b). Thus, in unstressed HEK293T cells, γ-glutamylcysteine may not contribute to the basal O2·– regulation that can occur in primary neurons, in which we previously reported a significant increase in O2·– by shGCL13. It therefore appears that the impact of γ-glutamylcysteine as a physiological redox regulator differs among cell types. Next, we knocked down GSS (Fig. 2c), which resulted in endogenous γ-glutamylcysteine accumulation and GSH decrease (Supplementary Table S1). We found that GSS knockdown abolished the increase in rotenone-induced O2·–, both in the absence (Supplementary Fig. S1f) and in the presence of mitoGCL (Fig. 2d).

Bottom Line: Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases.In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment.Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Institute of Neurosciences of Castile and Leon, University of Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain.

ABSTRACT
Reactive oxygen species regulate redox-signaling processes, but in excess they can cause cell damage, hence underlying the aetiology of several neurological diseases. Through its ability to down modulate reactive oxygen species, glutathione is considered an essential thiol-antioxidant derivative, yet under certain circumstances it is dispensable for cell growth and redox control. Here we show, by directing the biosynthesis of γ-glutamylcysteine-the immediate glutathione precursor-to mitochondria, that it efficiently detoxifies hydrogen peroxide and superoxide anion, regardless of cellular glutathione concentrations. Knocking down glutathione peroxidase-1 drastically increases superoxide anion in cells synthesizing mitochondrial γ-glutamylcysteine. In vitro, γ-glutamylcysteine is as efficient as glutathione in disposing of hydrogen peroxide by glutathione peroxidase-1. In primary neurons, endogenously synthesized γ-glutamylcysteine fully prevents apoptotic death in several neurotoxic paradigms and, in an in vivo mouse model of neurodegeneration, γ-glutamylcysteine protects against neuronal loss and motor impairment. Thus, γ-glutamylcysteine takes over the antioxidant and neuroprotective functions of glutathione by acting as glutathione peroxidase-1 cofactor.

Show MeSH
Related in: MedlinePlus