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Regulation of antioxidant metabolism by translation initiation factor 2alpha.

Tan S, Somia N, Maher P, Schubert D - J. Cell Biol. (2001)

Bottom Line: The phosphorylation of eIF2alpha also results in resistance to oxidative stress.In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+).Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Cellular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

ABSTRACT
Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stress-induced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2alpha and express a low amount of eIF2alpha. Sensitivity is restored when the clones are transfected with full-length eIF2alpha; transfection of wild-type cells with the truncated eIF2alpha gene confers resistance. The phosphorylation of eIF2alpha also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+). In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca(2+) when stressed, and the GSH synthetic enzyme gamma-glutamyl cysteine synthetase (gammaGCS) is elevated. The change in gammaGCS is regulated by a translational mechanism. Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

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HT22 cells become glutamate resistant when the eIF2α S51D mutant is stably expressed. HT22 cells were infected with virus containing either the pCLBABEpuro empty vector (♦), the S51A mutant of eIF2α (-○), or the eIF2α S51D mutant (▴). Cell viability was measured by the MTT assay. Samples were prepared in triplicate (n = 4).
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Figure 3: HT22 cells become glutamate resistant when the eIF2α S51D mutant is stably expressed. HT22 cells were infected with virus containing either the pCLBABEpuro empty vector (♦), the S51A mutant of eIF2α (-○), or the eIF2α S51D mutant (▴). Cell viability was measured by the MTT assay. Samples were prepared in triplicate (n = 4).

Mentions: To confirm that the loss of eIF2α activity is linked to glutamate resistance, a second method was employed which utilizes a dominant negative approach to regulate eIF2α function. The phosphorylated form of eIF2α sequesters the guanine nucleotide exchange factor, eIF2β, resulting in a decrease in protein translation (Ernst et al. 1987). The S51D mutant of eIF2α mimics constitutive phosphorylation when serine 51 in eIF2α is replaced with an aspartic acid (Kaufman et al. 1989). The S51A mutant cannot be phosphorylated when serine 51 in eIF2α is replaced with alanine (Pathak et al. 1988). Thus, the S51D mutant inhibits protein synthesis while the S51A mutant prevents the shutdown of protein translation by the phosphorylation of eIF2α. To assay the effect of eIF2α phosphorylation on glutamate sensitivity, wild-type HT22 cells were infected with virus that contained either the S51D or S51A mutant or an empty vector, and the cells were tested for glutamate resistance. HT22 cells infected with virus containing the mutant S51D become more resistant to glutamate (Fig. 3). The S51A mutant of eIF2α did not have any effect on the response of the cells to glutamate relative to empty vector (Fig. 3). These data show that the downregulation of eIF2α activity by protein phosphorylation can lead to glutamate resistance and that eIF2α phosphorylation may play an important role in cell death or survival after glutamate exposure. However, we could not directly assay eIF2α phosphorylation after glutamate exposure because none of the available antibodies immunoprecipitate or distinguish phosphorylated from unphosphorylated eIF2α in HT22 cells.


Regulation of antioxidant metabolism by translation initiation factor 2alpha.

Tan S, Somia N, Maher P, Schubert D - J. Cell Biol. (2001)

HT22 cells become glutamate resistant when the eIF2α S51D mutant is stably expressed. HT22 cells were infected with virus containing either the pCLBABEpuro empty vector (♦), the S51A mutant of eIF2α (-○), or the eIF2α S51D mutant (▴). Cell viability was measured by the MTT assay. Samples were prepared in triplicate (n = 4).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198799&req=5

Figure 3: HT22 cells become glutamate resistant when the eIF2α S51D mutant is stably expressed. HT22 cells were infected with virus containing either the pCLBABEpuro empty vector (♦), the S51A mutant of eIF2α (-○), or the eIF2α S51D mutant (▴). Cell viability was measured by the MTT assay. Samples were prepared in triplicate (n = 4).
Mentions: To confirm that the loss of eIF2α activity is linked to glutamate resistance, a second method was employed which utilizes a dominant negative approach to regulate eIF2α function. The phosphorylated form of eIF2α sequesters the guanine nucleotide exchange factor, eIF2β, resulting in a decrease in protein translation (Ernst et al. 1987). The S51D mutant of eIF2α mimics constitutive phosphorylation when serine 51 in eIF2α is replaced with an aspartic acid (Kaufman et al. 1989). The S51A mutant cannot be phosphorylated when serine 51 in eIF2α is replaced with alanine (Pathak et al. 1988). Thus, the S51D mutant inhibits protein synthesis while the S51A mutant prevents the shutdown of protein translation by the phosphorylation of eIF2α. To assay the effect of eIF2α phosphorylation on glutamate sensitivity, wild-type HT22 cells were infected with virus that contained either the S51D or S51A mutant or an empty vector, and the cells were tested for glutamate resistance. HT22 cells infected with virus containing the mutant S51D become more resistant to glutamate (Fig. 3). The S51A mutant of eIF2α did not have any effect on the response of the cells to glutamate relative to empty vector (Fig. 3). These data show that the downregulation of eIF2α activity by protein phosphorylation can lead to glutamate resistance and that eIF2α phosphorylation may play an important role in cell death or survival after glutamate exposure. However, we could not directly assay eIF2α phosphorylation after glutamate exposure because none of the available antibodies immunoprecipitate or distinguish phosphorylated from unphosphorylated eIF2α in HT22 cells.

Bottom Line: The phosphorylation of eIF2alpha also results in resistance to oxidative stress.In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+).Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

View Article: PubMed Central - PubMed

Affiliation: Cellular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

ABSTRACT
Oxidative stress and highly specific decreases in glutathione (GSH) are associated with nerve cell death in Parkinson's disease. Using an experimental nerve cell model for oxidative stress and an expression cloning strategy, a gene involved in oxidative stress-induced programmed cell death was identified which both mediates the cell death program and regulates GSH levels. Two stress-resistant clones were isolated which contain antisense gene fragments of the translation initiation factor (eIF)2alpha and express a low amount of eIF2alpha. Sensitivity is restored when the clones are transfected with full-length eIF2alpha; transfection of wild-type cells with the truncated eIF2alpha gene confers resistance. The phosphorylation of eIF2alpha also results in resistance to oxidative stress. In wild-type cells, oxidative stress results in rapid GSH depletion, a large increase in peroxide levels, and an influx of Ca(2+). In contrast, the resistant clones maintain high GSH levels and show no elevation in peroxides or Ca(2+) when stressed, and the GSH synthetic enzyme gamma-glutamyl cysteine synthetase (gammaGCS) is elevated. The change in gammaGCS is regulated by a translational mechanism. Therefore, eIF2alpha is a critical regulatory factor in the response of nerve cells to oxidative stress and in the control of the major intracellular antioxidant, GSH, and may play a central role in the many neurodegenerative diseases associated with oxidative stress.

Show MeSH
Related in: MedlinePlus