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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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γGCS protein expression is regulated at the level of translation. (A) γGCS, actin protein, and mRNA expression was measured in wild-type HT22 cells (lane 1), cells infected with the empty vector (pCLBABE) (lane 2), and the resistant clones 8 (lane 3) and 15 (lane 4) by Western and Northern blot analysis, respectively. (B) The Western blot from A was analyzed using the program NIH Image to determine the densities of each band. The densities were measured in four experiments, averaged, and normalized first to actin and then to the level of γGCS in pCLBABE, set as 1.0. Actin served as a loading control and showed that there was an equal amount of protein in each lane. Northern blots were quantitated on a PhosphorImager. The ratio of the catalytic subunit of γGCS to actin is presented normalized to γGCS in pCLBABE as 1.0. The results were confirmed by reverse transcription PCR analysis (data not shown). (C) Proteolytic breakdown of γGCS and P27 in wild-type and resistant cells. HT22 cells and resistant clone 15 were treated with 100 μg/ml cycloheximide and the amount of γGCS and P27 was quantitated by Western blot at 2-h intervals. The values are normalized to 0 time and are the mean ± SEM of triplicate experiments. Inset, Western blots of γGCS, wild-type cells (a); γGCS, clone 15 (b); P27, Cl 15 (c). Lanes 1, 2, 3, 4, and 5 are 0, 2, 4, 6, and 8 h after cycloheximide. x, γGCS, wild-type; •, γGCS Cl 15; ○, P27. (D) Resistant clones 8 and 15 were infected with wild-type eIF2α, the wild-type HT22 clone was infected with S51A or S51D, and the levels of γGCS and actin were determined by Western blotting. The amounts of γGCS and actin were quantitated and the amount of γGCS was normalized to the actin loading control. In each set of cells, the transfected cells were then normalized to γGCS in their parental line pCLBABE, resistant clone 8 or 15, which was set at 100%. The data are presented as the mean ± SEM, n = 4. *Significantly different from parental cells (P < 0.01); **significantly different from parental cells (P < 0.05).
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Figure 7: γGCS protein expression is regulated at the level of translation. (A) γGCS, actin protein, and mRNA expression was measured in wild-type HT22 cells (lane 1), cells infected with the empty vector (pCLBABE) (lane 2), and the resistant clones 8 (lane 3) and 15 (lane 4) by Western and Northern blot analysis, respectively. (B) The Western blot from A was analyzed using the program NIH Image to determine the densities of each band. The densities were measured in four experiments, averaged, and normalized first to actin and then to the level of γGCS in pCLBABE, set as 1.0. Actin served as a loading control and showed that there was an equal amount of protein in each lane. Northern blots were quantitated on a PhosphorImager. The ratio of the catalytic subunit of γGCS to actin is presented normalized to γGCS in pCLBABE as 1.0. The results were confirmed by reverse transcription PCR analysis (data not shown). (C) Proteolytic breakdown of γGCS and P27 in wild-type and resistant cells. HT22 cells and resistant clone 15 were treated with 100 μg/ml cycloheximide and the amount of γGCS and P27 was quantitated by Western blot at 2-h intervals. The values are normalized to 0 time and are the mean ± SEM of triplicate experiments. Inset, Western blots of γGCS, wild-type cells (a); γGCS, clone 15 (b); P27, Cl 15 (c). Lanes 1, 2, 3, 4, and 5 are 0, 2, 4, 6, and 8 h after cycloheximide. x, γGCS, wild-type; •, γGCS Cl 15; ○, P27. (D) Resistant clones 8 and 15 were infected with wild-type eIF2α, the wild-type HT22 clone was infected with S51A or S51D, and the levels of γGCS and actin were determined by Western blotting. The amounts of γGCS and actin were quantitated and the amount of γGCS was normalized to the actin loading control. In each set of cells, the transfected cells were then normalized to γGCS in their parental line pCLBABE, resistant clone 8 or 15, which was set at 100%. The data are presented as the mean ± SEM, n = 4. *Significantly different from parental cells (P < 0.01); **significantly different from parental cells (P < 0.05).

Mentions: Resistant clones 8 and 15 have decreased eIF2α activity and increased basal levels of GSH. Furthermore, the resistant clones and the cells expressing the phosphorylation mutant, S51D, maintain GSH levels 50% of their basal levels after glutamate exposure. To determine if there is a causal relationship between eIF2α protein levels and GSH production, the expression of the rate-limiting enzyme for GSH synthesis, γGCS, was examined in the wild-type cells and the resistant clones. Protein expression and mRNA levels of the catalytic subunit of γGCS were measured by Western and Northern blotting, respectively. Western blotting shows that the level of the catalytic subunit of γGCS is threefold higher in the resistant clones than in the wild-type HT22 cells (Fig. 7A and Fig. B). In contrast, when both γGCS and actin mRNA were quantitated and their ratio normalized to cells expressing the empty pCLBABE retroviral vector, the amount of γGCS mRNA remained relatively constant (Fig. 7A and Fig. B). To rule out the possibility that eIF2α activity changes the rate of γGCS breakdown, resistant clone 15 and wild-type cells were treated with cycloheximide and the rate of protein loss followed by Western blotting. This method gives values of protein turnover identical to pulse–chase experiments (Soucek et al. 1998). The rapidly turned over cell cycle protein, P27, served as a positive control (Soucek et al. 1998). Fig. 7 C shows that in contrast to P27, γGCS was degraded more slowly but at the same rate in resistant and wild-type cells. These results indicate that a decrease in eIF2α wild-type protein levels leads to an increase in production of the catalytic subunit of γGCS by a translational mechanism, resulting in significantly higher levels of GSH.


Regulation of antioxidant metabolism by translation initiation factor 2alpha.

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

γGCS protein expression is regulated at the level of translation. (A) γGCS, actin protein, and mRNA expression was measured in wild-type HT22 cells (lane 1), cells infected with the empty vector (pCLBABE) (lane 2), and the resistant clones 8 (lane 3) and 15 (lane 4) by Western and Northern blot analysis, respectively. (B) The Western blot from A was analyzed using the program NIH Image to determine the densities of each band. The densities were measured in four experiments, averaged, and normalized first to actin and then to the level of γGCS in pCLBABE, set as 1.0. Actin served as a loading control and showed that there was an equal amount of protein in each lane. Northern blots were quantitated on a PhosphorImager. The ratio of the catalytic subunit of γGCS to actin is presented normalized to γGCS in pCLBABE as 1.0. The results were confirmed by reverse transcription PCR analysis (data not shown). (C) Proteolytic breakdown of γGCS and P27 in wild-type and resistant cells. HT22 cells and resistant clone 15 were treated with 100 μg/ml cycloheximide and the amount of γGCS and P27 was quantitated by Western blot at 2-h intervals. The values are normalized to 0 time and are the mean ± SEM of triplicate experiments. Inset, Western blots of γGCS, wild-type cells (a); γGCS, clone 15 (b); P27, Cl 15 (c). Lanes 1, 2, 3, 4, and 5 are 0, 2, 4, 6, and 8 h after cycloheximide. x, γGCS, wild-type; •, γGCS Cl 15; ○, P27. (D) Resistant clones 8 and 15 were infected with wild-type eIF2α, the wild-type HT22 clone was infected with S51A or S51D, and the levels of γGCS and actin were determined by Western blotting. The amounts of γGCS and actin were quantitated and the amount of γGCS was normalized to the actin loading control. In each set of cells, the transfected cells were then normalized to γGCS in their parental line pCLBABE, resistant clone 8 or 15, which was set at 100%. The data are presented as the mean ± SEM, n = 4. *Significantly different from parental cells (P < 0.01); **significantly different from parental cells (P < 0.05).
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Figure 7: γGCS protein expression is regulated at the level of translation. (A) γGCS, actin protein, and mRNA expression was measured in wild-type HT22 cells (lane 1), cells infected with the empty vector (pCLBABE) (lane 2), and the resistant clones 8 (lane 3) and 15 (lane 4) by Western and Northern blot analysis, respectively. (B) The Western blot from A was analyzed using the program NIH Image to determine the densities of each band. The densities were measured in four experiments, averaged, and normalized first to actin and then to the level of γGCS in pCLBABE, set as 1.0. Actin served as a loading control and showed that there was an equal amount of protein in each lane. Northern blots were quantitated on a PhosphorImager. The ratio of the catalytic subunit of γGCS to actin is presented normalized to γGCS in pCLBABE as 1.0. The results were confirmed by reverse transcription PCR analysis (data not shown). (C) Proteolytic breakdown of γGCS and P27 in wild-type and resistant cells. HT22 cells and resistant clone 15 were treated with 100 μg/ml cycloheximide and the amount of γGCS and P27 was quantitated by Western blot at 2-h intervals. The values are normalized to 0 time and are the mean ± SEM of triplicate experiments. Inset, Western blots of γGCS, wild-type cells (a); γGCS, clone 15 (b); P27, Cl 15 (c). Lanes 1, 2, 3, 4, and 5 are 0, 2, 4, 6, and 8 h after cycloheximide. x, γGCS, wild-type; •, γGCS Cl 15; ○, P27. (D) Resistant clones 8 and 15 were infected with wild-type eIF2α, the wild-type HT22 clone was infected with S51A or S51D, and the levels of γGCS and actin were determined by Western blotting. The amounts of γGCS and actin were quantitated and the amount of γGCS was normalized to the actin loading control. In each set of cells, the transfected cells were then normalized to γGCS in their parental line pCLBABE, resistant clone 8 or 15, which was set at 100%. The data are presented as the mean ± SEM, n = 4. *Significantly different from parental cells (P < 0.01); **significantly different from parental cells (P < 0.05).
Mentions: Resistant clones 8 and 15 have decreased eIF2α activity and increased basal levels of GSH. Furthermore, the resistant clones and the cells expressing the phosphorylation mutant, S51D, maintain GSH levels 50% of their basal levels after glutamate exposure. To determine if there is a causal relationship between eIF2α protein levels and GSH production, the expression of the rate-limiting enzyme for GSH synthesis, γGCS, was examined in the wild-type cells and the resistant clones. Protein expression and mRNA levels of the catalytic subunit of γGCS were measured by Western and Northern blotting, respectively. Western blotting shows that the level of the catalytic subunit of γGCS is threefold higher in the resistant clones than in the wild-type HT22 cells (Fig. 7A and Fig. B). In contrast, when both γGCS and actin mRNA were quantitated and their ratio normalized to cells expressing the empty pCLBABE retroviral vector, the amount of γGCS mRNA remained relatively constant (Fig. 7A and Fig. B). To rule out the possibility that eIF2α activity changes the rate of γGCS breakdown, resistant clone 15 and wild-type cells were treated with cycloheximide and the rate of protein loss followed by Western blotting. This method gives values of protein turnover identical to pulse–chase experiments (Soucek et al. 1998). The rapidly turned over cell cycle protein, P27, served as a positive control (Soucek et al. 1998). Fig. 7 C shows that in contrast to P27, γGCS was degraded more slowly but at the same rate in resistant and wild-type cells. These results indicate that a decrease in eIF2α wild-type protein levels leads to an increase in production of the catalytic subunit of γGCS by a translational mechanism, resulting in significantly higher levels of GSH.

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