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Alkbh8 Regulates Selenocysteine-Protein Expression to Protect against Reactive Oxygen Species Damage.

Endres L, Begley U, Clark R, Gu C, Dziergowska A, Małkiewicz A, Melendez JA, Dedon PC, Begley TJ - PLoS ONE (2015)

Bottom Line: Here we detail basal and damage-induced translational regulation of a group of oxidative-stress response enzymes by the tRNA methyltransferase Alkbh8.We demonstrate that Alkbh8 is induced in response to ROS and is required for the efficient expression of selenocysteine-containing ROS detoxification enzymes belonging to the glutathione peroxidase (Gpx1, Gpx3, Gpx6 and likely Gpx4) and thioredoxin reductase (TrxR1) families.We also show that, in response to oxidative stress, the tRNA modification 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) increases in normal MEFs to drive the expression of ROS detoxification enzymes, with this damage-induced reprogramming of tRNA and stop-codon recoding corrupted in Alkbh8-/- MEFS.

View Article: PubMed Central - PubMed

Affiliation: Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, New York 12203, United States of America; RNA Institute and Cancer Research Center, University at Albany, State University of New York, Albany, New York 12222, United States of America.

ABSTRACT
Environmental and metabolic sources of reactive oxygen species (ROS) can damage DNA, proteins and lipids to promote disease. Regulation of gene expression can prevent this damage and can include increased transcription, translation and post translational modification. Cellular responses to ROS play important roles in disease prevention, with deficiencies linked to cancer, neurodegeneration and ageing. Here we detail basal and damage-induced translational regulation of a group of oxidative-stress response enzymes by the tRNA methyltransferase Alkbh8. Using a new gene targeted knockout mouse cell system, we show that Alkbh8-/- embryonic fibroblasts (MEFs) display elevated ROS levels, increased DNA and lipid damage and hallmarks of cellular stress. We demonstrate that Alkbh8 is induced in response to ROS and is required for the efficient expression of selenocysteine-containing ROS detoxification enzymes belonging to the glutathione peroxidase (Gpx1, Gpx3, Gpx6 and likely Gpx4) and thioredoxin reductase (TrxR1) families. We also show that, in response to oxidative stress, the tRNA modification 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) increases in normal MEFs to drive the expression of ROS detoxification enzymes, with this damage-induced reprogramming of tRNA and stop-codon recoding corrupted in Alkbh8-/- MEFS. These studies define Alkbh8 and tRNA modifications as central regulators of cellular oxidative stress responses in mammalian systems. In addition they highlight a new animal model for use in environmental and cancer studies and link translational regulation to the prevention of DNA and lipid damage.

No MeSH data available.


Related in: MedlinePlus

Compromised ROS response and stop-codon recoding with decreased Gpx and TrxR1 protein levels and activity in Alkbh8-/- MEFs.Wt and Alkbh8-/- MEFs were treated as indicated and then prepared for western blot analysis with A) anti-Alkbh8, anti-Nrf2, anti-ATM S15 phosphorylated B) anti-Gpx, C) and anti-TrxR antibodies. Anti-TrXR1 antibody (Peirce PAS-28886) detects TrxR1 at 67 kD and a cross-reacting (higher MW) band in mouse cells. For A—C, anti-GAPDH antibodies were used to as loading controls. D) qRT-PCR analysis of Gpx1 transcript levels. E) qRT-PCR analysis of TrxR1 transcript levels. F) Lipid peroxidation levels were measured in whole cell lysates prepared from wt and Alkbh8-/- MEFs under basal and H2O2-treated conditions. G) Stop-codon recoding was measured using the DualLuc-Gpx1 reporter system in wt and Alkbh8-/- MEFs under basal and H2O2-treated conditions. Statistical significance (p < 0.05) of biological replicates (N = 3) was measured using the Student’s t-Test.
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pone.0131335.g005: Compromised ROS response and stop-codon recoding with decreased Gpx and TrxR1 protein levels and activity in Alkbh8-/- MEFs.Wt and Alkbh8-/- MEFs were treated as indicated and then prepared for western blot analysis with A) anti-Alkbh8, anti-Nrf2, anti-ATM S15 phosphorylated B) anti-Gpx, C) and anti-TrxR antibodies. Anti-TrXR1 antibody (Peirce PAS-28886) detects TrxR1 at 67 kD and a cross-reacting (higher MW) band in mouse cells. For A—C, anti-GAPDH antibodies were used to as loading controls. D) qRT-PCR analysis of Gpx1 transcript levels. E) qRT-PCR analysis of TrxR1 transcript levels. F) Lipid peroxidation levels were measured in whole cell lysates prepared from wt and Alkbh8-/- MEFs under basal and H2O2-treated conditions. G) Stop-codon recoding was measured using the DualLuc-Gpx1 reporter system in wt and Alkbh8-/- MEFs under basal and H2O2-treated conditions. Statistical significance (p < 0.05) of biological replicates (N = 3) was measured using the Student’s t-Test.

Mentions: We used immunoblot approaches to determine if Alkbh8 gene expression is regulated in response to H2O2 exposure. Using wild type MEFs (both immortalized and primary) we demonstrate that Alkbh8 protein levels are induced by H2O2, further supporting a role for this enzyme in oxidative-induced stress response (Fig 5A). As Alkbh8-catalyzed tRNA modifications play a role in stop-codon reprogramming for efficient Gpx1 translation [15, 27, 29] we next analyzed protein expression levels for multiple selenocysteine containing glutathione peroxidase proteins (Gpx1, Gpx3 and Gpx6) in wild type and Alkbh8-/- MEFs. Notably, the protein expression of several Gpx family members was affected by Alkbh8 status (Fig 5B). Specifically, the protein levels of Gpx1 and Gpx6 were decreased in Alkbh8-/- cells under basal growth conditions; Gpx1, Gpx3 and 6 expression were all induced by oxidative-stress (i.e., H2O2), and this induction was markedly attenuated in Alkbh8-/- MEFs, suggesting that Gpx3 in MEFs is also acting in antioxidant defense in addition to playing a housekeeping role. Moreover, the decreased Gpx protein levels correlated with a decrease in Gpx activity in Alkbh8-/- MEFs relative to wt MEFs (S4 Fig). The cytosolic TrxR1 and mitochondrial TrxR2 proteins levels were also analyzed in wt and Alkbh8-/- MEFs under basal and H2O2 induction conditions. While the levels of both TrxR1 and TrxR2 were similar in wt and Alkbh8-/- MEFs under untreated conditions, we did observe a modest decrease in TrxR1 in Alkbh8-/- MEFs three and six hours after H2O2 treatment (Fig 5C). We did not observe a noticeable decrease in TrxR2 in Alkbh8-/- MEFs under any condition, relative to wt, thus we can only link Alkbh8 to the regulation of TrxR1 in the cytosol. To further link Alkbh8 to the regulation of Gpx1 and TrxR1 we introduced a control plasmid (pcDNA3.1-EV) and an Alkbh8 expression plasmid (pcDNA3.1-Alkbh8) into our Alkbh8-/- MEFs and analyzed for protein levels under basal conditions. We determined that re-expression of Alkbh8 in the Alkbh8-/- MEFs rescued Gpx1 and TrxR1 protein levels, while having little effect on TrxR2 levels (S5 Fig). qRT-PCR analysis of Gpx1 and TrxR1 transcripts in our MEF models demonstrates little difference in mRNA levels between wt and Alkbh8-/- (Fig 5D and 5E). Together the data support the idea that the observed decreases in selenocysteine protein expression in Alkbh8-/- MEFs is due an Alkbh8 deficiency that leads to a defect in post-transcriptional regulation of gene expression.


Alkbh8 Regulates Selenocysteine-Protein Expression to Protect against Reactive Oxygen Species Damage.

Endres L, Begley U, Clark R, Gu C, Dziergowska A, Małkiewicz A, Melendez JA, Dedon PC, Begley TJ - PLoS ONE (2015)

Compromised ROS response and stop-codon recoding with decreased Gpx and TrxR1 protein levels and activity in Alkbh8-/- MEFs.Wt and Alkbh8-/- MEFs were treated as indicated and then prepared for western blot analysis with A) anti-Alkbh8, anti-Nrf2, anti-ATM S15 phosphorylated B) anti-Gpx, C) and anti-TrxR antibodies. Anti-TrXR1 antibody (Peirce PAS-28886) detects TrxR1 at 67 kD and a cross-reacting (higher MW) band in mouse cells. For A—C, anti-GAPDH antibodies were used to as loading controls. D) qRT-PCR analysis of Gpx1 transcript levels. E) qRT-PCR analysis of TrxR1 transcript levels. F) Lipid peroxidation levels were measured in whole cell lysates prepared from wt and Alkbh8-/- MEFs under basal and H2O2-treated conditions. G) Stop-codon recoding was measured using the DualLuc-Gpx1 reporter system in wt and Alkbh8-/- MEFs under basal and H2O2-treated conditions. Statistical significance (p < 0.05) of biological replicates (N = 3) was measured using the Student’s t-Test.
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Related In: Results  -  Collection

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Show All Figures
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pone.0131335.g005: Compromised ROS response and stop-codon recoding with decreased Gpx and TrxR1 protein levels and activity in Alkbh8-/- MEFs.Wt and Alkbh8-/- MEFs were treated as indicated and then prepared for western blot analysis with A) anti-Alkbh8, anti-Nrf2, anti-ATM S15 phosphorylated B) anti-Gpx, C) and anti-TrxR antibodies. Anti-TrXR1 antibody (Peirce PAS-28886) detects TrxR1 at 67 kD and a cross-reacting (higher MW) band in mouse cells. For A—C, anti-GAPDH antibodies were used to as loading controls. D) qRT-PCR analysis of Gpx1 transcript levels. E) qRT-PCR analysis of TrxR1 transcript levels. F) Lipid peroxidation levels were measured in whole cell lysates prepared from wt and Alkbh8-/- MEFs under basal and H2O2-treated conditions. G) Stop-codon recoding was measured using the DualLuc-Gpx1 reporter system in wt and Alkbh8-/- MEFs under basal and H2O2-treated conditions. Statistical significance (p < 0.05) of biological replicates (N = 3) was measured using the Student’s t-Test.
Mentions: We used immunoblot approaches to determine if Alkbh8 gene expression is regulated in response to H2O2 exposure. Using wild type MEFs (both immortalized and primary) we demonstrate that Alkbh8 protein levels are induced by H2O2, further supporting a role for this enzyme in oxidative-induced stress response (Fig 5A). As Alkbh8-catalyzed tRNA modifications play a role in stop-codon reprogramming for efficient Gpx1 translation [15, 27, 29] we next analyzed protein expression levels for multiple selenocysteine containing glutathione peroxidase proteins (Gpx1, Gpx3 and Gpx6) in wild type and Alkbh8-/- MEFs. Notably, the protein expression of several Gpx family members was affected by Alkbh8 status (Fig 5B). Specifically, the protein levels of Gpx1 and Gpx6 were decreased in Alkbh8-/- cells under basal growth conditions; Gpx1, Gpx3 and 6 expression were all induced by oxidative-stress (i.e., H2O2), and this induction was markedly attenuated in Alkbh8-/- MEFs, suggesting that Gpx3 in MEFs is also acting in antioxidant defense in addition to playing a housekeeping role. Moreover, the decreased Gpx protein levels correlated with a decrease in Gpx activity in Alkbh8-/- MEFs relative to wt MEFs (S4 Fig). The cytosolic TrxR1 and mitochondrial TrxR2 proteins levels were also analyzed in wt and Alkbh8-/- MEFs under basal and H2O2 induction conditions. While the levels of both TrxR1 and TrxR2 were similar in wt and Alkbh8-/- MEFs under untreated conditions, we did observe a modest decrease in TrxR1 in Alkbh8-/- MEFs three and six hours after H2O2 treatment (Fig 5C). We did not observe a noticeable decrease in TrxR2 in Alkbh8-/- MEFs under any condition, relative to wt, thus we can only link Alkbh8 to the regulation of TrxR1 in the cytosol. To further link Alkbh8 to the regulation of Gpx1 and TrxR1 we introduced a control plasmid (pcDNA3.1-EV) and an Alkbh8 expression plasmid (pcDNA3.1-Alkbh8) into our Alkbh8-/- MEFs and analyzed for protein levels under basal conditions. We determined that re-expression of Alkbh8 in the Alkbh8-/- MEFs rescued Gpx1 and TrxR1 protein levels, while having little effect on TrxR2 levels (S5 Fig). qRT-PCR analysis of Gpx1 and TrxR1 transcripts in our MEF models demonstrates little difference in mRNA levels between wt and Alkbh8-/- (Fig 5D and 5E). Together the data support the idea that the observed decreases in selenocysteine protein expression in Alkbh8-/- MEFs is due an Alkbh8 deficiency that leads to a defect in post-transcriptional regulation of gene expression.

Bottom Line: Here we detail basal and damage-induced translational regulation of a group of oxidative-stress response enzymes by the tRNA methyltransferase Alkbh8.We demonstrate that Alkbh8 is induced in response to ROS and is required for the efficient expression of selenocysteine-containing ROS detoxification enzymes belonging to the glutathione peroxidase (Gpx1, Gpx3, Gpx6 and likely Gpx4) and thioredoxin reductase (TrxR1) families.We also show that, in response to oxidative stress, the tRNA modification 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) increases in normal MEFs to drive the expression of ROS detoxification enzymes, with this damage-induced reprogramming of tRNA and stop-codon recoding corrupted in Alkbh8-/- MEFS.

View Article: PubMed Central - PubMed

Affiliation: Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, New York 12203, United States of America; RNA Institute and Cancer Research Center, University at Albany, State University of New York, Albany, New York 12222, United States of America.

ABSTRACT
Environmental and metabolic sources of reactive oxygen species (ROS) can damage DNA, proteins and lipids to promote disease. Regulation of gene expression can prevent this damage and can include increased transcription, translation and post translational modification. Cellular responses to ROS play important roles in disease prevention, with deficiencies linked to cancer, neurodegeneration and ageing. Here we detail basal and damage-induced translational regulation of a group of oxidative-stress response enzymes by the tRNA methyltransferase Alkbh8. Using a new gene targeted knockout mouse cell system, we show that Alkbh8-/- embryonic fibroblasts (MEFs) display elevated ROS levels, increased DNA and lipid damage and hallmarks of cellular stress. We demonstrate that Alkbh8 is induced in response to ROS and is required for the efficient expression of selenocysteine-containing ROS detoxification enzymes belonging to the glutathione peroxidase (Gpx1, Gpx3, Gpx6 and likely Gpx4) and thioredoxin reductase (TrxR1) families. We also show that, in response to oxidative stress, the tRNA modification 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) increases in normal MEFs to drive the expression of ROS detoxification enzymes, with this damage-induced reprogramming of tRNA and stop-codon recoding corrupted in Alkbh8-/- MEFS. These studies define Alkbh8 and tRNA modifications as central regulators of cellular oxidative stress responses in mammalian systems. In addition they highlight a new animal model for use in environmental and cancer studies and link translational regulation to the prevention of DNA and lipid damage.

No MeSH data available.


Related in: MedlinePlus