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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

Increased DNA damage and activated DNA damage response in Alkbh8-/- MEFs.A) Comet assay of wt and Alkbh8-/- nuclei isolated from MEFs that were cultured as described above. B) Comet tails were quantified using CometScore and are shown as the mean % of DNA in the comet tail (±STDV, n = 3). Significant differences in the % of DNA in the comet tails of wt and Alkbh8-/- MEFs were determined by Student t-test *p < 0.001). C) Single cell suspensions were fixed and stained with a FITC conjugated antibody specific for γ-H2AX and the DRAQ5 nuclear stain followed by quantitative imaging with an ISX100 flow cytometer. Representative γ-H2AX foci images are shown for cell batches taken from the peak frequency of Spot Count distributions for wt and Alkbh8-/- sample runs. D) γ-H2AX foci were scored for intensity and number using the IDEAS Spot Count analysis program, presented as the percentage frequency of cells with 0–10 Spot Counts, and those with a Spot counts less than or greater than 3 (inset, n = 6).
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pone.0131335.g002: Increased DNA damage and activated DNA damage response in Alkbh8-/- MEFs.A) Comet assay of wt and Alkbh8-/- nuclei isolated from MEFs that were cultured as described above. B) Comet tails were quantified using CometScore and are shown as the mean % of DNA in the comet tail (±STDV, n = 3). Significant differences in the % of DNA in the comet tails of wt and Alkbh8-/- MEFs were determined by Student t-test *p < 0.001). C) Single cell suspensions were fixed and stained with a FITC conjugated antibody specific for γ-H2AX and the DRAQ5 nuclear stain followed by quantitative imaging with an ISX100 flow cytometer. Representative γ-H2AX foci images are shown for cell batches taken from the peak frequency of Spot Count distributions for wt and Alkbh8-/- sample runs. D) γ-H2AX foci were scored for intensity and number using the IDEAS Spot Count analysis program, presented as the percentage frequency of cells with 0–10 Spot Counts, and those with a Spot counts less than or greater than 3 (inset, n = 6).

Mentions: Slow cell growth and an increased incidence of cell death are potential phenotypes for cells compromised for damage prevention, stress- and DDR-systems, defects that potentially manifest as unrepaired single or double strand DNA breaks. We used the Comet assay to determine whether Alkbh8-/- MEFs had DNA strand breaks. We found that under basal growth conditions, the nuclei derived from Alkbh8-/- MEFs had a much higher percentage of strand breaks compared to nuclei derived from wt MEFs (Fig 2A and 2B). An early event at sites of DSBs is the recruitment of the Mre11-Rad51-Nbs1 (MRN) complex, a process that promotes the phosphorylation of the histone protein H2AX on Ser129, (denoted γ-H2AX), which accumulates as distinct foci at sites of double strand breaks [39, 40]. We stained MEFs with a γ-H2AX specific antibody conjugated to FITC and used imaging flow cytometry to quantitate the number of γ-H2AX positive foci. In these experiments the number of cells containing 0–10 γ-H2AX positive foci (i.e., using a spot count algorithm) were calculated for both wt and Alkbh8-/- MEFs for 20,000 cells, with each spot count number represented as a total frequency. In general, the number of γ-H2AX foci per cell was increased in Alkbh8-/- MEFs, relative to wild type MEFs, under basal growth conditions. Specifically, 40% of the Alkbh8-/- MEF population had greater than three foci, while the wt population had only 18%. Our results demonstrate that an Alkbh8 deficiency leads to the activation of the DNA damage response, which is most likely due to an increase in DSBs (Fig 2C and 2D).


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)

Increased DNA damage and activated DNA damage response in Alkbh8-/- MEFs.A) Comet assay of wt and Alkbh8-/- nuclei isolated from MEFs that were cultured as described above. B) Comet tails were quantified using CometScore and are shown as the mean % of DNA in the comet tail (±STDV, n = 3). Significant differences in the % of DNA in the comet tails of wt and Alkbh8-/- MEFs were determined by Student t-test *p < 0.001). C) Single cell suspensions were fixed and stained with a FITC conjugated antibody specific for γ-H2AX and the DRAQ5 nuclear stain followed by quantitative imaging with an ISX100 flow cytometer. Representative γ-H2AX foci images are shown for cell batches taken from the peak frequency of Spot Count distributions for wt and Alkbh8-/- sample runs. D) γ-H2AX foci were scored for intensity and number using the IDEAS Spot Count analysis program, presented as the percentage frequency of cells with 0–10 Spot Counts, and those with a Spot counts less than or greater than 3 (inset, n = 6).
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pone.0131335.g002: Increased DNA damage and activated DNA damage response in Alkbh8-/- MEFs.A) Comet assay of wt and Alkbh8-/- nuclei isolated from MEFs that were cultured as described above. B) Comet tails were quantified using CometScore and are shown as the mean % of DNA in the comet tail (±STDV, n = 3). Significant differences in the % of DNA in the comet tails of wt and Alkbh8-/- MEFs were determined by Student t-test *p < 0.001). C) Single cell suspensions were fixed and stained with a FITC conjugated antibody specific for γ-H2AX and the DRAQ5 nuclear stain followed by quantitative imaging with an ISX100 flow cytometer. Representative γ-H2AX foci images are shown for cell batches taken from the peak frequency of Spot Count distributions for wt and Alkbh8-/- sample runs. D) γ-H2AX foci were scored for intensity and number using the IDEAS Spot Count analysis program, presented as the percentage frequency of cells with 0–10 Spot Counts, and those with a Spot counts less than or greater than 3 (inset, n = 6).
Mentions: Slow cell growth and an increased incidence of cell death are potential phenotypes for cells compromised for damage prevention, stress- and DDR-systems, defects that potentially manifest as unrepaired single or double strand DNA breaks. We used the Comet assay to determine whether Alkbh8-/- MEFs had DNA strand breaks. We found that under basal growth conditions, the nuclei derived from Alkbh8-/- MEFs had a much higher percentage of strand breaks compared to nuclei derived from wt MEFs (Fig 2A and 2B). An early event at sites of DSBs is the recruitment of the Mre11-Rad51-Nbs1 (MRN) complex, a process that promotes the phosphorylation of the histone protein H2AX on Ser129, (denoted γ-H2AX), which accumulates as distinct foci at sites of double strand breaks [39, 40]. We stained MEFs with a γ-H2AX specific antibody conjugated to FITC and used imaging flow cytometry to quantitate the number of γ-H2AX positive foci. In these experiments the number of cells containing 0–10 γ-H2AX positive foci (i.e., using a spot count algorithm) were calculated for both wt and Alkbh8-/- MEFs for 20,000 cells, with each spot count number represented as a total frequency. In general, the number of γ-H2AX foci per cell was increased in Alkbh8-/- MEFs, relative to wild type MEFs, under basal growth conditions. Specifically, 40% of the Alkbh8-/- MEF population had greater than three foci, while the wt population had only 18%. Our results demonstrate that an Alkbh8 deficiency leads to the activation of the DNA damage response, which is most likely due to an increase in DSBs (Fig 2C and 2D).

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