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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

Alkbh8-/- cells have elevated reactive oxygen species (ROS).A) Intracellular ROS was measured in immortalized MEFs by DCFDA staining. Representative images are shown for cell batches taken from the peak frequency of DCFDA intensities for wt and Alkbh8-/- MEFs. B) The frequency of wt (blue) and Alkbh8-/- (green) cells exhibiting specific DCFDA intensities is plotted for a population of 6,000 cells. C) Intracellular ROS was measured in immortalized MEFs by DCFDA staining 6 h after the indicated treatments and is shown as median fluorescence intensity emitted by oxidized DCFDA at 517–527 nm +STDV, n = 3).
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pone.0131335.g004: Alkbh8-/- cells have elevated reactive oxygen species (ROS).A) Intracellular ROS was measured in immortalized MEFs by DCFDA staining. Representative images are shown for cell batches taken from the peak frequency of DCFDA intensities for wt and Alkbh8-/- MEFs. B) The frequency of wt (blue) and Alkbh8-/- (green) cells exhibiting specific DCFDA intensities is plotted for a population of 6,000 cells. C) Intracellular ROS was measured in immortalized MEFs by DCFDA staining 6 h after the indicated treatments and is shown as median fluorescence intensity emitted by oxidized DCFDA at 517–527 nm +STDV, n = 3).

Mentions: Our DNA damage, cell sensitivity and transcriptional analysis all support a role for Alkbh8 in the regulation of ROS levels or the repair of the resulting damage. To determine if the regulation of ROS levels is perturbed, we next measured overall levels of intracellular ROS in wt and Alkbh8-/- MEFs by staining cells with dichlorofluorescein diacetate (DCFDA) and used imaging flow cytometry to determine the median fluorescence intensity of our various cell populations. Alkbh8-/- MEFs had a higher median oxidized DCFDA fluorescence \intensity when compared to wt MEFs, indicating that these cells display increased intracellular ROS (Fig 4A and 4B). This effect was exacerbated after treatment of the cells with H2O2. Notably the increased ROS observed in Alkbh8-/- MEFs could be rescued by antioxidant treatment with N-acetylcysteine (NAC) (Fig 4C). The increased ROS phenotype was also apparent in immortalized Alkbh8-/- cells, which were established concurrent with primary MEF experiments (results not shown). Our DCFDA results support the idea that Alkbh8 plays an important role in the detoxification of ROS.


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)

Alkbh8-/- cells have elevated reactive oxygen species (ROS).A) Intracellular ROS was measured in immortalized MEFs by DCFDA staining. Representative images are shown for cell batches taken from the peak frequency of DCFDA intensities for wt and Alkbh8-/- MEFs. B) The frequency of wt (blue) and Alkbh8-/- (green) cells exhibiting specific DCFDA intensities is plotted for a population of 6,000 cells. C) Intracellular ROS was measured in immortalized MEFs by DCFDA staining 6 h after the indicated treatments and is shown as median fluorescence intensity emitted by oxidized DCFDA at 517–527 nm +STDV, n = 3).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492958&req=5

pone.0131335.g004: Alkbh8-/- cells have elevated reactive oxygen species (ROS).A) Intracellular ROS was measured in immortalized MEFs by DCFDA staining. Representative images are shown for cell batches taken from the peak frequency of DCFDA intensities for wt and Alkbh8-/- MEFs. B) The frequency of wt (blue) and Alkbh8-/- (green) cells exhibiting specific DCFDA intensities is plotted for a population of 6,000 cells. C) Intracellular ROS was measured in immortalized MEFs by DCFDA staining 6 h after the indicated treatments and is shown as median fluorescence intensity emitted by oxidized DCFDA at 517–527 nm +STDV, n = 3).
Mentions: Our DNA damage, cell sensitivity and transcriptional analysis all support a role for Alkbh8 in the regulation of ROS levels or the repair of the resulting damage. To determine if the regulation of ROS levels is perturbed, we next measured overall levels of intracellular ROS in wt and Alkbh8-/- MEFs by staining cells with dichlorofluorescein diacetate (DCFDA) and used imaging flow cytometry to determine the median fluorescence intensity of our various cell populations. Alkbh8-/- MEFs had a higher median oxidized DCFDA fluorescence \intensity when compared to wt MEFs, indicating that these cells display increased intracellular ROS (Fig 4A and 4B). This effect was exacerbated after treatment of the cells with H2O2. Notably the increased ROS observed in Alkbh8-/- MEFs could be rescued by antioxidant treatment with N-acetylcysteine (NAC) (Fig 4C). The increased ROS phenotype was also apparent in immortalized Alkbh8-/- cells, which were established concurrent with primary MEF experiments (results not shown). Our DCFDA results support the idea that Alkbh8 plays an important role in the detoxification of ROS.

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