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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

Model for Alkbh8 as a translational regulator of an ROS response node.Alkbh8-directed cytoprotective responses through stop codon reprogramming for selenoprotein expression, and implications for disease prevention.
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pone.0131335.g007: Model for Alkbh8 as a translational regulator of an ROS response node.Alkbh8-directed cytoprotective responses through stop codon reprogramming for selenoprotein expression, and implications for disease prevention.

Mentions: We are the first group to demonstrate that Alkbh8-/- MEFs have elevated intracellular reactive oxygen species (ROS) and increased DNA damage levels, with these phenotypes linked to reduced Gpx1, Gpx3, Gpx6 and TrxR1 protein levels. Further we report that lipid peroxidation products are higher in Alkbh8-/- MEFs after H2O2 treatment, relative to wt, supporting a regulatory link between Alkbh8 and Gpx4. Importantly we demonstrate that there is an increase in Alkbh8 protein and mcm5Um levels in response to H2O2 and identify a key regulatory pathway in the response to ROS. The ROS stress phenotype observed in our Alkbh8-/- MEFs is due to defective stop codon reprogramming, leading to sub-optimal selenocysteine protein expression. Alkbh8-regulated ROS and lipid detoxification systems provide cytoprotection against ROS-stress (Fig 7). Faced with increased ROS levels, it makes sense for the cell to increase Alkbh8 and mcm5Um levels to increase the amount of Gpx1, Gpx3, Gpx4, Gpx6 and TrxR1, so as to increase H2O2 and lipid peroxidation detoxification capacity. Reducing the levels of H2O2 and other ROS will clearly prevent DNA and protein damage and reduce cell death. Alkbh8 can thus be thought of as a regulator of a damage mitigation system that protects DNA from ROS offenders. Further, the removal of lipid peroxidation products should prevent DNA mutation, since lipid peroxidation products like malondialdehyde have been demonstrated to cause DNA damage that promotes insertion, deletion and basepair mutations in human cells [45]. Thus, we have established a cytoprotective role for Alkbh8. Based on the increased DNA damage, lipid peroxidation and the mcm5Um-dependent regulation of Gpx1, Gpx3 and Gpx6, there are suggested anti-mutagenic roles for Alkbh8 via the prevention of DNA damage such as 8-oxoguanine and M1G (pyrimidopurin-10(3H)-one). Our suggestion is supported by the documented increase in mutagenesis that is observed in catalase deficient bacterial cells [46].


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)

Model for Alkbh8 as a translational regulator of an ROS response node.Alkbh8-directed cytoprotective responses through stop codon reprogramming for selenoprotein expression, and implications for disease prevention.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492958&req=5

pone.0131335.g007: Model for Alkbh8 as a translational regulator of an ROS response node.Alkbh8-directed cytoprotective responses through stop codon reprogramming for selenoprotein expression, and implications for disease prevention.
Mentions: We are the first group to demonstrate that Alkbh8-/- MEFs have elevated intracellular reactive oxygen species (ROS) and increased DNA damage levels, with these phenotypes linked to reduced Gpx1, Gpx3, Gpx6 and TrxR1 protein levels. Further we report that lipid peroxidation products are higher in Alkbh8-/- MEFs after H2O2 treatment, relative to wt, supporting a regulatory link between Alkbh8 and Gpx4. Importantly we demonstrate that there is an increase in Alkbh8 protein and mcm5Um levels in response to H2O2 and identify a key regulatory pathway in the response to ROS. The ROS stress phenotype observed in our Alkbh8-/- MEFs is due to defective stop codon reprogramming, leading to sub-optimal selenocysteine protein expression. Alkbh8-regulated ROS and lipid detoxification systems provide cytoprotection against ROS-stress (Fig 7). Faced with increased ROS levels, it makes sense for the cell to increase Alkbh8 and mcm5Um levels to increase the amount of Gpx1, Gpx3, Gpx4, Gpx6 and TrxR1, so as to increase H2O2 and lipid peroxidation detoxification capacity. Reducing the levels of H2O2 and other ROS will clearly prevent DNA and protein damage and reduce cell death. Alkbh8 can thus be thought of as a regulator of a damage mitigation system that protects DNA from ROS offenders. Further, the removal of lipid peroxidation products should prevent DNA mutation, since lipid peroxidation products like malondialdehyde have been demonstrated to cause DNA damage that promotes insertion, deletion and basepair mutations in human cells [45]. Thus, we have established a cytoprotective role for Alkbh8. Based on the increased DNA damage, lipid peroxidation and the mcm5Um-dependent regulation of Gpx1, Gpx3 and Gpx6, there are suggested anti-mutagenic roles for Alkbh8 via the prevention of DNA damage such as 8-oxoguanine and M1G (pyrimidopurin-10(3H)-one). Our suggestion is supported by the documented increase in mutagenesis that is observed in catalase deficient bacterial cells [46].

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