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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-/- MEFs are sensitive to DNA damaging agents.MEFs were either treated with (A) MMS, (B) exposed to γ-irradiation or treated with (C) H2O2 or (D) Rotenone and viability was assessed by enumerating PI or trypan blue negative cells 48 (MMS, irradiation, and Rotenone) or 72 (H2O2) h later. In all panels, error bars represent standard deviation (±STDV, n = 3) and significant differences in viability of wt and Alkbh8-/- MEFs were determined by Student’s t-test (τp < 0.005, *p < 0.05, ψp < 0.01, ωp < 0.001).
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pone.0131335.g003: Alkbh8-/- MEFs are sensitive to DNA damaging agents.MEFs were either treated with (A) MMS, (B) exposed to γ-irradiation or treated with (C) H2O2 or (D) Rotenone and viability was assessed by enumerating PI or trypan blue negative cells 48 (MMS, irradiation, and Rotenone) or 72 (H2O2) h later. In all panels, error bars represent standard deviation (±STDV, n = 3) and significant differences in viability of wt and Alkbh8-/- MEFs were determined by Student’s t-test (τp < 0.005, *p < 0.05, ψp < 0.01, ωp < 0.001).

Mentions: The increased strand breaks and activated DNA damage response in Alkbh8-/- MEFs suggested that Alkbh8 deficiency leads to increased levels of damaging agents and/or a compromised DNA damage response downstream of γ-H2AX phosphorylation. Often, defects in damage prevention or DNA damage-responses sensitize cells to DNA damaging agents. For example, ATM and BRCA1 mutant cell lines are highly sensitive to ionizing radiation [41, 42] and p53-deficient cells undergo greater DNA damage induced apoptosis in response to chemotherapeutic drugs that induce DNA strand breaks (i.e., topoisomerase inhibitors) in comparison to wild type p53 cells [43]. To help identify the type of damaging agent or the defective repair pathway in Alkbh8-/- MEFs we treated the cells with various DNA damaging agents. We observed an increased sensitivity of Alkbh8-/- MEFs, relative to wt MEFs, to MMS, ionizing irradiation, H2O2 and Rotenone (Fig 3). This sensitivity was particularly pronounced for the latter two compounds, which are both known to increase intracellular ROS, with Rotenone being an endogenous ROS inducer through its ability to perturb mitochondrial function. Taken together, these observations support a role for mammalian Alkbh8 in preventing ROS, oxidative DNA damage, or both, or participating in some aspect of the repair of oxidatively damaged DNA.


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-/- MEFs are sensitive to DNA damaging agents.MEFs were either treated with (A) MMS, (B) exposed to γ-irradiation or treated with (C) H2O2 or (D) Rotenone and viability was assessed by enumerating PI or trypan blue negative cells 48 (MMS, irradiation, and Rotenone) or 72 (H2O2) h later. In all panels, error bars represent standard deviation (±STDV, n = 3) and significant differences in viability of wt and Alkbh8-/- MEFs were determined by Student’s t-test (τp < 0.005, *p < 0.05, ψp < 0.01, ωp < 0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131335.g003: Alkbh8-/- MEFs are sensitive to DNA damaging agents.MEFs were either treated with (A) MMS, (B) exposed to γ-irradiation or treated with (C) H2O2 or (D) Rotenone and viability was assessed by enumerating PI or trypan blue negative cells 48 (MMS, irradiation, and Rotenone) or 72 (H2O2) h later. In all panels, error bars represent standard deviation (±STDV, n = 3) and significant differences in viability of wt and Alkbh8-/- MEFs were determined by Student’s t-test (τp < 0.005, *p < 0.05, ψp < 0.01, ωp < 0.001).
Mentions: The increased strand breaks and activated DNA damage response in Alkbh8-/- MEFs suggested that Alkbh8 deficiency leads to increased levels of damaging agents and/or a compromised DNA damage response downstream of γ-H2AX phosphorylation. Often, defects in damage prevention or DNA damage-responses sensitize cells to DNA damaging agents. For example, ATM and BRCA1 mutant cell lines are highly sensitive to ionizing radiation [41, 42] and p53-deficient cells undergo greater DNA damage induced apoptosis in response to chemotherapeutic drugs that induce DNA strand breaks (i.e., topoisomerase inhibitors) in comparison to wild type p53 cells [43]. To help identify the type of damaging agent or the defective repair pathway in Alkbh8-/- MEFs we treated the cells with various DNA damaging agents. We observed an increased sensitivity of Alkbh8-/- MEFs, relative to wt MEFs, to MMS, ionizing irradiation, H2O2 and Rotenone (Fig 3). This sensitivity was particularly pronounced for the latter two compounds, which are both known to increase intracellular ROS, with Rotenone being an endogenous ROS inducer through its ability to perturb mitochondrial function. Taken together, these observations support a role for mammalian Alkbh8 in preventing ROS, oxidative DNA damage, or both, or participating in some aspect of the repair of oxidatively damaged DNA.

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