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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

Ribonucleoside modifications in wild type and Alkbh8-/- mice after oxidative stress.Wt and Alkbh8-/- MEFs were either left untreated (UT) or exposed to 1.2 mM of H2O2 for 1 hour. The levels of mcm5s2U, mcm5Um and mcm5U ribonucleoside modifications were identified by HPLC-coupled mass spectrometry and quantified by integrating the normalized peak area intensity for each signal at the indicated post-exposure time points. Significant differences in mcm5Um modifications were determined by the Student’s t-test: UT, Alkbh8-/- (n = 6) versus wt (n = 5), *p < 0.05; 3 h, Alkbh8-/- (n = 3) versus wt (n = 3), **p < 0.02; 6 h, Alkbh8-/- (n = 3) versus wt (n = 3), ***p < 0.002; 20 h, Alkbh8-/- (n = 2) versus wt (n = 2), ****p < 0.01; 24 h, Alkbh8-/- (n = 3) versus wt (n = 2), *****p < 0.001. UT, 6 h, wt (n = 3) versus 0 hours, wt γp < 0.02; 20 hours, wt (n = 2) versus 0 h, wt (n = 5) γγp < 0.01.
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pone.0131335.g006: Ribonucleoside modifications in wild type and Alkbh8-/- mice after oxidative stress.Wt and Alkbh8-/- MEFs were either left untreated (UT) or exposed to 1.2 mM of H2O2 for 1 hour. The levels of mcm5s2U, mcm5Um and mcm5U ribonucleoside modifications were identified by HPLC-coupled mass spectrometry and quantified by integrating the normalized peak area intensity for each signal at the indicated post-exposure time points. Significant differences in mcm5Um modifications were determined by the Student’s t-test: UT, Alkbh8-/- (n = 6) versus wt (n = 5), *p < 0.05; 3 h, Alkbh8-/- (n = 3) versus wt (n = 3), **p < 0.02; 6 h, Alkbh8-/- (n = 3) versus wt (n = 3), ***p < 0.002; 20 h, Alkbh8-/- (n = 2) versus wt (n = 2), ****p < 0.01; 24 h, Alkbh8-/- (n = 3) versus wt (n = 2), *****p < 0.001. UT, 6 h, wt (n = 3) versus 0 hours, wt γp < 0.02; 20 hours, wt (n = 2) versus 0 h, wt (n = 5) γγp < 0.01.

Mentions: The observed H2O2-induced increases in Alkbh8, Gpx1, Gpx3, and Gpx6 proteins in wt MEFs suggest that the associated tRNA modifications required for the translation of selenocysteine containing proteins are also increased in response to elevated ROS levels. Further the decreased Gpx1, Gpx3 and Gpx6 in in Alkbh8-/- MEFs suggests that tRNA modification is affected, relative to wt. To investigate these possibilities, we used our established LC-MS/MS approach [14, 17, 19, 36] with mass transitions specified in Table 2 to quantitate the basal and H2O2-induced levels of mcm5U, mcm5s2U and mcm5Um in wt and Alkbh8-/- MEFs (Fig 6). When comparing wt and Alkbh8-/- MEFs, we observed little difference in mcm5U and mcm5s2U levels under both basal and H2O2 treated conditions. In contrast, we observed a significant (p < 0.05) difference in mcm5Um in wt and Alkbh8-/- MEFs under basal conditions and after H2O2 treatment. We have also quantitated mcm5-base modifications in the livers of wt and Alkbh8-/- animals and observed analogous results to our MEF’s. Specifically we observed similar levels of the mcm5U and mcm5s2U modifications in wt and Alkbh8-/- livers and significantly decreased (p < 0.05) levels of the mcm5Um modification in the Alkbh8-/- vs. wt livers (S6 Fig). After exposure to ROS, the most obvious difference in mcm5Um levels in MEFs was seen 20 h after H2O2 treatment, when the wt cells had ~3.5-fold higher levels of this wobble base modification only found on tRNAUGA-SEC, relative to Alkbh8-/- MEFs. The 20-hour post-H2O2 time point also represents the peak levels of mcm5Um for wt MEFs, and lowest levels for Alkbh8-/- MEFs, further demonstrating a significant Alkbh8-dependent increase.


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)

Ribonucleoside modifications in wild type and Alkbh8-/- mice after oxidative stress.Wt and Alkbh8-/- MEFs were either left untreated (UT) or exposed to 1.2 mM of H2O2 for 1 hour. The levels of mcm5s2U, mcm5Um and mcm5U ribonucleoside modifications were identified by HPLC-coupled mass spectrometry and quantified by integrating the normalized peak area intensity for each signal at the indicated post-exposure time points. Significant differences in mcm5Um modifications were determined by the Student’s t-test: UT, Alkbh8-/- (n = 6) versus wt (n = 5), *p < 0.05; 3 h, Alkbh8-/- (n = 3) versus wt (n = 3), **p < 0.02; 6 h, Alkbh8-/- (n = 3) versus wt (n = 3), ***p < 0.002; 20 h, Alkbh8-/- (n = 2) versus wt (n = 2), ****p < 0.01; 24 h, Alkbh8-/- (n = 3) versus wt (n = 2), *****p < 0.001. UT, 6 h, wt (n = 3) versus 0 hours, wt γp < 0.02; 20 hours, wt (n = 2) versus 0 h, wt (n = 5) γγp < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131335.g006: Ribonucleoside modifications in wild type and Alkbh8-/- mice after oxidative stress.Wt and Alkbh8-/- MEFs were either left untreated (UT) or exposed to 1.2 mM of H2O2 for 1 hour. The levels of mcm5s2U, mcm5Um and mcm5U ribonucleoside modifications were identified by HPLC-coupled mass spectrometry and quantified by integrating the normalized peak area intensity for each signal at the indicated post-exposure time points. Significant differences in mcm5Um modifications were determined by the Student’s t-test: UT, Alkbh8-/- (n = 6) versus wt (n = 5), *p < 0.05; 3 h, Alkbh8-/- (n = 3) versus wt (n = 3), **p < 0.02; 6 h, Alkbh8-/- (n = 3) versus wt (n = 3), ***p < 0.002; 20 h, Alkbh8-/- (n = 2) versus wt (n = 2), ****p < 0.01; 24 h, Alkbh8-/- (n = 3) versus wt (n = 2), *****p < 0.001. UT, 6 h, wt (n = 3) versus 0 hours, wt γp < 0.02; 20 hours, wt (n = 2) versus 0 h, wt (n = 5) γγp < 0.01.
Mentions: The observed H2O2-induced increases in Alkbh8, Gpx1, Gpx3, and Gpx6 proteins in wt MEFs suggest that the associated tRNA modifications required for the translation of selenocysteine containing proteins are also increased in response to elevated ROS levels. Further the decreased Gpx1, Gpx3 and Gpx6 in in Alkbh8-/- MEFs suggests that tRNA modification is affected, relative to wt. To investigate these possibilities, we used our established LC-MS/MS approach [14, 17, 19, 36] with mass transitions specified in Table 2 to quantitate the basal and H2O2-induced levels of mcm5U, mcm5s2U and mcm5Um in wt and Alkbh8-/- MEFs (Fig 6). When comparing wt and Alkbh8-/- MEFs, we observed little difference in mcm5U and mcm5s2U levels under both basal and H2O2 treated conditions. In contrast, we observed a significant (p < 0.05) difference in mcm5Um in wt and Alkbh8-/- MEFs under basal conditions and after H2O2 treatment. We have also quantitated mcm5-base modifications in the livers of wt and Alkbh8-/- animals and observed analogous results to our MEF’s. Specifically we observed similar levels of the mcm5U and mcm5s2U modifications in wt and Alkbh8-/- livers and significantly decreased (p < 0.05) levels of the mcm5Um modification in the Alkbh8-/- vs. wt livers (S6 Fig). After exposure to ROS, the most obvious difference in mcm5Um levels in MEFs was seen 20 h after H2O2 treatment, when the wt cells had ~3.5-fold higher levels of this wobble base modification only found on tRNAUGA-SEC, relative to Alkbh8-/- MEFs. The 20-hour post-H2O2 time point also represents the peak levels of mcm5Um for wt MEFs, and lowest levels for Alkbh8-/- MEFs, further demonstrating a significant Alkbh8-dependent increase.

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