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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 have slow growth and increased apoptosis phenotypes.A) 4 x 104 wt or Alkbh8-/- MEFs were seeded per 9.6 cm2 well and viable (trypan blue negative) cells were counted over a period of 12 days. Error bars represent standard deviation (±STDV, n = 3) and significant differences in growth was determined by Student t-test (τp < 0.005, *p < 0.05). B) 1 x 104 cells were seeded per 58.1 cm2 dish, stained with crystal violet after 2 weeks in culture and colonies with a diameter > 2 mm were counted. Representative pictures of crystal violet stained cells. C-D) MEFs were allowed to grow to confluence and 2.5 x 105 cells were plated per 58.1 cm2 dish. Cells were fixed and assessed for apoptosis by TUNEL assay using a dUTP-FITC conjugate, 4 and 7 days after plating. Apoptotic cells were enumerated by flow cytometry and appear in the FITC-A channel. The percentage of TUNEL-positive cells is shown as the mean ± standard deviation (n = 3, right panel, *p < 0.05, **p < 0.001). E-F) RNA or whole protein extracts were prepared from sub-confluent cultures of wt and Alkbh8-/- MEFs and assessed for (E) PERP gene expression by qRT-PCR, in which each sample was normalized to internal Gapdh levels and (F) p53 protein expression by Western blotting
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pone.0131335.g001: Alkbh8-/- MEFs have slow growth and increased apoptosis phenotypes.A) 4 x 104 wt or Alkbh8-/- MEFs were seeded per 9.6 cm2 well and viable (trypan blue negative) cells were counted over a period of 12 days. Error bars represent standard deviation (±STDV, n = 3) and significant differences in growth was determined by Student t-test (τp < 0.005, *p < 0.05). B) 1 x 104 cells were seeded per 58.1 cm2 dish, stained with crystal violet after 2 weeks in culture and colonies with a diameter > 2 mm were counted. Representative pictures of crystal violet stained cells. C-D) MEFs were allowed to grow to confluence and 2.5 x 105 cells were plated per 58.1 cm2 dish. Cells were fixed and assessed for apoptosis by TUNEL assay using a dUTP-FITC conjugate, 4 and 7 days after plating. Apoptotic cells were enumerated by flow cytometry and appear in the FITC-A channel. The percentage of TUNEL-positive cells is shown as the mean ± standard deviation (n = 3, right panel, *p < 0.05, **p < 0.001). E-F) RNA or whole protein extracts were prepared from sub-confluent cultures of wt and Alkbh8-/- MEFs and assessed for (E) PERP gene expression by qRT-PCR, in which each sample was normalized to internal Gapdh levels and (F) p53 protein expression by Western blotting

Mentions: Mice were made Alkbh8 deficient using an insertional mutagenic approach that introduced a "gene trap" into intron 7 [34]. This insertion creates a fusion transcript with a premature stop codon upstream of coding exons 8–11 and thus truncates not only the entire methyltransferase domain but also a portion of the 2OG-Fe(II) (S1 Fig). In culturing Alkbh8-/- MEFs, it became apparent that they grew more slowly relative to their wt counterparts. As a first step in phenotype characterization studies, the growth and colony forming potential of Alkbh8-/- MEFs were assessed and compared to wt MEFs. During a 10-day period of culture, the Alkbh8-/- MEFs grew approximately 2-times slower than did the wt MEFs (Fig 1A). Further, Alkbh8-/- MEFs plated at low density formed half the number of colonies than did the wt MEFs after two weeks of culturing (Fig 1B). The slow growth and diminished colony forming phenotypes suggest that Alkbh8-/- were surviving under stress, a trait common to other stress response and DNA repair-deficient cells [38].


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 have slow growth and increased apoptosis phenotypes.A) 4 x 104 wt or Alkbh8-/- MEFs were seeded per 9.6 cm2 well and viable (trypan blue negative) cells were counted over a period of 12 days. Error bars represent standard deviation (±STDV, n = 3) and significant differences in growth was determined by Student t-test (τp < 0.005, *p < 0.05). B) 1 x 104 cells were seeded per 58.1 cm2 dish, stained with crystal violet after 2 weeks in culture and colonies with a diameter > 2 mm were counted. Representative pictures of crystal violet stained cells. C-D) MEFs were allowed to grow to confluence and 2.5 x 105 cells were plated per 58.1 cm2 dish. Cells were fixed and assessed for apoptosis by TUNEL assay using a dUTP-FITC conjugate, 4 and 7 days after plating. Apoptotic cells were enumerated by flow cytometry and appear in the FITC-A channel. The percentage of TUNEL-positive cells is shown as the mean ± standard deviation (n = 3, right panel, *p < 0.05, **p < 0.001). E-F) RNA or whole protein extracts were prepared from sub-confluent cultures of wt and Alkbh8-/- MEFs and assessed for (E) PERP gene expression by qRT-PCR, in which each sample was normalized to internal Gapdh levels and (F) p53 protein expression by Western blotting
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131335.g001: Alkbh8-/- MEFs have slow growth and increased apoptosis phenotypes.A) 4 x 104 wt or Alkbh8-/- MEFs were seeded per 9.6 cm2 well and viable (trypan blue negative) cells were counted over a period of 12 days. Error bars represent standard deviation (±STDV, n = 3) and significant differences in growth was determined by Student t-test (τp < 0.005, *p < 0.05). B) 1 x 104 cells were seeded per 58.1 cm2 dish, stained with crystal violet after 2 weeks in culture and colonies with a diameter > 2 mm were counted. Representative pictures of crystal violet stained cells. C-D) MEFs were allowed to grow to confluence and 2.5 x 105 cells were plated per 58.1 cm2 dish. Cells were fixed and assessed for apoptosis by TUNEL assay using a dUTP-FITC conjugate, 4 and 7 days after plating. Apoptotic cells were enumerated by flow cytometry and appear in the FITC-A channel. The percentage of TUNEL-positive cells is shown as the mean ± standard deviation (n = 3, right panel, *p < 0.05, **p < 0.001). E-F) RNA or whole protein extracts were prepared from sub-confluent cultures of wt and Alkbh8-/- MEFs and assessed for (E) PERP gene expression by qRT-PCR, in which each sample was normalized to internal Gapdh levels and (F) p53 protein expression by Western blotting
Mentions: Mice were made Alkbh8 deficient using an insertional mutagenic approach that introduced a "gene trap" into intron 7 [34]. This insertion creates a fusion transcript with a premature stop codon upstream of coding exons 8–11 and thus truncates not only the entire methyltransferase domain but also a portion of the 2OG-Fe(II) (S1 Fig). In culturing Alkbh8-/- MEFs, it became apparent that they grew more slowly relative to their wt counterparts. As a first step in phenotype characterization studies, the growth and colony forming potential of Alkbh8-/- MEFs were assessed and compared to wt MEFs. During a 10-day period of culture, the Alkbh8-/- MEFs grew approximately 2-times slower than did the wt MEFs (Fig 1A). Further, Alkbh8-/- MEFs plated at low density formed half the number of colonies than did the wt MEFs after two weeks of culturing (Fig 1B). The slow growth and diminished colony forming phenotypes suggest that Alkbh8-/- were surviving under stress, a trait common to other stress response and DNA repair-deficient cells [38].

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