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Alkbh8 Regulates Selenocysteine-Protein Expression to Protect against Reactive Oxygen Species Damage.

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.

SUBMITTER: Endres L

PROVIDER: S-EPMC4492958 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Rid (YjgF/YER057c/UK114) protein superfamily was first defined by sequence homology with available protein sequences from bacteria, archaea, and eukaryotes (L. Parsons, N. Bonander, E. Eisenstein, M. Gilson, et al., Biochemistry 42:80-89, 2003, https://doi.org/10.1021/bi020541w). The archetypal subfamily, RidA (reactive intermediate deaminase A), is found in all domains of life, with the vast majority of free-living organisms carrying at least one RidA homolog. In over 2 decades, close to 100 reports have implicated Rid family members in cellular processes in prokaryotes, yeast, plants, and mammals. Functional roles have been proposed for Rid enzymes in amino acid biosynthesis, plant root development and nutrient acquisition, cellular respiration, and carcinogenesis. Despite the wealth of literature and over a dozen high-resolution structures of different RidA enzymes, their biochemical function remained elusive for decades. The function of the RidA protein was elucidated in a bacterial model system despite (i) a minimal phenotype of ridA mutants, (ii) the enzyme catalyzing a reaction believed to occur spontaneously, and (iii) confusing literature on the pleiotropic effects of RidA homologs in prokaryotes and eukaryotes. Subsequent work provided the physiological framework to support the RidA paradigm in Salmonella enterica by linking the phenotypes of mutants lacking ridA to the accumulation of the reactive metabolite 2-aminoacrylate (2AA), which damaged metabolic enzymes. Conservation of enamine/imine deaminase activity of RidA enzymes from all domains raises the likelihood that, despite the diverse phenotypes, the consequences when RidA is absent are due to accumulated 2AA (or a similar reactive enamine) and the diversity of metabolic phenotypes can be attributed to differences in metabolic network architecture. The discovery of the RidA paradigm in S. enterica laid a foundation for assessing the role of Rid enzymes in diverse organisms and contributed fundamental lessons on metabolic network evolution and diversity in microbes. This review describes the studies that defined the conserved function of RidA, the paradigm of enamine stress in S. enterica, and emerging studies that explore how this paradigm differs in other organisms. We focus primarily on the RidA subfamily, while remarking on our current understanding of the other Rid subfamilies. Finally, we describe the current status of the field and pose questions that will drive future studies on this widely conserved protein family to provide fundamental new metabolic information.

Project description:KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1(-/-) cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1(-/-) cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45alpha, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the cell capacity to scavenge intracellular ROS through an antioxidant pathway involving FoxO1 and SOD2, thus providing novel and useful insights into the understanding of KRIT1 molecular and cellular functions.

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Alkbh8 Regulates Selenocysteine-Protein Expression to Protect against Reactive Oxygen Species Damage.

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