Selenium is an essential micronutrient in the diet of humans and other mammals and many health benefits have been ascribed to this element including preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process, and having roles in mammalian development, male reproduction and immune function. We proposed previously that the health benefits of selenium are due in large part to the presence of selenium in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec), and since the pathway for Sec biosynthesis had not been determined in archaea and eukaryotes, we undertook a project to elucidate how this amino acid, which is the 21st amino acid in the genetic code, was synthesized and to identify and characterize each of the components involved. We previously established the biosynthetic pathway of Sec in eukaryotes and archaea and are now focusing our attention on the two Sec tRNA isoforms that we have shown are responsible for the synthesis of the two subclasses of selenoproteins designated housekeeping and stress-related selenoproteins;and on the methylase, designated Um34 methylase, that synthesizes the methyl group at the 2?-O-postion on the ribosyl moiety at nucleotide 34 of Sec tRNA. We previously confirmed that addition of Um34 to the isoform, 5-methylcarobxymethyluridine (mcmU), to form 5-methylcarboxymethyl, 2?-O-methyluridine (mcmUm) requires that mcmU is aminoacylated with Sec, i.e., that the substrate for the methylase (designated Um34 methylase) which carries out this reaction is selenocysteyl-tRNA. In the past year, the aminoacylation study was completed and published and it provided important insights into our understanding of how the subclass of selenoproteins, designated stress-related selenoproteins, is biosynthesized specifically by the mcmUm isoform.We identified the methylase, designated Um34 methylase, that is responsible for the addition of the 2?-O-methyl group to the ribosyl moiety at position 34. The Um34 methylase gene has been cloned into expression vectors, expressed in bacterial and mammalian cells, the protein product isolated and it is currently being characterized. Um34 methylase has a mitochondrial signal at its amino terminus. We have shown that the methylase is localized in the mitochondria, wherein the approximately 20 amino acid mitochondrial signal is cleaved when the protein enters this organelle. We are currently developing an assay to further characterize Um34 methylase using the mcmU Sec tRNA isoform as substrate and S-adenosylmethionine as the methyl donor.In addition, we previously demonstrated that cysteine could replace Sec in thioredoxin reductase 1 (TR1) and have expanded these studies extensively in the past year. We are examining the replacement of Sec by cysteine and the mistranslation of the Sec UGA codon by other amino acids in cancer cells and in cells treated with antibiotics. We found that the antibiotics doxycycline (Dox), chloramphenicol (Cp) and Geneticin (G418) block selenoprotein synthesis by inhibiting the Sec insertion step in eukaryotic cells. In this study, we focused on three selenoproteins, TR1, glutathione peroxidase 1 (GPx1) and glutathione peroxidase 4 (GPx4), because the Sec codon is found at a different position in each protein and the proteins show differential use of the Sec tRNA isoforms, mcmU and mcmUm. All three antibiotics induced the protein expression of TR1 and GPx4 as measured by western blot analysis, while GPx1 expression decreased upon treatment. However, selenium-75 labeling and enzymatic assays showed a decrease in expression and activity of all three selenoproteins. Mass spec analysis of purified, His-tagged recombinant TR1, GPx1 and GPx4 revealed that Sec residues were replaced by other amino acids, such as cysteine, arginine and tryptophan, in an antibiotic-dependent manner and thus misreading was occurring and not Cys/Sec replacement. In human cancer tissues, we found that TR1 protein expression was increased in both liver and lung tumor tissues compared with surrounding normal tissues, whereas the activities of TR1 in tumors were slightly decreased compared with surrounding normal tissue. Mass spec analysis of purified TR1 from tumors and surrounding normal tissue revealed that cysteine is inserted in place of Sec in tumor tissues or the protein was truncated at the Sec codon.

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National Cancer Institute (NCI)
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Hatfield, Dolph L; Tsuji, Petra A; Carlson, Bradley A et al. (2014) Selenium and selenocysteine: roles in cancer, health, and development. Trends Biochem Sci 39:112-20
Seeher, Sandra; Atassi, Tarik; Mahdi, Yassin et al. (2014) Secisbp2 is essential for embryonic development and enhances selenoprotein expression. Antioxid Redox Signal 21:835-49
Barroso, Madalena; Florindo, Cristina; Kalwa, Hermann et al. (2014) Inhibition of cellular methyltransferases promotes endothelial cell activation by suppressing glutathione peroxidase 1 protein expression. J Biol Chem 289:15350-62
Turanov, Anton A; Lobanov, Alexei V; Hatfield, Dolph L et al. (2013) UGA codon position-dependent incorporation of selenocysteine into mammalian selenoproteins. Nucleic Acids Res 41:6952-9
Howard, Michael T; Carlson, Bradley A; Anderson, Christine B et al. (2013) Translational redefinition of UGA codons is regulated by selenium availability. J Biol Chem 288:19401-13
Tobe, Ryuta; Naranjo-Suarez, Salvador; Everley, Robert A et al. (2013) High error rates in selenocysteine insertion in mammalian cells treated with the antibiotic doxycycline, chloramphenicol, or geneticin. J Biol Chem 288:14709-15
Kim, Mijin; Chen, Zifan; Shim, Myoung Sup et al. (2013) SUMO modification of NZFP mediates transcriptional repression through TBP binding. Mol Cells 35:70-8
Lee, Byung Cheon; Lobanov, Alexey V; Marino, Stefano M et al. (2011) A 4-selenocysteine, 2-selenocysteine insertion sequence (SECIS) element methionine sulfoxide reductase from Metridium senile reveals a non-catalytic function of selenocysteines. J Biol Chem 286:18747-55
Kim, Jin Young; Carlson, Bradley A; Xu, Xue-Ming et al. (2011) Inhibition of selenocysteine tRNA[Ser]Sec aminoacylation provides evidence that aminoacylation is required for regulatory methylation of this tRNA. Biochem Biophys Res Commun 409:814-9
Shim, Myoung Sup; Kim, Jin Young; Lee, Kwang Hee et al. (2011) l(2)01810 is a novel type of glutamate transporter that is responsible for megamitochondrial formation. Biochem J 439:277-86

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