Abstract
Antibiotics target bacteria by interfering with essential processes such as translation, but their effects on translation in mammalian cells are less well characterized. We found that doxycycline, chloramphenicol, and Geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). However, these proteins were differentially affected due to varying errors in Sec insertion at UGA. In the presence of doxycycline, chloramphenicol, or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most affected and GPx1 was the least affected by these translation errors. These observations were consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment.
Highlights
Antibiotics produce errors in bacterial protein synthesis, but their effects on translation in mammalian cells are poorly understood
Chloramphenicol, and Geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells
Cp, and G418 Affect Sec Insertion—To assess the effect of Dox, Cp, and G418 on selenoprotein synthesis, EMT6 cells were labeled with 75Se, grown in the presence of these antibiotics, and compared with cells grown in their absence (Fig. 1A, upper panel)
Summary
Antibiotics produce errors in bacterial protein synthesis, but their effects on translation in mammalian cells are poorly understood. This phenomenon would rule out Cys/ Sec replacement in those selenoproteins that are synthesized exclusively by the Um34 isoform, methylcarboxymethyl-5Јuridine-2Ј-O-methylribose Antibiotics, such as Geneticin (G418), doxycycline (Dox), and chloramphenicol (Cp), are widely used in mammalian cell culture and have cytotoxic effects on the host cell. This reduction in activity was due to substitution of Sec with Arg, premature termination of TR1 at UGA, and insertion of Cys and Trp in the latter protein Both GPx1 and GPx4 had significant levels of Cys in place of Sec following G418 treatment, but no detectable amounts of Trp following treatment with any of these antibiotics. This study exposed an unexpected level of vulnerability in Sec insertion under conditions of antibiotic treatment
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