Aminoglycosides Decrease Glutathione Peroxidase-1 Activity by Interfering with Selenocysteine Incorporation

Diane E Handy,Gaozhen Hang,John Scolaro,Nicole Metes,Nadia Razaq,Yi Yang,Joseph Loscalzo

doi:10.1074/jbc.m511295200

Diane E Handy, Gaozhen Hang + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m511295200

Copy DOI

Abstract

Cellular glutathione peroxidase is a key intracellular antioxidant enzyme that contains a selenocysteine residue at its active site. Selenium, a selenocysteine incorporation sequence in the 3'-untranslated region of the glutathione peroxidase mRNA, and other translational cofactors are necessary for "read-through" of a UGA stop codon that specifies selenocysteine incorporation. Aminoglycoside antibiotics facilitate read-through of premature stop codons in prokayotes and eukaryotes. We studied the effects of G418, an aminoglycoside, on cellular glutathione peroxidase expression and function in mammalian cells. Insertion of a selenocysteine incorporation element along with a UGA codon into a reporter construct allows for read-through only in the presence of selenium. G418 increased read-through in selenium-replete cells as well as in the absence of selenium. G418 treatment increased immunodetectable endogenous or recombinant glutathione peroxidase but reduced the specific activity of the enzyme. Tandem mass spectrometry experiments indicated that G418 caused a substitution of l-arginine for selenocysteine. These data show that G418 can affect the biosynthesis of this key antioxidant enzyme by promoting substitution at the UGA codon.

Highlights

Cellular glutathione peroxidase-1 (GPx-1)2 is one of several mammalian glutathione peroxidases that reduce hydrogen and lipid peroxides using glutathione (GSH) as a reductant [1]
Addition of a SECIS element in the sense orientation within the 3Ј-untranslated region (3Ј-UTR) of the luciferase gene allowed for a low level of read-through dependent on the levels of selenium supplementation (Fig. 1B); by contrast, the presence of a SECIS element in the antisense orientation did not allow for an increase in luciferase activity
No other substituted peptides were identified in the LC-MS/MS analysis. These findings suggest that GPx-1 translation is highly sensitive to G418 in COS7 cells grown under conditions of selenium restriction

Summary

Introduction

Cellular glutathione peroxidase-1 (GPx-1) is one of several mammalian glutathione peroxidases that reduce hydrogen and lipid peroxides using glutathione (GSH) as a reductant [1]. Each monomer of the tetrameric enzyme contains a selenocysteine (SEC) residue at the active site of the protein [2, 3] This selenium-containing amino acid is incorporated during translation through the recognition of a UGA codon as a site for SEC incorporation rather than as a site for termination of translation. Prior studies have shown that insertion of a UGA within the protein coding region of heterologous transcripts can be properly decoded as a selenocysteine codon provided that the SECIS element and surrounding sequences are present in the 3Ј-UTR of the transcript [10, 11]. Because of the specialized mechanism of translation required for recoding a UGA as a site for selenocysteine incorporation, we tested whether G418, an AMG, could alter SEC incorporation, protein expression, and enzyme activity of the antioxidant enzyme GPx-1 in mammalian cells

Methods

Results

Conclusion