Homocysteine Down-regulates Cellular Glutathione Peroxidase (GPx1) by Decreasing Translation

Abstract

Hyperhomocysteinemia contributes to vascular dysfunction and an increase in the risk of cardiovascular disease. An elevated level of homocysteine in vivo and in cell culture systems results in a decrease in the activity of cellular glutathione peroxidase (GPx1), an intracellular antioxidant enzyme that reduces hydrogen peroxide and lipid peroxides. In this study, we show that homocysteine interferes with GPx1 protein expression without affecting transcript levels. Expression of the selenocysteine (SEC)-containing GPx1 protein requires special translational cofactors to "read-through" a UGAstop codon that specifies SEC incorporation at the active site of the enzyme. These factors include a selenocysteine incorporation sequence (SECIS) in the 3-untranslated region of the GPx1 mRNA and cofactors involved in the biosynthesis and translational insertion of SEC. To monitor SEC incorporation, we used a reporter gene system that has a UGA codon within the protein-coding region of the luciferase mRNA. Addition of either the GPx1 or GPx3 SECIS element in the 3-untranslated region of the luciferase gene stimulated read-through by 6 -11-fold in selenium-replete cells; absence of selenium prevented translation. To alter cellular homocysteine production, we used methionine in the presence of aminopterin, a folate antagonist, co-administered with hypoxanthine and thymidine (HAT/Met). This treatment increased homocysteine levels in the media by 30% (p < 0.01) and decreased GPx1 enzyme activity by 45% (p ‫؍‬ 0.0028). HAT/Met treatment decreased selenium-mediated read-through significantly (p < 0.001) in luciferase constructs containing the GPx1 or GPx3 SECIS element; most importantly, the suppression of selenium-dependent read-through was similar whether an SV40 promoter or the GPx1 promoter was used to drive transcription of the SECIS-containing constructs. Furthermore, HAT/Met had no effect on steadystate GPx1 mRNA levels but decreased GPx1 protein levels, suggesting that this effect is not transcriptionally mediated. These data support the conclusion that homocysteine decreases GPx1 activity by altering the translational mechanism essential for the synthesis of this selenocysteine-containing protein.

Selective homocysteine-lowering gene transfer attenuates pressure overload-induced cardiomyopathy via reduced oxidative stress

“…Handy et al [34] previously demonstrated that homocysteine decreases glutathione peroxidase activity by altering the specific translational mechanism essential for the synthesis of this selenocysteine-containing protein. Superoxide dismutase protein levels in the heart were reduced in a dog model [35] and in a rat model of chronic hyperhomocysteinemia [36].…”

Aldosterone Increases Oxidant Stress to Impair Guanylyl Cyclase Activity by Cysteinyl Thiol Oxidation in Vascular Smooth Muscle Cells

“…COS-7 cells, which do not express endogenous GC, were plated in 6-well tissue culture dishes and transfected with 5 g of DNA for 4 h with Lipofectamine 2000 TM (Invitrogen) (24). After this time, medium was replaced with full growth media, and experiments were performed after 48 h.…”

The Roles of Two miRNAs in Regulating the Immune Response of Sea Cucumber

“…ROS are important intracellular second messengers that mediate the production of inflammatory cytokines (Gorowara et al 1998). Abundant evidence indicated that excessive Hcy in endothelial cells induces endothelial dysfunction through the inhibition of arginine transport (Jin et al 2007), elevates ROS levels through autooxidation, and diminishes the activities of antioxidative enzymes, such as glutathione peroxidase-1 (Handy et al 2005) and superoxide dismutase (Yamamoto et al 2000). Furthermore, recent studies indicate that Hcy induces the production of superoxide anion (AlvarezMaqueda et al 2004;Au-Yeung et al 2006), exerts important effects on cellular NO production (Tsen et al 2003;Zhang et al 2000), and interferes with de novo GSH synthesis Figure 11 Schematic representation of the involvement of miR-137 and miR-2008 in the host-pathogen interaction by cotargeting AjBHMT.…”