The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was timeand dose-dependent, with an apparent K m of 22 M and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.
The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxRoverexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1-or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.Thioredoxin reductases (TrxRs) 1 are members of the nucleotide-disulfide oxidoreductase family and are ubiquitously found in mammalian tissues. Two main isoforms of thioredoxin reductases exist in mammals: the classical cytosolic form (TrxR1, the TXNRD1 gene product) (1, 2), and the mitochondrial form (TrxR2) (3, 4). A third form, thioredoxin and glutathione reductase, is mainly expressed in testis (5). All mammalian TrxRs are homodimeric selenocysteine-containing enzymes (6) that share high sequence homology between each other and contain an NADPH binding domain and a FAD binding domain per subunit (7). TrxRs reduce and thereby activate thioredoxins (Trxs), which are small ubiquitous proteins with a conserved active site sequence (-Trp-Cys-Gly-ProCys-) that catalyze many redox reactions through the reversible oxidation of the active site dithiol to a disulfide. Trxs are involved in a variety of reactions, such as redox regulation of transcription factors (8, 9), reduction of hydroperoxides (4, 10), and modulation of cell growth (11) and apoptosis (12, 13). The selenocysteine residue (Sec) that is found at the C terminus in TrxRs (-Gly-Cys-Sec-Gly-COOH) is encoded by a UGA codon that is normally identified as a stop codon, but which can be transcribed as Sec in the presence of a selenocysteine insertion sequence (SECIS) element in the 3Ј-UTR of the corresponding mRNA (14). The C-terminal Cys-Sec motif is located on a presumably flexible arm of the enzyme that is kept reduced by the N-terminal redox active motif (-Cys-Val-Asn-Val-Gly...
Lipoamide dehydrogenase belongs to a family of pyridine nucleotide disulfide oxidoreductases and is ubiquitous in aerobic organisms. This enzyme also reduces ubiquinone (the only endogenously synthesized lipid-soluble antioxidant) to ubiquinol, the form in which it functions as an antioxidant. The reduction of ubiquinone was linear with time and exhibited turnover numbers of 5 and 1.2 min 21 in the presence and absence of zinc, respectively. The reaction was stimulated by zinc and cadmium but not by the other divalent ions tested. The zinc/cadmium-dependent stimulation of the reaction increased rapidly and linearly up to a concentration of 0.1 mm and was even further increased at 0.5 mm. At pH 6, the activity was three times higher than at physiological pH. Alteration of the NADPH : NADP 1 ratio revealed that the reaction is inhibited by higher concentrations of the oxidized cofactors. FAD reduced ubiquinone in a dose-dependent manner at a considerably lower rate, suggesting that the reduction of ubiquinone by lipoamide dehydrogenase involves the FAD moiety of the enzyme.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.