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.
Ubiquinol is an endogenously synthesized lipid-soluble antioxidant. Regeneration of ubiquinol from the oxidized form is essential to the maintenance of its antioxidant function. We demonstrated that lipoamide dehydrogenase can reduce ubiquinone to ubiquinol. Zinc increased the rate of the NADPHdependent reduction more than 10-fold. The concentration ubiquinone resulting in the half-maximal rate of reduction was approximately 5 W WM in the presence and 4 W WM in the absence of zinc. These data may explain how ubiquinone is reduced to the active antioxidant ubiquinol, which plays such an important role in protecting against oxidative stress and lipid peroxidation.z 1999 Federation of European Biochemical Societies.Key words: Ubiquinone; Lipoamide dehydrogenase; Antioxidant; Zinc IntroductionReactive oxygen species (ROS) can cause oxidative damage to living cells due to their high level of reactivity. The polyunsaturated fatty acids in biological membranes are particularly sensitive to oxidative damage by ROS. Reaction of these species with unsaturated fatty acids initiates a chain-reaction leading to extensive damage to the membrane, a process known as lipid peroxidation [1]. This process can be prevented by lipid-soluble antioxidants such as vitamin E (K-tocopherol) and ubiquinol [1], which function as antioxidants only in the reduced state. Therefore, when tocopherol and ubiquinol are oxidized by ROS their reduced forms must be regenerated.Ubiquinone (Q10) is an obligatory participant in the mitochondrial respiratory chain, where it is present in large excess over the other components involved in electron transport in order to achieve maximal respiration [2]. In recent years ubiquinol (reduced Q10) has been the object of renewed interest, since accumulating evidence indicates that this substance is our only endogenously synthesized lipid-soluble antioxidant and is present in all membranes [1,3^5]. The mechanism by which Q10 is reduced to ubiquinol is not known although di¡erent cytosolic enzymes have been proposed to be involved [6,7]. However, no speci¢c enzyme catalyzing this reaction has been identi¢ed and therefore, we have investigated the wellknown enzyme, lipoamide dehydrogenase (EC 1.8.1.4), in this regard.Lipoamide dehydrogenase is a component of the 3-K-ketoacid dehydrogenase complex which oxidizes pyruvate, K-ketoglutarate and the branched-chain K-ketoacids. This enzyme complex is present at the matrix surface of the inner mitochondrial membrane in all eukaryotic organisms studied to date. It catalyzes the oxidative decarboxylation of pyruvate with concomitant formation of CO P , acetyl-CoA and NADH. In this complex (dihydro)lipoamide dehydrogenase catalyzes NAD-dependent oxidation of the dihydrolipoyl cofactor covalently linked to the components catalyzing acetyl transferase.Lipoamide dehydrogenase belongs to a family of pyridine nucleotide-disul¢de oxidoreductases [8], which are homodimeric proteins with subunits approximately 50 kDa in size, each containing a FAD moiety and a redox-active ...
Ubiquinol is a powerful antioxidant, which is oxidized in action and needs to be replaced or regenerated to be capable of a sustained effort. This article summarises current knowledge of extramitochondrial reduction of ubiquinone by three flavoenzymes, i.e. lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase, belonging to the same pyridine nucleotide-disulfide oxidoreductase family. These three enzymes are the most efficient extramitochondrial ubiquinone reductases so far described. The reduction of ubiquinone by lipoamide dehydrogenase and glutathione reductase is potently stimulated by zinc and the highest rate of reduction is achieved at acidic pH and the rates are equal with either NADPH or NADH as co-factors. The most efficient ubiquinone reductases are mammalian cytosolic thioredoxin reductases, which are selenoenzymes with a number of biological functions. Reduction of ubiquinone by thioredoxin reductase is in contrast to the other two enzymes investigated, inhibited by zinc and shows a sharp physiological pH optimum at pH 7.5. Furthermore, the reaction is selenium dependent as revealed from experiments using truncated and mutant forms of the enzyme and also in a cellular context by selenium treatment of transfected thioredoxin reductase overexpressing stable cell lines. The reduction of ubiquinone by the three enzymes offers a multifunctional system for extramitochondrial regeneration of an important antioxidant.
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