Thioredoxin reductase specificity

Furman, C., Rundlof, A.K., Larigauderie, G., Jaye, M., Bricca, G., Copin, C., Kandoussi, A.M., Fruchart, J.C., Amer, E.S., and Rouis, M. 2004. Thioredoxin reductase 1 is upregulated in atherosclerotic plaques specific induction of the promoter in human macrophages by oxidized low-density lipoproteins. Free Radical Biol. Med. 37 71-85. 

In the search for possible targets the inhibition activity of a range of RAPTA compounds was studied for two specific enzymes - namely thioredoxin reductases (TrxR) and cathepsin B (cat B), important targets in cancer chemotherapy [28]. TrxR was chosen as it is highly relevant with respect to gold-based drugs (see below) and cat B is implicated in various stages of metastasis which correlate to the observed in vitro effects of RAPTA-T [29], It turned out that while the selected compounds (Fig. 3) are not inhibitors of TrxR with the exception of carboRAPTA-C, they are active inhibitors of cat B [28],... 

The discovery of non-specific disulfide reductases which are labile in aerobic cellular extracts suggests that kinetic constraints of thiol/disulfide exchange in vivo are very complex. One of such proteins is thioredoxin which behaves as a non-specific protein-disulfide reductase. Thioredoxin also works as a cofactor of sulfoxide reductases. The dithiol active site of thioredoxin sits on a protrusion of the protein surface [274], Thioredoxin is an ubiquitous protein whose molecular weight is about 12 KDa [274,275], It has been found in cytosolic and mitochondrial [276] compartments of animal cells, and it is partly bound to membranes. High contents in thioredoxin have been found in neurons, secretory and epithelial cells. Redox recycling of thioredoxin is insured by thioredoxin reductase, which has been identified in a variety of mammalian cells as a symmetrical dimer with a molecular weight of 116KDa[274]. Thioredoxin reductase is NADPH-specific, but it exhibits a very wide disulfide substrate specificity. 

Glutaredoxin is another small ubiquitous protein with a different dithiol-active center which catalyzes GSH-disulfide transhydrogenase reactions. It is GSH-specific and cannot be reduced by thioredoxin reductase. It uses GSH and an NADPH-coupled glutaredoxin reductase to catalyze the reduction of a variety of disulfide substrates, including 2-hydroxyethyl-disulfide and ribonucleotide reductase [281]. Since GSSG inhibits the latter reaction, a high ratio of GSH to GSSG will promote the synthesis of deoxyribonucleotides, which is a likely control mechanism of DNA synthesis. 

The specificity of these enzymes toward their disulfide substrates is quite remarkable There is virtually no cross-reactivity. Since it is quite difficult to separate glutathione reductase from thioredoxin reductase it... 

The three enzymes are quite specific for their respective pyridine nucleotide substrates. Under conditions normally used for assay, lipoamide dehydrogenase is less than % as active with NADPH as with NADH IS) and thioredoxin reductase is less than 1% as active with NADH as with NADPH 36, Sff). Lipoamide dehydrogenase can transfer electrons to a number of NAD analogs 37). Yeast glutathione reductase is quite specific for NADPH 60), but the erythrocyte enzyme is 20% as active with NADH as with NADPH under the conditions of the standard assay 30,40,61). 

Thioredoxin reductase is specific for NADPH and moreover for the hydrogen from the B side of the nicotinamide ring (38B). 

Figure 1 Reactions involved in the glycine reduction pathway hy Clostridia. Pa, Pb, and Pc denote the components of the glycine reduction complex where Pb is specific for the substrate (e.g. glycine, betaine, sarcosine). The reduction of oxidized protein Pa occurs via a redox chain involving thioredoxin reductase (TR) and thioredoxin (TRX). The involvement of ketene as an intermediate has not yet been proven...

In 1996, Tamura and Stadtman showed that thioredoxin reductase from mammalia-in contrast to the bacterial homologs-is a selenoprotein, and that selenocysteine is the penultimate C-terminal amino acid residue. Selenium-containing isoenzymes, which are tissue specific, have later on been characterized biochemically, or the existence of such isoforms was suggested by in-sihco methods. The substrate of thioredoxin reductase is thioredoxin, which is the key regulator of the redox status within cells. Thioredoxin catalyzes thiol-disulfide exchange reactions in proteins and peptides it is involved as redox mediator in the... 

Little is known about the specific biochemical mechanism(s) by which selenium and selenium compounds exert their acute toxic effects. Long-term effects on the hair, skin, nails, liver, and nervous system are also well documented, and a general theory has been developed to explain the toxicity of exposure to excess selenium, as discussed below. Generally, water-soluble forms are more easily absorbed and are generally of greater acute toxicity. Mechanisms of absorption and distribution for dermal and pulmonary uptake are unknown and subject to speculation, but an active transport mechanism for selenomethionine absorption in the intestine has been described (Spencer and Blau 1962). The mechanisms by which selenium exerts positive effects as a component of glutathione peroxidase, thioredoxin reductase, and the iodothyronine 5 -deiodinases are better understood, but the roles of other selenium-containing proteins in mammalian metabolism have not been clarified. 

The metabolism of selenium is now fairly well understood. To become incorporated into selenium-specific proteins (e.g., glutathione peroxidase, thioredoxin reductase, iodothyronine 5 -deiodinase) through a cotranslational mechanism requires that selenium be in the form of selenide (Sunde 1990). All forms of selenium can be transformed to selenide, although the rates of transformation vary. For example, selenate is not converted to selenide as readily as selenite. The formation of selenide from selenocysteine requires a specific enzyme, selenocysteine (3-lyase, which catalyzes the decomposition of selenocysteine to alanine and hydrogen selenide. Excess selenium is methylated and exhaled or excreted in the urine in both humans and animals. Further research is required to determine which selenium metabolites or intermediates lead to toxicity. 

The reduction of ribonucleotides to deoxyribonucleotides is linked to NADPH by thioredoxin and thioredoxin reductase. Thioredoxin is a small protein which is oxidized from a dithiol to a disulfide form during ribonucleotide reduction. The dithiol form of thioredoxin is regenerated by NADPH and a specific flavoprotein, thioredoxin reductase. The thioredoxin system consisting of thioredoxin and thioredoxin reductase was first identified as the reducing system from E. coli by Reichard and co-workers (35, 36) and both proteins have since been purified to homogeneity (37, 38). 

Thioredoxin reductase from calf liver was found to reduce 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) at the expense of NADPH even in the absence of thioredoxin as an intermediate electron carrier, indicating that this mammalian reductase may have a wider substrate specificity than the corresponding enzymes from E. coli and yeast. 

The thioredoxin reductases are highly specific for their thioredoxin substrate. For instance, yeast thioredoxin reductase is specific for the reduction of the homologous thioredoxins and does not interact with thioredoxins from E. coli or from T4-infected E. coli. Furthermore E. coli thioredoxin reductase does not use the thioredoxins from Novikoff hepatoma, yeast, and L. leichmannii. 

P. falciparum has been shown to contain a thioredoxin redox system— thioredoxin reductase (TrxR), thioredoxin and thioredoxin peroxidases (Nickel et ah, 2006). P. falciparum thioredoxin reductase is similar to GR in that it is homodimeric, NADPH dependent and binds one FAD per subunit. TrxR appears to be essential to parasite survival as knockout of the gene is lethal (Krnajski et ah, 2002). In a screen of 350,000 compounds, several unsaturated Mannich bases were identified as potential inhibitors of TrxR. The screen, as well as structure-activity relationship studies, resulted in the development of the first specific PfTrxR inhibitors (Andricopulo et ah, 2006 Davioud-Charvet et ah, 2003). 

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