John R. Gasdaska

Crystal structures of reduced, oxidized, and mutated human thioredoxins: evidence for a regulatory homodimer.

BACKGROUND Human thioredoxin reduces the disulfide bonds of numerous proteins in vitro, and can activate transcription factors such as NFkB in vivo. Thioredoxin can also act as a growth factor, and is overexpressed and secreted in certain tumor cells. RESULTS Crystal structures were determined for reduced and oxidized wild type human thioredoxin (at 1.7 and 2.1 A nominal resolution, respectively), and for reduced mutant proteins Cys73-->Ser and Cys32-->Ser/Cys35-->Ser (at 1.65 and 1.8 A, respectively). Surprisingly, thioredoxin is dimeric in all four structures; the dimer is linked through a disulfide bond between Cys73 of each monomer, except in Cys73-->Ser where a hydrogen bond occurs. The thioredoxin active site is blocked by dimer formation. Conformational changes in the active site and dimer interface accompany oxidation of the active-site cysteines, Cys32 and Cys35. CONCLUSIONS It has been suggested that a reduced pKa in the first cysteine (Cys32 in human thioredoxin) of the active-site sequence is important for modulation of the redox potential in thioredoxin. A hydrogen bond between the sulfhydryls of Cys32 and Cys35 may reduce the pKa of Cys32 and this pKa depression probably results in increased nucleophilicity of the Cys32 thiolate group. This nucleophilicity, in tum, is thought to be necessary for the role of thioredoxin in disulfide-bond reduction. The physiological role, if any, of thioredoxin dimer formation remains unknown. It is possible that dimerization may provide a mechanism for regulation of the protein, or a means of sensing oxidative stress.

Cloning and sequencing of a human thioredoxin reductase

The DNA sequence encoding human placental thioredoxin reductase has been determined. Of the 3826 base pairs sequenced, 1650 base pairs were in an open reading frame encoding a mature protein with 495 amino acids and a calculated molecular mass of 54,171. Sequence analysis showed strong similarity to glutathione reductases and other NADPH‐dependent reductases. Human thioredoxin reductase contains the redoxactive cysteines in the putative FAD binding domain and has a dimer interface domain not previously seen with prokaryote and lower eukaryote thioredoxin reductases.

Redox Signaling and the Control of Cell Growth and Death

Publisher Summary Cells maintain an intracellular environment that is reducing in the face of a highly oxidizing extracellular environment. Regulated alterations in the intracellular redox state (redox signaling) can modulate events such as DNA synthesis, enzyme activation, selective gene expression, and regulation of the cell cycle. The primary consequence of intracellular redox signaling is a change in the oxidation state of cysteine residues of key proteins. This form of posttranslational modification of protein is difficult to follow because it lacks a convenient marker and is readily reversed when the cell contents are exposed to extracellular oxidizing conditions. For this reason, knowledge of the role that redox signaling may play in cell function has lagged behind that of protein modification produced. This chapter discusses the components of the cellular machinery used for the redox regulation of protein activity and its consequences for cell growth and death. Discussed here are the cellular redox systems—glutathione/glutathione reductase, thioredoxin reductase/thioredoxin, protein disulfide isomerase, glutaredoxin, ref-I, and metallothionein. NADPH reduces oxidized glutathione (GSSG) and thioredoxin [Trx (ox)] through glutathione reductase and thioredoxin reductase, respectively, giving reduced glutathione (GSH) and reduced thioredoxin Trx (red). GSH reduces oxidized glutaredoxin [Grx (ox)], and Trx reduces oxidized Ref-1.Trx and Grx provide reducing equivalents to ribonucleotide reductase (RR) for deoxyribonucleotide synthesis. The chapter details the redox targets—ribonucleotide reductase, receptor proteins, and the transcription factors: OxyR, NF-KB/Rel, AP-1—and protein folding and degradation. Mentioned are the cellular responses to redox changes—cell proliferation and activities of thioredoxin related to cell proliferation, oxidant signaling and apoptosis, oxidative stress, and hypoxia. Compelling evidence from in vitro studies shows that alterations in the redox state of proteins involving key cysteine residues lead to conformational changes. Recent studies have identified several redox systems in cells that appear to respond to external stimuli.

Effect of selenium on rat thioredoxin reductase activity: increase by supranutritional selenium and decrease by selenium deficiency.

Thioredoxin reductase is a newly identified selenocysteine-containing enzyme that catalyzes the NADPH-dependent reduction of the redox protein thioredoxin. Thioredoxin stimulates cell growth, is found in dividing normal cells, and is over-expressed in a number of human cancers. Redox activity is essential for the growth effects of thioredoxin; thus, thioredoxin reductase could be involved in regulating cell growth through its reduction of thioredoxin. In rats fed a selenium-deficient diet (<0.01 ppm) for up to 98 days, thioredoxin reductase activity was decreased, compared with that of rats fed a normal selenium diet (0.1 ppm), in lung, liver, and kidney, while thioredoxin reductase activity in the spleen and prostate was unaltered. Rats fed a high selenium diet (1.0 ppm) exhibited a 1.5-fold increase in kidney and a 2.0-fold increase in lung thioredoxin reductase activity that began to return to control values after 20 and 69 days, respectively. Liver showed a 2.1-fold increase in thioredoxin reductase activity at 20 days only. Thioredoxin reductase protein levels measured by western blotting using an antibody to human thioredoxin reductase were decreased in rats fed the selenium-deficient diet and did not increase in rats fed the high selenium diet. Rat thioredoxin reductase was shown to incorporate 75Selenium. Thus, in some tissues at least, the increase in thioredoxin reductase activity of rats fed a high selenium diet appears to be due to an increase in the specific activity of the enzyme, possibly caused by increased selenocysteine incorporation without an increase in thioredoxin reductase protein synthesis.

Regulation of Human Thioredoxin Reductase Expression and Activity by 3′-Untranslated Region Selenocysteine Insertion Sequence and mRNA Instability Elements

Thioredoxin reductases function in regulating cellular redox and function through their substrate, thioredoxin, in the proper folding of enzymes and redox regulation of transcription factor activity. These enzymes are overexpressed in certain tumors and cancer cells and down-regulated in apoptosis and may play a role in regulating cell growth. Mammalian thioredoxin reductases contain a selenocysteine residue, encoded by a UGA codon, as the penultimate carboxyl-terminal amino acid. This amino acid has been proposed to carry reducing equivalents from the active site to substrates. We report expression of a wild-type thioredoxin reductase selenoenzyme, a cysteine mutant enzyme, and the UGA-terminated protein in mammalian cells and overexpression of the cysteine mutant and UGA-terminated proteins in the baculovirus insect cell system. We show that substitution of cysteine for selenocysteine decreases enzyme activity for thioredoxin by 2 orders magnitude, and that termination at the UGA codon abolishes activity. We further demonstrate the presence of a functional selenocysteine insertion sequence element that is highly active but only moderately responsive to selenium supplementation. Finally, we show that thioredoxin reductase mRNA levels are down-regulated by other sequences in the 3′-untranslated region, which contains multiple AU-rich instability elements. These sequences are found in a number of cytokine and proto-oncogene mRNAs and have been shown to confer rapid mRNA turnover.

Oxidative inactivation of thioredoxin as a cellular growth factor and protection by a Cys73 → Ser mutation

Thioredoxin (Trx) is a widely distributed redox protein that regulates several intracellular redox-dependent processes and stimulates the proliferation of both normal and tumor cells. We have found that when stored in the absence of reducing agents, human recombinant Trx undergoes spontaneous oxidation, losing its ability to stimulate cell growth, but is still a substrate for NADPH-dependent reduction by human thioredoxin reductase. There is a slower spontaneous conversion of Trx to a homodimer that is not a substrate for reduction by thioredoxin reductase and that does not stimulate cell proliferation. Both conversions can be induced by chemical oxidants and are reversible by treatment with the thiol reducing agent dithiothreitol. SDS-PAGE suggests that Trx undergoes oxidation to monomeric form(s) preceding dimer formation. We have recently shown by X-ray crystallography that Trx forms a dimer that is stabilized by an intermolecular Cys73-Cys73 disulfide bond. A Cys73-->Ser mutant Trx (C73S) was prepared to determine the role of Cys73 in oxidative stability and growth stimulation. C73S was as effective as Trx in stimulating cell growth and was a comparable substrate for thioredoxin reductase. C73S did not show spontaneous or oxidant-induced loss of activity and did not form a dimer. The results suggest that Trx can exist in monomeric forms, some of which are mediated by Cys73 that do not stimulate cell proliferation but can be reduced by thioredoxin reductase. Cys73 is also involved in formation of an enzymatically inactive homodimer, which occurs on long term storage or by chemical oxidation. Thus, although clearly involved in protein inactivation, Cys73 is not necessary for the growth stimulating activity of Trx.

Cockroach transferrin closely resembles vertebrate transferrins in its metal ion-binding properties: A spectroscopic study

The optical and electron paramagnetic resonance (EPR) spectroscopic properties of a transferrin from the cockroach Blaberus discoidalis have been investigated to determine the relation of this protein to vertebrate transferrins. Difference spectrophotometry substantiates the involvement of tyrosyl residues in iron binding, and confirms the specific binding of two equivalents of iron per molecule. The far-UV CD spectrum also indicates a secondary structure with marked similarity to those of vertebrate transferrins. EPR studies show a dependence of iron binding on (bi)carbonate, consistent with the absolute requirement of transferrins for a synergistic anion in binding iron. Continuous wave (CW) and pulsed EPR studies of the cupric complex of the protein implicate a histidyl nitrogen ligand in metal coordination, as in human transferrin. Additional studies establish that the pH-dependent release of iron is similar to that of human serum transferrin. The present data confirm cockroach transferrin as an authentic member of the transferrin superfamily, thereby suggesting an ancestral relationship of insect to vertebrate transferrins.