Thioredoxin

The thioredoxin system, consisting of thioredoxin and thioredoxin reductase, was originally discovered as the hydrogen carrier system, which provides, with NADPH, the reducing potential for the reduction of ribonucleotides (5, 35). Since then considerable evidence has been accumulated to indicate that this or a closely related system also participates in a variety of other enzymatic reductions. For instance thioredoxin can function as an electron carrier between NADPH and several disulfides, such as insulin, lipoate and oxidized glutathione. Furthermore Porque et al. (114) have shown that thioredoxin and thioredoxin reductase from yeast can function as hydrogen carriers in the reduction of methionine sulfoxide and sulfate. 

Thioredoxin from E. coli is a heat stable small acidic protein (pi 4.4—4.5) with a molecular weight of about 12,000 (37, 115—118). It consists of a single polypeptide chain with 108 amino acid residues the complete amino acid sequence has been determined (Fig. 2). Thioredoxin can occur in the oxidized or reduced state. Reduced thioredoxin contains two cysteine residues in positions 32 and 35 which can be oxidized enzymatically or chemically to the disulfide bridge of cystine. Only two 

Both E. coli and T4 thioredoxin have recently been purified by immu-noabsorbent affinity chromatography. The specific immunoadsorbents were prepared by coupling the a-globular fractions of rabbit anti-thio-redoxin antisera to Sepharose. This technique makes it possible to isolate both thioredoxins from phage-infected cells by two consecutive chromatography steps on the specific adsorbents (125, 126). 

Thioredoxin from yeast has been obtained in two forms (I and II) of which thioredoxin II has been purified to homogeneity (43). Both yeast thioredoxins are able to serve as hydrogen donors for the ribonucleotide reductase from E. coli. The molecular weight (12,600) of thioredoxin II is similar to that of thioredoxin from E. coli. Although both yeast thioredoxins contain only one tryptophan residue and although their amino acid compositions differ markedly from that of E. coli thioredoxin, the amino acid sequences around the disulfide bridge of these three thioredoxins are identical  

The oxidation reduction potential of the yeast thioredoxin-II (SH)2/ thioredoxin-S2 couple is —0.24 V at pH 7. 

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Figure 2.7 (a) Illustration of the twist of (3 sheefs. Befa sfrands are drawn as arrows from the amino end to the carboxy end of the p strand in this schematic drawing of fhe protein thioredoxin from E. coli, fhe sfrucfure of which was defermined in the laboratory of Carl Branden, Uppsala, Sweden, fo 2.8 A resolution. The mixed p sheet is viewed from one of ifs ends, (b) The hydrogen bonds between the P strands in the mixed p sheet of fhe same profein. [(a) Adapfed from B. Furugren.]... 

The thioredoxin domain (see Figure 2.7) has a central (3 sheet surrounded by a helices. The active part of the molecule is a Pa(3 unit comprising p strands 2 and 3 joined by a helix 2. The redox-active disulfide bridge is at the amino end of this a helix and is formed by a Cys-X-X-Cys motif where X is any residue in DsbA, in thioredoxin, and in other members of this family of redox-active proteins. The a-helical domain of DsbA is positioned so that this disulfide bridge is at the center of a relatively extensive hydrophobic protein surface. Since disulfide bonds in proteins are usually buried in a hydrophobic environment, this hydrophobic surface in DsbA could provide an interaction area for exposed hydrophobic patches on partially folded protein substrates. 

Figure 6.8 Schematic diagram of the enzyme DsbA which catalyzes disulfide bond formation and rearrangement. The enzyme is folded into two domains, one domain comprising five a helices (green) and a second domain which has a structure similar to the disulfide-containing redox protein thioredoxin (violet). The N-terminal extension (blue) is not present in thioredoxin. (Adapted from J.L. Martin et al.. Nature 365 464-468, 1993.)...

The C-terminal domain of phosducin is a five-stranded mixed p sheet with a helices on both sides, similar to the thioredoxin fold of disulfide iso-merase DsbA described in Chapter 6. Despite significant sequence homology to thioredoxin, the phosducin domain, unlike other members of this family. 

Figure 13.16 Schematic diagram of the phosducin molecule. Helices are blue, p strands are red and loop regions are orange. The structure folds into two separate domains, a N-terminal helical domain and a C-terminal domain that has the thioredoxin fold. Some of the loop regions in the helical domain are not well defined. (Adapted from R. Gaudet et al.. Cell 87 577-588, 1996.)...

Dithiothreitol plus 2-mercaptoethanol Dithiothreitol plus NADPH, thioredoxin, and 1.0... 

There are other substrates for the E. coli Met(0) peptide reductase, one of which is Met(0)-a-l-PI. The native protein is the major serum elastase inhibitor that functions by forming a binary complex with elastase which inhibits its activity. Met(0)-a-l-PI, on the other hand, which can be formed by treatment of the protein with TV-chlorosuccinimide, cannot form a complex with elastase and therefore is not able to inhibit elastase activity117,118. Table 6 shows, however, that when Met(0)-a-l-PI is incubated in the presence of Met(0)-peptide reductase and dithiothreitol the protein regains its ability to form a complex with elastase and inhibit elastase activity119. Similar to results found with Met(0)-L12 reduced thioredoxin could replace the dithiothreitol as reductant in the enzymatic reaction. 

Details of the incubation and assay are described in the legend to Figure 8. Reproduced by permission of Raven Press, New York from Brot and coworkers in Holmgren (ed.) Thioredoxin and Glutaredoxin Systems. 1986. 

As an example of purification via the ELP fusion approach Meyer and Chilkoti (Fig. 9, left), purified the proteins thioredoxin and tendamistat. For this purpose these target proteins and ELP were genetically fused via a short peptide sequence that included a thrombin cleavage site, which allows the removal of the ELP tag after the purification is completed. The general outline of the purification procedure... 

In a subsequent study, the effect of reducing the ELP molecular weight on the expression and purification of a fusion protein was investigated. Two ELPs, ELP [V-20] and ELP[VsA2G3-90], both with a transition temperature at 40°C in phosphate-buffered saline (PBS) containing 1 M NaCl, were applied for the purification of thioredoxin. Similar yields were observed for both fusion proteins, resulting in a higher thioredoxin yield for the ELP[V-20] fusion, since the ELP fraction was smaller. However, a more complex phase transition behavior was observed for this ELP and therefore a selection of an appropriate combination of salt concentration and solution temperature was required [39]. 

Nordberg J et ai Mammalian thioredoxin reductase is irreversibly inhibited by dinitrohaiobenzenes by alkylation of both the redox active selenocysteine and its neighboring cysteine residue.) Biol Chem 1998 273 10835. 

Fig. 2.2 Simplified scheme of oxidant/antioxidant regulation ofNF-KB activation. Different stimuli, leading to an increase of ROS generation inside the ceU, activate the phosphorylation of IkB inhibitory protein and the subsequent proteolysis. Thioredoxin (Trx) may reduce activated NF-kB proteins facilitating nuclear translocation.Qnce released from IkB, the NF-kB complex translocates into the nucleus and the binding to DNA domain in the promoters and enhancers of genes such as TNF-a, IL-1, proliferation and chemotactic factors, adhesion molecule. Some of these genes, in turn, may further induce NF-kB activation, leading to a vicious circle if the regulatory cellular system escapes from...

PiaRigobello, M., Messori, L., Marcon, G., Cinellu, M.A., Bragadin, M., Folda, A., Scutari, G. and Bindoli, A. (2004) Gold complexes inhibit mitochondrial thioredoxin reductase consequences on mitochondrial functions. Journal of Inorganic Biochemistry, 98, 1634—1641. 

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