NEBRASKA REDOX BIOLOGY CENTER EDUCATIONAL PORTAL

Nebraska Redox Biology Center Educational Portal


Thioredoxins

The thioredoxin protein is a small ubiquitous molecule classified as a general protein disulphide reductant [ 1, 2, 3]. Thioredoxins are present in nearly all known organisms and are essential for complex organisms including humans [ 2, 3, 4]. The thioredoxin fold, named after the protein in which it was first observed, is a distinct structural motif consisting of a four-stranded β-sheet and three flanking α-helices [ 1]. It has been identified in the three-dimensional structures of more than 30 protein. families. There are 769 solved thioredoxin proten structure in Protein Data Bank at this time (June 2015).

3D structure of Human thioredoxin.


Nearly all thioredoxin fold protein are thiloxidoreductases [ 1, 2, 4]. They are involved in redox processes in the cell. Several thioredoxin forld thiol oxidoreductase families, such as thioredoxins, glutaredoxins, peroxiredoxins, glutathione peroxidases, and protein disulfide isomerases, are represented by multiple copies even when such proteins coexisted in the same organism and cellular compartment. Such redundancy in the set of thioredoxin-like proteins in an organism is typical for all branches of life. The structural similaryty between thioredoxin fold proteins does not suggest a common function. They can work as reductases, oxydases, disulfide isomerases, be involved in protein modification and redox signaling [ 4, 5].

One of the best studied redox motifs involving cysteines is the CxxC motif, in which two cysteines are separated by two other residues. This motif is employed by thioredoxin-like proteins for reduction of intermolecular and intramolecular disulfide bonds and other forms of oxidized cysteines, by protein disulfide isomerases and oxidases for disulfide bond formation and isomerization [ 1, 5]. The CxxC motif is located after the β-sheet 2 and forms the N-terminal portion of α-helix 2 in thioredoxin fold core motif
β-α-β-(CxxC)-α-β-β-α . The place of the one cysteine in this motif may be occupied by selenocysteine, serine, thrionine or, in rare case, by othe amino acids, modifying the functional properties of thioredoxin-like prteins. The CxxC motif has been called a "rheostat at the active center", because changes in residues that separate the two cysteines dramatically influence redox properties, configuring proteins for a particular redox function. [ 1, 6, 7].

Yeast Saccharomyces Cerevisiae Thioredoxins:

>gi|6323072|ref|NP_013144.1| Trx1 [Saccharomyces cerevisiae]
MVTQFKTASEFDSAIAQDKLVVVDFYATWCGPCKMIAPMIEKFSEQYPQADFYKLDVDELGDVAQKNEVSAMPTLLLFKNGKEVAKVVGANPAAIKQAIAANA

>gi|6321648|ref|NP_011725.1| Trx2 [Saccharomyces cerevisiae]
MVTQLKSASEYDSALASGDKLVVVDFFATWCGPCKMIAPMIEKFAEQYSDAAFYKLDVDEVSDVAQKAEVSSMPTLIFYKGGKEVTRVVGANPAAIKQAIASNV

>gi|6319925|ref|NP_010006.1| Trx3 [Saccharomyces cerevisiae]
MLFYKPVMRMAVRPLKSIRFQSSYTSITKLTNLTEFRNLIKQNDKLVIDFYATWCGPCKMMQPHLTKLIQAYPDVRFVKCDVDESPDIAKECEVTAMPTFVLGKDG
QLIGKIIGANPTALEKGIKDL



Thioredoxins are typical reductases. Basic mechanism of thioredoxin system functioning is base on electron flow from NADPH to thioredoxin reductase, than to thioredoxin and from thioredoxin to target molecules. [ 1, 2, 3].


Thioredoxin, is a key component of antioxidant system protecting against oxidative stress through its disulfide reductase activity. The thioredoxin system provides the electrons to thiol-dependent peroxidases to remove reactive oxygen and nitrogen species with a fast reaction rate. Antioxidant functions of thioredoxins is shown DNA and protein repair, reducing ribonucleotide reductase, methionine sulfoxide reductases and regulating the activity of many redox-sensitive transcription factors. Moreover, Trx systems play critical roles in the immune response and cell death via interaction with redox signaling machinary components. [ 2, 3, 5, 8, 9].

The thioredoxin-mediated reaction involves several steps: (1) deprotonation of thiol (reactive thiolate formation), (2) formation of intermolecular disulfide bond, (3) attack of the resolving cysteine thiol (the second cysteine in CxxC redox mitif) on the intermolecular disulfide, and the release of two proteins after the thiol-disulfide exchange reaction [ 1, 2, 3, 7].

Schematic presentation of the thioredoxin reachtion mechanism.


The major and commonly used assay for thioredoxin activity measurement is based on insulin reduction with subsequent thiol measurement with DTNB [10, 11]. Thioredoxin in the samples is first reduced by dithiothreitol (DTT) (reaction 2). Then, thioredoxnTrx reduces insulin disulfides (reaction 2). In the presence of TrxR and at the expense of NADPH, Trx can be reduced and brought back into the system. When the reaction is stopped the sulfhydryl groups are measured using 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) (reaction 3) which gives an intense yellow colour with maximum absorbance at 412nm.

1) Trx-S2 + DTTred → Trx-(SH)2 + DTTred ;

2) Trx-(SH)2 + Insulin-S2 → Trx-S2 + Insulin-(SH)2 ;

3) Insulin-(SH)2 + DTNB → Insulin-S2 +2TNBbright yellow ;

Fluorecsently labeled insulin could be used instead of regular [ 11, 12 13]. There are lot of commertially avaliable thioredoxin assay kits. Most of them are based on reduction of insulin in the presence of Dithiothreitol (DTT), resulting in the formation of insulin aggregates which then bind fluorogenic detection dye.

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