An Atlas of the Thioredoxin Fold Class Reveals the Complexity of Function-Enabling Adaptations

The group of proteins that contain a thioredoxin (Trx) fold is huge and diverse. Assessment of the variation in catalytic machinery of Trx fold proteins is essential in providing a foundation for understanding their functional diversity and predicting the function of the many uncharacterized members of the class. The proteins of the Trx fold class retain common features—including variations on a dithiol CxxC active site motif—that lead to delivery of function. We use protein similarity networks to guide an analysis of how structural and sequence motifs track with catalytic function and taxonomic categories for 4,082 representative sequences spanning the known superfamilies of the Trx fold. Domain structure in the fold class is varied and modular, with 2.8% of sequences containing more than one Trx fold domain. Most member proteins are bacterial. The fold class exhibits many modifications to the CxxC active site motif—only 56.8% of proteins have both cysteines, and no functional groupings have absolute conservation of the expected catalytic motif. Only a small fraction of Trx fold sequences have been functionally characterized. This work provides a global view of the complex distribution of domains and catalytic machinery throughout the fold class, showing that each superfamily contains remnants of the CxxC active site. The unifying context provided by this work can guide the comparison of members of different Trx fold superfamilies to gain insight about their structure-function relationships, illustrated here with the thioredoxins and peroxiredoxins.     

Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif

Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E0) of the Trx1 active site (-230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an alpha helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals.     

Alleviation of oxidative stress by potent and selective thioredoxin-mimetic peptides

One of the major enzymatic cell defenses providing protection from oxidative injury is the TrxR-Trx system. It consists of NADPH and thioredoxin reductase (TrxR), which maintain thioredoxin (Trx) in a reduced state. Perturbing the TrxR-Trx system with the selective TrxR inhibitor auranofin (AuF; 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranosato-S-(triethylphosphine) gold) induces oxidative stress by keeping Trx in its oxidized state. We have prepared a family of tri- and tetra-oligopeptides derived from the canonical CxxC motif of the Trx active site and a modified CxC motif. These Trx-mimetic compounds are N- and C-terminal-blocked peptides that consist of two cysteine residues that flank the two-amino-acid CxxC motif (CB4 and CB6) or the single-amino-acid CxC motif (CB3). Catecholamine (CA) secretion in bovine chromaffin cells, which is a highly redox sensitive process, is abolished by AuF. The Trx-mimetic peptides effectively restore CA secretion, as monitored by amperometry in single cells. They also prevent the AuF-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase. In PC12 cells, the alleviation of AuF-induced ERK1/2-MAPK phosphorylation by Trx-like peptides parallels their effect of restoring CA secretion. CB3, CB4, and CB6 act intracellularly and are significantly more potent than the traditional antioxidants NAC, GSH, DTT, AD4 (NAC-amide), and ascorbic acid. Taken together, the CxxC and CxC peptides represent a new family of potent and selective redox compounds that could serve as potential candidates for prevention and treatment of oxidative-stress-related disorders.     

Identification and characterization of TRP14, a thioredoxin-related protein of 14 kDa. New insights into the specificity of thioredoxin function

We have identified and characterized a 14-kDa human thioredoxin (Trx)-related protein designated TRP14. This cytosolic protein was expressed in all tissues and cell types examined, generally in smaller amounts than Trx1. Although TRP14 contains five cysteines, only the two Cys residues in its WCPDC motif were exposed and redox sensitive. Unlike Trx1, which was an equally good substrate for both Trx reductase 1 (TrxR1) and TrxR2, oxidized TRP14 was reduced by TrxR1 but not by TrxR2. Biochemical characterization of TRP14 suggested that, like Trx1, TRP14 is a disulfide reductase; its active site cysteine is sufficiently nucleophilic with the pK(a) value of 6.1; and its redox potential (-257 mV) is similar to those of other cellular thiol reductants. However, although TRP14 reduced small disulfide-containing peptides, it did not reduce the disulfides of known Trx1 substrates, ribonucleotide reductase, peroxiredoxin, and methionine sulfoxide reductase. These results suggest that TRP14 and Trx1 might act on distinct substrate proteins.     

Identity and functions of CxxC-derived motifs

Two cysteines separated by two other residues (the CxxC motif) are employed by many redox proteins for formation, isomerization, and reduction of disulfide bonds and for other redox functions. The place of the C-terminal cysteine in this motif may be occupied by serine (the CxxS motif), modifying the functional repertoire of redox proteins. Here we found that the CxxC motif may also give rise to a motif, in which the C-terminal cysteine is replaced with threonine (the CxxT motif). Moreover, in contrast to a view that the N-terminal cysteine in the CxxC motif always serves as a nucleophilic attacking group, this residue could also be replaced with threonine (the TxxC motif), serine (the SxxC motif), or other residues. In each of these CxxC-derived motifs, the presence of a downstream alpha-helix was strongly favored. A search for conserved CxxC-derived motif/helix patterns in four complete genomes representing bacteria, archaea, and eukaryotes identified known redox proteins and suggested possible redox functions for several additional proteins. Catalytic sites in peroxiredoxins were major representatives of the TxxC motif, whereas those in glutathione peroxidases represented the CxxT motif. Structural assessments indicated that threonines in these enzymes could stabilize catalytic thiolates, suggesting revisions to previously proposed catalytic triads. Each of the CxxC-derived motifs was also observed in natural selenium-containing proteins, in which selenocysteine was present in place of a catalytic cysteine.     

Structural basis of cellular redox regulation by human TRP14

Thioredoxin-related protein 14 (TRP14) is involved in regulating tumor necrosis factor-alpha-induced signaling pathways in a different manner from human thioredoxin 1 (Trx1). Here, we report the crystal structure of human TRP14 determined at 1.8-A resolutions. The structure reveals a typical thioredoxin fold with characteristic structural features that account for the substrate specificity of the protein. The surface of TRP14 in the vicinity of the active site includes an extended loop and an additional alpha-helix, and the distribution of charged residues in the surface is different from Trx1. The distinctive dipeptide between the redox-active cysteines contributes to stabilizing the thiolate anion of the active site cysteine 43, increasing reactivity of the cysteine toward substrates. These structural differences in the active site suggest that TRP14 has evolved to regulate cellular redox signaling by recognizing a distinctive group of substrates that would complement the group of proteins regulated by Trx1.     

Distinction of thioredoxin transnitrosylation and denitrosylation target proteins by the ICAT quantitative approach

S-Nitrosylation is a reversible PTM for regulating protein function. Thioredoxin-1 (Trx1) catalyzes either transnitrosylation or denitrosylation of specific proteins, depending on the redox status of the cysteines within its conserved oxidoreductase CXXC motif. With a disulfide bond formed between the two catalytic cysteines, Trx1 is not only inactive as a denitrosylase, but it may also be nitrosylated at Cys73 and serve as a transnitrosylating agent. Identification of Trx1-mediated transnitrosylation or denitrosylation targets will contribute to a better understanding of Trx1's function. Previous experimental approaches based on the attenuation of CXXC oxidoreductase activity cannot readily distinguish Trx1 transnitrosylation targets from denitrosylation targets. In this study, we used the ICAT method in conjunction with the biotin switch technique to differentiate Trx1 transnitrosylation targets from denitrosylation target proteins from neuroblastoma cells. We demonstrate that the ICAT approach is effective for quantitative identification of putative Trx1 transnitrosylation and denitrosylation target peptides. From these analyses, we confirmed reports that peroxiredoxin 1 is a Trx1 transnitrosylation, but not a denitrosylation target, and we found several other proteins, including cyclophilin A to be modulated in this manner. Unexpectedly, we found that many nitrosylation sites are reversibly regulated by Trx1, suggesting a more prominent role for Trx1 in regulating S-nitrosylation.     

The Escherichia coli thioredoxin homolog YbbN/Trxsc is a chaperone and a weak protein oxidoreductase

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN functions with two redox cysteines. Accordingly, YbbN catalyzes the oxidation of reduced RNase and the isomerization of scrambled RNase. Moreover, upon oxidation, its oligomeric state changes from dimers to tetramers and higher oligomers. YbbN also possesses chaperone properties, promoting protein folding after urea denaturation and forming complexes with unfolded proteins. This is the first biochemical characterization of a member of the YbbN class of bacterial thioredoxin-like proteins, and in vivo experiments will allow to determine the importance of its redox and chaperone properties in the cellular physiology.     

The conserved active site tryptophan of thioredoxin has no effect on its redox properties

In Staphylococcus aureus thioredoxin (Trx) it has been shown that mutation of the conserved active site tryptophan residue (Trp28) has a large effect on the protein stability, on the pKa of the nucleophilic cysteine and on the redox potential. Since these effects can either be due to the partially unfolding of the Trp28Ala mutant or to the absence of the indole side chain of Trp28 as possible interaction partner for the active site cysteines, the origin of the experimentally observed effects is not known and is beyond experimental approach. With theoretical pKa and density functional theory reactivity analysis on model systems where Trp28 has been replaced by an alanine within the structural environment of Trx it is shown that Trp28 does not affect the redox parameters of Trx. As such, the experimentally observed redox effects of the Trx W28A mutant might be due to structural changes induced by partial unfolding.     

Crystal structures of two functionally different thioredoxins in spinach chloroplasts

Thioredoxins are small ubiquitous proteins which act as general protein disulfide reductases in living cells. Chloroplasts contain two distinct thioredoxins ( f and m) with different phylogenetic origin. Both act as enzyme regulatory proteins but have different specificities towards target enzymes. Thioredoxin f (Trx f), which shares only low sequence identity with thioredoxin m (Trx m) and with all other known thioredoxins, activates enzymes of the Calvin cycle and other photosynthetic processes. Trx m shows high sequence similarity with bacterial thioredoxins and activates other chloroplast enzymes. The here described structural studies of the two chloroplast thioredoxins were carried out in order to gain insight into the structure/function relationships of these proteins. Crystal structures were determined for oxidized, recombinant thioredoxin f (Trx f-L) and at the N terminus truncated form of it (Trx f-S), as well as for oxidized and reduced thioredoxin m (at 2.1 and 2.3 A resolution, respectively). Whereas thioredoxin f crystallized as a monomer, both truncated thioredoxin f and thioredoxin m crystallized as non-covalent dimers. The structures of thioredoxins f and m exhibit the typical thioredoxin fold consisting of a central twisted five-stranded beta-sheet surrounded by four alpha-helices. Thioredoxin f contains an additional alpha-helix at the N terminus and an exposed third cysteine close to the active site. The overall three-dimensional structures of the two chloroplast thioredoxins are quite similar. However, the two proteins have a significantly different surface topology and charge distribution around the active site. An interesting feature which might significantly contribute to the specificity of thioredoxin f is an inherent flexibility of its active site, which has expressed itself crystallographically in two different crystal forms.     

Isolation and characterization of a thioredoxin-dependent peroxidase from Chlamydomonas reinhardtii.

All living organisms contain redox systems involving thioredoxins (Trx), proteins featuring an extremely conserved and reactive active site that perform thiol-disulfide interchanges with disulfide bridges of target proteins. In photosynthetic organisms, numerous isoforms of Trx coexist, as revealed by sequencing of Arabidopsis genome. The specific functions of many of them are still unknown. In an attempt to find new molecular targets of Trx in Chlamydomonas reinhardtii, an affinity column carrying a cytosolic Trx h mutated at the less reactive cysteine of its active site was used to trap Chlamydomonas proteins that form mixed disulfides with Trx. The major protein bound to the column was identified by amino-acid sequencing and mass spectrometry as a thioredoxin-dependent 2Cys peroxidase. Isolation and sequencing of its gene revealed that this peroxidase is most likely a chloroplast protein with a high homology to plant 2Cys peroxiredoxins. It is shown that the Chlamydomonas peroxiredoxin (Ch-Prx1) is active with various thioredoxin isoforms, functions as an antioxidant toward reactive oxygen species (ROS), and protects DNA against ROS-induced degradation. Expression of the peroxidase gene in Chlamydomonas was found to be regulated by light, oxygen concentration, and redox state. The data suggest a role for the Chlamydomonas Prx in ROS detoxification in the chloroplast.     

Thioredoxin-dependent enzymatic activation of mercaptopyruvate sulfurtransferase. An intersubunit disulfide bond serves as a redox switch for activation

Rat 3-mercaptopyruvate sulfurtransferase (MST) contains three exposed cysteines as follows: a catalytic site cysteine, Cys(247), in the active site and Cys(154) and Cys(263) on the surface of MST. The corresponding cysteine to Cys(263) is conserved in mammalian MSTs, and Cys(154) is a unique cysteine. MST has monomer-dimer equilibrium with the assistance of oxidants and reductants. The monomer to dimer ratio is maintained at approximately 92:8 in 0.2 m potassium phosphate buffer containing no reductants under air-saturated conditions; the dimer might be symmetrical via an intersubunit disulfide bond between Cys(154) and Cys(154) and between Cys(263) and Cys(263), or asymmetrical via an intersubunit disulfide bond between Cys(154) and Cys(263). Escherichia coli reduced thioredoxin (Trx) cleaved the intersubunit disulfide bond to activate MST to 2.3- and 4.9-fold the levels of activation of dithiothreitol (DTT)-treated and DTT-untreated MST, respectively. Rat Trx also activated MST. On the other hand, reduced glutathione did not affect MST activity. E. coli C35S Trx, in which Cys(35) was replaced with Ser, formed some adducts with MST and activated MST after treatment with DTT. Thus, Cys(32) of E. coli Trx reacted with the redox-active cysteines, Cys(154) and Cys(263), by forming an intersubunit disulfide bond and a sulfenyl Cys(247). A consecutively formed disulfide bond between Trx and MST must be cleaved for the activation. E. coli C32S Trx, however, did not activate MST. Reduced Trx turns on a redox switch for the enzymatic activation of MST, which contributes to the maintenance of cellular redox homeostasis.     

A conserved cis-proline precludes metal binding by the active site thiolates in members of the thioredoxin family of proteins

Many thioredoxin-fold proteins possess a conserved cis-proline located in their C-terminal portions. This residue, as well as catalytic and resolving cysteines, is a key functional group in the active sites of these thiol-disulfide oxidoreductases. However, the specific function of the proline is poorly understood, and some thioredoxin-fold proteins lack this residue. Herein, we found that mutation of a cis-proline, Pro75, in human thioredoxin to serine, threonine, or alanine leads to the formation of an Fe2-S2 cluster in this protein. Further mutagenesis studies revealed that the first cysteine in the CxxC motif and a cysteine in the C-terminal region of the protein were responsible for metal binding. Replacement of Pro75 with arginine, a residue that occurs in place of Pro in peroxiredoxins, also led to the formation of the cluster in the thioredoxin. In addition, we found that mutation of the TxxC active site in a peroxiredoxin to the CxxC form could lead to coordination of an Fe2-S2 cluster in these proteins in vitro. Sco1, a distantly related thioredoxin-fold protein, has histidine in place of the cis-proline, and this residue binds copper. The Pro75His mutation led to increased copper binding by human thioredoxin when cells were grown in the presence of this trace element. Taken together, our data suggest that an important function of Pro75 in human thioredoxin, and likely other members of this superfamily, is to prevent metal binding by the reactive thiolate-based active site.     

Selective redox regulation of cytokine receptor signaling by extracellular thioredoxin-1

The thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of Trx1 require a functional active site, suggesting a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular Trx1 redox activity to cellular responses have not been identified so far. Using a mechanism-based kinetic trapping technique to identify disulfide exchange interactions on the intact surface of living lymphocytes, we found that Trx1 catalytically interacts with a single principal target protein. This target protein was identified as the tumor necrosis factor receptor superfamily member 8 (TNFRSF8/CD30). We demonstrate that the redox interaction is highly specific for both Trx1 and CD30 and that the redox state of CD30 determines its ability to engage the cognate ligand and transduce signals. Furthermore, we confirm that Trx1 affects CD30-dependent changes in lymphocyte effector function. Thus, we conclude that receptor-ligand signaling interactions can be selectively regulated by an extracellular redox catalyst.     

Thioredoxin-dependent regulation of AIF-mediated DNA damage

The thioredoxin (Trx) system is one major redox system in mammalian cells. One of its component, Trx, is involved in redox homeostasis and many cellular biological processes through participating in disulfide reduction, S-nitrosylation/S-denitrosylation reactions and protein-protein interactions. In this study, we report the identification of a novel interaction between cytosolic/nuclear Trx1 and apoptosis inducing factor (AIF), and the redox sensitivity and biological significance of the Trx-AIF interaction was characterized. Cytosolic Trx1 but not mitochondrial Trx2 was observed to interact with AIF under physiological conditions and Trx1's active site cysteines were crucial for the interaction. Under oxidative stress conditions, Trx-AIF interaction was disrupted. When the treated cells were allowed to recover from oxidative stress by means of removal of the oxidants, interaction between Trx1 and AIF was re-established time-dependently, which underpins the biological relevance of a Trx-dependent redox regulation of AIF-mediated cell death. Indeed, in times of oxidative stress, nuclear translocation of AIF was found to occur concurrently with perturbations to the Trx-AIF interaction. Once localized in the nucleus, reduced Trx1 hindered the interaction between AIF and DNA, thereby bringing about an attenuation of AIF-mediated DNA damage. In conclusion, characterization of the Trx-AIF interaction has led to an understanding of the effect of reduced Trx1 on possibly regulating AIF-dependent cell death through impeding AIF-mediated DNA damage. Importantly, identification of the novel interaction between Trx1 and AIF has provided opportunities to design and develop therapeutically relevant strategies that either promote or prevent this protein-protein interaction for the treatment of different disease states.     

A structural disulfide of yeast protein-disulfide isomerase destabilizes the active site disulfide of the N-terminal thioredoxin domain

Protein-disulfide isomerase (PDI) is an essential catalyst of disulfide formation and isomerization in the eukaryotic endoplasmic reticulum. PDI has two active sites at either end of the molecule, each containing two cysteines that facilitate thiol-disulfide exchange. In addition to its four catalytic cysteines, PDI possesses two non-active site cysteines whose location and separation distance varies by organism. In higher eukaryotes, the non-active site cysteines are located in the C-terminal half of the protein sequence and are separated by 30 amino acids. In contrast, the internal cysteines of PDI from lower eukaryotes are located near the N-terminal active site and are much closer together in sequence. The function of these cysteines and the significance of their unique location in yeast PDI have been unclear. Previous data (Xiao, R., Wilkinson, B., Solovyov, A., Winther, J. R., Holmgren, A., Lundstrom-Ljung, J., and Gilbert, H. F. (2004) J. Biol. Chem. 279, 49780-49786) suggest that the internal cysteines exist as a disulfide in the endoplasmic reticulum of Saccharomyces cerevisiae. By coupling mass spectrometry with a gel-shift technique that allows us to measure the redox potentials of the PDI active sites in the presence and absence of the non-active site cysteines, we find that the non-active site cysteines form a disulfide that is stable even in a very reducing environment and demonstrate that this disulfide exists to destabilize the N-terminal active site disulfide, making it a better oxidant by 18-fold. Consistent with this finding, we show that mutating the non-active site cysteines to alanines disrupts both the oxidase and isomerase activities of PDI in vitro.     

Identification of a new family of plant proteins loosely related to glutaredoxins with four CxxC motives

The annotation of the recently released Populus trichocarpa genome, has allowed us to characterize extensively the multigenic families of the redoxin proteins. Proteins with two cysteines separated by two amino acids (CxxC motif) are often involved in redox reactions by promoting the formation, reduction or isomerization of disulfide bonds or by binding prosthetic groups or metals. We report here the presence of a new protein family in higher plants, constituted of 19 members in Populus trichocarpa, 15 in Arabidopsis thaliana and 17 in Oryza sativa. These proteins are almost specific to higher plants, with only two homologous genes found in mammals and arthropoda but none in other kingdoms. While these proteins were predicted as glutaredoxin-like proteins (GRL) in the automatic annotation procedure, they do not share the major conserved features of glutaredoxins but instead they display four conserved CxxC motives. A classification of these proteins, based on sequence similarity, gene structure and predicted cellular localization is proposed. The expression of these genes was also investigated by analyzing EST databases and Arabidopsis microarray results.