Atypical Thioredoxin Research Articles

The atypical thioredoxin 'Alr2205', a newly identified partner of the typical 2-Cys-Peroxiredoxin, safeguards the cyanobacterium Anabaena from oxidative stress.

Thioredoxins (Trxs) are ubiquitous proteins that play vital roles in several physiological processes. Alr2205, a thioredoxin-like protein from Anabaena PCC 7120, was evolutionarily closer to the Trx-domain of the NADPH-Thioredoxin Reductase C than the other thioredoxins. The Alr2205 protein showed disulphide reductase activity despite the presence a non-canonical active site motif 'CPSC'. Alr2205 not only physically interacted with, but also acted as a physiological reductant of Alr4641 (2-Cys-Peroxiredoxin), supporting its peroxidase function. Structurally, Alr2205 was a monomeric protein that formed an intramolecular disulphide bond between the two active site cysteines. However, the Alr2205C41S protein, wherein the resolving cysteine was mutated to serine, was capable of forming intermolecular disulphide bond and exist as a dimer when treated with H2O2. Overproduction of Alr2205 in E. coli protected cells from heavy metals, but not oxidative stress. To delve into its physiological role, Alr2205/Alr2205C41S was overexpressed in Anabaena, and the ability of the corresponding strains (An2205+ or An2205C41S+) to withstand environmental stresses was assessed. An2205+ showed higher resistance to H2O2 than An2205C41S+, indicating that the disulphide reductase function of this protein was critical to protect cells from this peroxide. Although, An2205+ did not show increased capability to withstand cadmium stress, An2205C41S+ was more susceptible to this heavy metal. This is the first study that provides a vital understanding into the function of atypical thioredoxins in countering the toxic effects of heavy metals/H2O2 in prokaryotes.

Redox properties of the plant atypical thioredoxin DCC1

Redox properties of the plant atypical thioredoxin DCC1

Genome-wide analysis and transcriptional regulation of the typical and atypical thioredoxins in Arabidopsis thaliana.

Thioredoxins (TRXs), a large subclass of ubiquitous oxidoreductases, are involved in thiol redox regulation. Here, we performed a comprehensive analysis of TRXs in the Arabidopsis thaliana genome, revealing 41 genes encoding 18 typical and 23 atypical TRXs, and 6 genes encoding thioredoxin reductases (TRs). The high number of atypical TRXs indicates special functions in plants that mostly await elucidation. We identified an atypical class of thioredoxins called TRX-c in the genomes of photosynthetic eukaryotes. Localized to the chloroplast, TRX-c displays atypical CPLC, CHLC and CNLC motifs in the active sites. In silico analysis of the transcriptional regulations of TRXs revealed high expression of TRX-c in leaves and strong regulation under cold, osmotic, salinity and metal ion stresses.

Genome-Wide Analysis of the Thioredoxin Gene Family in Gossypium hirsutum L. and the Role of the Atypical Thioredoxin Gene GhTRXL3-2 in Flowering

Thioredoxin (TRX) is a highly conserved low-molecular-weight protein and a ubiquitous antioxidant enzyme that plays key role in the regulation of plant growth and development. Here, using the whole-genome sequence, we performed a systematic analysis for the TRX gene family in upland cotton (Gossypium hirsutum L.) and analyzed their structural characteristics, evolution, and expression profiles during growth and development. At least 86 GhTRX members, 40 typical and 46 atypical, were identified in the cotton genome, and they were unevenly distributed on the 26 chromosomes. Conserved domains and phylogenic tree construction classified the typical TRX gene family into seven subfamilies and the atypical TRX into nine subfamilies. An evolutionary analysis revealed that the TRX gene family underwent purification selection during evolution. In addition, an RNA-Seq analysis showed that, during vegetative and reproductive development, the differences in transcript abundance levels and organ-specific expression patterns suggest functional diversity. Biochemical assays demonstrated that the atypical TRX protein GhTRXL3-2 interacted with the cotton FLOWERING LOCUS T protein GhFT. The overexpression of GhTRXL3-2 in Arabidopsis thaliana resulted in early flowering compared with control plants. Additionally, the silencing of GhTRXL3-2 in cotton delayed maturation, suggesting that it has important roles in cotton’s flowering regulation. These results help clarify the evolution of the TRX genes and elucidate their biological functions in cotton flowering regulation.

Structural insight into the biological functions of Arabidopsis thaliana ACHT1

The Arabidopsis thaliana atypical Cys His-rich thioredoxins (ACHTs) are a small class of atypical thioredoxins (TRXs) located in chloroplasts thylakoids and are characterized by a noncanonical motif at their redox active site, C (G/S)(S/G)C. Previous studies have reported that ACHT1 can interact with A. thaliana 2-Cys peroxiredoxins (2-Cys Prxs, including PrxA and PrxB) to transmit oxidation signals in response to illumination with normal light intensity. In this study, we reported the crystal structure of ACHT1 and show that ACHT1 adopts a canonical TRX fold. Comparison of the structures of ACHT1 in both reducing and oxidizing environments revealed that while the redox environment did not influence the overall structure of ACHT1, it did change the conformation of its catalytic residues. We found that the catalytic C125 of ACHT1 is the target residue for PrxA in vitro. In addition, we found that ACHT1 can reduce the peroxidase activity of PrxA, and further confirmed that the ability of ACHT1 to restore the peroxidase function of PrxA was due to the interaction between the two. Our results provide a structural basis for studying the function of atypical TRXs and the oxidative regulation mechanism of ACHT1 and 2-Cys Prxs in chloroplasts.

Characterization of TrxC, an Atypical Thioredoxin Exclusively Present in Cyanobacteria.

Cyanobacteria form a diverse group of oxygenic photosynthetic prokaryotes considered to be the antecessor of plant chloroplast. They contain four different thioredoxins isoforms, three of them corresponding to m, x and y type present in plant chloroplast, while the fourth one (named TrxC) is exclusively found in cyanobacteria. TrxC has a modified active site (WCGLC) instead of the canonical (WCGPC) present in most thioredoxins. We have purified it and assayed its activity but surprisingly TrxC lacked all the classical activities, such as insulin precipitation or activation of the fructose-1,6-bisphosphatase. Mutants lacking trxC or over-expressing it were generated in the model cyanobacterium Synechocystis sp. PCC 6803 and their phenotypes have been analyzed. The ΔtrxC mutant grew at similar rates to WT in all conditions tested although it showed an increased carotenoid content especially under low carbon conditions. Overexpression strains showed reduced growth under the same conditions and accumulated lower amounts of carotenoids. They also showed lower oxygen evolution rates at high light but higher Fv’/Fm’ and Non-photochemical-quenching (NPQ) in dark adapted cells, suggesting a more oxidized plastoquinone pool. All these data suggest that TrxC might have a role in regulating photosynthetic adaptation to low carbon and/or high light conditions.

Biochemical Function, Molecular Structure and Evolution of an Atypical Thioredoxin Reductase from Desulfovibrio vulgaris.

Thioredoxin reductase (TR) regulates the intracellular redox environment by reducing thioredoxin (Trx). In anaerobes, recent findings indicate that the Trx redox network is implicated in the global redox regulation of metabolism but also actively participates in protecting cells against O2. In the anaerobe Desulfovibrio vulgaris Hildenborough (DvH), there is an intriguing redundancy of the Trx system which includes a classical system using NADPH as electron source, a non-canonical system using NADH and an isolated TR (DvTRi). The functionality of DvTRi was questioned due to its lack of reactivity with DvTrxs. Structural analysis shows that DvTRi is a NAD(P)H-independent TR but its reducer needs still to be identified. Moreover, DvTRi reduced by an artificial electron source is able to reduce in turn DvTrx1 and complexation experiments demonstrate a direct interaction between DvTRi and DvTrx1. The deletion mutant tri exhibits a higher sensitivity to disulfide stress and the gene tri is upregulated by O2 exposure. Having DvTRi in addition to DvTR1 as electron source for reducing DvTrx1 must be an asset to combat oxidative stress. Large-scale phylogenomics analyses show that TRi homologs are confined within the anaerobes. All TRi proteins displayed a conserved TQ/NGK motif instead of the HRRD motif, which is selective for the binding of the 2′-phosphate group of NADPH. The evolutionary history of TRs indicates that tr1 is the common gene ancestor in prokaryotes, affected by both gene duplications and horizontal gene events, therefore leading to the appearance of TRi through subfunctionalization over the evolutionary time.

An Atypical Thioredoxin Imparts Early Resistance to Sugarcane Mosaic Virus in Maize

Sugarcane mosaic virus (SCMV) causes substantial losses of grain yield and forage biomass in susceptible maize worldwide. A major quantitative trait locus, Scmv1, has been identified to impart strong resistance to SCMV at the early infection stage. Here, we demonstrate that ZmTrxh, encoding an atypical h-type thioredoxin, is the causal gene at Scmv1, and that its transcript abundance correlated strongly with maize resistance to SCMV. ZmTrxh alleles, whether they are resistant or susceptible, share the identical coding/proximal promoter regions, but vary in the upstream regulatory regions. ZmTrxh lacks two canonical cysteines in the thioredoxin active-site motif and exists uniquely in the maize genome. Because of this, ZmTrxh is unable to reduce disulfide bridges but possesses a strong molecular chaperone-like activity. ZmTrxh is dispersed in maize cytoplasm to suppress SCMV viral RNA accumulation. Moreover, ZmTrxh-mediated maize resistance to SCMV showed no obvious correlation with the salicylic acid- and jasmonic acid-related defense signaling pathways. Taken together, our results indicate that ZmTrxh exhibits a distinct defense profile in maize resistance to SCMV, differing from previously characterized dominant or recessive potyvirus resistance genes.

ACHT4-driven oxidation of APS1 attenuates starch synthesis under low light intensity in Arabidopsis plants

The regulatory mechanisms that use signals of low levels of reactive oxygen species (ROS) could be obscured by ROS produced under stress and thus are better investigated under homeostatic conditions. Previous studies showed that the chloroplastic atypical thioredoxin ACHT1 is oxidized by 2-Cys peroxiredoxin (2-Cys Prx) in Arabidopsis plants illuminated with growth light and in turn transmits a disulfide-based signal via yet unknown target proteins in a feedback regulation of photosynthesis. Here, we studied the role of a second chloroplastic paralog, ACHT4, in plants subjected to low light conditions. Likewise, ACHT4 reacted in planta with 2-Cys Prx, indicating that it is oxidized by a similar disulfide exchange reaction. ACHT4 further reacted uniquely with the small subunit (APS1) of ADP-glucose pyrophosphorylase (AGPase), the first committed enzyme of the starch synthesis pathway, suggesting that it transfers the disulfides it receives from 2-Cys Prx to APS1 and turns off AGPase. In accordance, ACHT4 participated in an oxidative signal that quenched AGPase activity during the diurnal transition from day to night, and also in an attenuating oxidative signal of AGPase in a dynamic response to small fluctuations in light intensity during the day. Increasing the level of expressed ACHT4 or of ACHT4ΔC, a C terminus-deleted form that does not react with APS1, correspondingly decreased or increased the level of reduced APS1 and decreased or increased transitory starch content. These findings imply that oxidative control mechanisms act in concert with reductive signals to fine tune starch synthesis during daily homeostatic conditions.

Atypical Thioredoxins in Poplar: The Glutathione-Dependent Thioredoxin-Like 2.1 Supports the Activity of Target Enzymes Possessing a Single Redox Active Cysteine

Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways.

Proteomic Analysis of Protein Expression Affected by Peroxiredoxin V Knock-Down in Hypoxic Kidney

Peroxiredoxin V, an atypical thioredoxin peroxidase, is widely expressed in mammalian tissues. In addition, Prdx V is localized in mitochondria, peroxisome, cytosol, and the nucleus. Prdx V has been reported to protect a wide range of cellular environments as an antioxidant enzyme, and its dysfunctions may be implicated in several diseases, such as cancer, inflammation, and neurodegenerative disease. Identification and relative quantification of proteins affected by Prdx V may help identify novel signaling mechanisms that are important for oxidative stress response. However, the role of Prdx V in the modulation of hypoxia-related cellular response is not studied yet. To examine the function of endogenous Prdx V in hypoxic condition in vivo, we generated a transgenic mouse model with Prdx V siRNA expression controlled by U6 promoter. Of many tissues, the knockdown of Prdx V expression was displayed in the kidney, lung, and liver but not the spleen and skin. We conducted on the basis of nano-UPLC-MS(E) proteomic study to identify the Prdx V-affected protein networks in hypoxic kidneys. In this study, we identified protein networks associated with oxidative stress, fatty acid metabolism, and mitochondrial dysfunction. Our results indicated that Prdx V affected to regulation of kidney homeostasis under hypoxia stress.

Comparative Genomic Study of the Thioredoxin Family in Photosynthetic Organisms with Emphasis on Populus trichocarpa

The recent genome sequencing of Populus trichocarpa and Vitis vinifera, two models of woody plants, of Sorghum bicolor, a model of monocot using C4 metabolism, and of the moss Physcomitrella patens, together with the availability of photosynthetic organism genomes allows performance of a comparative genomic study with organisms having different ways of life, reproduction modes, biological traits, and physiologies. Thioredoxins (Trxs) are small ubiquitous proteins involved in the reduction of disulfide bridges in a variety of target enzymes present in all sub-cellular compartments and involved in many biochemical reactions. The genes coding for these enzymes have been identified in these newly sequenced genomes and annotated. The gene content, organization and distribution were compared to other photosynthetic organisms, leading to a refined classification. This analysis revealed that higher plants and bryophytes have a more complex family compared to algae and cyanobacteria and to non-photosynthetic organisms, since poplar exhibits 49 genes coding for typical and atypical thioredoxins and thioredoxin reductases, namely one-third more than monocots such as Oryza sativa and S. bicolor. The higher number of Trxs in poplar is partially explained by gene duplication in the Trx m, h, and nucleoredoxin classes. Particular attention was paid to poplar genes with emphasis on Trx-like classes called Clot, thioredoxin-like, thioredoxins of the lilium type and nucleoredoxins, which were not described in depth in previous genomic studies.

A Small Family of Chloroplast Atypical Thioredoxins

The reduction and the formation of regulatory disulfide bonds serve as a key signaling element in chloroplasts. Members of the thioredoxin (Trx) superfamily of oxidoreductases play a major role in these processes. We have characterized a small family of plant-specific Trxs in Arabidopsis (Arabidopsis thaliana) that are rich in cysteine and histidine residues and are typified by a variable noncanonical redox active site. We found that the redox midpoint potential of three selected family members is significantly less reducing than that of the classic Trxs. Assays of subcellular localization demonstrated that all proteins are localized to the chloroplast. Selected members showed high activity, contingent on a dithiol electron donor, toward the chloroplast 2-cysteine peroxiredoxin A and poor activity toward the chloroplast NADP-malate dehydrogenase. The expression profile of the family members suggests that they have distinct roles. The intermediate redox midpoint potential value of the atypical Trxs might imply adaptability to function in modulating the redox state of chloroplast proteins with regulatory disulfides.

An Atypical Catalytic Mechanism Involving Three Cysteines of Thioredoxin

Unlike other thioredoxins h characterized so far, a poplar thioredoxin of the h type, PtTrxh4, is reduced by glutathione and glutaredoxin (Grx) but not NADPH:thioredoxin reductase (NTR). PtTrxh4 contains three cysteines: one localized in an N-terminal extension (Cys(4)) and two (Cys(58) and Cys(61)) in the classical thioredoxin active site ((57)WCGPC(61)). The property of a mutant in which Cys(58) was replaced by serine demonstrates that it is responsible for the initial nucleophilic attack during the catalytic cycle. The observation that the C4S mutant is inactive in the presence of Grx but fully active when dithiothreitol is used as a reductant indicates that Cys(4) is required for the regeneration of PtTrxh4 by Grx. Biochemical and x-ray crystallographic studies indicate that two intramolecular disulfide bonds involving Cys(58) can be formed, linking it to either Cys(61) or Cys(4). We propose thus a four-step disulfide cascade mechanism involving the transient glutathionylation of Cys(4) to convert this atypical thioredoxin h back to its active reduced form.

Identification of a novel thioredoxin‐related protein, PC‐TRP, which is preferentially expressed in plasma cells

Using cDNA subtraction we have identified a previously unknown cDNA, termed plasma cell thioredoxin-related protein (PC-TRP), which was overexpressed in plasma cells in vitro. The full-length 2636-bp PC-TRP cDNA contained an open reading frame coding for 417 amino acids. The cDNA sequence was mapped to a region on mouse chromosome 13 and the PC-TRP gene consists of ten exons distributed over a 28-kb region. The PC-TRP was found to contain three atypical thioredoxin domains and showed functional thioredoxin activity in vitro. A more detailed analysis revealed that PC-TRP was expressed at a high level in plasma cells, both at the mRNA and protein level, while present at very low levels in all other cells and tissues examined. Immunohistological analysis revealed that PC-TRP was expressed at high levels in all plasma cells isolated from bone marrow or lamina propria, irrespective of their Ig isotype. In the spleen, however, 60% of the IgG-expressing plasma cells showed only a dull staining at day 14 after immunization, while >90% of the IgG-secreting cells in the spleen expressed PC-TRP at day 21 after immunization. In addition, an IgG-secreting, PC-TRPdull plasma cell population could also be observed after B cell activation in vitro.