Thiol Redox Proteins Research Articles

Functional characterization of monothiol and dithiol glutaredoxins from Leptospira interrogans

Thiol redox proteins and low molecular mass thiols have essential functions in maintaining cellular redox balance in almost all living organisms. In the pathogenic bacterium Leptospira interrogans, several redox components have been described, namely, typical 2-Cys peroxiredoxin, a functional thioredoxin system, glutathione synthesis pathway, and methionine sulfoxide reductases. However, until now, information about proteins linked to GSH metabolism has not been reported in this pathogen. Glutaredoxins (Grxs) are GSH-dependent oxidoreductases that regulate and maintain the cellular redox state together with thioredoxins. This work deals with recombinant production at a high purity level, biochemical characterization, and detailed kinetic and structural study of the two Grxs (Lin1CGrx and Lin2CGrx) identified in L. interrogans serovar Copenhageni strain Fiocruz L1-130. Both recombinant LinGrxs exhibited the classical in vitro GSH-dependent 2-hydroxyethyl disulfide and dehydroascorbate reductase activity. Strikingly, we found that Lin2CGrx could serve as a substrate of methionine sulfoxide reductases A1 and B from L. interrogans. Distinctively, only recombinant Lin1CGrx contained a [2Fe2S] cluster confirming a homodimeric structure. The functionality of both LinGrxs was assessed by yeast complementation in null grx mutants, and both isoforms were able to rescue the mutant phenotype. Finally, our data suggest that protein glutathionylation as a post-translational modification process is present in L. interrogans. As a whole, our results support the occurrence of two new redox actors linked to GSH metabolism and iron homeostasis in L. interrogans.

28 - A guanylyl cyclase-thioredoxin transnitrosation complex

Nitric Oxide (NO) is best known for its ability to stimulate soluble guanylyl cyclase (sGC) to produce cGMP and stimulate its downstream signaling pathways. However, NO can also covalently modify cysteines via S-nitrosation (addition of a NO moiety to the cysteine of a protein, SNO). Although this reversible post-translational modification is increasingly recognized as an important regulatory mechanism of protein function, and to play a role in cardiac protection, dynamic regulation of protein nitrosation specificity is poorly understood. Combining proteomics quantification and molecular biological methods, we observed that sGC, the key NO receptor, modulates the level of nitrosation of specific proteins in cardiomyocytes and smooth muscle cells. Preliminary data showed that sGC increases nitrosation by a protein-protein interaction-driven SNO transfer (transnitrosation). Moreover, this increased nitrosation is due, for a specific subset of proteins, to the association of sGC with thioredoxin 1 (Trx1), a cardiac protective thiol-redox protein with both transnitrosation and denitrosation activities. Initial mass spectrometry and biochemical analyses showed that sGC transnitrosates Trx1, which in turn nitrosates a specific subset of targets in cardiomyocytes. These observations lead to the provocative idea that sGC modulates S-nitrosation specificity via a transnitrosation cascade that includes Trx1.

Stress-Induced Protein S-Glutathionylation and S-Trypanothionylation in African Trypanosomes-A Quantitative Redox Proteome and Thiol Analysis.

Aims: Trypanosomatids have a unique trypanothione-based thiol redox metabolism. The parasite-specific dithiol is synthesized from glutathione and spermidine, with glutathionylspermidine as intermediate catalyzed by trypanothione synthetase. In this study, we address the oxidative stress response of African trypanosomes with special focus on putative protein S-thiolation.Results: Challenging bloodstream Trypanosoma brucei with diamide, H2O2 or hypochlorite results in distinct levels of reversible overall protein S-thiolation. Quantitative proteome analyses reveal 84 proteins oxidized in diamide-stressed parasites. Fourteen of them, including several essential thiol redox proteins and chaperones, are also enriched when glutathione/glutaredoxin serves as a reducing system indicating S-thiolation. In parasites exposed to H2O2, other sets of proteins are modified. Only three proteins are S-thiolated under all stress conditions studied in accordance with a highly specific response. H2O2 causes primarily the formation of free disulfides. In contrast, in diamide-treated cells, glutathione, glutathionylspermidine, and trypanothione are almost completely protein bound. Remarkably, the total level of trypanothione is decreased, whereas those of glutathione and glutathionylspermidine are increased, indicating partial hydrolysis of protein-bound trypanothione. Depletion of trypanothione synthetase exclusively induces protein S-glutathionylation. Total mass analyses of a recombinant peroxidase treated with T(SH)2 and either diamide or hydrogen peroxide verify protein S-trypanothionylation as stable modification.Innovation: Our data reveal for the first time that trypanosomes employ protein S-thiolation when exposed to exogenous and endogenous oxidative stresses and trypanothione, despite its dithiol character, forms protein-mixed disulfides.Conclusion: The stress-specific responses shown here emphasize protein S-trypanothionylation and S-glutathionylation as reversible protection mechanism in these parasites. Antioxid. Redox Signal. 27, 517–533.

The Myeloablative Drug Busulfan Converts Cysteine to Dehydroalanine and Lanthionine in Redoxins.

The myeloablative agent busulfan (1,4-butanediol dimethanesulfonate) is an old drug that is used routinely to eliminate cancerous bone marrow prior to hematopoietic stem cell transplant. The myeloablative activity and systemic toxicity of busulfan have been ascribed to its ability to cross-link DNA. In contrast, here we demonstrate that incubation of busulfan with the thiol redox proteins glutaredoxin or thioredoxin at pH 7.4 and 37 °C results in the formation of putative S-tetrahydrothiophenium adducts at their catalytic Cys residues, followed by β-elimination to yield dehydroalanine. Both proteins contain a second Cys, in their catalytic C-X-X-C motif, which reacts with the dehydroalanine, the initial Cys adduct with busulfan, or the S-tetrahydrothiophenium, to form novel intramolecular cross-links. The reactivity of the dehydroalanine (DHA) formed is further demonstrated by adduction with glutathione to yield a lanthionine and by a novel reaction with the reducing agent tris(2-carboxyethyl)phosphine (TCEP), which yields a phosphine adduct via Michael addition to the DHA. Formation of a second quaternary organophosphonium salt via nucleophilic substitution with TCEP on the initial busulfan-protein adduct or on the THT(+)-Redoxin species is also observed. These results reveal a rich potential for reactions of busulfan with proteins in vitro, and likely in vivo. It is striking that several of the chemically altered protein products retain none of the atoms of busulfan, in contrast to typical drug-protein adducts or traditional protein modification reagents. In particular, the ability of a clinically used drug to convert Cys to dehydrolanine in intact proteins, and its subsequent reaction with biological thiols, is unprecedented.

Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction.

Soluble guanylyl cyclase (sGC) is a heterodimeric nitric oxide (NO) receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI) interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT) and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed.

Stable Isotope Metabolic Labeling-Based Quantitative Thiol Redox Proteomic Analysis of Hydrogen Peroxide-treated Arabidopsis plant

Homeostasis of protein thiol redox plays an important role in numerous cellular processes and stress responses. The thiol moiety of cysteine residue is easily altered to various redox isoforms that play critical roles in regulation of both structure and function of proteins. In the present study, a quantitative thiol redox proteomic method, termed OxNSIL, has been developed, which integrates the chemical labeling by biotin-tagged alkylating reagents with the heavy nitrogen stable isotope-coded salt metabolic labeling and acquires both advantages of biotin-avidin enrichment and MS-based quantitation of cysteine residue-containing peptides. Both the reduced and the reversibly oxidized cysteine thiol moieties are finally identified in a site-specific manner and measured in a single experiment. Hydrogen peroxide, H2O2, was applied on the whole Arabidopsis plant to trigger thiol redox state alteration in order to obtain a proof-of-concept result for application of OxNSIL approach in plants. A total of 438 non-redundant biotin-tagged thiolalkylated peptides representing 391 different cysteine-containing proteins were eventually obtained, among which 17 redox-sensitive biotin tag-labeled peptides were significantly altered by H2O2. Some well-known ROS-related proteins, such as Ferredoxins 1/2, RuBisCO large subunit, RuBisCO activase, and Fructose-bisphosphate aldolase 6/8 were also detected as the oxidation- sensitive thiol redox proteins. These results substantiate the usefulness of OxNSIL approach in study of in planta ROS-induced cellular thiol redox state alterations.

Thiol-redox proteins interact with soluble guanylyl cyclase and modulate its activity

Background Nitric oxide (NO) signalling through activation of soluble guanylyl cyclase (sGC) is key for the physiology of the cardiovascular and neuronal systems. Discrepancies in sGC activation and deactivation in vitro vs. in vivo have led to a search for endogenous factors that regulate sGC or assist in cellular localization. In our previous work, which identified Hsp70 as a modulator of sGC, we determined that protein disulfide isomerase (PDI) bound to an sGC-affinity matrix [1]. As several studies have reported that thiol oxidants and reductants modulate sGC activity, we sought to characterize the potential interaction between sGC and PDI, an enzyme that not only isomerases disulfide bonds but also regulates some proteins activity via interaction with its cysteine (Cys) active sites [2,3]. Results Using protein chemistry and purified sGC and PDI, we first showed that reduced sGC was modified by oxidized PDI in non-reducing Western blot and that sGC thiol titer was altered under these conditions. Moreover a PDI-Flag resin retains specifically sGC and this complex was resolved in the presence of a thiol-reductant. These data suggested that the mechanism of interaction between sGC and PDI is by mixed-disulfide exchange. In addition, PDI modulated NO-stimulated sGC activity while a PDI mutated in its two Cys active sites did not. A relatively novel method, in situ PLA (proximity ligation assay) established that endogenous PDI and sGC also interact in the CRL 2018 smooth muscle cells suggesting that the interaction between sGC and PDI is biologically relevant. Notably, both a 1a ndb 1s GC subunits of sGC showed potential co-localization with PDI [4]. Discussion These results underlined the significance of redox-thiol modulation of sGC and support past and current research showing that certain sGC Cys are necessary for such functions as enzyme activation, dimerization, and desensitization by S-nitrosation. A novel Mass Spectrometry analysis is currently applied to identify sGC Cys disulfide bonds and combined with mutational analysis and molecular dynamics simulation should unveil their involvement in interaction with PDI and their relevance as a new mechanism of sGC modulation.

Staphylococcus aureusNrdH Redoxin Is a Reductant of the Class Ib Ribonucleotide Reductase

Staphylococci contain a class Ib NrdEF ribonucleotide reductase (RNR) that is responsible, under aerobic conditions, for the synthesis of deoxyribonucleotide precursors for DNA synthesis and repair. The genes encoding that RNR are contained in an operon consisting of three genes, nrdIEF, whereas many other class Ib RNR operons contain a fourth gene, nrdH, that determines a thiol redoxin protein, NrdH. We identified a 77-amino-acid open reading frame in Staphylococcus aureus that resembles NrdH proteins. However, S. aureus NrdH differs significantly from the canonical NrdH both in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that S. aureus NrdH is a thiol redox protein. It is not essential for aerobic or anaerobic growth and appears to have a marginal role in protection against oxidative stress. In vitro, S. aureus NrdH was found to be an efficient reductant of disulfide bonds in low-molecular-weight substrates and proteins using dithiothreitol as the source of reducing power and an effective reductant for the homologous class Ib RNR employing thioredoxin reductase and NADPH as the source of the reducing power. Its ability to reduce NrdEF is comparable to that of thioredoxin-thioredoxin reductase. Hence, S. aureus contains two alternative thiol redox proteins, NrdH and thioredoxin, with both proteins being able to function in vitro with thioredoxin reductase as the immediate hydrogen donors for the class Ib RNR. It remains to be clarified under which in vivo physiological conditions the two systems are used.

Glutathionylation of Trypanosomal Thiol Redox Proteins

Trypanosomatids, the causative agents of several tropical diseases, lack glutathione reductase and thioredoxin reductase but have a trypanothione reductase instead. The main low molecular weight thiols are trypanothione (N(1),N(8)-bis-(glutathionyl)spermidine) and glutathionyl-spermidine, but the parasites also contain free glutathione. To elucidate whether trypanosomes employ S-thiolation for regulatory or protection purposes, six recombinant parasite thiol redox proteins were studied by ESI-MS and MALDI-TOF-MS for their ability to form mixed disulfides with glutathione or glutathionylspermidine. Trypanosoma brucei mono-Cys-glutaredoxin 1 is specifically thiolated at Cys(181). Thiolation of this residue induced formation of an intramolecular disulfide bridge with the putative active site Cys(104). This contrasts with mono-Cys-glutaredoxins from other sources that have been reported to be glutathionylated at the active site cysteine. Both disulfide forms of the T. brucei protein were reduced by tryparedoxin and trypanothione, whereas glutathione cleaved only the protein disulfide. In the glutathione peroxidase-type tryparedoxin peroxidase III of T. brucei, either Cys(47) or Cys(95) became glutathionylated but not both residues in the same protein molecule. T. brucei thioredoxin contains a third cysteine (Cys(68)) in addition to the redox active dithiol/disulfide. Treatment of the reduced protein with GSSG caused glutathionylation of Cys(68), which did not affect its capacity to catalyze reduction of insulin disulfide. Reduced T. brucei tryparedoxin possesses only the redox active Cys(32)-Cys(35) couple, which upon reaction with GSSG formed a disulfide. Also glyoxalase II and Trypanosoma cruzi trypanothione reductase were not sensitive to thiolation at physiological GSSG concentrations.

An introduction to thiol redox proteins in the endoplasmic reticulum and a review of current electrochemical methods of detection of thiols

This aim of this paper is to expound the complexity of thiol redox systems in the endoplasmic reticulum of eukaryotic cells to the electroanalytical community. A summary of the state of the art in electrochemical methods for detection of thiols gives an insight into the challenges that need to be addressed to bridge the disparity between current analytical techniques and applications in a 'real' biological scenario.

Expression of the Vaccinia Virus A2.5L Redox Protein Is Required for Virion Morphogenesis

In this article we report the initial biochemical, genetic, and electron microscopic analysis of a previously uncharacterized, 8.9-kDa, predicted thiol-redox protein. The name A2.5L was assigned to the corresponding vaccinia virus gene, which is conserved in all sequenced poxviruses. Multiple alignment analysis and secondary structure prediction indicated that the A2.5L gene product is an all-α-helical protein with a conserved Cxx(x)C motif in the N-terminal α-helix. The DNA replication requirement and kinetics of A2.5L protein accumulation in virus-infected cells were typical of a late gene product, in agreement with the predicted promoter sequence. The A2.5L protein was a monomer under reducing conditions, but was mostly associated with the vaccinia virus E10R redox protein as a heterodimer under nonreducing conditions. The A2.5L protein was detected in virus particles at various stages of assembly, suggesting that it is an integral component of intracellular virions. An inducer-dependent A2.5L null mutant was constructed: in the absence of inducer, infectious virus formation was abolished and electron microscopy revealed an assembly block with an accumulation of crescent membranes and immature virions. This stage of assembly block was similar to that occurring when the E10R and G4L redox proteins were repressed, which is compatible with the involvement of E10R, A2.5L, and G4L in the same redox pathway.

Thioredoxin h is one of the major proteins in rice phloem sap

Sieve tubes play important roles in the transfer of nutrients as well as signals. Hundreds of proteins were found in pure phloem sap collected from rice (Oryza sativa L. cv. Kantou) plants through the cut ends of insect stylets. These proteins may be involved in nutrient transfer and signal transduction. To characterize the nature of these proteins, the partial amino-acid sequence of a 13-kDa protein, named RPP13-1, that was abundant in the pure phleom sap was determined. A cDNA clone of 687 bp, containing an open reading frame of 122 amino acids, was isolated using corresponding oligonucleotides as a probe. The deduced amino-acid sequence was very similar to that of the ubiquitous thiol redox protein, thioredoxin. The consensus sequences of thioredoxins are highly conserved. No putative signal peptide was identified. Antiserum against wheat thioredoxin h cross-reacted with RPP13-1 in the phloem sap of rice plants. RPP13-1 produced in Escherichia coli was reactive to antiserum against wheat thioredoxin h. Both E. coli-produced RPP13-1 and the phloem sap proteins catalyzed the reduction of the disulfide bonds of insulin in the presence of dithiothreitol. These results indicate that an active thioredoxin is a major protein translocating in rice sieve tubes.

Copper complex of a new ribonucleotide reductase inhibitor characterized by antitumoral properties

Ribonucleotide reductase (RNR) catalyzes the rate-limiting de novo synthesis of 2′-deoxyribonucleotides from the corresponding ribonucleotides and thereby provides balanced deoxyribonucleotide pools required for error-free DNA replication and repair. The essential role of RNR in DNA synthesis and the use of DNA as genetic material has made it an important target for the development of anticancer and antiviral agents. The most well known feature of the universal RNR reaction in all kingdoms of life is the involvement of protein free radicals. Redox-active cysteines, thiyl radicals, and thiol redox proteins of the thioredoxin superfamily play major roles in the catalytic mechanism. The involvement of cysteine residues in catalysis is common to all three classes of RNR. Taking account of the recent progress in this field of research, this review focuses on the use of thiols in the redox mechanism of RNR enzymes.