Disulfide Oxidoreductase Research Articles

Exploration of glutathione reductase for abiotic stress response in bread wheat (Triticum aestivum L.)

A total of seven glutathione reductase (GR) genes were identified in Triticum aestivum, which were used for comparative structural characterization, phylogenetic analysis and expression profiling with the GR genes of other cereal plants. The modulated gene expression and enzyme activity revealed the role of GRs in abiotic stress response in T. aestivum. Glutathione reductase (GR) is an enzymatic antioxidant that converts oxidized glutathione (GSSG) into reduced glutathione (GSH) through the ascorbate-glutathione cycle. In this study, a total of seven GR genes forming two homeologous groups were identified in the allohexaploid genome of bread wheat (Triticum aestivum). Besides, we identified three GR genes in each Aegilops tauschii, Brachypodium distachyon, Triticum urartu and Sorghum bicolor, which were used for comparative characterization. Phylogenetic analysis revealed the clustering of GR proteins into two groups; class I and class II, which were predicted to be localized in cytoplasm and chloroplast, respectively. The exon-intron and conserved motif patterns were almost conserved in each group, in which a maximum of 10 and 17 exons were present in chloroplastic and cytoplasmic GRs, respectively. The protein structure analysis confirmed the occurrence of conserved pyridine nucleotide disulfide oxidoreductase (Pyr_redox) and pyridine nucleotide disulfide oxidoreductase dimerization (Pyr_redox_dim) domains in each GR. The active site of GR proteins consisted of two conserved cysteine residues separated by four amino acid residues. Promoter analysis revealed the occurrence of growth and stress-related cis-active elements. Tissue-specific expression profiling suggested the involvement of GRs in both vegetative and reproductive tissue development in various plants. The differential expression of TaGR genes and enhanced GR enzyme activity suggested their roles under drought, heat, salt and arsenic stress. Interaction of GRs with other proteins and chemical compounds of the ascorbate-glutathione cycle revealed their coordinated functioning. The current study will provide a foundation for the validation of the precise role of each GR gene in future studies.

Crystal structure of chloroplastic thioredoxin z defines a type-specific target recognition.

Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant TRXs evolved into seven types with diverse subcellular localization and distinct protein target selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows thioredoxin z (TRXz) with a capacity to activate the photosynthetic Calvin-Benson cycle enzyme phosphoribulokinase. The distinct electronegative surface of TRXz thereby extends the repertoire of TRX-target recognitions.

AccPDIA6 from Apis cerana cerana plays important roles in antioxidation

PDIA6 is a member of the protein disulfide isomerase (PDI) family, shows disulfide isomerase activity and oxidoreductase activity, and can act as a molecular chaperone. Its biological functions include modulating apoptosis, regulating the proliferation and invasion of cancer cells, supporting thrombosis and modulating insulin secretion. However, the roles of PDIA6 in Apis cerana cerana are poorly understood. Herein, we obtained the PDIA6 gene from A. cerana cerana (AccPDIA6). We investigated the expression patterns of AccPDIA6 in response to oxidative stress induced by H2O2, UV, HgCl2, extreme temperatures (4 °C, 42 °C) and pesticides (thiamethoxam and hexythiazox) and found that AccPDIA6 was upregulated by these treatments. Western blot analysis indicated that AccPDIA6 was also upregulated by oxidative stress at the protein level. In addition, a survival test demonstrated that the survival rate of E. coli cells expressing AccPDIA6 increased under oxidative stress, suggesting a possible antioxidant function of AccPDIA6. In addition, we tested the transcripts of other antioxidant genes and found that some of them were downregulated in AccPDIA6 knockdown samples. It was also discovered that the antioxidant enzymatic activity of superoxide dismutase (SOD) decreased in AccPDIA6-silenced bees. Moreover, the survival rate of AccPDIA6 knockdown bees decreased under oxidative stress, implying that AccPDIA6 may function in the oxidative stress response by enhancing the viability of honeybees. Taken together, these results indicated that AccPDIA6 may play an essential role in counteracting oxidative stress.

Genome-wide identification, characterization and expression profile of glutaredoxin gene family in relation to fruit ripening and response to abiotic and biotic stresses in banana (Musa acuminata)

Glutaredoxins (GRXs) are disulfide oxidoreductases that are involved in various biological processes. However, little information on the role of GRXs in the regulation of fruit ripening and the response to stress is available. In this study, we isolated 64 GRX genes from banana genome. Their encoded GRX proteins could be classified into four classes: CC, CGFS, CPYC and GRL types. The distribution and synteny of these GRXs on chromosomes, the gene structures, the promoter sequences, and the possible protein subcellular localizations were characterized. Molecular interaction network analysis suggested that MaGRX might interact with glutathione reductase (GR), sulfiredoxin, peroxiredoxin (Prx), and NADPH-dependent thioredoxin reductase C (NTRC), contributing to the antioxidative defense of banana fruit. MicroRNA prediction showed that MaGRX genes might be targeted by different miRNAs. Transcriptome analysis characterized the expression profiles of different MaGRX genes during banana fruit ripening, and in response to different storage stresses. The results suggested that CC-type, CPYC-type and GRL-type MaGRXs might be more active than CGFS-type MaGRXs during banana fruit ripening and the response to stress. Moreover, MaGRX6/7/9/11/17/23/28 and MaGRL3/16/19 might play important roles in regulating fruit ripening or in response to low and high temperature, or Fusarium proliferatum infection.

Monohaloacetic Acids and Monohaloacetamides Attack Distinct Cellular Proteome Thiols.

Disinfection byproduct (DBP) exposure has been linked to multiple adverse health outcomes. However, the molecular initiating events by which DBPs induce their toxicities remain unclear. Herein, we combined reporter cell lines and activity-based protein profiling (ABPP) chemical proteomics to identify the protein targets of three monohaloacetic acids (mHAAs) and three monohaloacetamides (mHAMs), at the proteome-wide level. While mHAAs and mHAMs have similar potencies in reducing MTT activity, mHAMs induced greater Nrf2-mediated oxidative stress responses, demonstrating their distinct toxicity pathways. ABPP on crude cell lysates suggested that general proteome thiol reactivity correlates with cytotoxicity. Interestingly, live cell ABPP results revealed class-specific proteins attacked by mHAMs or mHAAs. Subsequent proteomic analysis identified >100 unique targets per DBP. mHAMs preferentially react with redox proteins including disulfide oxidoreductase enzymes, accounting for their stronger Nrf2 responses. To further probe alkylation mechanisms, we directly monitored protein adducts and identified 120 and 37 unique peptides with iodoacetamide and iodoacetic acid adducts, respectively. Of the latter, we confirmed glyceraldehyde-3-phosphate dehydrogenase as a key target of IAA, specifically attacking the catalytic Cys 152. This is the first study reporting the distinct cellular protein targets of mHAAs and mHAMs at the proteome-wide level, which highlights their different toxicity pathways despite their similar structures.

Molecular mechanisms of Mycoredoxin-1 in resistance to oxidative stress in Corynebacterium glutamicum.

Glutaredoxins (Grxs) with Cys-Pro-Phe (Tyr)-Cys motif and a thioredoxin fold structure play an important role in the anti-oxidant system of bacteria by catalyzing a variety of thiol-disulfide exchange reactions with a 2-Cys mechanism or a 1-Cys mechanism. However, the catalytic and physiological mechanism of Corynebacterium glutamicum Mycoredoxin 1 (Mrx1) that shares a high amino acid sequence similarity to Grxs has not been fully elucidated. Here, we report that Mrx1 has a protective function against various adverse conditions, and the decrease of cell viability to various stress conditions by deletion of the Mrx1 in C. glutamicum was confirmed in the mrx1 mutant. The physiological roles of Mrx1 in defence to oxidative stress were corroborated by its induced expression under various stresses, regulated directly by the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. As well as reducing mycothiol (MSH) mixed disulfide bonds via a 1-Cys mechanism, C. glutamicum Mrx1 catalytically reduced the disulfides in the Ib RNR, insulin and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) by exclusively linking the MSH/Mtr (mycothiol disulfide reductase)/NADPH electron pathway via a 2-Cys mechanism. Thus, we present the first evidence that the Mrx1 is able to protect against the damaging effects of various exogenous stresses by acting as a disulfide oxidoreductase, thereby giving a new insight in how C. glutamicum survives oxidative stressful conditions.

Corynebacterium glutamicum Mycoredoxin 3 protects against multiple oxidative stresses and displays thioredoxin-like activity.

Glutaredoxins (Grxs) and thioredoxins (Trxs) play a critical role in resistance to oxidative conditions. However, physiological and biochemical roles of Mycoredoxin 3 (Mrx3) that shared a high amino acid sequence similarity to Grxs remain unknown in Corynebacterium glutamicum. Here we showed that mrx3 deletion strains of C. glutamicum was involved in the protection against oxidative stress. Recombinant Mrx3 not only catalytically reduced the disulfide bonds in ribonucleotide reductase (RNR), insulin and 5,5'-dithiobis-(2-nitro-benzoicacid) (DTNB), but also reduced the mixed disulphides between mycothiol (MSH) and substrate, which was exclusively linked to the thioredoxin reductase (TrxR) electron transfer pathway by a dithiol mechanism. Site-directed mutagenesis confirmed that the conserved Cys17 and Cys20 in Mrx3 were necessary to maintain its activity. The mrx3 deletion mutant showed decreased resistance to various stress, and these sensitive phenotypes were almost fully restored in the complementary strain. The physiological roles of Mrx3 in resistance to various stress were further supported by the induced expression of mrx3 under various stress conditions, directly under the control of the stress-responsive extracytoplasmic function-sigma (ECF-σ) factor SigH. Thus, we presented the first evidence that Mrx3 protected against various oxidative stresses by acting as a disulfide oxidoreductase behaving like Trx.

Enzymatic Antioxidant Signatures in Hyperthermophilic Archaea.

To fight reactive oxygen species (ROS) produced by both the metabolism and strongly oxidative habitats, hyperthermophilic archaea are equipped with an array of antioxidant enzymes whose role is to protect the biological macromolecules from oxidative damage. The most common ROS, such as superoxide radical (O2•−) and hydrogen peroxide (H2O2), are scavenged by superoxide dismutase, peroxiredoxins, and catalase. These enzymes, together with thioredoxin, protein disulfide oxidoreductase, and thioredoxin reductase, which are involved in redox homeostasis, represent the core of the antioxidant system. In this review, we offer a panorama of progression of knowledge on the antioxidative system in aerobic or microaerobic (hyper)thermophilic archaea and possible industrial applications of these enzymes.

Reversal of Alpha-Synuclein Fibrillization by Protein Disulfide Isomerase.

Aggregates of α-synuclein contribute to the etiology of Parkinson’s Disease. Protein disulfide isomerase (PDI), a chaperone and oxidoreductase, blocks the aggregation of α-synuclein. An S-nitrosylated form of PDI that cannot function as a chaperone is associated with elevated levels of aggregated α-synuclein and is found in brains afflicted with Parkinson’s Disease. The protective role of PDI in Parkinson’s Disease and other neurodegenerative disorders is linked to its chaperone function, yet the mechanism of neuroprotection remains unclear. Using Thioflavin-T fluorescence and transmission electron microscopy, we show here for the first time that PDI can break down nascent fibrils of α-synuclein. Mature fibrils were not affected by PDI. Another PDI family member, ERp57, could prevent but not reverse α-synuclein aggregation. The disaggregase activity of PDI was effective at a 1:50 molar ratio of PDI:α-synuclein and was blocked by S-nitrosylation. PDI could not reverse the aggregation of malate dehydrogenase, which indicated its disaggregase activity does not operate on all substrates. These findings establish a previously unrecognized disaggregase property of PDI that could underlie its neuroprotective function.

Insights into the unique carboxylation reactions in the metabolism of propylene and acetone.

Alkenes and ketones are two classes of ubiquitous, toxic organic compounds in natural environments produced in several biological and anthropogenic processes. In spite of their toxicity, these compounds are utilized as primary carbon and energy sources or are generated as intermediate metabolites in the metabolism of other compounds by many diverse bacteria. The aerobic metabolism of some of the smallest and most volatile of these compounds (propylene, acetone, isopropanol) involves novel carboxylation reactions resulting in a common product acetoacetate. Propylene is metabolized in a four-step pathway involving five enzymes where the penultimate step is a carboxylation reaction catalyzed by a unique disulfide oxidoreductase that couples reductive cleavage of a thioether linkage with carboxylation to produce acetoacetate. The carboxylation of isopropanol begins with conversion to acetone via an alcohol dehydrogenase. Acetone is converted to acetoacetate in a single step by an acetone carboxylase which couples the hydrolysis of MgATP to the activation of both acetone and bicarbonate, generating highly reactive intermediates that are condensed into acetoacetate at a Mn2+ containing the active site. Acetoacetate is then utilized in central metabolism where it is readily converted to acetyl-coenzyme A and subsequently converted into biomass or utilized in energy metabolism via the tricarboxylic acid cycle. This review summarizes recent structural and biochemical findings that have contributed significant insights into the mechanism of these two unique carboxylating enzymes.

A Disulfide Oxidoreductase (CHU_1165) Is Essential for Cellulose Degradation by Affecting Outer Membrane Proteins in Cytophaga hutchinsonii.

Cytophaga hutchinsonii cells can bind to the surface of insoluble cellulose and degrade it by utilizing a novel cell contact-dependent mechanism, in which the outer membrane proteins may play important roles. In this study, the deletion of a gene locus, chu_1165, which encodes a hypothetical protein with 32% identity with TlpB, a disulfide oxidoreductase in Flavobacterium psychrophilum, caused a complete cellulolytic defect in C. hutchinsonii Further study showed that cells of the Δ1165 strain could not bind to cellulose, and the levels of many outer membrane proteins that can bind to cellulose were significantly decreased. The N-terminal region of CHU_1165 is anchored to the cytoplasmic membrane with five predicted transmembrane helices, and the C-terminal region is predicted to stretch to the periplasm and has a similar thioredoxin (Trx) fold containing a Cys-X-X-Cys motif that is conserved in disulfide oxidoreductases. Recombinant CHU_1165His containing the Cys-X-X-Cys motif was able to reduce the disulfide bonds of insulin in vitro Site-directed mutation showed that the cysteines in the Cys-X-X-Cys motif and at residues 106 and 108 were indispensable for the function of CHU_1165. Western blotting showed that CHU_1165 was in an oxidized state in vivo, suggesting that it may act as an oxidase to catalyze disulfide bond formation. However, many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of the cysteine in these proteins did not affect cellulose degradation, indicating that CHU_1165 may have an indirect or pleiotropic effect on the function of these outer membrane proteins.IMPORTANCECytophaga hutchinsonii can rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation by affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies show that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate the formation of a disulfide bond in these proteins. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist in the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii.

A peroxiredoxin of Thermus thermophilus HB27: Biochemical characterization of a new player in the antioxidant defence

To fight oxidative damage due to reactive oxygen species (ROS), cells are equipped of different enzymes, among which Peroxiredoxins (Prxs) (EC 1.11.1.15) play a key role. Prxs are thiol-based enzymes containing one (1-Cys Prx) or two (2-Cys Prx) catalytic cysteine residues. In 2-Cys Prxs the cysteine residues form a disulfide bridge following reduction of peroxide which is in turn reduced by Thioredoxin reductase (Tr) /Thioredoxin (Trx) disulfide reducing system to regenerate the enzyme. In this paper we investigated on Prxs of Thermus thermophilus whose genome contains an ORF TT_C0933 encoding a putative Prx, belonging to the subfamily of Bacterioferritin comigratory protein (Bcp): the synthetic gene was produced and expressed in E. coli and the recombinant protein, TtBcp, was biochemically characterized. TtBcp was active on both organic and inorganic peroxides and showed stability at high temperatures. To get insight into disulfide reducing system involved in the recycling of the enzyme we showed that TtBcp catalically eliminates hydrogen peroxide using an unusual partner, the Protein Disulfide Oxidoreductase (TtPDO) that could replace regeneration of the enzyme. Altogether these results highlight not only a new anti-oxidative pathway but also a promising molecule for possible future biotechnological applications.

The Oxidoreductase DsbA1 negatively influences 2,4-diacetylphloroglucinol biosynthesis by interfering the function of Gcd in Pseudomonas fluorescens 2P24

BackgroundThe polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG), produced by Pseudomonas fluorescens 2P24, is positively regulated by the GacS-GacA two-component system.ResultsHere we reported on the characterization of DsbA1 (disulfide oxidoreductase) as novel regulator of biocontrol activity in P. fluorescens. Our data showed that mutation of dsbA1 caused the accumulation of 2,4-DAPG in a GacA-independent manner. Further analysis indicated that DsbA1 interacts with membrane-bound glucose dehydrogenase Gcd, which positively regulates the production of 2,4-DAPG. Mutation of cysteine (C)-235, C275, and C578 of Gcd, significantly reduced the interaction with DsbA1, enhanced the activity of Gcd and increased 2,4-DAPG production.ConclusionsOur results suggest that DsbA1 regulates the 2,4-DAPG concentration via fine-tuning the function of Gcd in P. fluorescens 2P24.

Oxidization of TGFβ-activated kinase by MPT53 is required for immunity to Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb)-derived components are usually recognized by pattern recognition receptors to initiate a cascade of innate immune responses. One striking characteristic of Mtb is their utilization of different type VII secretion systems to secrete numerous proteins across their hydrophobic and highly impermeable cell walls, but whether and how these Mtb-secreted proteins are sensed by host immune system remains largely unknown. Here, we report that MPT53 (Rv2878c), a secreted disulfide-bond-forming-like protein of Mtb, directly interacts with TGF-β-activated kinase 1 (TAK1) and activates TAK1 in a TLR2- or MyD88-independent manner. MPT53 induces disulfide bond formation at C210 on TAK1 to facilitate its interaction with TRAFs and TAB1, thus activating TAK1 to induce the expression of pro-inflammatory cytokines. Furthermore, MPT53 and its disulfide oxidoreductase activity is required for Mtb to induce the host inflammatory responses via TAK1. Our findings provide an alternative pathway for host signalling proteins to sense Mtb infection and may favour the improvement of current vaccination strategies.

Implications of the mitochondrial interactome of mammalian thioredoxin 2 for normal cellular function and disease

Multiple thioredoxin isoforms exist in all living cells. To explore the possible functions of mammalian mitochondrial thioredoxin 2 (Trx2), an interactome of mouse Trx2 was initially created using (i) a monothiol mouse Trx2 species for capturing protein partners from different organs and (ii) yeast two hybrid screens on human liver and rat brain cDNA libraries. The resulting interactome consisted of 195 proteins (Trx2 included) plus the mitochondrial 16S RNA. 48 of these proteins were classified as mitochondrial (MitoCarta2.0 human inventory). In a second step, the mouse interactome was combined with the current four-membered mitochondrial sub-network of human Trx2 (BioGRID) to give a 53-membered human Trx2 mitochondrial interactome (52 interactor proteins plus the mitochondrial 16S RNA). Although thioredoxins are thiol-employing disulfide oxidoreductases, approximately half of the detected interactions were not due to covalent disulfide bonds. This finding reinstates the extended role of thioredoxins as moderators of protein function by specific non-covalent, protein-protein interactions. Analysis of the mitochondrial interactome suggested that human Trx2 was involved potentially in mitochondrial integrity, formation of iron sulfur clusters, detoxification of aldehydes, mitoribosome assembly and protein synthesis, protein folding, ADP ribosylation, amino acid and lipid metabolism, glycolysis, the TCA cycle and the electron transport chain. The oxidoreductase functions of Trx2 were verified by its detected interactions with mitochondrial peroxiredoxins and methionine sulfoxide reductase. Parkinson's disease, triosephosphate isomerase deficiency, combined oxidative phosphorylation deficiency, and lactate dehydrogenase b deficiency are some of the diseases where the proposed mitochondrial network of Trx2 may be implicated.

The reactive form of a C–S bond–cleaving, CO2-fixing flavoenzyme

2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO2 fixation. 2-KPCC differs from other DSORs by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site. Here, using site-directed mutagenesis and stopped-flow kinetics, we examined the reactive form of 2-KPCC and its single turnover reactions with a suicide substrate and CO2 The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. In the absence of the histidine, the flavin and disulfide moieties were no longer coupled via a covalent or charge transfer interaction as in typical DSORs. Similar to thioredoxins, the pKa between 7.5 and 8.1 that controls reactivity appeared to be due to a single proton shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C-S bond of the substrate. The lack of a histidine protected 2-KPCC's reactive intermediate from unwanted protonation; however, without its input as a catalytic acid-base, the oxidative half-reaction where carboxylation takes place was remarkably slow, limiting the overall reaction rate. We conclude that stringent regulation of protons in the DSOR active site supports C-S bond cleavage and selectivity for CO2 fixation.

Glutaredoxin 1 up-regulates deglutathionylation of α4 integrin and thereby restricts neutrophil mobilization from bone marrow

α4 integrin plays a crucial role in retention and release of neutrophils from bone marrow. Although α4 integrin is known to be a potential target of reactive oxygen species (ROS)-induced cysteine glutathionylation, the physiological significance and underlying regulatory mechanism of this event remain elusive. Here, using in vitro and in vivo biochemical and cell biology approaches, we show that physiological ROS-induced glutathionylation of α4 integrin in neutrophils increases the binding of neutrophil-associated α4 integrin to vascular cell adhesion molecule 1 (VCAM-1) on human endothelial cells. This enhanced binding was reversed by extracellular glutaredoxin 1 (Grx1), a thiol disulfide oxidoreductase promoting protein deglutathionylation. Furthermore, in a murine inflammation model, Grx1 disruption dramatically elevated α4 glutathionylation and subsequently enhanced neutrophil egress from the bone marrow. Corroborating this observation, intravenous injection of recombinant Grx1 into mice inhibited α4 glutathionylation and thereby suppressed inflammation-induced neutrophil mobilization from the bone marrow. Taken together, our results establish ROS-elicited glutathionylation and its modulation by Grx1 as pivotal regulatory mechanisms controlling α4 integrin affinity and neutrophil mobilization from the bone marrow under physiological conditions.

23 - Control of protein cysteine oxidation in lung fibrosis via a coordinated network of protein disulfide oxidoreductases

Pulmonary fibrosis (IPF) is a devastating progressive disease that is fatal within 3-5 years of diagnosis. We recently demonstrated increases in protein S-glutathionylation (PSSG) in lungs from patients with IPF which correlated with loss of lung function (Anathy, Nature Medicine 2018). We have also shown that PSSG in lung epithelial cells is controlled by glutaredoxin (GLRX) glutathione S-transferase P (GSTP), and protein disulfide isomerase A3 (PDIA3). Assessment of transcripts of these oxidoreductases in IPF lung tissues and controls revealed small but statistically significant increases in mRNA levels of PDIA3, whereas expression of GSTPand GLRXmRNAs was unchanged in IPF patients compared to controls. Despite the lack of GSTPmRNA expression changes in IPF, we have observed robust expression of GSTP in hyperplastic type II epithelial cells and in regions of disease progression in IPF.Pharmacological inhibition of GSTP, using TLK117, a clinically applicable inhibitor of GSTP, protected against the progression of bleomycin-induced fibrosis in association with decreased S-glutathionylation (McMillan JCI Insight 2016). Despite slight changes in GLRXmRNA, GLRX enzymatic activity was strongly attenuated in lungs from patients with IPF. Administration of recombinant active Glrx to mice with pre-existing bleomycin or TGFB1-induced fibrosis reversed the existing increases in fibrosis in association with decreased S-glutathionylation. Similarly, epithelial specific ablation of Pdia3, or pharmacological inhibition of PDIs also attenuated bleomycin-induced lung epithelial apoptosis and fibrosis in mice. Collectively these findings suggest that multiple redox enzymes (PDIA3, GSTP, GLRX) act in concert to control the extent of S-glutathionylation, apoptosis and fibrogenesis. Coordinated targeting of this enzyme axis may provide a powerful strategy to restore redox homeostasis and reverse fibrosis. Our findings also indicate that global transcriptomics analyses in lung tissue homogenates may not identify relevant targets for pharmacological manipulation, given that PDIA3, GSTP, and GLRX activities, were found be a strong determinants of disease progression, instead of mRNA expression.

Molecular Mechanisms of the Methionine Sulfoxide Reductase System from Neisseria meningitidis.

Neisseria meningitidis, an obligate pathogenic bacterium in humans, has acquired different defense mechanisms to detect and fight the oxidative stress generated by the host’s defense during infection. A notable example of such a mechanism is the PilB reducing system, which repairs oxidatively-damaged methionine residues. This review will focus on the catalytic mechanism of the two methionine sulfoxide reductase (MSR) domains of PilB, which represent model enzymes for catalysis of the reduction of a sulfoxide function by thiols through sulfenic acid chemistry. The mechanism of recycling of these MSR domains by various “Trx-like” disulfide oxidoreductases will also be discussed.

Function of alkyl hydroperoxidase AhpD in resistance to oxidative stress in Corynebacterium glutamicum.

Alkyl hydroperoxidase reductase AhpD, which is functionally equivalent to the bacterial flavin-containing disulfide reductase AhpF, acts as a proton donor for the organic peroxide-scavenging alkyl hydroperoxidase AhpC. Although AhpD has long been demonstrated in Mycobacterium tuberculosis, its physiological and biochemical functions remain largely unknown in other actinobacteria, including Corynebacterium glutamicum, Streptomyces, and Mycobacterium smegmatis. Here, we report that C. glutamicum AhpD contributed to regenerate a variety of thiol-dependent peroxidase in the decomposition of peroxide by linking a dihydrolipoamide dehydrogenase (Lpd)/dihydrolipoamide succinyltransferase (SucB)/NADH system through the cyclization of their own active site dithiol to the oxidized disulphide. The CXXC motif of AhpD was essential to maintain the peroxides reduction activity of thiol-dependent peroxidase. ΔahpD1ΔahpD2 mutants exhibited significantly decreased resistance to adverse stress conditions and obviously increased the accumulation of reactive oxygen species (ROS). The physiological roles of AhpD in resistance to adverse stresses, were corroborated by their induced expression under various stresses and their direct regulation under the stress-responsive ECF-sigma factor SigH. C. glutamicum AhpDs were disulfide oxidoreductases behaving like thioredoxin (Trx) in regenerating thiol-dependent peroxidase for stress response, which provides the theoretical basis for an in-depth study of the reduction system in ahpC-lacking bacteria.