Disulphide Reductase Research Articles

Protein Disulphide Isomerase 6 (PDIA6) Attenuates Platelet Endoplasmic Reticulum Stress and Secretion in a Mouse Model

Background:Platelet hyperreactivity involves increased secretion of their granule content which promotes platelet aggregation and thrombosis. Platelet hyperreactivity is observed in conditions such as diabetes mellitus and is associated with decreased cardioprotective effect from antiplatelet agents in this patient group. Diabetes is associated with increased endoplasmic reticulum (ER) stress from hyperglycemia and hyperlipidemia. Protein disulphide isomerase 6 (PDIA6) is an endoplasmic reticulum protein which folds nascent proteins by reduction and oxidation of their disulphide bonds. PDIA6 has been shown to inhibit downstream ER stress pathways by inhibiting the phosphorylation of IRE-1 in fibroblasts (Eletto, Mol Cell, 2014). We hypothesized that ER stress pathways are functional in platelets and that PDIA6 may inhibit ER stress pathways leading to platelet secretion.Methods:We generated conditional PDIA6 knockout mice (PF4Cre+ Pdia6 fl/fl) (CKO) in the megakaryocyte/platelet lineage by CRISPR-Cas9 technology (Fig.1A). Megakaryopoiesis and haemostasis was assessed by bone marrow histology, coagulation assays, platelet aggregation and tail bleeding studies. We induced ER stress of purified platelets by incubation with thapsigargin and tunicamycin. Activation of the PERK and IRE1 pathways was measured by Western blot. Thrombosis was assessed in vitro by microfluidic devices and in vivo by electrolytic injury of the carotid artery.Results:PDIA6 CKO mice displayed a mild macrothrombocytopenia: the mean (+/-SD) platelet count in Pf4Cre+/Pdia6fl/fl was 775 +/- 98 x10 3/ul compared with 874 +/- 55 x10 3/ul in Pdia6fl/fl (p<0.005). The median platelet volume was 6.3 fL in Pf4Cre+/Pdia6fl/fl compared with 5.7 fL in Pdia6fl/fl (p<0.005). Megakaryopoiesis was normal at baseline. However, PDIA6 CKO mice showed significant upregulation of intracellular platelet PDIs including PDIA1, PDIA3 and PDIA4. PDIA6 deficient platelets displayed significant increase of disulphide reductase activity and the generation of free thiols on the platelet surface. Activation of the PERK and IRE-1 pathway at baseline and after induction of ER stress was increased in PDIA6 deficient platelets (Figure 1B, C). There was striking hypersecretion of PDIA1 (Figure 1D) and α-granule proteins (Figure 1E, F) in response to shear and stimulation with thrombin. PDIA6 CKO mice displayed a prothrombotic phenotype with increased platelet adhesion to fibrinogen under shear (500 s-1) and decreased time to carotid artery occlusion (mean+/SD: 10.8 +/-3.2 min in Pf4Cre+/Pdia6fl/fl compared with 15.3 +/-5.2 min in Pdia6fl/fl, n=8-10, p<0.05).Conclusion:We have identified a role for platelet PDIA6 in attenuating platelet ER stress and secretion. This opens avenues for further study into the role of platelet PDIs in conditions with increased ER stress, such as obesity and diabetes.Figure 1: PDIA6 deficient platelets have increased endoplasmic reticulum (ER) stress and are hypersecretory. A. Western blot of PDIA6 protein in platelets from Pf4Cre+/Pdia6fl/fl mice and control mice (Pdia6fl/fl) showing efficient deletion of PDIA6 in platelets. B. PDIA6 deficient platelets have increased phosphorylation of pEIF2a (PERK phosphorylation pathway) at baseline and after induction of ER stress by thapsigargin, representative image. C. Normalized band intensity (peIF2a/beta actin) in platelets treated with DMSO control or thapsigargin. D. Increased secretion of thiol isomerase PDIA1. E. alpha granule proteins: platelet factor 4 (PF4) and F. von Willebrand factor (vWF) from PDIA6 deficient platelets compared with controls after stimulation with thrombin 0.5 U/ml. n=3-5 Pf4Cre+/Pdia6fl/fl (red boxes) and n=3-5 Pdia6fl/fl mice (grey boxes). Columns are presented as mean+/-SD, *p<0.05, ** p<0.001 by Mann Whitney. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chloroplast Lipids Metabolism and Function. A Redox Perspective.

Plant productivity is determined by the conversion of solar energy into biomass through oxygenic photosynthesis, a process performed by protein-cofactor complexes including photosystems (PS) II and I, and ATP synthase. These complexes are embedded in chloroplast thylakoid membrane lipids, which thus function as structural support of the photosynthetic machinery and provide the lipid matrix to avoid free ion diffusion. The lipid and fatty acid composition of thylakoid membranes are unique in chloroplasts and cyanobacteria, which implies that these molecules are specifically required in oxygenic photosynthesis. Indeed, there is extensive evidence supporting a relevant function of glycerolipids in chloroplast biogenesis and photosynthetic efficiency in response to environmental stimuli, such as light and temperature. The rapid acclimation of higher plants to environmental changes is largely based on thiol-based redox regulation and the disulphide reductase activity thioredoxins (Trxs), which are reduced by ferredoxin (Fdx) via an Fdx-dependent Trx reductase. In addition, chloroplasts harbour an NADPH-dependent Trx reductase C, which allows the use of NADPH to maintain the redox homeostasis of the organelle. Here, we summarise the current knowledge of chloroplast lipid metabolism and the function of these molecules as structural basis of the complex membrane network of the organelle. Furthermore, we discuss evidence supporting the relevant role of lipids in chloroplast biogenesis and photosynthetic performance in response to environmental cues in which the redox state of the organelle plays a relevant role.

A novel soybean protein disulphide isomerase family protein possesses dithiol oxidation activity: identification and characterization of GmPDIL6.

Secretory and membrane proteins synthesized in the endoplasmic reticulum (ER) are folded with intramolecular disulphide bonds, viz. oxidative folding, catalysed by the protein disulphide isomerase (PDI) family proteins. Here, we identified a novel soybean PDI family protein, GmPDIL6. GmPDIL6 has a single thioredoxin-domain with a putative N-terminal signal peptide and an active centre (CKHC). Recombinant GmPDIL6 forms various oligomers binding iron. Oligomers with or without iron binding and monomers exhibited a dithiol oxidase activity level comparable to those of other soybean PDI family proteins. However, they displayed no disulphide reductase and extremely low oxidative refolding activity. Interestingly, GmPDIL6 was mainly expressed in the cotyledon during synthesis of seed storage proteins and GmPDIL6 mRNA was up-regulated under ER stress. GmPDIL6 may play a role in the formation of disulphide bonds in nascent proteins for oxidative folding in the ER.

Functional Assays of Thiol Isomerase ERp5.

Endoplasmic reticulum protein 5 (ERp5) is a member of the thiol isomerase family of enzymes, whose prototype member is protein disulphide isomerase (PDI). Thiol isomerases catalyze reduction/oxidation (redox) reactions which lead to the cleavage, formation, or isomerization of disulphide bonds in protein substrates. Thiol isomerase reactions on protein disulphides are important for the correct folding of proteins in the endoplasmic reticulum and for the regulation of various protein functions in the extracellular space. Apart from the disulphide reactions, thiol isomerases assist protein folding by chaperone activity.The disulphide redox activity of ERp5 can be measured with functional assays involving artificial or natural substrates containing disulphide bonds. Herein we describe step-by-step assays of ERp5 reductase, isomerization, and de-nitrosylation activity. Disulphide reductase assays include insulin or di-eosin-GSSG as substrates whereas the isomerization assay includes RNase as substrate. The reduction of natural substrates, i.e., integrin αIIbβ3, can be detected using maleimide labels of free thiols and Western blotting. The biotin switch assay is used to measure the de-nitrosylation of S-nitrosylated substrates. These assays can measure the activity of purified ERp5 protein but can also be applied for the measurement of thiol isomerase activity in cellular samples.

Inhibition of mycothione disulphide reductase and mycobacterial biofilm by selected South African plants

The causative agent of tuberculosis is the bacterium Mycobacterium tuberculosis. It is the second leading cause of death worldwide. The development of resistance associated with this bacterium is of great concern, and the ability of the bacteria to form protective biofilms and persist for longer periods of time needs to be considered as a possible target. Plants have been considered as an additive to current treatments as an adjuvant, to aid the body in fighting the disease and as a source for new drug candidates to aid in the development of resistance of mycobacteria to available drugs. Twenty plants were selected based on their traditional usage against tuberculosis and related symptoms. The antimycobacterial activity of the ethanolic extracts from different genera and variable plant parts were evaluated using the microplate Alamar Blue assay. A biofilm formation inhibition assay was adapted and used to determine the inhibitory activity against Mycobacterium smegmatis biofilm formation. The cytotoxic effect of the plants was determined on U937 human macrophage cells using the XTT (2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) cell proliferation kit. ADTNB-coupled Glutathione/Mycothiol disulfide reductase assay was used to determine the inhibitory activity against the human and bacterial reductases. Of the 20 plant extracts tested, it was found that Salvia africana-lutea, Ficus sur and Sphedamnocarpus pruriens showed moderate antimycobacterial activity with minimum inhibitory concentration values of 31.25, 62.5 and 62.5 μg/ml, respectively. Sphedamnocarpus pruriens (BF SI 1.00) and S.africana-lutea (BF SI 0.33) showed potential biofilm formation inhibition at concentrations, 62.2 and 95.8 μg/ml, respectively when compared to Ciprofloxacin (BF SI 0.32), which inhibited at a concentration of 1.9 μg/ml. Most of the plant extracts showed low to moderate cellular toxicity except Withania somnifera that showed a higher toxicity with an IC50 of 6.8 μg/ml (Actinomycin D0.0009 μg/ml). F.sur showed the highest selectivity index of 3.36. During mechanistic studies, S.africana-lutea showed inhibitory activity against Mycothiol disulfide reductase (Mtr) exhibiting an inhibitory concentration (IC50) of 102.3 μg/ml and showed lower inhibitory activity against Glutathione reductase (Gtr) with an IC50 of 224.1 μg/ml. This study indicates that South African plant extracts traditionally used to treat tuberculosis-related symptoms have reasonable antimycobacterial, antibiofilm and enzyme inhibitory activity with relatively low toxicity.

Antibacterial and Anticancer Activity of Ethnomedicinal Plants Used in the Jongilanga Community, Mpumalanga

Seventy-four ethanolic extracts were prepared from traditionally used medicinal plants in the Jongilanga community in Mpumalanga South Africa. The aim was to determine the biological activity of the selected plants against cancer, mycobacteria species and acne. From the results, it was evident that Mundulea sericea was able to inhibit the proliferation of human melanoma cells (A375) with a fifty percent inhibitory concentration (IC50) ranging between 50 and 100 µg/ml as well as the ability to inhibit Mycobacterium tuberculosis, Mycobacterium smegmatis and Propionibacterium acnes with minimum inhibitory concentrations (MIC) of 125, 31.25 and 7.9 µg/ml respectively. This further led to the investigation of the antioxidant and anti-inflammatory activity as well as the influence of the extract on mycothiol disulphide reductase (Mtr) and glutathione reductase enzymes (Gtr) as potential targets against the above-mentioned diseases. M. sericea inhibited the COX-2 enzyme, responsible for inflammation, with an IC50 value of 10.70 ± 1.14 µg/ml, furthermore compounds previously isolated from M. sericea showed potential inhibition of COX-2 in molecular docking studies. Low radical scavenging capacity against the DPPH free radical with an IC50 value of 60.52 ± 2.40 µg/ml was obtained, however, M. sericea showed a higher affinity towards Mtr as compared to Gtr, which makes it an ideal plant for use as an antimycobacterial agent.

Feather-Degrading Bacteria: Their Biochemical and Genetic Characteristics

Feather-degrading bacteria are a group of micro-organisms with the ability to degrade feather. They have been identified ubiquitously both in wild birds and poultry, as well as the feather waste produced by the food industry. In wild birds, they play a major role in influencing the colour of plumage and sexual selection. However, their significance for the poultry industry seems to be even greater. This branch of the food industry produces significant amounts of feather waste, which is difficult to process using the traditional chemical and physical methods. Due to the specific biochemical structure of feather, dominated by beta-keratin strengthened by numerous disulphide bonds, bacterial cultures seem to be the most economical and environment-friendly agent in the degradation of feather waste. The ability to degrade feathers is dependent on production of keratinase and disulphide reductase. The present review characterizes in detail a wide range of keratinases isolated from feather-degrading bacteria with potential application in feather waste processing. Furthermore, molecular methods of identifying and analysing the species of feather-degrading bacteria have been presented.

Structural and mechanistic insights into Mycothiol Disulphide Reductase and the Mycoredoxin-1-alkylhydroperoxide reductase E assembly of Mycobacterium tuberculosis

Mycobacteria employ a versatile machinery of the mycothiol-dependent system, containing the proteins mycothiol disulfide reductase (Mtr), the oxido-reductase Mycoredoxin-1 (Mrx-1) and the alkyl-hydroperoxide subunit E (AhpE). The mycothiol-dependent protein ensemble regulates the balance of oxidized-reduced mycothiol, to ensure a reductive intracellular environment for optimal functioning of its proteins even upon exposure to oxidative stress. Here, we determined the first low-resolution solution structure of Mycobacterium tuberculosis Mtr (MtMtr) derived from small-angle X-ray scattering data, which provides insight into its dimeric state. The solution shape reveals the two NADPH-binding domains inside the dimeric MtMtr in different conformations. NMR-titration shows that the MtMtr-MtMrx-1 interaction is characterized by a fast exchange regime and critical residues involved in the protein-protein interaction were identified. Using NMR spectroscopy and docking studies, the epitopes of MtMrx-1 and MtAhpE interaction are described, shedding new light into the interaction interface and mechanism of action. Finally, the essential residue of MtMrx-1 identified in the interaction with MtMtr and MtAhpE form a platform for structure-guided drug design against the versatile enzyme machinery of the mycothiol-dependent system inside M. tuberculosis.

Proteomic and functional analyses reveal pleiotropic action of the anti-tumoral compound NBDHEX in Giardia duodenalis

Giardiasis, a parasitic diarrheal disease caused by Giardia duodenalis, affects one billion people worldwide. Treatment relies only on a restricted armamentarium of drugs. The disease burden and the increase in treatment failure highlight the need for novel, safe and well characterized drug options. The antitumoral compound NBDHEX is effective in vitro against Giardia trophozoites and inhibits glycerol-3-phosphate dehydrogenase. Aim of this work was to search for additional NBDHEX protein targets. The intrinsic NBDHEX fluorescence was exploited in a proteomic analysis to select and detect modified proteins in drug treated Giardia. In silico structural analysis, intracellular localization and functional assays were further performed to evaluate drug effects on the identified targets. A small subset of Giardia proteins was covalently bound to the drug at specific cysteine residues. These proteins include metabolic enzymes, e.g. thioredoxin reductase (gTrxR), as well as elongation factor 1B-γ (gEF1Bγ), and structural proteins, e.g. α-tubulin. We showed that NBDHEX in vitro binds to recombinant gEF1Bγ and gTrxR, but only the last one could nitroreduce NBDHEX leading to drug modification of gTrxR catalytic cysteines, with concomitant disulphide reductase activity inhibition and NADPH oxidase activity upsurge. Our results indicate that NBDHEX reacts with multiple targets whose roles and/or functions are specifically hampered. In addition, NBDHEX is in turn converted to reactive intermediates extending its toxicity. The described NBDHEX pleiotropic action accounts for its antigiardial activity and encourages the use of this drug as a promising alternative for the future treatment of giardiasis.

Evaluation of Giardia lamblia thioredoxin reductase as drug activating enzyme and as drug target

The antioxidative enzyme thioredoxin reductase (TrxR) has been suggested to be a drug target in several pathogens, including the protist parasite Giardia lamblia. TrxR is also believed to catalyse the reduction of nitro drugs, e.g. metronidazole and furazolidone, a reaction required to render these compounds toxic to G. lamblia and other microaerophiles/anaerobes. It was the objective of this study to assess the potential of TrxR as a drug target in G. lamblia and to find direct evidence for the role of this enzyme in the activation of metronidazole and other nitro drugs.TrxR was overexpressed approximately 10-fold in G. lamblia WB C6 cells by placing the trxR gene behind the arginine deiminase (ADI) promoter on a plasmid. Likewise, a mutant TrxR with a defective disulphide reductase catalytic site was strongly expressed in another G. lamblia WB C6 cell line. Susceptibilities to five antigiardial drugs, i.e. metronidazole, furazolidone, nitazoxanide, albendazole and auranofin were determined in both transfectant cell lines and compared to wildtype. Further, the impact of all five drugs on TrxR activity in vivo was measured.Overexpression of TrxR rendered G. lamblia WB C6 more susceptible to metronidazole and furazolidone but not to nitazoxanide, albendazole, and auranofin. Of all five drugs tested, only auranofin had an appreciably negative effect on TrxR activity in vivo, albeit to a much smaller extent than expected. Overexpression of TrxR and mutant TrxR had hardly any impact on growth of G. lamblia WB C6, although the enzyme also exerts a strong NADPH oxidase activity which is a source of oxidative stress.Our results constitute first direct evidence for the notion that TrxR is an activator of metronidazole and furazolidone but rather question that it is a relevant drug target of presently used antigiardial drugs.

Cotesia vestalis parasitization suppresses expression of a Plutella xylostella thioredoxin.

Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins involved in protecting organisms against toxic reactive oxygen species. In this study, a typical thioredoxin gene, PxTrx, was isolated from Plutella xylostella. The full-length cDNA sequence is composed of 959 bp containing a 321 bp open reading frame that encodes a predicted protein of 106 amino acids, a predicted molecular weight of 11.7 kDa and an isoelectric point of 5.03. PxTrx was mainly expressed in larval Malpighian tubules and the fat body. An enriched recombinant PxTrx had insulin disulphide reductase activity and stimulated Human Embryonic Kidney 293 (HEK293) cell proliferation. It also protected supercoiled DNA and living HEK293 cells from H2 O2 -induced damage. Parasitization by Cotesia vestalis and injections of 0.05 and 0.01 equivalents of C. vestalis Bracovirus (CvBv), the symbiotic virus carried by the parasitoid, led to down-regulation of PxTrx expression in host fat body. Taken together, our results indicate that PxTrx contributes to the maintenance of P. xylostella cellular haemostasis. Host fat body expression of PxTrx is strongly attenuated by parasitization and by injections of CvBv.

Increased thioredoxin levels are related to insulin resistance in familial combined hyperlipidaemia.

Thioredoxins (TRX) are major cellular protein disulphide reductases that are critical for redox regulation. Oxidative stress and inflammation play promoting roles in the genesis and progression of atherosclerosis, but until now scarce data are available considering the influence of TRX activity in familial combined hyperlipidaemia (FCH). Since FCH is associated with high risk of cardiovascular disease, the objective of the present study was to assess oxidative stress status in FCH patients, and evaluate the influence of insulin resistance (IR). A cohort of 35 control subjects and 35 non-related FCH patients were included, all of them nondiabetic, normotensive and nonsmokers. We measured lipid profile, glucose and insulin levels in plasma, and markers of oxidative stress and inflammation such as oxidized glutathione (GSSG), reduced glutathione (GSH) and TRX. Familial combined hyperlipidaemia subjects showed significantly higher levels of GSSG, GSSG/GSH ratio and TRX than controls. In addition, FCH individuals with IR showed the worst profile of oxidative stress status compared to controls and FCH patients without IR (P < 0·01). TRX levels correlated with higher insulin resistance. Familial combined hyperlipidaemia patients showed increased TRX levels. TRX was positively correlated with IR. These data could partially explain the increased risk of cardiovascular events in primary dyslipidemic patients.

Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens

The application of forward genetic screens to cultured human cells represents a powerful method to study gene function. The repurposing of the bacterial CRISPR/Cas9 system provides an effective method to disrupt gene function in mammalian cells, and has been applied to genome-wide screens. Here, we compare the efficacy of genome-wide CRISPR/Cas9-mediated forward genetic screens versus gene-trap mutagenesis screens in haploid human cells, which represent the existing ‘gold standard' method. This head-to-head comparison aimed to identify genes required for the endoplasmic reticulum-associated degradation (ERAD) of MHC class I molecules. The two approaches show high concordance (>70%), successfully identifying the majority of the known components of the canonical glycoprotein ERAD pathway. Both screens also identify a role for the uncharacterized gene TXNDC11, which we show encodes an EDEM2/3-associated disulphide reductase. Genome-wide CRISPR/Cas9-mediated screens together with haploid genetic screens provide a powerful addition to the forward genetic toolbox.

Trichinella spiralis: genome database searches for the presence and immunolocalization of protein disulphide isomerase family members.

The formation of nurse cells in host muscle cells during Trichinella spiralis infection is a key step in the infective mechanism. Collagen trimerization is set up via disulphide bond formation, catalysed by protein disulphide isomerase (PDI). In T. spiralis, some PDI family members have been identified but no localization is described and no antibodies specific for T. spiralis PDIs are available. In this work, computational approaches were used to search for non-described PDIs in the T. spiralis genome database and to check the cross-reactivity of commercial anti-human antibodies with T. spiralis orthologues. In addition to a previously described PDI (PDIA2), endoplasmic reticulum protein (ERp57/PDIA3), ERp72/PDIA4, and the molecular chaperones calreticulin (CRT), calnexin (CNX) and immunoglobulin-binding protein/glucose-regulated protein (BIP/GRP78), we identified orthologues of the human thioredoxin-related-transmembrane proteins (TMX1, TMX2 and TMX3) in the genome protein database, as well as ERp44 (PDIA10) and endoplasmic reticulum disulphide reductase (ERdj5/PDIA19). Immunocytochemical staining of paraffin sections of muscle infected by T. spiralis enabled us to localize some orthologues of the human PDIs (PDIA3 and TMX1) and the chaperone GRP78. A theoretical three-dimensional model for T. spiralis PDIA3 was constructed. The localization and characteristics of the predicted linear B-cell epitopes and amino acid sequence of the immunogens used for commercial production of anti-human PDIA3 antibodies validated the use of these antibodies for the immunolocalization of T. spiralis PDIA3 orthologues. These results suggest that further study of the role of the PDIs and chaperones during nurse cell formation is desirable.

Hydrolyzing proficiency of keratinases in feather degradation.

The keratinase degrade highly rigid, cross linked structural polypeptides with different efficiency depending on the type of source. Two newly isolated strains of Bacillus subtilis (RSE163 and RSE165; NCBI Accession no JQ887983 and JQ887982) were found to be efficient keratinase producers with unusual catalytic activity result in different morphological changes in degradation pattern of feather, confirmed by their scanned electron micrographs. Maximum keratinolytic activity of both the strains B. subtilis RSE163 and RSE165 were found to be 366±15.79 and 194±7.26U after 72h of incubation. While the disulphide reductase activity of RSE163 and RSE165 estimated 0.24±0.05 and 0.15±0.03U/ml of enzyme after 24h of incubation. A total of 16 free amino acids of variable concentration were also analyzed in the cell free supernatant of hydrolyzed feather from two strains. Present study demonstrates the action of two different keratinases in feather degradation.

Bacterial mechanisms of reversible protein S‐thiolation: structural and mechanistic insights into mycoredoxins

Mycobacteria produce millimolar concentrations of mycothiol (MSH) as their major low molecular weight thiol redox buffer. MSH-deficient mutants display increased sensitivity towards reactive oxygen, nitrogen and electrophilic species as well as alkylating agents and antibiotics. MSH is maintained in its reduced thiol state by the NADPH-dependent mycothiol disulphide reductase (Mtr). However, the redoxin that uses the MSH/Mtr/NADPH pathway for reduction of MSH-mixed protein disulphides, formed during oxidative stress, has long remained unknown. In this issue, Van Laer et al. report that MSH provides the reducing power for mycoredoxin-1 (Mrx1) in reduction of synthetic MSH-mixed disulphides. The reduced (dithiol) and oxidized (disulphide) solution structures of Mrx1 have been solved by nuclear magnetic resonance (NMR) spectroscopy. NMR time course experiments have also demonstrated the transient S-mycothiolation of the active site Cys14 of oxidized Mrx1 during reduction by the MSH/Mtr/NADPH electron pathway. The paper opens a new era of research to identify S-mycothiolated Mrx1 substrates and the function of MSH in redox regulation and virulence in Mycobacterium tuberculosis.

The flavoprotein Cyc2p, a mitochondrial cytochrome c assembly factor, is a NAD(P)H‐dependent haem reductase

Cytochrome c assembly requires sulphydryls at the CXXCH haem binding site on the apoprotein and also chemical reduction of the haem co-factor. In yeast mitochondria, the cytochrome haem lyases (CCHL, CC(1) HL) and Cyc2p catalyse covalent haem attachment to apocytochromes c and c(1) . An in vivo indication that Cyc2p controls a reductive step in the haem attachment reaction is the finding that the requirement for its function can be bypassed by exogenous reductants. Although redox titrations of Cyc2p flavin (E(m) = -290 mV) indicate that reduction of a disulphide at the CXXCH site of apocytochrome c (E(m) = -265 mV) is a thermodynamically favourable reaction, Cyc2p does not act as an apocytochrome c or c(1) CXXCH disulphide reductase in vitro. In contrast, Cyc2p is able to catalyse the NAD(P)H-dependent reduction of hemin, an indication that the protein's role may be to control the redox state of the iron in the haem attachment reaction to apocytochromes c. Using two-hybrid analysis, we show that Cyc2p interacts with CCHL and also with apocytochromes c and c(1) . We postulate that Cyc2p, possibly in a complex with CCHL, reduces the haem iron prior to haem attachment to the apoforms of cytochrome c and c(1) .

Regulation and properties of glucose-6-phosphate dehydrogenase: A review

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway that catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconolactone in presence of NADP+. G6PD is an enzyme of vital importance because of its role in various haemolytic disorders and its potential as a regulator for various biosynthetic pathways. NADPH is the important product of the reaction and is used for the reductive biosynthesis of fatty acids, isoprenoids, aromatic amino acids, etc. NADPH produced also plays important function in the protection of the cell against oxidative agents by transferring its reductive power to glutathione disulphide via glutathione disulphide reductase. The present review deals with the importance, occurrence, structure, physico-chemical properties, genetics, and regulation of G6PD. Key words: Glucose-6-phosphate dehydrogenase, NADPH, pentose phosphate pathway, oxidative stress.

The coenzyme A disulphide reductase of Borrelia burgdorferi is important for rapid growth throughout the enzootic cycle and essential for infection of the mammalian host

In a microarray analysis of the RpoS regulon in mammalian host-adapted Borrelia burgdorferi, bb0728 (cdr) was found to be dually transcribed by the sigma factors σ(70) and RpoS. The cdr gene encodes a coenzyme A disulphide reductase (CoADR) that reduces CoA-disulphides to CoA in an NADH-dependent manner. Based on the abundance of CoA in B. burgdorferi and the biochemistry of the enzyme, CoADR has been proposed to play a role in the spirochaete's response to reactive oxygen species. To better understand the physiologic function(s) of BbCoADR, we generated a B. burgdorferi mutant in which the cdr gene was disrupted. RT-PCR and 5'-RACE analysis revealed that cdr and bb0729 are co-transcribed from a single transcriptional start site upstream of the bb0729 coding sequence; a shuttle vector containing the bb0729-cdr operon and upstream promoter element was used to complement the cdr mutant. Although the mutant was no more sensitive to hydrogen peroxide than its parent, it did exhibit increased sensitivity to high concentrations of t-butyl-hydroperoxide, an oxidizing compound that damages spirochetal membranes. Characterization of the mutant during standard (15% oxygen, 6% CO(2)) and anaerobic (< 1% O(2) , 9-13% CO(2)) cultivation at 37°C revealed a growth defect under both conditions that was particularly striking during anaerobiosis. The mutant was avirulent by needle inoculation and showed decreased survival in feeding nymphs, but displayed no survival defect in unfed flat nymphs. Based on these results, we propose that BbCoADR is necessary to maintain optimal redox ratios for CoA/CoA-disulphide and NAD(+) /NADH during periods of rapid replication throughout the enzootic cycle, to support thiol-disulphide homeostasis, and to indirectly protect the spirochaete against peroxide-mediated membrane damage; one or more of these functions are essential for infection of the mammalian host by B. burgdorferi.

Chaperone-like properties of tobacco plastid thioredoxins f and m

Thioredoxins (Trxs) are ubiquitous disulphide reductases that play important roles in the redox regulation of many cellular processes. However, some redox-independent functions, such as chaperone activity, have also been attributed to Trxs in recent years. The focus of our study is on the putative chaperone function of the well-described plastid Trxs f and m. To that end, the cDNA of both Trxs, designated as NtTrxf and NtTrxm, was isolated from Nicotiana tabacum plants. It was found that bacterially expressed tobacco Trx f and Trx m, in addition to their disulphide reductase activity, possessed chaperone-like properties. In vitro, Trx f and Trx m could both facilitate the reactivation of the cysteine-free form of chemically denatured glucose-6 phosphate dehydrogenase (foldase chaperone activity) and prevent heat-induced malate dehydrogenase aggregation (holdase chaperone activity). Our results led us to infer that the disulphide reductase and foldase chaperone functions prevail when the proteins occur as monomers and the well-conserved non-active cysteine present in Trx f is critical for both functions. By contrast, the holdase chaperone activity of both Trxs depended on their oligomeric status: the proteins were functional only when they were associated with high molecular mass protein complexes. Because the oligomeric status of both Trxs was induced by salt and temperature, our data suggest that plastid Trxs could operate as molecular holdase chaperones upon oxidative stress, acting as a type of small stress protein.