<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignment of the cytosolic dithiol glutaredoxin 1 from the pathogen <Emphasis Type="Italic">Trypanosoma brucei</Emphasis>

Trypanosomatids are parasites responsible for several tropical and subtropical diseases, such as Chaga’s disease, sleeping sickness and Leishmaniasis. In contrast to the mammalian host, the thiol-redox metabolism of these pathogens depends on trypanothione [bis-glutathionylspermidine, T(SH)₂] instead of glutathione (GSH) providing a set of lineage-specific proteins as drug target candidates. Glutaredoxins (Grx) are ubiquitous small thiol–disulfide oxidoreductases that belong to the thioredoxin-fold family. They play a central role in redox homeostasis and iron sulfur-cluster biogenesis. Each species, including trypanosomes, possesses its own set of isoforms distributed in different subcellular compartments. The genome of trypanosomatids encodes for two class I (dithiolic) Grxs named 2-C-Grx1 and 2-C-Grx2. Both proteins were shown to efficiently reduce different disulfides at the expenses of T(SH)₂ using a mechanism that involves the two cysteines in the active site. Moreover, the cytosolic Trypanosoma brucei 2-C-Grx1 but not the mitochondrial 2-C-Grx2 was able to coordinate an iron–sulfur cluster with T(SH)₂ or GSH as ligand. As a first step to unravel the structural basis for the specificity observed in the trypanosomal glutaredoxins, we present here the NMR resonance assignment of 2-C-Grx1 from the parasite T. brucei brucei.     

Gene knockdown of gamma-glutamylcysteine synthetase by RNAi in the parasitic protozoa Trypanosoma brucei demonstrates that it is an essential enzyme

The parasitic protozoa Trypanosoma brucei utilizes a novel cofactor (trypanothione, T(SH)2), which is a conjugate of GSH and spermidine, to maintain cellular redox balance. gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the first step in the biosynthesis of GSH. To evaluate the importance of thiol metabolism to the parasite, RNAi methods were used to knock down gene expression of gamma-GCS in procyclic T. brucei cells. Induction of gamma-GCS RNAi with tetracycline led to cell death within 4-6 days post-induction. Cell death was preceded by the depletion of the gamma-GCS protein and RNA and by the loss of the cellular pools of GSH and T(SH)2. The addition of GSH (80 microM) to cell cultures rescued the RNAi cell death phenotype and restored the intracellular thiol pools to wild-type levels. Treatment of cells with buthionine sulfoximine (BSO), an enzyme-activated inhibitor of gamma-GCS, also resulted in cell death. However, the toxicity of the inhibitor was not reversed by GSH, suggesting that BSO has more than one cellular target. BSO depletes intracellular thiols to a similar extent as gamma-GCS RNAi; however, addition of GSH did not restore the pools of GSH and T(SH)2. These data suggest that BSO also acts to inhibit the transport of GSH or its peptide metabolites into the cell. The ability of BSO to inhibit both synthesis and transport of GSH likely makes it a more effective cytotoxic agent than an inhibitor with a single mode of action. Finally the potential for the T(SH)2 biosynthetic enzymes to be regulated in response to reduced thiol levels was studied. The expression levels of ornithine decarboxylase and of S-adenosylmethionine decarboxylase, two essential enzymes in spermidine biosynthesis, remained constant in induced gamma-GCS RNAi cell lines.     

Validation of Trypanosoma brucei trypanothione synthetase as drug target

In trypanosomes, the parasite-specific thiol trypanothione [T(SH)2] fulfills various functions, the best established being detoxification of H2O2 and organic hydroperoxides and ribonucleotide reduction. Recently, a trypanothione synthetase (Tb-TryS) gene from Trypanosoma brucei was isolated and the heterologously expressed Tb-TryS catalyzed the entire synthesis of T(SH)2 from glutathione (GSH) and spermidine in vitro. To confirm the in situ function of the complex Tb-TryS activities and to evaluate the importance of T(SH)2 metabolism in T. brucei, TryS suppression by double-stranded RNA interference was performed. Knockdown of TryS led to depletion of both T(SH)2 and glutathionylspermidine (Gsp) and accumulation of GSH, while concomitantly impairment of viability and arrest of proliferation were observed. TryS-downregulated cells displayed a significantly increased sensitivity to H2O2 and tert.-butyl hydroperoxide. These data verify the hypothesis that in T. brucei, a single enzyme synthesizes the spermidine-conjugated thiols (Gsp and T(SH)2) and further confirms the significance of trypanothione in the defense against oxidative stress and the maintenance of viability and proliferation in unstressed parasites.     

Leishmania infantum: provision of reducing equivalents to the mitochondrial tryparedoxin/tryparedoxin peroxidase system

Within the mitochondrion of Leishmania infantum, hydroperoxide metabolism relies on the activity of tryparedoxin-dependent peroxidases (TXNPxs). Tryparedoxins (TXNs) are thioredoxin-related oxidoreductases, which in vitro are reduced by the trypanothione reductase/trypanothione [TR/T(SH)₂] redox couple. Still, there is no evidence that this actually occurs in the mitochondrion. This communication addresses the question of how the mitochondrial TXN/TXNPx system is reduced. First, using a digitonin fractionation assay, we show that TR activity is absent from the L. infantum mitochondrion. The possibility that this organelle possesses alternative electron sources for TXN/TXNPx is then investigated. Biochemical assays performed with purified recombinant enzymes, revealed that TR and T(SH)₂ can be replaced, albeit less efficiently, by the dihydrolipoamide dehydrogenase/lipoamide redox system as TXN/TXNPx electron donor. This result challenges the classical view that T(SH)₂ is the only reductant for TXNs and add new prospects regarding the involvement of 2-oxo acid dehydrogenase complexes in L. infantum mitochondrial hydroperoxide metabolism.     

Thiol-induced reduction of antimony(V) into antimony(III): A comparative study with trypanothione, cysteinyl-glycine, cysteine and glutathione

Gluthathione (GSH) has been previously shown to promote the reduction of pentavalent antimony (Sb(V)) into the more toxic trivalent antimony (Sb(III)) in the antimonial drug, meglumine antimonate. However, this reaction occurred at acidic pH (pH 5) but not at the pH of the cytosol (pH 7.2) in which GSH is encountered. The aim of the present study was to further characterize the reaction between thiols and antimonial drugs, addressing the following aspects: (i) the reducing activity of cysteine (Cys) and cysteinyl-glycine (Cys-Gly), expected to be the predominant thiols in the acidic compartiments of mammalian cells; (ii) the reducing activity of trypanothione (T(SH)₂), the main intracelular thiol in Leishmania parasites; (iii) the influence of the state of complexation of Sb(V) on the rate of Sb(V) reduction. We report here that Cys, Cys-Gly and T(SH)₂ did promote the reduction of Sb(V) into Sb(III) at 37 °C. Strikingly, the initial rates of reduction of Sb(V) were much greater in the presence of Cys-Gly, Cys and T(SH)₂ than in the presence of GSH. These reactions occurred at both pH 5 and pH 7 but were favored at acidic pH. Moreover, our data shows that Sb(V) is reduced more slowly in the form of meglumine antimonate than in its non-complexed form, indicating that the complexation of Sb(V) tends to slow down the rate of its reduction. In conclusion, our data supports the hypothesis that Sb(V) is reduced in vivo by T(SH)₂ within Leishmania parasites and by Cys or Cys-Gly within the acidic compartments of mammalian cells.     

Benznidazole Biotransformation and Multiple Targets in Trypanosoma cruzi Revealed by Metabolomics

Background

The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. In the present work we used a non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.

Methodology/Principal findings

Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols trypanothione, homotrypanothione and cysteine were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment. These metabolites included reduction products, fragments and covalent adducts of reduced Bzn linked to each of the major low molecular weight thiols: trypanothione, glutathione, γ-glutamylcysteine, glutathionylspermidine, cysteine and ovothiol A. Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI, were found within the parasites, but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.

Conclusions/significance

Our data is indicative of a major role of the thiol binding capacity of Bzn reduction products in the mechanism of Bzn toxicity against T. cruzi.     

Dissecting the Catalytic Mechanism of Trypanosoma brucei Trypanothione Synthetase by Kinetic Analysis and Computational Modeling

In pathogenic trypanosomes, trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and trypanothione (bis(glutathionyl)spermidine (T(SH)₂)). Here we present a thorough kinetic analysis of Trypanosoma brucei TryS in a newly developed phosphate buffer system at pH 7.0 and 37 °C, mimicking the physiological environment of the enzyme in the cytosol of bloodstream parasites. Under these conditions, TryS displays K_m values for GSH, ATP, spermidine, and Gsp of 34, 18, 687, and 32 μm, respectively, as well as K_i values for GSH and T(SH)₂ of 1 mm and 360 μm, respectively. As Gsp hydrolysis has a K_m value of 5.6 mm, the in vivo amidase activity is probably negligible. To obtain deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained for different substrate/product combinations under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This system''s biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps, and (iii) T(SH)₂ inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex. The newly detected concerted substrate and product inhibition suggests that TryS activity is tightly regulated.     

Polyamine metabolism in <Emphasis Type="Italic">Leishmania</Emphasis>: from arginine to trypanothione

Polyamines (PAs) are essential metabolites in eukaryotes, participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. The PAs are synthesized by a metabolic process which involves arginase (ARG), which catalyzes the enzymatic hydrolysis of l-arginine (l-Arg) to l-ornithine and urea, and ornithine decarboxylase (ODC), which catalyzes the enzymatic decarboxylation of l-ornithine in putrescine. The S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the irreversible decarboxylation of S-adenosylmethionine (AdoMet), generating the decarboxylated S-adenosylmethionine (dAdoMet), which is a substrate, together with putrescine, for spermidine synthase (SpdS). Leishmania parasites and all the other members of the trypanosomatid family depend on spermidine for growth and survival. They can synthesize PAs and polyamine precursors, and also scavenge them from the microenvironment, using specific transporters. In addition, Trypanosomatids have a unique thiol-based metabolism, in which trypanothione (N1-N8-bis(glutathionyl)spermidine, T(SH)₂) and trypanothione reductase (TR) replace many of the antioxidant and metabolic functions of the glutathione/glutathione reductase (GR) and thioredoxin/thioredoxin reductase (TrxR) systems present in the host. Trypanothione synthetase (TryS) and TR are necessary for the protozoa survival. Consequently, enzymes involved in spermidine synthesis and its utilization, i.e. ARG, ODC, AdoMetDC, SpdS and, in particular, TryS and TR, are promising targets for drug development.     

Changes in low-molecular-weight thiol-disulphide redox couples are part of bread wheat seed germination and early seedling growth

The tripeptide antioxidant glutathione (γ-l-glutamyl-l-cysteinyl-glycine; GSH) essentially contributes to thiol-disulphide conversions, which are involved in the control of seed development, germination, and seedling establishment. However, the relative contribution of GSH metabolism in different seed structures is not fully understood. We studied the GSH/glutathione disulphide (GSSG) redox couple and associated low-molecular-weight (LMW) thiols and disulphides related to GSH metabolism in bread wheat (Triticum aestivum L.) seeds, focussing on redox changes in the embryo and endosperm during germination. In dry seeds, GSH was the predominant LMW thiol and, 15?h after the onset of imbibition, embryos of non-germinated seeds contained 12 times more LMW thiols than the endosperm. In germinated seeds, the embryo contained 17 and 11 times more LMW thiols than the endosperm after 15 and 48?h, respectively. This resulted in the embryo having significantly more reducing half-cell reduction potentials of GSH/GSSG and cysteine (Cys)/cystine (CySS) redox couples (E_GSSG/2GSH and E_CySS/2Cys, respectively). Upon seed germination and early seedling growth, Cys and CySS concentrations significantly increased in both embryo and endosperm, progressively contributing to the cellular LMW thiol-disulphide redox environment (E_{thiol-disulphide}). The changes in E_CySS/2Cys could be related to the mobilisation of storage proteins in the endosperm during early seedling growth. We suggest that E_GSSG/2GSH and E_CySS/2Cys can be used as markers of the physiological and developmental stage of embryo and endosperm. We also present a model of interaction between LMW thiols and disulphides with hydrogen peroxide (H₂O₂) in redox regulation of bread wheat germination and early seedling growth.     

Thiols and polyamines in the cytoprotective effect of taurine on carbon tetrachloride‐induced hepatotoxicity

The mechanism by which taurine (2‐aminoethanesulfonic acid) protects hepatocytes injury induced by carbon tetrachloride (CCl₄) is not fully understood. In a previous study, we reported that cellular polyamines play an important role in this mechanism. The relationship between cellular glutathione (GSH), protein‐SH levels, and lactate dehydrogenase (LDH), with respect to the effect of polyamine on the cytoprotective ability of taurine in CCl₄‐induced toxicity in isolated rat hepatocytes, was examined. CCl₄ induced a LDH release and decreased cellular thiols and polyamine levels. Treating with taurine reversed these depletions. The effect of CCl₄ was also reversed by the addition of exogenous polyamines. Pretreating with α‐difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, which is a key enzyme in polyamine biosynthesis and therefore used to deplete cellular polyamine, prevented the protective effect of taurine. Adding diethyl maleate, a cellular glutathione‐depleting agent, reduced the effect of exogenous polyamines. The role of polyamine in the cytoprotective effect of taurine in CCl₄‐induced toxicity may therefore be by preventing, among others, GSH and protein‐SH depletions. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 71–76, 1999     

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin

Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H₂O₂ and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m⁻¹ s⁻¹) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.     

Multiparametric protocol for the determination of thiol redox state in living matter

Thiol redox state (TRS) evaluation is mostly restricted to the estimation of GSH and GSSG. However, these TRS parameters can estimate the GSSG/GSH potential, which might be useful for indicating abnormalities in redox metabolism. Nonetheless, evaluation of the multiparameric nature of TRS is required for a more accurate assessment of its physiological role. The present protocol extends the partial assessment of TRS by current methodologies. It measures 15 key parameters of TRS by two modular subprotocols: one for the glutathione (GSH)- and cysteine (CSH)-based nonprotein (NP) thiols/mixed disulfides (i.e., GSH, GSSG, GSSNP, CSH, CSSNP, NPSH, NPSSNP, NP_xSH_NPSSNP, NP_xSH_NPSH), and the other for their protein (P) thiols/mixed disulfides (i.e., PSH, PSSG, PSSC, PSSNP, PSSP, NP_xSH_PSSNP). The protocol eliminates autoxidation of GSH and CSH (and thus overestimation of GSSG and CSSNP). Its modularity allows the determination GSH and GSSG also by other published specific assays. The protocol uses three assays; two are based on the photometric reagents 4,4′-dithiopyridine (DTP) and ninhydrin (NHD), and the third on the fluorometric reagent o-phthaldialdehyde (OPT). The initial assays employing these reagents have been extensively modified and redesigned for increased specificity, sensitivity, and simplicity. TRS parameter values and their standard errors are estimated automatically by sets of Excel-adapted algebraic equations. Protocol sensitivity for NPSH, PSH, NPSSNP, PSSP, PSSNP, CSH, CSSNP, PSSC, NP_xSH_NPSSNP, and NP_xSH_NPSH is 1 nmol –SH/CSH, for GSSNP 0.2 nmol, for GSH and GSSG 0.4 nmol, and for PSSG 0.6 nmol. The protocol was applied on human plasma, a sample of high clinical value, and can be also applied in any organism.     

High throughput screening against the peroxidase cascade of African trypanosomes identifies antiparasitic compounds that inactivate tryparedoxin

In African trypanosomes, the detoxification of broad spectrum hydroperoxides relies on a unique cascade composed of trypanothione (T(SH)(2)), trypanothione reductase, tryparedoxin (Tpx), and nonselenium glutathione peroxidase-type enzymes. All three proteins are essential for Trypanosoma brucei. Here, we subjected the complete system to a high throughput screening approach with nearly 80,000 chemicals. Twelve compounds inhibited the peroxidase system. All but one carried chloroalkyl substituents. The detailed kinetic analysis showed that two compounds weakly inhibited trypanothione reductase, but none of them specifically interacted with the peroxidase. They proved to be time-dependent inhibitors of Tpx-modifying Cys-40, the first cysteine of its active site WCPPC motif. Importantly, gel shift assays verified Tpx as a target in the intact parasites. T(SH)(2), present in the in vitro assays and in the cells in high molar excess, did not interfere with Tpx inactivation. The compounds inhibited the proliferation of bloodstream T. brucei with EC(50) values down to <1 μM and exerted up to 83-fold lower toxicity toward HeLa cells. Irreversible inhibitors are traditionally regarded as unfavorable. However, a large number of antimicrobials and anticancer therapeutics acts covalently with their target protein. The compounds identified here also interacted with recombinant human thioredoxin, a distant relative of Tpx. This finding might even be exploited for thioredoxin-based anticancer drug development approaches reported recently. The fact that the T(SH)(2)/Tpx couple occupies a central position within the trypanosomal thiol metabolism and delivers electrons also for the synthesis of DNA precursors renders the parasite-specific oxidoreductase an attractive drug target molecule.     

In-silico Investigation of Antitrypanosomal Phytochemicals from Nigerian Medicinal Plants

Background

Human African trypanosomiasis (HAT), a parasitic protozoal disease, is caused primarily by two subspecies of Trypanosoma brucei. HAT is a re-emerging disease and currently threatens millions of people in sub-Saharan Africa. Many affected people live in remote areas with limited access to health services and, therefore, rely on traditional herbal medicines for treatment.

Methods

A molecular docking study has been carried out on phytochemical agents that have been previously isolated and characterized from Nigerian medicinal plants, either known to be used ethnopharmacologically to treat parasitic infections or known to have in-vitro antitrypanosomal activity. A total of 386 compounds from 19 species of medicinal plants were investigated using in-silico molecular docking with validated Trypanosoma brucei protein targets that were available from the Protein Data Bank (PDB): Adenosine kinase (TbAK), pteridine reductase 1 (TbPTR1), dihydrofolate reductase (TbDHFR), trypanothione reductase (TbTR), cathepsin B (TbCatB), heat shock protein 90 (TbHSP90), sterol 14α-demethylase (TbCYP51), nucleoside hydrolase (TbNH), triose phosphate isomerase (TbTIM), nucleoside 2-deoxyribosyltransferase (TbNDRT), UDP-galactose 4′ epimerase (TbUDPGE), and ornithine decarboxylase (TbODC).

Results

This study revealed that triterpenoid and steroid ligands were largely selective for sterol 14α-demethylase; anthraquinones, xanthones, and berberine alkaloids docked strongly to pteridine reductase 1 (TbPTR1); chromenes, pyrazole and pyridine alkaloids preferred docking to triose phosphate isomerase (TbTIM); and numerous indole alkaloids showed notable docking energies with UDP-galactose 4′ epimerase (TbUDPGE). Polyphenolic compounds such as flavonoid gallates or flavonoid glycosides tended to be promiscuous docking agents, giving strong docking energies with most proteins.

Conclusions

This in-silico molecular docking study has identified potential biomolecular targets of phytochemical components of antitrypanosomal plants and has determined which phytochemical classes and structural manifolds likely target trypanosomal enzymes. The results could provide the framework for synthetic modification of bioactive phytochemicals, de novo synthesis of structural motifs, and lead to further phytochemical investigations.     

Glutathione and infection

Background

The tripeptide γ-glutamylcysteinylglycine or glutathione (GSH) has demonstrated protective abilities against the detrimental effects of oxidative stress within the human body, as well as protection against infection by exogenous microbial organisms.

Scope of review

In this review we describe how GSH works to modulate the behavior of many cells including the cells of the immune system, augmenting the innate and the adaptive immunity as well as conferring protection against microbial, viral and parasitic infections. This article unveils the direct antimicrobial effects of GSH in controlling Mycobacterium tuberculosis (M. tb) infection within macrophages. In addition, we summarize the effects of GSH in enhancing the functional activity of various immune cells such as natural killer (NK) cells and T cells resulting in inhibition in the growth of M. tb inside monocytes and macrophages. Most importantly we correlate the decreased GSH levels previously observed in individuals with pulmonary tuberculosis (TB) with an increase in the levels of pro-inflammatory cytokines which aid in the growth of M. tb.

Major conclusions

In conclusion, this review provides detailed information on the protective integral effects of GSH along with its therapeutic effects as they relate to the human immune system and health.

General significance

It is important to note that the increases in the levels of pro-inflammatory cytokines are not only detrimental to the host due to the sequel that follow such as fever and cachexia, but also due to the alteration in the functions of immune cells. The additional protective effects of GSH are evident after sequel that follows the depletion of this antioxidant. This is evident in a condition such as Cystic Fibrosis (CF) where an increased oxidant burden inhibits the clearance of the affecting organism and results in oxidant-induced anti-protease inhibition. GSH has a similar protective effect in protozoans as it does in human cells. Thus GSH is integral to the survival of some of the protozoans because some protozoans utilize the compound trypanothione [T(SH)₂] as their main antioxidant. T(SH)₂ in turn requires GSH for its production. Hence a decrease in the levels of GSH (by a known inhibitor such as buthionine sulfoximine [BSO] can have adverse effects of the protozoan parasites. This article is part of a Special Issue entitled Cellular functions of glutathione.     

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

Trypanosoma brucei, the causative agent of African sleeping sickness, possesses two dithiol glutaredoxins (Grx1 and Grx2). Grx1 occurs in the cytosol and catalyzes protein deglutathionylations with k_cat/K_m-values of up to 2 × 10⁵ m⁻¹ s⁻¹. It accelerates the reduction of ribonucleotide reductase by trypanothione although less efficiently than the parasite tryparedoxin and has low insulin disulfide reductase activity. Despite its classical CPYC active site, Grx1 forms dimeric iron-sulfur complexes with GSH, glutathionylspermidine, or trypanothione as non-protein ligands. Thus, contrary to the generally accepted assumption, replacement of the Pro is not a prerequisite for cluster formation. T. brucei Grx2 shows an unusual CQFC active site, and orthologues occur exclusively in trypanosomatids. Grx2 is enriched in mitoplasts, and fractionated digitonin lysis resulted in a co-elution with cytochrome c, suggesting localization in the mitochondrial intermembrane space. Grx2 catalyzes the reduction of insulin disulfide but not of ribonucleotide reductase and exerts deglutathionylation activity 10-fold lower than that of Grx1. RNA interference against Grx2 caused a growth retardation of procyclic cells consistent with an essential role. Grx1 and Grx2 are constitutively expressed with cellular concentrations of about 2 μm and 200 nm, respectively, in both the mammalian bloodstream and insect procyclic forms. Trypanothione reduces the disulfide form of both proteins with apparent rate constants that are 3 orders of magnitude higher than those with glutathione. Grx1 and, less efficiently, also Grx2 catalyze the reduction of GSSG by trypanothione. Thus, the Grxs play exclusive roles in the trypanothione-based thiol redox metabolism of African trypanosomes.     

Preliminary studies on the involvement of glutathione metabolism and redox status against zinc toxicity in radish seedlings by 28-Homobrassinolide

The effect of exogenous application of 28-Homobrassinolide (HBR) on radish (Raphanus sativus L.) seedlings under zinc (Zn²⁺) stress on glutathione (GSH) production, consumption and changes in redox status was investigated. Zinc toxicity resulted in oxidative burst as evidenced by increased accumulation of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) content. These stress indices were significantly decreased by HBR supplementation. Under Zn²⁺ stress, GSH pool was decreased, while the contribution of oxidized glutathione (GSSG) to total GSH increased (GSSH/GSH ratio), this translated into significant reduction of GSH redox homeostasis. In addition, an increase of phytochelatins (PCs) was observed. In radish seedlings under Zn²⁺ stress, the activities of gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GS), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and cysteine (Cys) levels increased but the activity of glutathione reductase (GR) decreased. However, application of HBR increased the GSH pool and maintained their redox ratio by increasing the enzyme activities of GSH biosynthesis (γ-ECS and GS) and GSH metabolism (GR, GPX and GST). The results of present study are novel in being the first to demonstrate that exogenous application of HBR modulates the GSH synthesis, metabolism and redox homeostasis to confer resistance against Zn²⁺ induced oxidative stress.     

Synthesis and enzymology of modifiedN-benzyloxycarbonyl-L-cysteinylglycyl-3,3-dimethylaminopropylamide disulphides as alternative substrates for trypanothione reductase fromTrypanosoma crud: Part 3

Summary Kinetic data for alternative substrates of recombinant trypanothione reductase fromTrypanosoma cruzi were measured for a series ofN-substituted-L-cysteinylglycyl-3-dimethylaminopropylamides, in which the cysteineN-substituent was either a variant of the benzyloxycarbonyl group or was L-phenylalanine or L-tryptophan. Replacing the benzylic ether oxygen atom by CH₂. or NH had relatively minor effects on k_cat, but raised the value of K_m, 4.5- and 10-fold, respectively. Similarly, relative to the carbobenzoxy group, anN-L-phenylalanyl orN-L-tryptophanyl replacement on the cysteine hardly altered k_cat, but increased K_m, values by 16.6 and 7.4 fold, respectively. These observations were consistent with the K_m, values referring primarily to binding for this series of nonspecific substrates.Abbreviations DCC N,N-dicyclohexylcarbodiimide - dmapa dimethylaminopropylamine - DMF dimethylformamide - GR glutathione reductase - GSSG glutathione disulphide - GSH reduced glutathione - T[S]₂ trypanothione disulphide - Hbt hydroxybenzotriazole - TFA trifluoroacetic acid - TLC thin layer chromatography - T[SH]₂ reduced trypanothione as dithiol - TR trypanothione reductase - Z.cys.gly.dmapa N-benzyloxycarbonyl-Lcysteinylglycyl-3-dimethylpropylamide     

Effect of oxygen radicals and hyperoxia on rat heart ornithine decarboxylase activity

Rat heart ornithine decarboxylase activity from isoproterenol-treated rats was inactivated in vitro by reactive species of oxygen generated by the reaction xanthine/xanthine oxidase. Reduced glutathione, dithiothreitol and superoxide dismutase had a protective effect in homogenates and in partially purified ornithine decarboxylase exposed to the xanthine/xanthine oxidase reaction, while diethyldithiocarbamate, which is an inhibitor of superoxide dismutase, potentiated the damage induced by O₂^? on enzyme activity. Dithiothreitol at concentrations above 1.25 mM had an inhibitory effect oupon supernatant ornithine decarboxylase activity, while at 2.5 mM it was most effective in the recovery of ornithine decarboxylase activity, after the purification of the enzyme by the ammonium sulphate precipitation procedure. The ornithine decarboxylase inactivated by the xanthine/xanthine oxidase reaction showed a higher value of K_m and a reduction of V_max with respect to control activity. The exposure of rates to 100% oxygen for 3 h reduced significantly the isoproterenol-induced heart ornithine decarboxylase activity. The injection with diethyldithiocarbamate 1 h before hyperoxic exposure further reduced heart ornithine decarboxylase activity.