Entamoeba histolytica thioredoxin reductase: Molecular and functional characterization of its atypical properties

Background

Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents between NADPH and thioredoxin, a small protein that plays key metabolic functions in maintaining the intracellular redox balance.

Methods

The present work deals with in vitro steady state kinetic studies aimed to reach a better understanding of the kinetic and structural properties of thioredoxin reductase from E. histolytica (EhTRXR).

Results

Our results support that native EhTRXR is a homodimeric covalent protein that is able to catalyze the NAD(P)H-dependent reduction of amoebic thioredoxins and S‐nitrosothiols. In addition, the enzyme exhibited NAD(P)H dependent oxidase activity, which generates hydrogen peroxide from molecular oxygen. The enzyme can reduce compounds like methylene blue, quinones, ferricyanide or nitro-derivatives; all alternative substrates displaying a relative high capacity to inhibit disulfide reductase activity of EhTRXR.

Conclusions and general significance

Interestingly, EhTRXR exhibited kinetic and structural properties that differ from other low molecular weight TRXR. The TRX system could play an important role in the parasite defense against reactive species. The latter should be critical during the extra intestinal phase of the amoebic infection. So far we know, this is the first in depth characterization of EhTRXR activity and functionality.     

Serum thioredoxin reductase levels increase in response to chemically induced acute liver injury

Background

Mammalian thioredoxin reductases (TrxR) are selenoproteins with important roles in antioxidant defense and redox regulation, principally linked to functions of their main substrates thioredoxins (Trx). All major forms of TrxR are intracellular while levels in serum are typically very low.

Methods

Serum TrxR levels were determined with immunoblotting using antibodies against mouse TrxR1 and total enzyme activity measurements were performed, with serum and tissue samples from mouse models of liver injury, as triggered by either thioacetamide (TAA) or carbon tetrachloride (CCl₄).

Results

TrxR levels in serum increased upon treatment and correlated closely with those of alanine aminotransferase (ALT), an often used serum biomarker for liver damage. In contrast, Trx1, glutathione reductase, superoxide dismutase or selenium-containing glutathione peroxidase levels in serum displayed much lower increases than TrxR or ALT.

Conclusions

Serum TrxR levels are robustly elevated in mouse models of chemically induced liver injury.

General significance

The exaggerated TrxR release to serum upon liver injury may reflect more complex events than a mere passive release of hepatic enzymes to the extracellular milieu. It can also not be disregarded that enzymatically active TrxR in serum could have yet unidentified physiological functions.     

Plasmodium falciparum antioxidant protein as a model enzyme for a special class of glutaredoxin/glutathione-dependent peroxiredoxins

Background

Peroxiredoxins are important heterogeneous thiol-dependent hydroperoxidases with a variety of isoforms and enzymatic mechanisms. A special subclass of glutaredoxin/glutathione-dependent peroxiredoxins has been discovered in bacteria and eukaryotes during the last decade, but the exact enzymatic mechanisms of these enzymes remain to be unraveled.

Methods

We performed a comprehensive analysis of the enzyme kinetics and redox states of one of these glutaredoxin/glutathione-dependent peroxiredoxins, the antioxidant protein from the malaria parasite Plasmodium falciparum, using steady-state kinetic measurements, site-directed mutagenesis, redox mobility shift assays, gel filtration, and mass spectrometry.

Results

P. falciparum antioxidant protein requires not only glutaredoxin but also glutathione as a true substrate for the reduction of hydroperoxides. One peroxiredoxin cysteine residue and one glutaredoxin cysteine residue are sufficient for catalysis, however, additional cysteine residues of both proteins result in alternative redox states and conformations in vitro with implications for redox regulation. Our data furthermore point to a glutathione-dependent peroxiredoxin activation and a negative subunit cooperativity.

Conclusions

The investigated glutaredoxin/glutathione/peroxiredoxin system provides numerous new insights into the mechanism and redox regulation of peroxiredoxins.

General significance

As a member of the special subclass of glutaredoxin/glutathione-dependent peroxiredoxins, the P. falciparum antioxidant protein could become a reference protein for peroxiredoxin catalysis and regulation.     

Association of PARP-1, NF-κB,NF-κBIA and IL-6, IL-1β and TNF-α with Graves Disease and Graves Ophthalmopathy

Background

Graves Disease (GD) is an autoimmune disorder affected by an interaction of multiple genes such as Nuclear Factor-κB (NF-κB), Nuclear Factor-κB Inhibitor (NF-κBIA), Poly (ADP-ribose) polymerase-1 (PARP-1) and cytokines like Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α) and mostly accompanied by an ocular disorder, Graves Ophthalmopathy (GO). We hypothesize that there is a relationship between GD, GO, polymorphisms of inflammatory related genes and their association with cytokines, which may play important roles in autoimmune and inflammatory processes.

Subjects and methods

To confirm our hypothesis, we studied the polymorphisms and cytokine levels of 120 patients with GD and GO using PCR-RFLP and ELISA methods, respectively.

Results

We found that patients with GG genotype and carriers of G allele of PARP-1 G1672A polymorphism are at risk in the group having GD (p = 0.0007) while having GA genotype may be protective against the disease. PARP-1 C410T polymorphism was found to be associated with GO by increasing the risk by 1.7 times (p = 0.004). Another risk factor for development of GO was the polymorphism of del/ins of NFkB1 gene (p = 0.032) that increases the risk by 39%. Levels of cytokines were also elevated in patients with GD, but no association was found between levels of cytokines and the development of GO as there was no change in levels of cytokines.

Conclusions

We suggest that, PARP-1 and NFkB1 gene polymorphisms may be risk factors for developing Graves Disease and Ophthalmopathy.     

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells

Background

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.

Methods

The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.

Results

We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.

Conclusions

Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.

General significance

Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.     

S-nitrosylation of surfactant protein D as a modulator of pulmonary inflammation

Background

Surfactant protein D (SP-D) is a member of the family of proteins termed collagen-like lectins or “collectins” that play a role in non-antibody-mediated innate immune responses [1]. The primary function of SP-D is the modulation of host defense and inflammation [2].

Scope of review

This review will discuss recent findings on the physiological importance of SP-D S-nitrosylation in biological systems and potential mechanisms that govern SP-D mediated signaling.

Major conclusions

SP-D appears to have both pro- and anti-inflammatory signaling functions.SP-D multimerization is a critical feature of its function and plays an important role in efficient innate host defense. Under baseline conditions, SP-D forms a multimer in which the N-termini are hidden in the center and the C-termini are on the surface. This multimeric form of SP-D is limited in its ability to activate inflammation. However, NO can modify key cysteine residues in the hydrophobic tail domain of SP-D resulting in a dissociation of SP-D multimers into trimers, exposing the S-nitrosylated N-termini. The exposed S-nitrosylated tail domain binds to the calreticulin/CD91 receptor complex and initiates a pro-inflammatory response through phosphorylation of p38 and NF-κB activation [3,4]. In addition, the disassembled SP-D loses its ability to block TLR4, which also results in activation of NF-κB.

General significance

Recent studies have highlighted the capability of NO to modify SP-D through S-nitrosylation, causing the activation of a pro-inflammatory role for SP-D [3]. This represents a novel mechanism both for the regulation of SP-D function and NO's role in innate immunity, but also demonstrates that the S-nitrosylation can control protein function by regulating quaternary structure. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation.     

Trypanothione: A unique bis-glutathionyl derivative in trypanosomatids

Background

Trypanosomatids are early-diverging eukaryotes devoid of the major disulfide reductases – glutathione reductase and thioredoxin reductase – that control thiol-redox homeostasis in most organisms. These protozoans have evolved a unique thiol-redox system centered on trypanothione, a bis-glutathionyl conjugate of spermidine. Notably, the trypanothione system is capable to sustain several cellular functions mediated by thiol-dependent (redox) processes.

Scope of review

This review provides a summary of some historical and evolutionary aspects related to the discovery and appearance of trypanothione in trypanosomatids. It also addresses trypanothione's biosynthesis, physicochemical properties and reactivity towards biologically-relevant oxidants as well as its participation as a cofactor for metal binding. In addition, the role of the second most abundant thiol of trypanosomatids, glutathione, is revisited in light of the putative glutathione-dependent activities identified in these organisms.

Major conclusions

Based on biochemical and genome data, the occurrence of a thiol-redox system that is strictly dependent on trypanothione appears to be a feature unique to the order Kinetoplastida. The properties of trypanothione, a dithiol, are the basis for its unique reactivity towards a wide diversity of oxidized and/or electrophilic moieties in proteins and low molecular weight compounds from endogenous or exogenous sources. Novel functions have emerged for trypanothione as a potential cofactor in iron metabolism.

General significance

The minimalist thiol-redox system, developed by trypanosomatids, is an example of metabolic fitness driven by the remarkable physicochemical properties of a glutathione derivative. From a pharmacological point of view, such specialization is the Achilles' heel of these ancient and deadly parasites. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Bioinformatics analysis reveals disturbance mechanism of MAPK signaling pathway and cell cycle in Glioblastoma multiforme

Background & objectives

To analyze the reversal gene pairs and identify featured reversal genes related to mitogen-activated protein kinases (MAPK) signaling pathway and cell cycle in Glioblastoma multiforme (GBM) to reveal its pathogenetic mechanism.

Methods

We downloaded the gene expression profile GSE4290 from the Gene Expression Omnibus database, including 81 gene chips of GBM and 23 gene chips of controls. The t test was used to analyze the DEGs (differentially expressed genes) between 23 normal and 81 GBM samples. Then some perturbing metabolic pathways, including MAPK (mitogen-activated protein kinases) and cell cycle signaling pathway, were extracted from KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database. Cancer genes were obtained from the database of Cancer Gene Census. The reversal gene pairs between DEGs and cancer genes were further analyzed in MAPK and cell cycle signaling pathway.

Results

A total 8523 DEGs were obtained including 4090 up-regulated and 4433 down-regulated genes. Among them, ras-related protein rab-13(RAB13), neuroblastoma breakpoint family member 10 (NBPF10) and disks large homologue 4 (DLG4) were found to be involved in GBM for the first time. We obtained MAPK and cell cycle signaling pathways from KEGG database. By analyzing perturbing mechanism in these two pathways, we identified several reversal gene pairs, including NRAS (neuroblastoma RAS) and CDK2 (cyclin-dependent kinase 2), CCND1 (cyclin D1) and FGFR (fibroblast growth factor receptor). Further analysis showed that NRAS and CDK2 were positively related with GBM. However, FGFR2 and CCND1 were negatively related with GBM.

Interpretation & conclusions

These findings suggest that newly identified DEGs and featured reversal gene pairs participated in MAPK and cell cycle signaling pathway may provide a new therapeutic line of approach to GBM.     

Thioredoxin and glutaredoxin system proteins—immunolocalization in the rat central nervous system

Background

The oxidoreductases of the thioredoxin (Trx) family of proteins play a major role in the cellular response to oxidative stress. Redox imbalance is a major feature of brain damage. For instance, neuronal damage and glial reaction induced by a hypoxic–ischemic episode is highly related to glutamate excitotoxicity, oxidative stress and mitochondrial dysfunction. Most animal models of hypoxia–ischemia in the central nervous system (CNS) use rats to study the mechanisms involved in neuronal cell death, however, no comprehensive study on the localization of the redox proteins in the rat CNS was available.

Methods

The aim of this work was to study the distribution of the following proteins of the thioredoxin and glutathione/glutaredoxin (Grx) systems in the rat CNS by immunohistochemistry: Trx1, Trx2, TrxR1, TrxR2, Txnip, Grx1, Grx2, Grx3, Grx5, and γ-GCS, peroxiredoxin 1 (Prx1), Prx2, Prx3, Prx4, Prx5, and Prx6. We have focused on areas most sensitive to a hypoxia–ischemic insult: Cerebellum, striatum, hippocampus, spinal cord, substantia nigra, cortex and retina.

Results and conclusions

Previous studies implied that these redox proteins may be distributed in most cell types and regions of the CNS. Here, we have observed several remarkable differences in both abundance and regional distribution that point to a complex interplay and crosstalk between the proteins of this family.

General significance

We think that these data might be helpful to reveal new insights into the role of thiol redox pathways in the pathogenesis of hypoxia–ischemia insults and other disorders of the CNS.This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.     

Redox atlas of the mouse : Immunohistochemical detection of glutaredoxin-, peroxiredoxin-, and thioredoxin-family proteins in various tissues of the laboratory mouse

Background

Oxidoreductases of the thioredoxin family of proteins have been thoroughly studied in numerous cellular and animal models mimicking human diseases. Despite of their well documented role in various disease conditions, no systematic information on the presence of these proteins is available.

Methods

Here, we have systematically analyzed the presence of some of the major constituents of the glutaredoxin (Grx)-, peroxiredoxin (Prx)-, and thioredoxin (Trx)-systems, i.e. Grx1, Grx2, Grx3 (TXNL-2/PICOT), Grx5, nucleoredoxin (Nrx), Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, Trx1, thioredoxin reductase 1 (TrxR1), Trx2, TrxR2, and γ-glutamyl cysteine synthetase (γ-GCS) in various tissues of the mouse using immunohistochemistry.

Results

The identification of the Trx family proteins in the central nervous system, sensory organs, digestive system, lymphatic system, reproductive system, urinary system, respiratory system, endocrine system, skin, heart, and muscle revealed a number of significant differences between these proteins with respect to their distribution in these tissues.

Conclusion

Our results imply more specific functions and interactions between the proteins of this family than previously assumed.

General significance

Crucial functions of Trx family proteins have been demonstrated in various disease conditions. A detailed overview on their distribution in various tissues will be helpful to fully comprehend their potential role and the interactions of these proteins in the most thoroughly studied model for human diseases—the laboratory mouse.This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.     

Heme-iron utilization by Leptospira interrogans requires a heme oxygenase and a plastidic-type ferredoxin-NADP reductase

Background

Heme oxygenase catalyzes the conversion of heme to iron, carbon monoxide and biliverdin employing oxygen and reducing equivalents. This enzyme is essential for heme-iron utilization and contributes to virulence in Leptospira interrogans.

Methods

A phylogenetic analysis was performed using heme oxygenases sequences from different organisms including saprophytic and pathogenic Leptospira species. L. interrogans heme oxygenase (LepHO) was cloned, overexpressed and purified. The structural and enzymatic properties of LepHO were analyzed by UV–vis spectrophotometry and ¹H NMR. Heme-degrading activity, ferrous iron release and biliverdin production were studied with different redox partners.

Results

A plastidic type, high efficiently ferredoxin-NADP⁺ reductase (LepFNR) provides the electrons for heme turnover by heme oxygenase in L. interrogans. This catalytic reaction does not require a ferredoxin. Moreover, LepFNR drives the heme degradation to completeness producing free iron and α-biliverdin as the final products. The phylogenetic divergence between heme oxygenases from saprophytic and pathogenic species supports the functional role of this enzyme in L. interrogans pathogenesis.

Conclusions

Heme-iron scavenging by LepHO in L. interrogans requires only LepFNR as redox partner. Thus, we report a new substrate of ferredoxin-NADP⁺ reductases different to ferredoxin and flavodoxin, the only recognized protein substrates of this flavoenzyme to date. The results presented here uncover a fundamental step of heme degradation in L. interrogans.

General significance

Our findings contribute to understand the heme-iron utilization pathway in Leptospira. Since iron is required for pathogen survival and infectivity, heme degradation pathway may be relevant for therapeutic applications.