Subcellular Localization of Thioredoxin/Thioredoxin Reductase System-A Missing Link in Endoplasmic Reticulum Redox Balance.

The lumen of the endoplasmic reticulum (ER) is usually considered an oxidative environment; however, oxidized thiol-disulfides and reduced pyridine nucleotides occur there parallelly, indicating that the ER lumen lacks components which connect the two systems. Here, we investigated the luminal presence of the thioredoxin (Trx)/thioredoxin reductase (TrxR) proteins, capable of linking the protein thiol and pyridine nucleotide pools in different compartments. It was shown that specific activity of TrxR in the ER is undetectable, whereas higher activities were measured in the cytoplasm and mitochondria. None of the Trx/TrxR isoforms were expressed in the ER by Western blot analysis. Co-localization studies of various isoforms of Trx and TrxR with ER marker Grp94 by immunofluorescent analysis further confirmed their absence from the lumen. The probability of luminal localization of each isoform was also predicted to be very low by several in silico analysis tools. ER-targeted transient transfection of HeLa cells with Trx1 and TrxR1 significantly decreased cell viability and induced apoptotic cell death. In conclusion, the absence of this electron transfer chain may explain the uncoupling of the redox systems in the ER lumen, allowing parallel presence of a reduced pyridine nucleotide and a probably oxidized protein pool necessary for cellular viability.

From ubiquity to specificity: The diverse functions of bacterial thioredoxin systems.

The thioredoxin (Trx) system, found universally, is responsible for the regeneration of reversibly oxidized protein thiols in living cells. This system is made up of a Trx and a Trx reductase, and it plays a central role in maintaining thiol-based redox homeostasis by reducing oxidized protein thiols, such as disulfide bonds in proteins. Some Trxs also possess a chaperone function that is independent of thiol-disulfide exchange, in addition to their thiol-disulfide reductase activity. These two activities of the Trx system are involved in numerous physiological processes in bacteria. This review describes the diverse physiological roles of the Trx system that have emerged throughout bacterial evolution. The Trx system is essential for responding to oxidative and nitrosative stress. Beyond this primary function, the Trx system also participates in redox regulation and signal transduction, and in controlling metabolism, motility, biofilm formation, and virulence. This range of functions has evolved alongside the diversity of bacterial lifestyles and their specific constraints. This evolution can be characterized by the multiplication of the systems and by the specialization of cofactors or targets to adapt to the constraints of atypical lifestyles, such as photosynthesis, insect endosymbiosis, or spore-forming bacteria.

Catalytic Activity of the Archetype from Group 4 of the FTR-like Ferredoxin:Thioredoxin Reductase Family Is Regulated by Unique S = 7/2 and S = 1/2 [4Fe-4S] Clusters.

Thioredoxin reductases (TrxR) activate thioredoxins (Trx) that regulate the activity of diverse target proteins essential to prokaryotic and eukaryotic life. However, very little is understood of TrxR/Trx systems and redox control in methanogenic microbes from the domain Archaea (methanogens), for which genomes are abundant with annotations for ferredoxin:thioredoxin reductases [Fdx/thioredoxin reductase (FTR)] from group 4 of the widespread FTR-like family. Only two from the FTR-like family are characterized: the plant-type FTR from group 1 and FDR from group 6. Herein, the group 4 archetype (AFTR) from Methanosarcina acetivorans was characterized to advance understanding of the family and TrxR/Trx systems in methanogens. The modeled structure of AFTR, together with EPR and Mössbauer spectroscopies, supports a catalytic mechanism similar to plant-type FTR and FDR, albeit with important exceptions. EPR spectroscopy of reduced AFTR identified a transient [4Fe-4S]1+ cluster exhibiting a mixture of S = 7/2 and typical S = 1/2 signals, although rare for proteins containing [4Fe-4S] clusters, it is most likely the on-pathway intermediate in the disulfide reduction. Furthermore, an active site histidine equivalent to residues essential for the activity of plant-type FTR and FDR was found dispensable for AFTR. Finally, a unique thioredoxin system was reconstituted from AFTR, ferredoxin, and Trx2 from M. acetivorans, for which specialized target proteins were identified that are essential for growth and other diverse metabolisms.

Thioredoxin system in colorectal cancer: Its role in carcinogenesis, disease progression, and response to treatment.

The thioredoxin system is essential for many physiological processes, including the maintenance of redox signalling pathways. Alterations in the activity, expression and interactions with other signalling pathways can lead to protective or pathophysiological responses. Thioredoxin and thioredoxin reductase, the two main components of this system, are often overexpressed in cancer, including colorectal cancer. This overexpression is often linked with tumour progression and poor outcomes. This review discusses the role of the Trx system in driving colorectal carcinogenesis and disease progression, as well as the challenges of targeting this system. Additionally, the recent advancements in the development of novel and effective thioredoxin inhibitors for colorectal cancer are also explored.

Silver Organometallics that are Highly Potent Thioredoxin and Glutathione Reductase Inhibitors: Exploring the Correlations of Solution Chemistry with the Strong Antibacterial Effects.

The antibacterial activity of silver species is well-established; however, their mechanism of action has not been adequately explored. Furthermore, issues of low-molecular silver compounds with cytotoxicity, stability, and solubility hamper their progress to drug leads. We have investigated silver N-heterocyclic carbene (NHC) halido complexes [(NHC)AgX, X = Cl, Br, and I] as a promising new type of antibacterial silver organometallics. Spectroscopic studies and conductometry established a higher stability for the complexes with iodide ligands, and nephelometry indicated that the complexes could be administered in solutions with physiological chloride levels. The complexes showed a broad spectrum of strong activity against pathogenic Gram-negative bacteria. However, there was no significant activity against Gram-positive strains. Further studies clarified that tryptone and yeast extract, as components of the culture media, were responsible for this lack of activity. The reduction of biofilm formation and a strong inhibition of both glutathione and thioredoxin reductases with IC50 values in the nanomolar range were confirmed for selected compounds. In addition to their improved physicochemical properties, the compounds with iodide ligands did not display cytotoxic effects, unlike the other silver complexes. In summary, silver NHC complexes with iodide secondary ligands represent a useful scaffold for nontoxic silver organometallics with improved physicochemical properties and a distinct mechanism of action that is based on inhibition of thioredoxin and glutathione reductases.

The redox-active defensive Selenoprotein T as a novel stress sensor protein playing a key role in the pathophysiology of heart failure.

Maladaptive cardiac hypertrophy contributes to the development of heart failure (HF). The oxidoreductase Selenoprotein T (SELENOT) emerged as a key regulator during rat cardiogenesis and acute cardiac protection. However, its action in chronic settings of cardiac dysfunction is not understood. Here, we investigated the role of SELENOT in the pathophysiology of HF: (i) by designing a small peptide (PSELT), recapitulating SELENOT activity via the redox site, and assessed its beneficial action in a preclinical model of HF [aged spontaneously hypertensive heart failure (SHHF) rats] and against isoproterenol (ISO)-induced hypertrophy in rat ventricular H9c2 and adult human AC16 cardiomyocytes; (ii) by evaluating the SELENOT intra-cardiomyocyte production and secretion under hypertrophied stimulation. Results showed that PSELT attenuated systemic inflammation, lipopolysaccharide (LPS)-induced macrophage M1 polarization, myocardial injury, and the severe ultrastructural alterations, while counteracting key mediators of cardiac fibrosis, aging, and DNA damage and restoring desmin downregulation and SELENOT upregulation in the failing hearts. In the hemodynamic assessment, PSELT improved the contractile impairment at baseline and following ischemia/reperfusion injury, and reduced infarct size in normal and failing hearts. At cellular level, PSELT counteracted ISO-mediated hypertrophy and ultrastructural alterations through its redox motif, while mitigating ISO-triggered SELENOT intracellular production and secretion, a phenomenon that presumably reflects the extent of cell damage. Altogether, these results indicate that SELENOT could represent a novel sensor of hypertrophied cardiomyocytes and a potential PSELT-based new therapeutic approach in myocardial hypertrophy and HF.

Development of New PCR Protocols to Detect Genetic Diversity in the Metronidazole Metabolism Genes in Susceptible and Refractory Clinical Samples of Giardia duodenalis.

PURPOSE: Investigating the genetic variation in thioredoxin reductase (TrxR) and nitroreductase (NR) genes in both treatment-resistant and -sensitive Giardia duodenalis isolates can provide valuable information in identifying potential markers of resistance to metronidazole. The rapid increase in metronidazole treatment failures suggests the presence of genetic resistance mechanisms. By analyzing these genes, researchers can gain insights into the efficacy of metronidazole against G. duodenalis and potentially develop alternative treatment strategies. In this regard, four G. duodenalis isolates (two clinically sensitive and two clinically resistant to metronidazole) were collected from various hospitals of Shiraz, southwestern Iran. METHODS: Parasitological methods including sucrose flotation and microscopy were employed for the primary confirmation of G. duodenalis cysts in stool samples. Microscopy-positive samples were approved by SSU-PCR amplification of the parasite DNA. All four positive G. duodenalis specimens at SSU-PCR were afterward analyzed utilizing designed primers based on important metronidazole metabolism genes including TrxR, NR1, and NR2. RESULTS: Unlike TrxR gene, the results of NR1 and NR2 genes showed that there are non-synonymous variations between sequences of treatment-sensitive and -resistant samples compared to reference sequences. Furthermore, the outcomes of molecular docking revealed that there is an interaction between the protein sequence and spatial shape of treatment-resistant samples and metronidazole in the position of serine amino acid based on the NR1 gene. CONCLUSION: This issue can be one of the possible factors involved in the resistance of Giardia parasites to metronidazole. To reach more accurate results, a large sample size along with simulation and advanced molecular dynamics investigations are needed.

Mechanism of Action of Antitumor Au(I) N-Heterocyclic Carbene Complexes: A Computational Insight on the Targeting of TrxR Selenocysteine.

The targeting of human thioredoxin reductase is widely recognized to be crucially involved in the anticancer properties of several metallodrugs, including Au(I) complexes. In this study, the mechanism of reaction between a set of five N-heterocyclic carbene Au(I) complexes and models of the active Sec residue in human thioredoxin reductase was investigated by means of density functional theory approaches. The study was specifically addressed to the kinetics and thermodynamics of the tiled process by aiming at elucidating and explaining the differential inhibitory potency in this set of analogous Au(I) bis-carbene complexes. While the calculated free energy profile showed a substantially similar reactivity, we found that the binding of these Au(I) bis-carbene at the active CysSec dyad in the TrxR enzyme could be subjected to steric and orientational restraints, underlining both the approach of the bis-carbene scaffold and the attack of the selenol group at the metal center. A new and detailed mechanistic insight to the anticancer activity of these Au(I) organometallic complexes was thus provided by consolidating the TrxR targeting paradigm.

Thioredoxin Reductase Inhibitor Suppresses the Local Progression of Rhabdomyosarcoma With PDX Models.

BACKGROUND/AIM: Chemoresistance in rhabdomyosarcoma (RMS) is associated with poor survival, necessitating the development of novel anticancer drugs. Auranofin (AUR), an anti-rheumatic drug, is a thioredoxin reductase (TXNRD) inhibitor with anticancer properties. Although patient-derived xenograft (PDX) models are essential for studying cancer biology, reports on sarcomas using the PDX model are scarce because of their rarity. This study aimed to investigate the effectiveness of AUR treatment in RMS using a PDX model to evaluate its impact on local progression. MATERIALS AND METHODS: A 20-year-old woman who was diagnosed with alveolar RMS was used to generate the PDX model. RMS PDX tumors were implanted in nude mice and divided into non-treated (vehicle) and treated (AUR) groups. Tumor volume and weight were evaluated, and immunohistochemical staining was performed to evaluate local progression of the sarcoma. The relationship between the TXNRD-1 expression and survival probability of patients with RMS was evaluated using publicly available expression cohorts. RESULTS: AUR significantly suppressed RMS tumor progression over time. It also significantly suppressed the tumor size and weight at the time of excision. Histological evaluation showed that AUR induced oxidative stress in the PDX mouse models and inhibited the local progression of RMS by inducing apoptosis. High TXNRD-1 expression was found to be a negative prognostic factor for overall survival in patients with RMS. CONCLUSION: AUR-induced inhibition of TXNRDs can significantly impede the local progression of RMS through the oxidative stress-apoptosis pathway as demonstrated in PDX models. Thus, targeting TXNRD inhibition may be a promising therapeutic strategy for the treatment of RMS.

HyPer as a tool to determine the reductive activity in cellular compartments.

A multitude of cellular metabolic and regulatory processes rely on controlled thiol reduction and oxidation mechanisms. Due to our aerobic environment, research preferentially focuses on oxidation processes, leading to limited tools tailored for investigating cellular reduction. Here, we advocate for repurposing HyPer1, initially designed as a fluorescent probe for H2O2 levels, as a tool to measure the reductive power in various cellular compartments. The response of HyPer1 depends on kinetics between thiol oxidation and reduction in its OxyR sensing domain. Here, we focused on the reduction half-reaction of HyPer1. We showed that HyPer1 primarily relies on Trx/TrxR-mediated reduction in the cytosol and nucleus, characterized by a second order rate constant of 5.8 × 102 M-1s-1. On the other hand, within the mitochondria, HyPer1 is predominantly reduced by glutathione (GSH). The GSH-mediated reduction rate constant is 1.8 M-1s-1. Using human leukemia K-562 cells after a brief oxidative exposure, we quantified the compartmentalized Trx/TrxR and GSH-dependent reductive activity using HyPer1. Notably, the recovery period for mitochondrial HyPer1 was twice as long compared to cytosolic and nuclear HyPer1. After exploring various human cells, we revealed a potent cytosolic Trx/TrxR pathway, particularly pronounced in cancer cell lines such as K-562 and HeLa. In conclusion, our study demonstrates that HyPer1 can be harnessed as a robust tool for assessing compartmentalized reduction activity in cells following oxidative stress.

The multiplicity of thioredoxin systems meets the specific lifestyles of Clostridia.

Cells are unceasingly confronted by oxidative stresses that oxidize proteins on their cysteines. The thioredoxin (Trx) system, which is a ubiquitous system for thiol and protein repair, is composed of a thioredoxin (TrxA) and a thioredoxin reductase (TrxB). TrxAs reduce disulfide bonds of oxidized proteins and are then usually recycled by a single pleiotropic NAD(P)H-dependent TrxB (NTR). In this work, we first analyzed the composition of Trx systems across Bacteria. Most bacteria have only one NTR, but organisms in some Phyla have several TrxBs. In Firmicutes, multiple TrxBs are observed only in Clostridia, with another peculiarity being the existence of ferredoxin-dependent TrxBs. We used Clostridioides difficile, a pathogenic sporulating anaerobic Firmicutes, as a model to investigate the biological relevance of TrxB multiplicity. Three TrxAs and three TrxBs are present in the 630Δerm strain. We showed that two systems are involved in the response to infection-related stresses, allowing the survival of vegetative cells exposed to oxygen, inflammation-related molecules and bile salts. A fourth TrxB copy present in some strains also contributes to the stress-response arsenal. One of the conserved stress-response Trx system was found to be present both in vegetative cells and in the spores and is under a dual transcriptional control by vegetative cell and sporulation sigma factors. This Trx system contributes to spore survival to hypochlorite and ensure proper germination in the presence of oxygen. Finally, we found that the third Trx system contributes to sporulation through the recycling of the glycine-reductase, a Stickland pathway enzyme that allows the consumption of glycine and contributes to sporulation. Altogether, we showed that Trx systems are produced under the control of various regulatory signals and respond to different regulatory networks. The multiplicity of Trx systems and the diversity of TrxBs most likely meet specific needs of Clostridia in adaptation to strong stress exposure, sporulation and Stickland pathways.

Thioredoxin Reductase-Mediated Reaction Evokes In Situ Surface Polarization Effect on BiOIO3: Toward a New Sensing Strategy for Cathodic Photoelectrochemistry.

We have witnessed the fast progress of cathodic photoelectrochemistry over the past decades, though its signal transduction tactic still lacks diversity. Exploring new sensing strategies for cathodic photoelectrochemistry is extremely demanding yet hugely challenging. This article puts forward a unique idea to incorporate an enzymatic reaction-invoked surface polarization effect (SPE) on the surface of BiOIO3 to implement an innovative cathodic photoelectrochemical (PEC) bioanalysis. Specifically, the thioredoxin reductase (TrxR)-mediated reaction produced the polar glutathione (GSH), which spontaneously coordinated to the surface of BiOIO3 and induced SPE by forming a polarized electric field, resulting in improved electron (e-) and hole (h+) pair separation efficiency and an enhanced photocurrent output. Correlating this phenomenon with the detection of TrxR exhibited a high performance in terms of sensitivity and selectivity, achieving a linear range of 0.007-0.5 µM and a low detection limit of 2.0 nM (S/N = 3). This study brings refreshing inspiration for the cathodic PEC signal transduction tactic through enzyme-mediated in situ reaction to introduce SPE, which enriches the diversity of available signaling molecules. Moreover, this study unveils the potential of in situ generated SPE for extended and futuristic applications.

The mechanism of action of auranofin analogs in B. cenocepacia revealed by chemogenomic profiling.

Drug repurposing efforts led to the discovery of bactericidal activity in auranofin, a gold-containing drug used to treat rheumatoid arthritis. Auranofin kills Gram-positive bacteria by inhibiting thioredoxin reductase, an enzyme that scavenges reactive oxygen species (ROS). Despite the presence of thioredoxin reductase in Gram-negative bacteria, auranofin is not always active against them. It is not clear whether the lack of activity in several Gram-negative bacteria is due to the cell envelope barrier or the presence of other ROS protective enzymes such as glutathione reductase (GOR). We previously demonstrated that chemical analogs of auranofin (MS-40 and MS-40S), but not auranofin, are bactericidal against the Gram-negative Burkholderia cepacia complex. Here, we explore the targets of auranofin, MS-40, and MS-40S in Burkholderia cenocepacia and elucidate the mechanism of action of the auranofin analogs by a genome-wide, randomly barcoded transposon screen (BarSeq). Auranofin and its analogs inhibited the B. cenocepacia thioredoxin reductase and induced ROS but did not inhibit the bacterial GOR. Genome-wide, BarSeq analysis of cells exposed to MS-40 and MS-40S compared to the ROS inducers arsenic trioxide, diamide, hydrogen peroxide, and paraquat revealed common and unique mediators of drug susceptibility. Furthermore, deletions of gshA and gshB that encode enzymes in the glutathione biosynthetic pathway led to increased susceptibility to MS-40 and MS-40S. Overall, our data suggest that the auranofin analogs kill B. cenocepacia by inducing ROS through inhibition of thioredoxin reductase and that the glutathione system has a role in protecting B. cenocepacia against these ROS-inducing compounds.IMPORTANCEThe Burkholderia cepacia complex is a group of multidrug-resistant bacteria that can cause infections in the lungs of people with the autosomal recessive disease, cystic fibrosis. Specifically, the bacterium Burkholderia cenocepacia can cause severe infections, reducing lung function and leading to a devastating type of sepsis, cepacia syndrome. This bacterium currently does not have an accepted antibiotic treatment plan because of the wide range of antibiotic resistance. Here, we further the research on auranofin analogs as antimicrobials by finding the mechanism of action of these potent bactericidal compounds, using a powerful technique called BarSeq, to find the global response of the cell when exposed to an antimicrobial.

The impact of light and thioredoxins on the plant thiol-disulfide proteome.

Thiol-based redox regulation is a crucial posttranslational mechanism to acclimate plants to changing light availability. Here, we conducted a biotin switch-based redox proteomics study in Arabidopsis (Arabidopsis thaliana) to systematically investigate dynamics of thiol-redox networks in response to temporal changes in light availability and across genotypes lacking parts of the thioredoxin (Trx) or NADPH-Trx-reductase C (NTRC) systems in the chloroplast. Time-resolved dynamics revealed light led to marked decreases in the oxidation states of many chloroplast proteins with photosynthetic functions during the first 10 min, followed by their partial reoxidation after 2 to 6 h into the photoperiod. This involved f, m, and x-type Trx proteins showing similar light-induced reduction-oxidation dynamics, while NTRC, 2-Cys peroxiredoxins, and Trx y2 showed an opposing pattern, being more oxidized in light than dark. In Arabidopsis trxf1f2, trxm1m2, or ntrc mutants, most proteins showed increased oxidation states in the light compared to wild type, suggesting their light-dependent dynamics were related to NTRC/Trx networks. While NTRC deficiency had a strong influence in all light conditions, deficiencies in f- or m-type Trxs showed differential impacts on the thiol-redox proteome depending on the light environment, being higher in constant or fluctuating light, respectively. The results indicate plant redox proteomes are subject to dynamic changes in reductive and oxidative pathways to cooperatively fine-tune photosynthetic and metabolic processes in the light. The importance of the individual elements of the NTRC/Trx networks mediating these responses depend on the extent of light variability, with NTRC playing a crucial role to balance protein-redox states in rapidly fluctuating light.

Guiding bar motif of thioredoxin reductase 1 modulates enzymatic activity and inhibitor binding by communicating with the co-factor FAD and regulating the flexible C-terminal redox motif.

Thioredoxin reductase (TXNRD) is a selenoprotein that plays a crucial role in cellular antioxidant defense. Previously, a distinctive guiding bar motif was identified in TXNRD1, which influences the transfer of electrons. In this study, utilizing single amino acid substitution and Excitation-Emission Matrix (EEM) fluorescence spectrum analysis, we discovered that the guiding bar communicates with the FAD and modulates the electron flow of the enzyme. Differential Scanning Fluorimetry (DSF) analysis demonstrated that the aromatic amino acid in guiding bar is a stabilizer for TXNRD1. Kinetic analysis revealed that the guiding bar is vital for the disulfide reductase activity but hinders the selenocysteine-independent reduction activity of TXNRD1. Meanwhile, the guiding bar shields the selenocysteine residue of TXNRD1 from the attack of electrophilic reagents. We also found that the inhibition of TXNRD1 by caveolin-1 scaffolding domain (CSD) peptides and compound LCS3 did not bind to the guiding bar motif. In summary, the obtained results highlight new aspects of the guiding bar that restrict the flexibility of the C-terminal redox motif and govern the transition from antioxidant to pro-oxidant.

Design, Synthesis, and Bioactivity Evaluations of 3-Methylenechroman-2-one Derivatives as Thioredoxin Reductase (TrxR) Inhibitors.

We aimed to design and synthesize 3-methylenechroman-2-one derivatives and test their potency as TrxR1 inhibitors. A convenient and easy-to-handle synthetic approach to 3-methylenechroman-2-ones was developed. The in vitro inhibitory activity towards recombinant TrxR1 was determined for the obtained compounds. The most potent representatives exhibited submicromolar TrxR1 inhibition activity (IC50 varied from 0.29 µM to 10.2 µM). Structure-activity relationship analysis indicates the beneficial role of the substituent at the position C-6 of the core of chroman-2-one, where the derivatives containing halogen are the most active among the scope of compounds obtained. The most potent TrxR1 inhibitor of the series was further examined in in vitro cell-based assays to assess cytotoxic effects on various cancer cell lines, and to evaluate their influence on cell apoptosis.

The effects of morin and methotrexate on pentose phosphate pathway enzymes and GR/GST/TrxR enzyme activities: An in vivo and in silico study.

In this study, the mechanisms by which the enzymes glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR), glutathione-S-transferase (GST), and thioredoxin reductase (TrxR) are inhibited by methotrexate (MTX) were investigated, as well as whether the antioxidant morin can mitigate or prevent these adverse effects in vivo and in silico. For 10 days, rats received oral doses of morin (50 and 100 mg/kg body weight). On the fifth day, a single intraperitoneal injection of MTX (20 mg/kg body weight) was administered to generate toxicity. Decreased activities of G6PD, 6PGD, GR, GST, and TrxR were associated with MTX-related toxicity while morin treatment increased the activity of the enzymes. The docking analysis indicated that H-bonds, pi-pi stacking, and pi-cation interactions were the dominant interactions in these enzyme-binding pockets. Furthermore, the docked poses of morin and MTX against GST were subjected to molecular dynamic simulations for 200 ns, to assess the stability of both complexes and also to predict key amino acid residues in the binding pockets throughout the simulation. The results of this study suggest that morin may be a viable means of alleviating the enzyme activities of important regulatory enzymes against MTX-induced toxicity.

Potent Anticancer Activity of a Dinuclear Gold(I) bis-N-Heterocyclic Imine Complex Related to Thioredoxin Reductase Inhibition in Vitro.

A dinuclear gold(I) complex featuring a strongly donating bis-N-heterocyclic imine ligand was synthesised and characterised by different methods, including single crystal X-ray diffraction (SC-XRD) analysis. The compound has been tested for its antiproliferative effects in a panel of human cancer cell lines in vitro, showing highly selective anticancer effects, particularly against human A549 non-small cell lung cancer cells (NSCLC), with respect to non-tumorigenic cells (VERO). The accumulation of the compound in A549 and VERO cells was studied by high-resolution continuum source atomic absorption spectrometry (HRCS-AAS), revealing that the anticancer effects are not particularly related to the different amounts of gold taken up by the cells over 72 h. Enzyme inhibition studies to evaluate the activity of the seleno-enzyme thioredoxin reductase (TrxR) in cancer cell extracts show that the gold(I) compound is a potent inhibitor (IC50=0.567±0.208 µM), while the free ligand is ineffective. This result correlates with the observed compound's selectivity towards A549 cells overexpressing the enzyme.

Biophysical and biochemical characterization of the thioredoxin system from Colwellia psychrerythraea.

The thioredoxin system is a ubiquitous oxidoreductase system consisting of the enzyme thioredoxin reductase, the protein thioredoxin, and the cofactor nicotinamide adenine dinucleotide phosphate. The system has been comprehensively studied from many organisms, such as Escherichia coli; however, structural and functional analysis of this system from psychrophilic bacteria has not been as extensive. In this study, the thioredoxin system proteins of a psychrophilic bacterium, Colwellia psychrerythraea, were characterized using biophysical and biochemical techniques. Analysis of the complete genome sequence of the C. psychrerythraea thioredoxin system suggested the presence of a putative thioredoxin reductase and at least three thioredoxin. In this study, these identified putative thioredoxin system components were cloned, overexpressed, purified, and characterized. Our studies have indicated that the thioredoxin system proteins from E. coli were more stable than those from C. psychrerythraea. Consistent with these results, kinetic assays indicated that the thioredoxin reductase from E. coli had a higher optimal temperature than that from C. psychrerythraea.

Purification and characterization of thioredoxin reductase enzyme from commercial Spirulina platensis tablets by affinity chromatography and investigation of the effects of some chemicals and drugs on enzyme activity.

Thioredoxin reductase (TrxR, enzyme code [E.C.] 1.6.4.5) is a widely distributed flavoenzyme that catalyzes nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of thioredoxin and many other physiologically important substrates. Spirulina platensis is a blue-green algae that is often used as a dietary supplement. S. platensis is rich in protein, lipid, polysaccharide, pigment, carotenoid, enzyme, vitamins and many other chemicals and exhibits a variety of pharmacological functions. In the present study, a simple and efficient method to purify TrxR from S. platensis tablets is reported. The extractions were carried out using two different methods: heat denaturation and 2',5'-adenosine diphosphate Sepharose 4B affinity chromatography. The enzyme was purified by 415.04-fold over the crude extract, with a 19% yield, and specific activity of 0.7640 U/mg protein. Optimum pH, temperature and ionic strength of the enzyme activity, as well as the Michaelis constant (Km ) and maximum velocity of enzyme (Vmax ) values for NADPH and 5,5'-dithiobis(2-nitrobenzoic acid) were determined. Tested metal ions, vitamins, and drugs showed inhibition effects, except Se4+ ion, cefazolin sodium, teicoplanin, and tobramycin that increased the enzyme activity in vitro. Ag+ , Cu2+ , Mg2+ , Ni2+ , Pb2+ , Zn2+ , Al3+ , Cr3+ , Fe3+ , and V4+ ions; vitamin B3 , vitamin B6 , vitamin C, and vitamin U and aciclovir, azithromycin, benzyladenine, ceftriaxone sodium, clarithromycin, diclofenac, gibberellic acid, glurenorm, indole-3-butyric acid, ketorolac, metformin, mupirocin, mupirocin calcium, paracetamol, and tenofovir had inhibitory effects on TrxR. Ag+ exhibited stronger inhibition than 1-chloro-2,4-dinitrobenzene (a positive control).