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While highly effective at killing Gram-positive bacteria, auranofin lacks significant activity against Gram-negative species for reasons that largely remain unclear. Here, we aimed to elucidate the molecular mechanisms underlying the low susceptibility of the Gram-negative model organism Escherichia coli to auranofin when compared to the Gram-positive model organism Bacillus subtilis . The proteome response of E. coli exposed to auranofin suggests a combination of inactivation of thiol-containing enzymes and the induction of systemic oxidative stress. Susceptibility tests in E. coli mutants lacking proteins upregulated upon auranofin treatment suggested that none of them are directly involved in E. coli 's high tolerance to auranofin. E. coli cells lacking the efflux pump component TolC were more sensitive to auranofin treatment, but not to an extent that would fully explain the observed difference in susceptibility of Gram-positive and Gram-negative organisms. We thus tested whether E. coli 's thioredoxin reductase (TrxB) is inherently less sensitive to auranofin than TrxB from B. subtilis , which was not the case. However, E. coli strains lacking the low-molecular-weight thiol glutathione, but not glutathione reductase, showed a high susceptibility to auranofin. Bacterial cells expressing the genetically encoded redox probe roGFP2 allowed us to observe the oxidation of cellular protein thiols in situ . Based on our findings, we hypothesize that auranofin leads to a global disturbance in the cellular thiol redox homeostasis in bacteria, but Gram-negative bacteria are inherently more resistant due to the presence of drug export systems and high cellular concentrations of glutathione.IMPORTANCEAuranofin is an FDA-approved drug for the treatment of rheumatoid arthritis. However, it has also high antibacterial activity, in particular against Gram-positive organisms. In the current antibiotics crisis, this would make it an ideal candidate for drug repurposing. However, its much lower activity against Gram-negative organisms prevents its broad-spectrum application. Here we show that, on the level of the presumed target, there is no difference in susceptibility between Gram-negative and Gram-positive species: thioredoxin reductases from both Escherichia coli and Bacillus subtilis are equally inhibited by auranofin. In both species, auranofin treatment leads to oxidative protein modification on a systemic level, as monitored by proteomics and the genetically encoded redox probe roGFP2. The single largest contributor to E. coli 's relative resistance to auranofin seems to be the low-molecular-weight thiol glutathione, which is absent in B. subtilis and other Gram-positive species., Competing Interests: The authors declare no conflict of interest.

Targeting inflammation and the molecules involved in the inflammatory process could be an effective cancer prevention and therapy strategy. Therefore, the use of anti-inflammatory strategies, such as NSAIDs and metal-based drugs, has become a promising approach for preventing and treating cancer by targeting multiple pathways involved in tumor progression. The present work describes new phosphane gold(I) complexes derived from nonsteroidal anti-inflammatory drugs as multitarget drugs against colon cancer. The antiproliferative effect of the most active complexes, [Au(L3)(JohnPhos)] ( 3b ), [Au(L4)(CyJohnPhos)] ( 4a ) and [Au(L4)(JohnPhos)] ( 4b ) against colon cancer cells (Caco2-/TC7) seems to be mediated by the inhibition of the enzyme cyclooxygenase-1/2, modulation of reactive oxygen species levels by targeting thioredoxin reductase (TrxR) activity, and induction of apoptosis in cancer cells. Additionally, the three complexes exhibit high selectivity index values toward noncancerous cells. The research highlights the importance of maintaining cellular redox balance and the role of TrxR in cancer cell survival.

Pyridine nucleotide-disulfide oxidoreductases are underexplored as drug targets, and thioredoxin reductases (TrxRs) stand out as compelling pharmacological targets. Selective TrxR inhibition is challenging primarily due to the reliance on covalent inhibition strategies. Recent studies identified a regulatory and druggable pocket in Schistosoma mansoni thioredoxin glutathione reductase (TGR), a TrxR-like enzyme, and an established drug target for schistosomiasis. This site is termed the "doorstop pocket" because compounds that bind there impede the movement of an aromatic side-chain necessary for the entry and exit of NADPH and NADP + during enzymatic turnover. This discovery spearheaded the development of new TGR inhibitors with efficacies surpassing those of current schistosomiasis treatment. Targeting the "doorstop pocket" is a promising strategy, as the pocket is present in all members of the pyridine nucleotide-disulfide oxidoreductase family, opening new avenues for exploring therapeutic approaches in diseases where the importance of these enzymes is established, including cancer and inflammatory and infectious diseases.

Hepatocellular carcinoma (HCC), the predominant type of liver cancer, is an aggressive malignancy with limited therapeutic options. In this study, we assess a collection of newly designed gold(I) phosphine complexes. Remarkably, the compound GC002 exhibits the greatest toxicity to HCC cells and outperforms established medications, such as sorafenib and auranofin, in terms of antitumor efficacy. GC002 triggers irreversible necroptosis in HCC cells by increasing the intracellular accumulation of reactive oxygen species (ROS). Mechanistically, GC002 significantly suppresses the activity of thioredoxin reductase (TrxR), which plays a crucial role in regulating redox homeostasis and is often overexpressed in HCC by binding directly to the enzyme. Our in vivo xenograft study confirms that GC002 possesses remarkable antitumor activity against HCC without severe side effects. These findings not only highlight the novel mechanism of controlling necroptosis via TrxR and ROS but also identify GC002 as a promising candidate for the further development of antitumor agents targeting HCC.

Cancer is a significant cause of death around the world, and for many varieties, treatment is not successful. Therefore, there is a need for the development of innovative, efficacious, and precisely targeted treatments. Here, we develop a series of Au(I) complexes (1-4) through rational manipulation of ligand structures, thereby achieving tumor cell specific targeting and orchestrated tumor eradication via chemo-phototherapy and induced immunogenic cell death. A comprehensive exploration based on in vitro and in vivo female mice experimentation shows that complex 4 exhibits proficiency in specific tumor imaging, endoplasmic reticulum targeting, and has robust therapeutic capabilities. Mechanistic elucidation indicates that the anticancer effect derives from the synergistic actions of thioredoxin reductase inhibition, highly efficient reactive oxygen species production and immunogenic cell death. This work presents a report on a robust Au(I) complex integrating three therapeutic modalities within a singular system. The strategy presented in this work provides a valuable reference for the development of high-performance therapeutic agents., (© 2024. The Author(s).)

Auranofin (AF), a former rheumatoid polyarthritis treatment, gained renewed interest for its use as an antimicrobial. AF is an inhibitor of thioredoxin reductase (TrxB), a thiol and protein repair enzyme, with an antibacterial activity against several bacteria including C. difficile, an enteropathogen causing post-antibiotic diarrhea. Several studies demonstrated the effect of AF on C. difficile physiology, but the crucial questions of resistance mechanisms and impact on microbiota remain unaddressed. We explored potential resistance mechanisms by studying the impact of TrxB multiplicity and by generating and characterizing adaptive mutations. We showed that if mutants inactivated for trxB genes have a lower MIC of AF, the number of TrxBs naturally present in clinical strains does not impact the MIC. All stable mutations isolated after AF long-term exposure were in the anti-sigma factor of σ B and strongly affect physiology. Finally, we showed that AF has less impact on human gut microbiota than vancomycin., (© 2024. The Author(s).)

A series of new gold(I) and silver(I) N-heterocyclic carbenes bearing a 1-thio-β-d-glucose tetraacetate moiety was synthesized and chemically characterized. The compounds' stability and solubility in physiological conditions were investigated employing a multitechnique approach. Interaction studies with biologically relevant proteins, such as superoxide dismutase (SOD) and human serum albumin (HSA), were conducted via UV-vis absorption spectroscopy and high-resolution ESI mass spectrometry. The biological activity of the compounds was evaluated in the A2780 and A2780R (cisplatin-resistant) ovarian cancer cell lines and the HSkMC (human skeletal muscle) healthy cell line. Inhibition studies of the selenoenzyme thioredoxin reductase (TrxR) were also carried out. The results highlighted that the gold complexes are more stable in aqueous environment and capable of interaction with SOD and HSA. Moreover, these carbenes strongly inhibited the TrxR activity. In contrast, the silver ones underwent structural alterations in the aqueous medium and showed greater antiproliferative activity.

Skeletal muscle aging in rats is a reduction in skeletal muscle mass caused by a decrease in the number or volume of skeletal muscle myofibers. Apoptosis has been recognized to play a key role in accelerating the process of skeletal muscle aging in rats. The thioredoxin (Trx) system is a widely expressed oxidoreductase system that controls the cellular reduction/oxidation state and has both potent anti-free radical damage and important pro-growth and apoptosis inhibitory functions. Previous studies have shown that exercise delays skeletal muscle aging. However, it is unclear whether exercise attenuates skeletal muscle aging via the Trx system. Therefore, the present study used the Trx system as an entry point to explore the effect of aerobic exercise to improve skeletal muscle aging in rats and its possible mechanisms, and to provide a theoretical basis for exercise to delay skeletal muscle aging in rats. It was shown that aerobic exercise in senescent rats resulted in increased gastrocnemius index, decreased body weight, increased endurance, decreased skeletal muscle cell apoptosis, increased activity and protein expression of the Trx system, and decreased expression of p38 and ASK1. Based on these findings, we conclude that 10 weeks of aerobic exercise may enhance the anti-apoptotic effect of Trx by up-regulating Trx and Trx reductase (TR) protein expression, which in turn increases Trx activity in rat skeletal muscle, and ultimately alleviates apoptosis in senescent skeletal muscle cells., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest in relation to this study., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Thioredoxin Reductase (TrxR) functions to recycle thioredoxin (Trx) during hydroperoxide metabolism mediated by peroxiredoxins and is currently being targeted using the FDA-approved anti-rheumatic drug, auranofin (AF), to selectively sensitize cancer cells to therapy. AF treatment decreased TrxR activity and clonogenic survival in small cell lung cancer (SCLC) cell lines (DMS273 and DMS53) as well as the H727 atypical lung carcinoid cell line. AF treatment also significantly sensitized DMS273 and H727 cell lines in vitro to sorafenib, an FDA-approved multi-kinase inhibitor that depleted intracellular glutathione (GSH). The pharmacokinetic, pharmacodynamic, and safety profile of AF was examined in nude mice with DMS273 xenografts administered AF intraperitoneally at 2 mg/kg or 4 mg/kg (IP) once (QD) or twice daily (BID) for 1-5 d. Plasma levels of AF were 10-20 μM (determined by mass spectrometry of gold), and the optimal inhibition of TrxR activity was obtained at 4 mg/kg once daily, with no effect on glutathione peroxidase 1 activity. This AF treatment extended for 14 d, inhibited TrxR (>75%), and resulted in a significant prolongation of median overall survival from 19 to 23 d ( p  = .04, N  = 30 controls, 28 AF). In this experiment, there were no observed changes in animal bodyweight, complete blood counts (CBCs), bone marrow toxicity, blood urea nitrogen, or creatinine. These results support the hypothesis that AF effectively inhibits TrxR both in vitro and in vivo in SCLC, sensitizes NETs and SCLC to sorafenib, and could be repurposed as an adjuvant therapy with targeted agents that induce disruptions in thiol metabolism.

Overexpression of thioredoxin reductase (TXNRD) plays crucial role in tumorigenesis. Therefore, designing TXNRD inhibitors is a promising strategy for targeted anticancer drug development. However, poor selectivity has always been a challenge, resulting in unavoidable toxicity in clinic. Herein we demonstrate a strategy to develop highly selective chiral metal complexes-based TXNRD inhibitors. By manipulating the conformation of two distinct weakly interacting groups, we optimize the compatibility between the drug and the electrophilic group within the active site of TXNRD to enhance their non-covalent interaction, thus effectively avoids the poor selectivity deriving from covalent drug interaction, on the basis of ensuring the strong inhibition. Detailed experimental and computational results demonstrate that the chiral isomeric drugs bind to the active site of TXNRD, and the interaction strength is well modulated by chirality. Especially, the meso-configuration, in which the two large sterically hindered active groups are positioned on opposite sides of the drug, exhibits the highest number of non-covalent interactions and most effective inhibition on TXNRD. Taken together, this work not only provides a novel approach for developing highly selective proteinase inhibitors, but also sheds light on possible underlying mechanisms for future application., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Aberrant activation of thioredoxin reductase (TrxR) is correlated with tumor occurrence and progression, suggesting that TrxR inhibitors can be used as antitumor agents. In this study, we evaluated the anticancer efficacy of eupalinilides B on colorectal cancer cells. Eupalinilides B primarily targeted the conserved selenocysteine 498 residues in TrxR. Besides, it inhibited the enzyme activity in an irreversible manner. After eupalinilides B was used to pharmacologically inhibit TrxR, reactive oxygen species accumulated, and the intracellular redox balance was broken, finally causing oxidative stress-induced tumor cell apoptosis. Significantly, eupalinilides B treatment inhibited in vivo tumor growth. Targeting TrxR by eupalinilides B reveals the new mechanism underlying eupalinilides B and provides insight in developing eupalinilides B as the candidate antitumor chemotherapeutic agent for the treatment of cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Antimicrobial resistance (AMR) has been emerging as a major global health threat and calls for the development of novel drug candidates. Metal complexes have been demonstrating high efficiency as antibacterial agents that differ substantially from the established types of antibiotics in their chemical structures and their mechanism of action. One strategy to exploit this potential is the design of metal-based hybrid organometallics that consist of an established antibiotic and a metal-based warhead that contributes an additional mechanism of action different from that of the parent antibiotic. In this communication, we describe the organometallic hybrid antibiotic 2c, in which the drug metronidazole is connected to a gold(I) N-heterocyclic carbene warhead that inhibits bacterial thioredoxin reductase (TrxR). Metronidazole can be used for the treatment with the obligatory anaerobic pathogen Clostridioides difficile (C. difficile), however, resistance to the drug hampers its clinical success. The gold organometallic conjugate 2c was an efficient inhibitor of TrxR and it was inactive or showed only minor effects against eucaryotic cells and bacteria grown under aerobic conditions. In contrast, a strong antibacterial effect was observed against both metronidazole-sensitive and -resistant strains of C. difficile. This report presents a proof-of-concept that the design of metal-based hybrid antibiotics can be a viable approach to efficiently tackle AMR., (© 2024. The Author(s).)

The thioredoxin system is involved in cancer development and therefore is a promising target for cancer chemotherapy. Thioredoxin reductase (TrxR) is a key component of the thioredoxin (Trx) system, and is overexpressed in many cancers to inhibit apoptosis-related proteins. Alternatively, inhibition of thioredoxin reductase and upregulation of apoptosis factors provide a therapeutic strategy for anti-tumor treatment. In this study, an ultrasound-activatable meso -organosilica nanomedicine was prepared by integrating chloroquine (CQ) into hollow mesoporous organosilica (CQ@MOS). The meso -organosilica nanomedicine can inhibit the activity of thioredoxin reductase, elevate cellular reactive oxygen species (ROS) levels, upregulate the pro-apoptotic factors in the c-Jun N-terminal kinase (JNK) apoptosis pathway and induce autophagy inhibition, further resulting in mitochondrial membrane potential (MMP) depolarization and cellular ATP content decrease, ultimately causing significant damage to tumor cells. Moreover, CQ@MOS can efficiently deliver chloroquine into cancer cells and promote an enhanced sonodynamic effect for effective anti-tumor chemotherapy and sonodynamic therapy. This study may enlighten us on a new anti-tumor strategy and suggest its promising applications in cancer treatments.

Using a multigram-scalable synthesis, we obtained nine dinuclear complexes based on nonendogenous iron(I) centers and featuring variable aminocarbyne and P-ligands. One compound from the series ( FEACYP ) emerged for its strong cytotoxicity in vitro against four human cancer cell lines, surpassing the activity of cisplatin by 3-6 times in three cell lines, with an average selectivity index of 6.2 compared to noncancerous HEK293 cells. FEACYP demonstrated outstanding water solubility (15 g/L) and stability in physiological-like solutions. It confirmed its superior antiproliferative activity when tested in 3D spheroids of human pancreatic cancer cells and showed a capacity to inhibit thioredoxin reductase (TrxR) similar to auranofin. In vivo treatment of murine LLC carcinoma with FEACYP (8 mg kg -1 dose) led to excellent tumor growth suppression (88%) on day 15, with no signs of systemic toxicity and only limited body weight loss.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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.

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.

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., (© 2024 John Wiley & Sons Ltd.)

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., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

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., (© 2024. The Author(s).)

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 (IC 50 =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., (© 2023 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and nicotinamide adenine dinucleotide phosphate (NADPH). Spirulina platensis, which is one of the blue-green algae in the form of spiral rings, belongs to the cyanobacteria class. Spirulina platensis can produce Trx under stress conditions. If it can produce Trx, it also has TrxR activity. Therefore, in this study, the TrxR enzyme was purified for the first time from Spirulina platensis, an algae the most grown and also used as a nutritional supplement in the world. A two-step purification process was used: preparation of the homogenate and 2',5'-ADP sepharose 4B affinity chromatography. The enzyme was purified with a purification fold of 1059.51, a recovery yield of 9.7 %, and a specific activity of 5.77 U/mg protein. The purified TrxR was tested for purity by SDS-PAGE. The molecular weight of its subunit was found to be about 45 kDa. Optimum pH, temperature and ionic strength of the enzyme were pH 7.0, 40 °C and 750 mM in phosphate buffer respectively. The Michaelis constant (K m ) and maximum velocity of enzyme (V max ) values for NADPH and 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) are 5 μM and 2.2 mM, and 0.0033 U/mL and 0.0044 U/mL, respectively. Storage stability of the purified enzyme was determined at several temperatures. The inhibition effects of Ag + , Cu 2+ , Al 3+ and Se 4+ metal ions on the purified TrxR activity were investigated in vitro. While Se 4+ ion increased the enzyme activity, other tested metal ions showed different type of inhibitory effects on the Lineweaver-Burk graphs., Competing Interests: Declaration of competing interest Author declare that they have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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 H 2 O 2 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 × 10 2  M -1 s -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 -1 s -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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

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., (© 2023 Wiley Periodicals LLC.)

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.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Anjou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2023 Deutsche Pharmazeutische Gesellschaft.)

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 (K m ) and maximum velocity of enzyme (V max ) values for NADPH and 5,5'-dithiobis(2-nitrobenzoic acid) were determined. Tested metal ions, vitamins, and drugs showed inhibition effects, except Se 4+ ion, cefazolin sodium, teicoplanin, and tobramycin that increased the enzyme activity in vitro. Ag + , Cu 2+ , Mg 2+ , Ni 2+ , Pb 2+ , Zn 2+ , Al 3+ , Cr 3+ , Fe 3+ , and V 4+ ions; vitamin B 3 , vitamin B 6 , 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)., (© 2023 International Union of Biochemistry and Molecular Biology, Inc.)

In the cytosol of human cells under low oxidative loads, hydrogen peroxide is confined to microdomains around its supply sites, due to its fast consumption by peroxiredoxins. So are the sulfenic and disulfide forms of the 2-Cys peroxiredoxins, according to a previous theoretical analysis [Travasso et al., Redox Biology 15 (2017) 297]. Here, an extended reaction-diffusion model that for the first time considers the differential properties of human peroxiredoxins 1 and 2 and the thioredoxin redox cycle predicts important new aspects of the dynamics of redox microdomains. The peroxiredoxin 1 sulfenates and disulfides are more localized than the corresponding peroxiredoxin 2 forms, due to the former peroxiredoxin's faster resolution step. The thioredoxin disulfides are also localized. As the H 2 O 2 supply rate (v sup ) approaches and then surpasses the maximal rate of the thioredoxin/thioredoxin reductase system (V), these concentration gradients become shallower, and then vanish. At low v sup the peroxiredoxin concentration determines the H 2 O 2 concentrations and gradient length scale, but as v sup approaches V, the thioredoxin reductase activity gains influence. A differential mobility of peroxiredoxin disulfide dimers vs. reduced decamers enhances the redox polarity of the cytosol: as v sup approaches V, reduced decamers are preferentially retained far from H 2 O 2 sources, attenuating the local H 2 O 2 buildup. Substantial total protein concentration gradients of both peroxiredoxins emerge under these conditions, and the concentration of reduced peroxiredoxin 1 far from the H 2 O 2 sources even increases with v sup . Altogether, the properties of 2-Cys peroxiredoxins and thioredoxin are such that localized H 2 O 2 supply induces a redox and functional polarization between source-proximal regions (redox microdomains) that facilitate peroxiredoxin-mediated signaling and distal regions that maximize antioxidant protection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

During photosynthesis, plants must manage strong fluctuations in light availability on different time scales, leading to long-term acclimation and short-term responses. However, little is known about the regulation and coordination of these processes and the modulators involved. In this study, we used proteomics, metabolomics, and reverse genetics to investigate how different light environmental factors, such as intensity or variability, affect long-term and short-term acclimation responses of Arabidopsis (Arabidopsis thaliana) and the importance of the chloroplast redox network in their regulation. In the wild type, high light, but not fluctuating light, led to large quantitative changes in the proteome and metabolome, accompanied by increased photosynthetic dynamics and plant growth. This finding supports light intensity as a stronger driver for acclimation than variability. Deficiencies in NADPH-thioredoxin reductase C (NTRC) or thioredoxins m1/m2, but not thioredoxin f1, almost completely suppressed the re-engineering of the proteome and metabolome, with both the induction of proteins involved in stress and redox responses and the repression of those involved in cytosolic and plastid protein synthesis and translation being strongly attenuated. Moreover, the correlations of protein or metabolite levels with light intensity were severely disturbed, suggesting a general defect in the light-dependent acclimation response, resulting in impaired photosynthetic dynamics. These results indicate a previously unknown role of NTRC and thioredoxins m1/m2 in modulating light acclimation at proteome and metabolome levels to control dynamic light responses. NTRC, but not thioredoxins m1/m2 or f1, also improves short-term photosynthetic responses by balancing the Calvin-Benson cycle in fluctuating light., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Thioredoxin (Trx) is a disulfide-containing redox protein that functions as a disulfide oxidoreductase. Myxococcus xanthus contains five Trxs (Trx1-Trx5) and one Trx reductase (TrxR). Trxs typically have a CGPC active-site motif; however, M. xanthus Trxs have slightly different active-site sequences, with the exception of Trx4. The five Trxs of M. xanthus exhibited reduced activities against insulin, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), cystine, glutathione disulfide (GSSG), S-nitrosoglutathione (GSNO), and H2O2 in the presence of TrxR. Myxococcus xanthus adenylate kinase and serine/threonine phosphatase activities, which were increased by the addition of dithiothreitol, were activated by the addition of Trxs and TrxR. Among these, Trx1, which has a CAPC sequence in its active site, exhibited the highest reducing activity with the exception of GSNO. Myxococcus xanthus TrxR showed weak reducing activity towards DTNB, GSSG, GSNO, and H2O2, suggesting that it has broad substrate specificity, unlike previously reported low-molecular-weight TrxRs. TrxR reduced oxidized Trx1 as the best substrate, with a kcat/Km value of 0.253 min-1 µM-1, which was 10-28-fold higher than that of the other Trxs. These results suggest that all Trxs possess reducing activity and that Trx1 may be the most functional in M. xanthus because TrxR most efficiently reduces oxidized Trx1., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Alzheimer's disease (AD) is the most common form of dementia and a public health problem. It exhibits significant oxidative stress and redox alterations. The antioxidant enzyme systems defend the cellular environment from oxidative stress. One of the redox systems is the thioredoxin system (TS), which exerts decisive control over the cellular redox environment. We aimed to review the protective effects of TS, which include thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH. In the following, we discussed the physiological functioning and the role of the TS in maintaining the cellular redox-homeostasis in the AD-damaged brain. Trx protects the cellular environment from oxidative stress, while TrxR is crucial for the cellular detoxification of reactive oxygen species in the brain. However, TS dysregulation increases the susceptibility to cellular death. The changes in Trx and TrxR levels are significantly associated with AD progression. Though the data from human, animal, and cellular models support the neuroprotective role of TS in the brain of AD patients, the translational potential of these findings to clinical settings is not yet applied. This review summarizes the current knowledge on the emerging role of the TrxR-Trx system in AD.

The review considers modern data on the mechanisms of activation and redox regulation of the NLRP3 inflammasome and gasdermins, as well as the role of selenium in these processes. Activation of the inflammasome and pyroptosis represent an evolutionarily conserved mechanism of the defense against pathogens, described for various types of cells and tissues (macrophages and monocytes, microglial cells and astrocytes, podocytes and parenchymal cells of the kidneys, periodontal tissues, osteoclasts and osteoblasts, as well as cells of the digestive and urogenital systems, etc.). Depending on the characteristics of redox regulation, the participants of NLRP3 inflammation and pyroptosis can be subdivided into 2 groups. Members of the first group block the mitochondrial electron transport chain, promote the formation of reactive oxygen species and the development of oxidative stress. This group includes granzymes, the mitochondrial antiviral signaling protein MAVS, and others. The second group includes thioredoxin interacting protein (TXNIP), erythroid-derived nuclear factor-2 (NRF2), Kelch-like ECH-associated protein 1 (Keap1), ninjurin (Ninj1), scramblase (TMEM16), inflammasome regulatory protein kinase NLRP3 (NEK7), caspase-1, gasdermins GSDM B, D and others. They have redox-sensitive domains and/or cysteine residues subjected to redox regulation, glutathionylation/deglutathionylation or other types of regulation. Suppression of oxidative stress and redox regulation of participants in NLRP3 inflammation and pyroptosis depends on the activity of the antioxidant enzymes glutathione peroxidase (GPX) and thioredoxin reductase (TRXR), containing a selenocysteine residue Sec in the active site. The expression of GPX and TRXR is regulated by NRF2 and depends on the concentration of selenium in the blood. Selenium deficiency causes ineffective translation of the Sec UGA codon, translation termination, and, consequently, synthesis of inactive selenoproteins, which can cause various types of programmed cell death: apoptosis of nerve cells and sperm, necroptosis of erythrocyte precursors, pyroptosis of infected myeloid cells, ferroptosis of T- and B-lymphocytes, kidney and pancreatic cells. In addition, suboptimal selenium concentrations in the blood (0.86 μM or 68 μg/l or less) have a significant impact on expression of more than two hundred and fifty genes as compared to the optimal selenium concentration (1.43 μM or 113 μg/l). Based on the above, we propose to consider blood selenium concentrations as an important parameter of redox homeostasis in the cell. Suboptimal blood selenium concentrations (or selenium deficiency states) should be used for assessment of the risk of developing inflammatory processes.

TXNL1 (also named TRP32, for thioredoxin related protein of 32 kDa) is a cytosolic thioredoxin-fold protein expressed in all cell types and conserved from yeast to mammals, but with yet poorly known function. Here, we expressed and purified human TXNL1 together with several Cys-to-Ser variants, characterizing their enzymatic properties. TXNL1 could reduce disulfides in insulin, cystine and glutathione disulfide (GSSG) in reactions coupled to thioredoxin reductase (TXNRD1, TrxR1) using NADPH, similarly to thioredoxin (TXN, Trx1), but with lower catalytic efficacy due to at least one order of magnitude higher K m of TrxR1 for TXNL1 compared to Trx1. However, in sharp contrast to Trx1, we found that TXNL1 also had efficient chaperone activity that did not require ATP. TXNL1 made non-covalent complexes with reduced insulin, thereby keeping it in solution, and TXNL1 provided chaperone function towards whole cell lysate proteins by preventing their aggregation during heating. The chaperone activities of TXNL1 did not require its redox activity or any dithiol-disulfide exchange reactions, as revealed using Cys-to-Ser substituted variants, as well as a maintained chaperone activity of TXNL1 also in the absence of TrxR1 and NADPH. These results reveal that TXNL1 has dual functions, supporting TrxR1-driven redox activities in disulfide reduction reactions, as well as being an ATP-independent chaperone that does not require involvement of its redox activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Selenium is an essential trace element in living organisms, and is present in selenoenzymes with antioxidant activity, like glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). The search for small selenium-containing molecules that mimic selenoenzymes is a strong field of research in organic and medicinal chemistry. In this review, we review the synthesis and bioassays of new and known organoselenium compounds with antioxidant activity, covering the last five years. A detailed description of the synthetic procedures and the performed in vitro and in vivo bioassays is presented, highlighting the most active compounds in each series.

Calredoxin (CRX) is a calcium (Ca2+)-dependent thioredoxin (TRX) in the chloroplast of Chlamydomonas (Chlamydomonas reinhardtii) with a largely unclear physiological role. We elucidated the CRX functionality by performing in-depth quantitative proteomics of wild-type cells compared with a crx insertional mutant (IMcrx), two CRISPR/Cas9 KO mutants, and CRX rescues. These analyses revealed that the chloroplast NADPH-dependent TRX reductase (NTRC) is co-regulated with CRX. Electron transfer measurements revealed that CRX inhibits NADPH-dependent reduction of oxidized chloroplast 2-Cys peroxiredoxin (PRX1) via NTRC and that the function of the NADPH-NTRC complex is under strict control of CRX. Via non-reducing SDS-PAGE assays and mass spectrometry, our data also demonstrated that PRX1 is more oxidized under high light (HL) conditions in the absence of CRX. The redox tuning of PRX1 and control of the NADPH-NTRC complex via CRX interconnect redox control with active photosynthetic electron transport and metabolism, as well as Ca2+ signaling. In this way, an economic use of NADPH for PRX1 reduction is ensured. The finding that the absence of CRX under HL conditions severely inhibited light-driven CO2 fixation underpins the importance of CRX for redox tuning, as well as for efficient photosynthesis., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The thioredoxin pathway is an antioxidant system present in most organisms. Electrons flow from a thioredoxin reductase to thioredoxin at the expense of a specific electron donor. Most known thioredoxin reductases rely on NADPH as a reducing cofactor. Yet, in 2016, a new type of thioredoxin reductase was discovered in Archaea which utilize instead a reduced deazaflavin cofactor (F 420 H 2 ). For this reason, the respective enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). To have a broader understanding of the biochemistry of DFTRs, we identified and characterized two other archaeal representatives. A detailed kinetic study, which included pre-steady state kinetic analyses, revealed that these two DFTRs are highly specific for F 420 H 2 while displaying marginal activity with NADPH. Nevertheless, they share mechanistic features with the canonical thioredoxin reductases that are dependent on NADPH (NTRs). A detailed structural analysis led to the identification of two key residues that tune cofactor specificity of DFTRs. This allowed us to propose a DFTR-specific sequence motif that enabled for the first time the identification and experimental characterization of a bacterial DFTR., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The increasing number of people dying from tuberculosis and the existence of extensively drug-resistant strains has led to an urgent need for new antituberculotic drugs with alternative modes of action. As part of the thioredoxin system, thioredoxin reductase (TrxR) is essential for the survival of Mycobacterium tuberculosis (Mtb) and shows substantial differences from human TrxR, making it a promising and most likely selective target. As a model organism for Mtb, crystals of Mycobacterium smegmatis TrxR that diffracted to high resolution were used in crystallographic fragment screening to discover binding fragments and new binding sites. The application of the 96 structurally diverse fragments from the F2X-Entry Screen revealed 56 new starting points for fragment-based drug design of new TrxR inhibitors. Over 200 crystal structures were analyzed using FragMAXapp, which includes processing and refinement by largely automated software pipelines and hit identification via the multi-data-set analysis approach PanDDA. The fragments are bound to 11 binding sites, of which four are positioned at binding pockets or important interaction sites and therefore show high potential for possible inhibition of TrxR., (open access.)

NADPH-dependent thioredoxin reductase C (NTRC) is a chloroplast redox regulator in algae and plants. Here, we used site-specific mutation analyses of the thioredoxin domain active site of NTRC in the green alga Chlamydomonas reinhardtii to show that NTRC mediates cold tolerance in a redox-dependent manner. By means of coimmunoprecipitation and mass spectrometry, a redox- and cold-dependent binding of the Calvin-Benson Cycle Protein 12 (CP12) to NTRC was identified. NTRC was subsequently demonstrated to directly reduce CP12 of C. reinhardtii as well as that of the vascular plant Arabidopsis thaliana in vitro. As a scaffold protein, CP12 joins the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to form an autoinhibitory supracomplex. Using size-exclusion chromatography, NTRC from both organisms was shown to control the integrity of this complex in vitro and thereby PRK and GAPDH activities in the cold. Thus, NTRC apparently reduces CP12, hence triggering the dissociation of the PRK/CP12/GAPDH complex in the cold. Like the ntrc::aphVIII mutant, CRISPR-based cp12::emx1 mutants also exhibited a redox-dependent cold phenotype. In addition, CP12 deletion resulted in robust decreases in both PRK and GAPDH protein levels implying a protein protection effect of CP12. Both CP12 functions are critical for preparing a repertoire of enzymes for rapid activation in response to environmental changes. This provides a crucial mechanism for cold acclimation.

NADPH:thioredoxin reductase (TrxR) is considered a potential target for anticancer agents. Several nitroheterocyclic sulfones, such as Stattic and Tri-1, irreversibly inhibit TrxR, which presumably accounts for their antitumor activity. However, it is necessary to distinguish the roles of enzymatic redox cycling, an inherent property of nitroaromatics (ArNO 2 ), and the inhibition of TrxR in their cytotoxicity. In this study, we calculated the previously unavailable values of single-electron reduction potentials of known inhibitors of TrxR (Stattic, Tri-1, and 1-chloro-2,4-dinitrobenzene (CDNB)) and inhibitors identified (nitrofuran NSC697923 and nitrobenzene BTB06584). These calculations were according to the rates of their enzymatic single-electron reduction (PMID: 34098820). This enabled us to compare their cytotoxicity with that of model redox cycling ArNO 2 . In MH22a and HCT-116 cells, Tri-1, Stattic, CDNB, and NSC697023 possessed at least 10-fold greater cytotoxicity than can be expected from their redox cycling activity. This may be related to TrxR inhibition. The absence of enhanced cytotoxicity in BTB06548 may be attributed to its instability. Another known inhibitor of TrxR, tetryl, also did not possess enhanced cytotoxicity, probably because of its detoxification by DT-diaphorase (NQO1). Apart from the reactions with NQO1, the additional mechanisms influencing the cytotoxicity of the examined inhibitors of TrxR are their reactions with cytochromes P-450. Furthermore, some inhibitors, such as Stattic and NSC697923, may also inhibit glutathione reductase. We suggest that these data may be instrumental in the search for TrxR inhibitors with enhanced cytotoxic/anticancer activity.

Plants contain three NADPH-thioredoxin reductases (NTR) located in the cytosol/mitochondria (NTRA/B) and the plastid (NTRC) with important metabolic functions. However, mutants deficient in all NTRs remained to be investigated. Here, we generated and characterised the triple Arabidopsis ntrabc mutant alongside with ntrc single and ntrab double mutants under different environmental conditions. Both ntrc and ntrabc mutants showed reduced growth and substantial metabolic alterations, especially in sink leaves and under high CO 2 (HC), as compared to the wild type. However, ntrabc showed higher effective quantum yield of PSII under both constant and fluctuating light conditions, altered redox states of NADH/NAD + and glutathione (GSH/GSSG) and lower potential quantum yield of PSII in sink leaves in ambient but not high CO 2 concentrations, as compared to ntrc, suggesting a functional interaction between chloroplastic and extra-chloroplastic NTRs in photosynthesis regulation depending on leaf development and environmental conditions. Our results unveil a previously unknown role of the NTR system in regulating sink leaf metabolism and plant acclimation to HC, while it is not affecting full plant development, indicating that the lack of the NTR system can be compensated, at least to some extent, by other redox mechanisms., (© 2023 John Wiley & Sons Ltd.)

The Se -nitrosation in selenoproteins such as glutathione peroxidase and thioredoxin reductase to produce Se -nitrososelenocysteines (Sec-SeNOs) has been proposed to play crucial roles in signaling processes mediated by reactive nitrogen species and nitrosative-stress responses, although chemical evidence for the formation of Sec-SeNOs has been elusive not only in proteins but also in small-molecule systems. Herein, we report the first synthesis of a Sec-SeNO by employing a selenocysteine model system that bears a protective molecular cradle. The Sec-SeNO was characterized using 1 H and 77 Se nuclear magnetic resonance as well as ultraviolet/visible spectroscopy and found to have persistent stability at room temperature in solution. The reaction processes involving the Sec-SeNO provide experimental information that serves as a chemical basis for elucidating the reaction mechanisms involving the SeNO species in biological functions, as well as in selenol-catalyzed NO generation from S -nitrosothiols.

Oxidizing signals mediated by the thiol-dependent peroxidase activity of 2-Cys peroxiredoxins (PRXs) plays an essential role in fine-tuning chloroplast redox balance in response to changes in light intensity, a function that depends on NADPH-dependent thioredoxin reductase C (NTRC). In addition, plant chloroplasts are equipped with glutathione peroxidases (GPXs), thiol-dependent peroxidases that rely on thioredoxins (TRXs). Despite having a similar reaction mechanism than 2-Cys PRXs, the contribution of oxidizing signals mediated by GPXs to the chloroplast redox homeostasis remains poorly known. To address this issue, we have generated the Arabidopsis (Arabidopsis thaliana) double mutant gpx1gpx7, which is devoid of the two GPXs, 1 and 7, localized in the chloroplast. Furthermore, to analyze the functional relationship of chloroplast GPXs with the NTRC-2-Cys PRXs redox system, the 2cpab-gpx1gpx7 and ntrc-gpx1gpx7 mutants were generated. The gpx1gpx7 mutant displayed wild type-like phenotype indicating that chloroplast GPXs are dispensable for plant growth at least under standard conditions. However, the 2cpab-gpx1gpx7 showed more retarded growth than the 2cpab mutant. The simultaneous lack of 2-Cys PRXs and GPXs affected PSII performance and caused higher delay of enzyme oxidation in the dark. In contrast, the ntrc-gpx1gpx7 mutant combining the lack of NTRC and chloroplast GPXs behaved like the ntrc mutant indicating that the contribution of GPXs to chloroplast redox homeostasis is independent of NTRC. Further supporting this notion, in vitro assays showed that GPXs are not reduced by NTRC but by TRX y2. Based on these results, we propose a role for GPXs in the chloroplast redox hierarchy., Competing Interests: Declaration of competing interest The authors have delcared that there is no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Thioredoxin reductase (TrxR) has been considered a potential target for cancer chemotherapy. It acts by controlling the redox homeostasis of human cells and, therefore, interfering in its function may trigger apoptosis, which is a crucial tumor suppression mechanism. Despite the great effort in the search for TrxR inhibitors, none was approved for human therapy. In the present study a virtual screening for natural organic compounds is discussed for a set of 72 compounds with known IC-50 for TrxR inhibition. The results suggest the Pleurotin, a naphthoquinone obtained from Hohenbuehelia grisea fungus, as a potential TrxR inhibitor, which acts by binding to the active site of the enzyme, between the N- and C-terminal domains. The presence of the ligand blocks the approximation of the C-terminal arm to the N-terminal, which is an essential step of the enzyme function. Besides, the two equivalent binding sites of TrxR were explored, by docking two ligands simultaneously. The results indicate that both sites have an allosteric correlation and, the presence of the ligand in one site may interfere, or even prevent, the binding of the second ligand at the other site. All these findings are quantitatively discussed based on the analysis of long molecular dynamics trajectories, which provides a full description of the ligand-receptor binding modes, average binding energies and conformational changes.Communicated by Ramaswamy H. Sarma.

Redox regulation of plastid gene expression and different metabolic pathways promotes many activities of redox-sensitive proteins. We address the question of how the plastid redox state and the contributing reducing enzymes control the enzymes of tetrapyrrole biosynthesis (TBS). In higher plants, this metabolic pathway serves to produce chlorophyll and heme, among other essential end products. Because of the strictly light-dependent synthesis of chlorophyll, tight control of TBS requires a diurnal balanced supply of the precursor 5-aminolevulinic acid (ALA) to prevent the accumulation of photoreactive metabolic intermediates in darkness. We report on some TBS enzymes that accumulate in a light intensity-dependent manner, and their contents decrease under oxidizing conditions of darkness, low light conditions, or in the absence of NADPH-dependent thioredoxin reductase (NTRC) and thioredoxin f1 (TRX-f1). Analysis of single and double trxf1 and ntrc mutants revealed a decreased content of the early TBS enzymes glutamyl-tRNA reductase (GluTR) and 5-aminolevulinic acid dehydratase (ALAD) instead of an exclusive decrease in enzyme activity. This effect was dependent on light conditions and strongly attenuated after transfer to high light intensities. Thus, it is suggested that a deficiency of plastid-localized thiol-redox transmitters leads to enhanced degradation of TBS enzymes rather than being directly caused by lower catalytic activity. The effects of the proteolytic activity of the Clp protease on TBS enzymes were studied by using Clp subunit-deficient mutants. The simultaneous lack of TRX and Clp activities in double mutants confirms the Clp-induced degradation of some TBS proteins in the absence of reductive activity of TRXs. In addition, we verified previous observations that decreased chlorophyll and heme levels in ntrc could be reverted to WT levels in the ntrc/Δ2cp triple mutant. The decreased synthesis of 5-aminolevulinic acid and porphobilinogen in ntrc was completely restored in ntrc/Δ2cp and correlated with WT-like levels of GluTR, ALAD, and other TBS proteins.

The redox couple formed by NADPH-dependent thioredoxin reductase C (NTRC) and 2-Cys peroxiredoxins (Prxs) allows fine-tuning chloroplast performance in response to light intensity changes. Accordingly, the Arabidopsis 2cpab mutant lacking 2-Cys Prxs shows growth inhibition and sensitivity to light stress. However, this mutant also shows defective post-germinative growth, suggesting a relevant role of plastid redox systems in seed development, which is so far unknown. To address this issue, we first analyzed the pattern of expression of NTRC and 2-Cys Prxs in developing seeds. Transgenic lines expressing GFP fusions of these proteins showed their expression in developing embryos, which was low at the globular stage and increased at heart and torpedo stages, coincident with embryo chloroplast differentiation, and confirmed the plastid localization of these enzymes. The 2cpab mutant produced white and abortive seeds, which contained lower and altered composition of fatty acids, thus showing the relevance of 2-Cys Prxs in embryogenesis. Most embryos of white and abortive seeds of the 2cpab mutant were arrested at heart and torpedo stages of embryogenesis suggesting an essential function of 2-Cys Prxs in embryo chloroplast differentiation. This phenotype was not recovered by a mutant version of 2-Cys Prx A replacing the peroxidatic Cys by Ser. Neither the lack nor the overexpression of NTRC had any effect on seed development indicating that the function of 2-Cys Prxs at these early stages of development is independent of NTRC, in clear contrast with the operation of these regulatory redox systems in leaves chloroplasts., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Main Conclusion: Arabidopsis GR1 and NTRA function in pollen tube penetrating the stigma into the transmitting tract during pollination. During pollination, recognition between pollen (tube) and stigma mediates the hydration and germination of pollen, as well as the growth of the pollen tube on the stigma. Arabidopsis glutathione reductase 1 (GR1) and NADPH-dependent thioredoxin reductase A (NTRA) are involved in regulating cell redox hemostasis. Both GR1 and NTRA are expressed in pollen, but their roles in pollen germination and the growth of the pollen tube need further investigation. In this study, we performed pollination experiments and found that the Arabidopsis gr1/ + ntra/- and gr1/- ntra/ + double mutation compromised the transmission of male gametophytes. Pollen morphology and viability of the mutants did not show obvious abnormalities. Additionally, the pollen hydration and germination of the double mutants on solid pollen germination medium were comparable to those of the wild type. However, the pollen tubes with gr1 ntra double mutation were unable to penetrate the stigma and enter the transmitting tract when they grew on the surface of the stigma. Our results indicate that GR1 and NTRA play a role in regulating the interaction between the pollen tube and the stigma during pollination., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Thioredoxin/glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both oxidized thioredoxin and glutathione with electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH). SmTGR is a drug target for the treatment of Schistosomiasis, an infection caused by Schistosoma platyhelminths residing in the blood vessels of the host. Schistosoma spp. are reliant on TGR enzymes as they lack catalase and so use reduced thioredoxin and glutathione to regenerate peroxiredoxins consumed in the detoxification of reactive oxygen species. SmTGR is a flavin adenine dinucleotide (FAD)-dependent enzyme, and we have used the flavin as a spectrophotometric reporter to observe the movement of electrons within the enzyme. The data show that NADPH fractionally reduces the active site flavin with an observed rate constant estimated in this study to be ∼3000 s -1 . The flavin then reoxidizes by passing electrons at a similar rate to the proximal Cys159-Cys154 disulfide pair. The dissociation of NADP + occurs with a rate of ∼180 s -1 , which induces the deprotonation of Cys159, and this coincides with the accumulation of an intense FAD-thiolate charge transfer band. It is proposed that the electrons then pass to the Cys596-Cys597 disulfide pair of the associated subunit in the dimer with a net rate constant of ∼2 s -1 . (Note: Cys597 is Sec597 in wild-type (WT) SmTGR.) From this position, the electrons can be passed to oxidized thioredoxin or further into the protein to reduce the Cys28-Cys31 disulfide pair of the originating subunit of the dimer. From the Cys28-Cys31 center, electrons can then pass to oxidized glutathione that has a binding site directly adjacent.