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Currently, the application of synthetic biology to artificially manipulate and utilize organisms for the synthesis of desired products such as nanomaterials with excellent fluorescence properties is attracting considerable attention. However, it is still difficult to obtain designed products efficiently due to insufficient knowledge of the biosynthetic mechanisms. The thioredoxin (TRX) and glutathione (GSH) pathways are generally conserved thiol-reductase systems that protect organisms from oxidative stress and are involved in selenium (Se) metabolism. In this study, we revealed the pivotal role of cytoplasmic TRX pathway in regulating the metabolism of Na2SeO3 during the live-cell synthesis of cadmium-selenium quantum dots (CdSe QDs) in Saccharomyces cerevisiae by regulating the expression level of genes related to TRX pathway and measuring the intracellular content of selenocysteine (SeCys). The determination of SeCys metabolism in yeast with GSH pathway-related genes deleted demonstrated that the TRX pathway played a more significant role in SeCys metabolism than GSH pathway. A 6.4-fold enhancement in the synthetic yield of CdSe QDs was achieved through the overexpression of TRX pathway-related genes, improvement of synthetic procedure, and supplementation of GSH based on the understanding of biological metabolism. Exploring the mechanism of CdSe QDs live-cell synthesis facilitates the precise manipulation of biological processes for the synthesis of inorganic nanomaterials. [ABSTRACT FROM AUTHOR]

The antitumor activity of various gold compounds is a promising field of investigation, attracting researchers seeking potential clinical candidates. To advance this research, they explore the complex mechanisms of action of these compounds. Since the discovery of the strong inhibition of thioredoxin reductase by auranofin, the primary mechanism explored has been the inhibition of this enzyme. This inhibition disrupts the redox balance in cells, promoting oxidative stress and triggering cell death. In this review, we analyzed studies from the past decade that measured cellular ROS increase and examined the coordination structures of gold compounds. We also correlate ROS increase with the inhibition of redox‐regulating enzymes, thioredoxin reductase, and glutathione reductase, to elucidate the relationship between these cellular effects and chemical structures. Our data compilation reveals that different structures exhibit varying efficacy: some significantly increase ROS production and inhibit thioredoxin reductase or glutathione reductase, while others elevate ROS levels without affecting these target enzymes, suggesting alternative mechanisms of action. This review consolidates critical evidence, enhancing our understanding of the mechanisms by which these gold complexes act and providing valuable insights for developing new therapeutic strategies against tumor cells. [ABSTRACT FROM AUTHOR]

Introduction Methods Results Conclusions Filifactor alocis is a newly appreciated member of the periodontal community with a strong periodontal disease correlation. Little is known about the survival mechanisms by which F. alocis copes with oxidative stress and establishes the infection within the local inflammatory microenvironment of the periodontal pocket. The aim of this study is to investigate if F. alocis putative peroxiredoxin/AhpC protein FA768 may constitute an alkyl hydroperoxide reductase system utilizing putative thioredoxin reductase protein FA608, and putative thioredoxin/glutaredoxin homolog FA1411/FA455.FA768, FA608, FA1411 and FA455 proteins from F. alocis were expressed and purified from Escherichia coli. Insulin and 5,5‐dithio‐bis‐2‐nitrobenzoic acid (DTNB) reduction assays were performed to determine if purified FA1411 and FA455 proteins could be a substrate for FA608. The peroxidase activity of FA768 was examined by measuring its ability to reduce hydrogen peroxide (H2O2) with FA608 and FA1411/FA455 provided as the reducing systems. Further, the hydroperoxide substrate specificity of FA768 was analyzed by monitoring the NADPH oxidation in the presence of different peroxides, including H2O2, cumyl hydroperoxide (CHP), and tert‐butyl hydroperoxide (t‐BHP).In this study, we have demonstrated the existence of a functioning thioredoxin‐dependent alkyl hydroperoxide system in F. alocis. This system is comprised of a thioredoxin reductase (FA608), a thioredoxin/glutaredoxin homolog (FA1411/FA455), and a typical 2‐cysteine peroxiredoxin/AhpC (FA768). FA608, together with FA1411/FA455, can function as a thioredoxin reductase system to reduce insulin, DTNB, and FA768. FA455 is a glutaredoxin‐like protein with thioredoxin functions in F. alocis. Both the FA768/FA608/FA1411 and FA768/FA608/FA455 reductase systems were NADPH‐dependent and exhibited specificity for broad hydroperoxide substrates H2O2, CHP, and t‐BHP.This is the first study of a thioredoxin dependent alkyl hydroperoxide system from a periodontal pathogen. This system is proposed to protect F. alocis against oxidative stress due to the likely absence of a catalase or an additional peroxiredoxin homolog. [ABSTRACT FROM AUTHOR]

Thioredoxin reductase 1 (TXNRD1) has been identified as one of the promising chemotherapeutic targets in cancer cells. Therefore, a novel TXNRD1 inhibitor could accelerate chemotherapy in clinical anticancer research. In this study, glaucocalyxin A (GlauA), a natural diterpene extracted from Rabdosia japonica var. glaucocalyx, was identified as a novel inhibitor of TXNRD1. We found that GlauA effectively inhibited recombinant TXNRD1 and reduced its activity in gastric cancer cells without affecting the enzyme's expression level. Mechanistically, the selenocysteine residue (U498) of TXNRD1 was irreversibly modified by GlauA through a Michael addition. Additionally, GlauA formed a covalent adduct with glutathione (GSH) and disrupted cellular redox balance by depleting cellular GSH. The inhibition of TXNRD1 and depletion of GSH by GlauA conferred its cytotoxic effects in spheroid culture and Transwell assays in AGS cells. The disulfide stress induced cytotoxicity of GlauA could be mitigated by adding reducing agents, such as DTT and β‐ME. Furthermore, the FDA‐approval drug auranofin, a TXNRD1 inhibitor, triggered oligomerization of the cytoskeletal protein Talin‐1 in AGS cells, indicating that inhibiting TXNRD1 triggered disulfide stress. In conclusion, this study uncovered GlauA as an efficient inhibitor of TXNRD1 and demonstrated the potential of TXNRD1 inhibition as an effective anticancer strategy by disrupting redox homeostasis and inducing disulfide stress. [ABSTRACT FROM AUTHOR]

State transitions are an acclimatory response by which plants, algae, and cyanobacteria counteract photosynthetic inefficiency caused by changes in incident light quality. In plants and green algae, state transition 7 (STN7/STT7) kinase promotes state 2 transition. Conserved cysteine residues are implicated in STN7/STT7 regulation, but the precise nature of their involvement remains unclear. Here, an analysis of the STN7 thiols in vitro and a determination of their midpoint redox potential indicate that the lumenal disulfide linkage is unlikely to be redox regulated while the stromal cysteines form a regulatory intramolecular disulfide. We further show that thioredoxin f1 (Trx‐f1) reduces the STN7 stromal disulfide linkage as consistent with a Trx‐f1‐mediated inhibition of the kinase under high light. [ABSTRACT FROM AUTHOR]

Simple Summary: The utilisation of oxygen for cellular respiration and other metabolic pathways in aerobic organisms is inherently associated with the production of reactive oxygen species, a group of highly reactive and oxidising small molecules. Reactive oxygen species can cause oxidative modifications of biomolecules and cellular components, thus compromising their native structure and biological functions. The role of the antioxidant defence is to tightly control levels of reactive species by connecting diverse antioxidant small molecules and antioxidant enzymes. This review summarises the actual knowledge on the role of the crucial part of the antioxidant system in insects termed the thioredoxin system. It is composed of redox-active protein thioredoxin, which interacts with and reduces the oxidised forms of multiple proteins. It is assisted by the enzyme thioredoxin reductase, catalysing the reductive regeneration of thioredoxins. Recent advances have uncovered how the functions of the thioredoxin system extend beyond its primary function in controlling the cell redox state to diverse developmental processes and the regulation of stress and immune responses. Increased levels of reactive oxygen species (ROS) produced during aerobic metabolism in animals can negatively affect the intracellular redox status, cause oxidative stress and interfere with physiological processes in the cells. The antioxidant defence regulates ROS levels by interplaying diverse enzymes and non-enzymatic metabolites. The thioredoxin system, consisting of the enzyme thioredoxin reductase (TrxR), the redox-active protein thioredoxin (Trx) and NADPH, represent a crucial component of antioxidant defence. It is involved in the signalling and regulation of multiple developmental processes, such as cell proliferation or apoptotic death. Insects have evolved unique variations of TrxR, which resemble mammalian enzymes in overall structure and catalytic mechanisms, but the selenocysteine–cysteine pair in the active site is replaced by a cysteine–cysteine pair typical of bacteria. Moreover, the role of the thioredoxin system in insects is indispensable due to the absence of glutathione reductase, an essential enzyme of the glutathione system. However, the functions of the Trx system in insects are still poorly characterised. In the present review, we provide a critical overview of the current knowledge on the insect Trx system, focusing mainly on TrxR's role in the antioxidant and immune system of model insect species. [ABSTRACT FROM AUTHOR]

Objective: Thioredoxin (TRX), a ubiquitous protein with strong antioxidant activity, decreases in the skin with age. A decrease in TRX is expected to induce cellular senescence, chronic inflammation, and degeneration and loss of extracellular matrix (ECM), such as collagen and elastin within the skin. In this study, we investigated the effects of TRX addition to excised skin or skin models to understand the role of TRX on cells and ECM within the skin. Methods: To evaluate its effect on skin cells, we cultured a three‐dimensional (3D) skin model in a medium containing TRX. The mRNA expression levels of proteins related to elastic and collagen fibres and the basement membrane were determined. Furthermore, 3D imaging and computational analysis were performed to evaluate the effect of TRX on the elastic fibres and extending COL VII structures in excised human skin after coculturing with TRX for 1, 4, 5 and 6 days. Results: Thioredoxin application to a 3D skin model upregulated elastin, COLI and COLVII mRNA expression. Applying TRX to the excised skin increased the number of linear elastic fibres. This effect of TRX demonstrated a daily increment in a dose‐dependent manner. Thioredoxin extended the fibrous structure of COL VII into the dermis, expanding its colocalization region with elastic fibres. These structural effects were confirmed using 3D imaging and computational methods. Conclusion: Thioredoxin elongates elastic fibres from the dermis to the basement membrane and extends the COL VII structure from the basement membrane to the dermis in excised human skin. These findings suggest the potential of TRX to protect the skin against age‐related alterations such as wrinkles and sagging. [ABSTRACT FROM AUTHOR]

Background: Poor survival outcomes in periampullary cancer highlight the need for improvement in biomarkers and the development of novel therapies. Redox proteins, including the thioredoxin system, play vital roles in cellular antioxidant systems. Methods: In this retrospective study, thioredoxin (Trx), thioredoxin-interacting protein (TxNIP), and thioredoxin reductase (TrxR) protein expression was assessed in 85 patients with pancreatic ductal adenocarcinoma (PDAC) and 145 patients with distal bile duct or ampullary carcinoma using conventional immunohistochemistry. Results: In patients with PDAC, high cytoplasmic TrxR expression was significantly associated with lymph node metastasis (p = 0.033). High cytoplasmic and nuclear Trx expression was significantly associated with better overall survival (p = 0.018 and p = 0.006, respectively), and nuclear Trx expression remained significant in multivariate Cox regression analysis (p < 0.0001). In distal bile duct and ampullary carcinomas, high nuclear TrxR expression was associated with vascular (p = 0.001) and perineural (p = 0.021) invasion, and low cytoplasmic TxNIP expression was associated with perineural invasion (p = 0.025). High cytoplasmic TxNIP expression was significantly associated with better overall survival (p = 0.0002), which remained significant in multivariate Cox regression analysis (p = 0.013). Conclusions: These findings demonstrate the prognostic importance of Trx system protein expression in periampullary cancers. [ABSTRACT FROM AUTHOR]

Background: Selenium and zinc are essential trace elements known to regulate cellular processes including redox homeostasis. During inflammation, circulating selenium and zinc concentrations are reduced in parallel, but underlying mechanisms are unknown. Accordingly, we modulated the zinc and selenium supply of HepG2 cells to study their relationship. Methods: HepG2 cells were supplied with selenite in combination with a short- or long-term zinc treatment to investigate intracellular concentrations of selenium and zinc together with biomarkers describing their status. In addition, the activation of the redox-sensitive transcription factor NRF2 was analyzed. Results: Zinc not only increased the nuclear translocation of NRF2 after 2 to 6 h but also enhanced the intracellular selenium content after 72 h, when the cells were exposed to both trace elements. In parallel, the activity and expression of the selenoprotein thioredoxin reductase 1 (TXNRD1) increased, while the gene expression of other selenoproteins remained unaffected or was even downregulated. The zinc effects on the selenium concentration and TXNRD activity were reduced in cells with stable NRF2 knockdown in comparison to control cells. Conclusions: This indicates a functional role of NRF2 in mediating the zinc/selenium crosstalk and provides an explanation for the observed unidirectional behavior of selenium and zinc. [ABSTRACT FROM AUTHOR]

Background Methods Results Conclusions Epithelial‐mesenchymal transition (EMT) is central to HCC metastasis, in which RNA‐binding proteins (RBPs) play a key role.To explore the role of RBPs in metastasis of hepatocellular carcinoma (HCC), whole transcriptome sequencing was conducted to identify differential RBPs between HCC with metastasis and HCC without metastasis. The influence of RBPs on metastasis of HCC was verified by in vitro and in vivo experiments. The interaction of RBPs with non‐coding RNAs was evaluated by RNA immunoprecipitation and pull‐down assays. RNA sequencing, whole‐genome sequencing, and alternative splicing analysis were further performed to clarify post‐transcriptional regulation mechanisms.Whole transcriptome sequencing results showed that expression of thioredoxin (Trx) was significantly upregulated in HCC patients with metastasis. Trx was also found to be associated with poor prognosis in HCC patients. Overexpression of Trx could promote migration and invasion of HCC cells in vitro and increase the rate of lung metastasis of HCC cells in vivo. Moreover, binding assays showed that Trx could bind to LINC00152. As a result, LINC00152 was verified to determine the pro‐metastasis function of Trx by knockdown assay. Furthermore, we revealed that Trx could regulate metastasis‐associated alternative splicing program. Specifically, angiopoietin 1 (ANGPT1) was the splicing target; the splicing isoform switching of ANGPT1 could activate the PI3K–Akt pathway, upregulate EMT‐associated proteins, and promote migration and invasion of HCC cells.We found that Trx could interact with LINC00152 and promote HCC metastasis via regulating alternative splicing, indicating that Trx may serve as a novel therapeutic target for HCC treatment. [ABSTRACT FROM AUTHOR]

The common octopus (Octopus vulgaris) is an excellent candidate for aquaculture diversification, due to its biological traits and high market demand. To ensure a high-quality product while maintaining welfare in captive environments, it is crucial to develop non-invasive methods for testing health biomarkers. Proteins found in skin mucus offer a non-invasive approach to monitoring octopus welfare. This study compares the protein profiles in the skin mucus of wild, aquarium-maintained, and senescent specimens to identify welfare biomarkers. A tandem mass tag (TMT) coupled with an Orbitrap Eclipse Tribrid mass spectrometer was used to create a reference dataset from octopus skin mucus, identifying 1496 non-redundant protein groups. Although similar profiles were observed, differences in relative abundances led to the identification of potential biomarkers, including caspase-3-like, protocadherin 4, deleted in malignant brain tumors, thioredoxin, papilin, annexin, cofilin and mucin-4 proteins. Some of these proteins also revealed potential as bioactive peptides. This investigation provides the most extensive analysis of the skin mucus proteome in the common octopus and is the first to explore how aquarium maintenance and senescence alter the mucus proteome. This research highlights the potential of skin mucus protein/peptides as non-invasive monitoring biomarkers in cultured animals. [ABSTRACT FROM AUTHOR]

Euglena gracilis , a fascinating organism in the scientific realm, exhibits characteristics of both animals and plants. It maintains redox homeostasis through a variety of enzymatic and non-enzymatic antioxidant molecules. In contrast to mammals, Euglena possesses nonselenocysteine glutathione peroxidase homologues that regulate its intracellular pools of reactive oxygen species. In the present study, a full-length cDNA of chloroplastic EgGPXL-1 was isolated and subjected to biochemical and functional characterization. Recombinant EgGPXL-1 scavenged H2O2 and t -BOOH, utilizing thioredoxin as an electron donor rather than glutathione. Despite its monomeric nature, EgGPXL-1 exhibits allosteric behavior with H2O2 as the electron acceptor and follows typical Michaelis–Menten kinetics with t -BOOH. Suppression of EgGPXL-1 gene expression under normal and high-light conditions did not induce critical situations in E. gracilis , suggesting the involvement of compensatory mechanisms in restoring normal conditions. [ABSTRACT FROM AUTHOR]

Cells constantly face the challenge of managing oxidants. In aerobic organisms, oxygen (O2) is used for energy production, generating reactive oxygen species (ROS) as byproducts of enzymatic reactions. To protect against oxidative damage, cells possess an intricate system of redox scavengers and antioxidant enzymes, collectively forming the antioxidant defense system. This system maintains the redox equilibrium and enables the generation of localized oxidative signals that regulate essential cellular functions. One key component of this defense is the thioredoxin (Trx) system, which includes Trx, thioredoxin reductase (TrxR), and NADPH. The Trx system reverses oxidation of macromolecules and indirectly neutralizes ROS via peroxiredoxin (Prx). This dual function protects cells from damage accumulation and supports physiological cell signaling. However, the Trx system also shields tumors from oxidative damage, aiding their survival. Due to elevated ROS levels from their metabolism, tumors often rely on the Trx system. In addition, the Trx system regulates critical pathways such as proliferation and neoangiogenesis, which tumors exploit to enhance growth and optimize nutrient and oxygen supply. Consequently, the Trx system is a potential target for cancer therapy. The challenge lies in selectively targeting malignant cells without disrupting the redox equilibrium in healthy cells. The aim of this review article is threefold: first, to elucidate the function of the Trx system; second, to discuss the Trx system as a potential target for cancer therapies; and third, to present the possibilities for inhibiting key components of the Trx system, along with an overview of the latest clinical studies on these inhibitors. [ABSTRACT FROM AUTHOR]

BACKGROUND: Studies have shown that cardiomyocyte apoptosis is closely related to cardiac decompensation and the cardiac aging process. Appropriate exercise can alter heart pump function in patients with heart failure as well as attenuate aging-induced cardiomyocyte apoptosis, hypertrophy, and fibrotic damage. OBJECTIVE: To investigate the effects of long-term aerobic exercise on cardiomyocyte apoptosis and the thioredoxin system in aging rats. METHODS: Thirty-six male Sprague-Dawley rats were selected and divided into three age groups: 3-month-old young group, 9-month-old middle-aged group, and 18-month-old elderly group, with 12 rats in each group. Within each age group, rats were randomly assigned to sedentary and exercise subgroups (n=6 per group). The sedentary groups did not undergo any exercise intervention. The exercise groups were acclimated to a treadmill environment and subsequently subjected to treadmill exercise for 45 minutes per day, at a speed of 15 m/min, 5 days per week for 10 weeks in total. At 24 hours after the final intervention, ELISA was employed to measure serum levels of cardiac troponin I and creatine kinase-MB in rats. TUNEL assay was utilized to detect cardiomyocyte apoptosis, while western blot assay was employed to assess the protein expression of Bax, Bcl-2, Caspase 3, thioredoxin-1, thioredoxin-2, thioredoxin reductase-1, thioredoxin reductase-2, thioredoxin-interacting protein, apoptosis signal-regulating kinase 1, and P38 mitogen-activated protein kinase in rat myocardial tissue. RESULTS AND CONCLUSION: Serum levels of cardiac troponin I and creatine kinase-MB in the elderly sedentary group were significantly higher than those in the young and middle-aged sedentary groups and elderly exercise group (P < 0.01). Serum levels of cardiac troponin I and creatine kinase-MB in the elderly sedentary group were significantly higher than those in the young and middle-aged exercise groups and elderly exercise group (P < 0.01). Positive apoptotic cells in rat myocardial tissue, along with increased protein expression of Bax and Caspase 3, exhibited an age-related upward trend, while Bcl-2 protein expression showed a declining trend. In comparison with the sedentary groups within each age category, the number of apoptotic cardiomyocytes and the expression of Bax and Caspase 3 proteins were reduced to different degrees, and the expression of Bcl-2 protein was increased to different degrees in the corresponding exercise groups. Compared with the young sedentary group, middle-aged sedentary group and elderly exercise group, elderly sedentary rats showed a significant decrease in the expression of myocardial thioredoxin 1, thioredoxin 2, thioredoxin reductase 1, and thioredoxin reductase 2 proteins (P < 0.05, P < 0.01). The expression of myocardial thioredoxin 1, thioredoxin 2, and thioredoxin reductase 2 proteins was lower in the elderly exercise group than in the young exercise group (P < 0.05, P < 0.01), while the expression of thioredoxin reductase 1 and thioredoxin reductase 2 proteins was lower in the elderly exercise group than in the middle-aged exercise group (P < 0.01). The protein expression of thioredoxin-interacting protein, apoptosis signal-regulating kinase 1, and P38 mitogen-activated protein kinase in rat myocardium was significantly higher in the elderly sedentary group than the young sedentary group, middleaged sedentary group and elderly exercise group (P < 0.01). The protein expression of thioredoxin-interacting protein, apoptosis signal-regulating kinase 1, and P38 mitogen-activated protein kinase in rat myocardium was significantly higher in the elderly exercise group than the young exercise group and middleaged exercise group (P < 0.01). To conclude, aerobic exercise may enhance the anti-apoptotic effects of thioredoxin by down-regulating the expression of thioredoxin-interacting protein in aging rat hearts, leading to the downregulation of apoptosis signal-regulated kinase 1 and P38 mitogen-activated kinase protein, thereby alleviating myocardial cell apoptosis in aging rat hearts. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by difficulties in social interaction and communication, repetitive behaviors, and restricted interests. Unfortunately, the underlying molecular mechanism behind ASD remains unknown. It has been reported that oxidative and nitrosative stress are strongly linked to ASD. We have recently found that nitric oxide (NO•) and its products play an important role in this disorder. One of the key proteins associated with NO• is thioredoxin (Trx). We hypothesize that the Trx system is altered in the Shank3 KO mouse model of autism, which may lead to a decreased activity of the nuclear factor erythroid 2-related factor 2 (Nrf2), resulting in oxidative stress, and thus, contributing to ASD-related phenotypes. To test this hypothesis, we conducted in vivo behavioral studies and used primary cortical neurons derived from the Shank3 KO mice and human SH-SY5Y cells with SHANK3 mutation. We showed significant changes in the levels and activity of Trx redox proteins in the Shank3 KO mice. A Trx1 inhibitor PX-12 decreased Trx1 and Nrf2 expression in wild-type mice, causing abnormal alterations in the levels of synaptic proteins and neurotransmission markers, and an elevation of nitrosative stress. Trx inhibition resulted in an ASD-like behavioral phenotype, similar to that of Shank3 KO mice. Taken together, our findings confirm the strong link between the Trx system and ASD pathology, including the increased oxidative/nitrosative stress, and synaptic and behavioral deficits. The results of this study may pave the way for identifying novel drug targets for ASD. [ABSTRACT FROM AUTHOR]

Background: Toxoplasma gondii (T. gondii) is a widespread, zoonotic protozoan intracellular parasite with a complex life cycle, which can cause toxoplasmosis, a potentially serious disease. During the invasion process, T. gondii proteins first bind to the relevant host cell receptors, such as glycosaminoglycan molecule (GAG-binding motif), which is one of the main receptors for parasites or virus to infect host cells. However, research on TGME49_216510 (T. gondii Trx21), a protein from Toxoplasma gondii, is limited. Methods: Bioinformatics analysis of the Trx21 protein was performed firstly. And specific primers were then designed using the conserved domain and GAG-binding motif to amplify, express, and purify a fragment of the Trx21 protein. The purified Trx21-GST protein was used for antioxidant and cell adhesion experiments. Simultaneously, mice were immunized with Trx21-His to generate specific polyclonal antibodies for subcellular localization analysis. Results: The Trx21 protein, consisting of 774 amino acids, included a transmembrane region, three GAG-binding motifs, and a Thioredoxin-like domain. The recombinant Trx21-His protein had a molecular mass of about 31 kDa, while the Trx21-GST protein had a molecular mass of about 55 kDa, which was analyzed by SDS-PAGE and Western blot. Subcellular localization analysis by IFA revealed that Trx21 is predominantly distributed in the cytoplasm of T. gondii. Furthermore, Trx21 exhibited a protective effect on supercoiled DNA against metal-catalyzed oxidation (MCO) and demonstrated adhesion abilities to Vero cells. Conclusions: These results indicate that Trx21 plays an important role in host cell interaction and oxidative damage. [ABSTRACT FROM AUTHOR]

Clostridioides (formerly Clostridium) difficile is a leading cause of infectious diarrhea associated with antibiotic therapy. The ability of this anaerobic pathogen to acquire enough iron to proliferate under iron limitation conditions imposed by the host largely determines its pathogenicity. However, since high intracellular iron catalyzes formation of deleterious reactive hydroxyl radicals, iron uptake is tightly regulated at the transcriptional level by the ferric uptake regulator Fur. Several studies relate lacking a functional fur gene in C. difficile cells to higher oxidative stress sensitivity, colonization defect and less toxigenicity, although Fur does not appear to directly regulate either oxidative stress response genes or pathogenesis genes. In this work, we report the functional characterization of C. difficile Fur and describe an additional oxidation sensing Fur‐mediated mechanism independent of iron, which affects Fur DNA‐binding. Using electrophoretic mobility shift assays, we show that Fur binding to the promoters of fur, feoA and fldX genes, identified as iron and Fur‐regulated genes in vivo, is specific and does not require co‐regulator metal under reducing conditions. Fur treatment with H2O2 produces dose‐dependent soluble high molecular weight species unable to bind to target promoters. Moreover, Fur oligomers are dithiotreitol sensitive, highlighting the importance of some interchain disulfide bond(s) for Fur oligomerization, and hence for activity. Additionally, the physiological electron transport chain NADPH‐thioredoxin reductase/thioredoxin from Escherichia coli reduces inactive oligomerized C. difficile Fur that recovers activity. In conjunction with available transcriptomic data, these results suggest a previously underappreciated complexity in the control of some members of the Fur regulon that is based on Fur redox properties and might be fundamental for the adaptive response of C. difficile during infection. [ABSTRACT FROM AUTHOR]

During land plant evolution, the number of genes encoding for components of the thiol redox regulatory network and the generator systems of reactive oxygen species (ROS) expanded, tentatively indicating that they have a role in tailored environmental acclimatization. This hypothesis has been validated both experimentally and theoretically during the last few decades. Recent developments of dynamic redox-sensitive GFP (roGFP)-based in vivo sensors for H2O2 and the redox potential of the glutathione pool have paved the way for dissecting the kinetics changes that occur in these crucial parameters in response to environmental stressors. The versatile cellular redox sensory and response regulatory system monitors alterations in redox metabolism and controls the activity of redox target proteins, and thereby affects most, if not all, cellular processes ranging from transcription to translation and metabolism. This review uses examples to describe the role of the redox- and ROS-dependent regulatory network in realising the appropriate responses to diverse environmental stresses. The selected case studies concern different environmental challenges, namely excess excitation energy, the heavy metal cadmium and the metalloid arsenic, nitrogen or phosphate shortages as examples for nutrient deficiency, wounding, and nematode infestation. Each challenge affects the redox-regulatory and ROS network, but our present state of knowledge also points toward pressing questions that remain open in relation to the translation of redox regulation to environmental acclimatization. [ABSTRACT FROM AUTHOR]

Thioredoxin (Trx), an important part of thioredoxin systems, plays crucial role in maintaining the intracellular redox balance by scavenging reactive oxygen species (ROS). However, few Trxs have been functionally characterized in Arma chinensis, especially in diapause. In this study, diapause induction condition promoted hydrogen peroxide accumulation and increased CAT enzymatic activity and ascorbate content, suggesting that A. chinensis was exposed to high level of ROS. Therefore, we identified AcTrx2 and AcTrx-like, and investigated the relationship with antioxidant defense. It was found that AcTrx2 expression was significantly induced, whereas AcTrx-like expression was the highest on day 10 under diapause conditions. The expression of AcTrx2 and AcTrx-like in fat body, a central metabolic organ of resisting oxidative stress, was significantly increased under diapause conditions, and was significantly improved by 5/15°C (diapause temperature).We investigated the knockdown of AcTrx2 and AcTrx-like in A. chinensis and found that some selected antioxidant genes were upregulated, indicating that the upregulated genes may be functional compensation for AcTrx2 and AcTrx-like silencing. We also found that the enzymatic activities of SOD and CAT, and the metabolite contents of hydrogen peroxide, ascorbate increased after AcTrx2 and AcTrx-like knockdown. These results suggested the AcTrx2 and AcTrx-like may play critical roles in antioxidant defense of A. chinensis diapause. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are important signalling molecules that influence many aspects of plant biology. One way in which ROS influence plant growth and development is by modifying intercellular trafficking through plasmodesmata (PD). Viruses have evolved to use PD for their local cell‐to‐cell spread between plant cells, so it is therefore not surprising that they have found ways to modulate ROS and redox signalling to optimise PD function for their benefit. This review examines how intracellular signalling via ROS and redox pathways regulate intercellular trafficking via PD during development and stress. The relationship between viruses and ROS‐redox systems, and the strategies viruses employ to control PD function by interfering with ROS‐redox in plants is also discussed. Summary statement: Reactive oxygen species (ROS) and redox are important players in pathways related to intercellular trafficking between plant cells. This reviews discussed the roles of ROS and redox in plant development and stress, focusing on how viruses redirect host ROS and redox signalling for their replication and spread during infection. [ABSTRACT FROM AUTHOR]

An arsenate reductase (Car1) from the Bacteroidetes species Rufibacter tibetensis 1351T was isolated from the Tibetan Plateau. The strain exhibits resistance to arsenite [As(III)] and arsenate [As(V)] and reduces As(V) to As(III). Here we shed light on the mechanism of enzymatic reduction by Car1. AlphaFold2 structure prediction, active site energy minimization, and steady‐state kinetics of wild‐type and mutant enzymes give insight into the catalytic mechanism. Car1 is structurally related to calcineurin‐like metallophosphoesterases (MPPs). It functions as a binuclear metal hydrolase with limited phosphatase activity, particularly relying on the divalent metal Ni2+. As an As(V) reductase, it displays metal promiscuity and is coupled to the thioredoxin redox cycle, requiring the participation of two cysteine residues, Cys74 and Cys76. These findings suggest that Car1 evolved from a common ancestor of extant phosphatases by incorporating a redox function into an existing MPP catalytic site. Its proposed mechanism of arsenate reduction involves Cys74 initiating a nucleophilic attack on arsenate, leading to the formation of a covalent intermediate. Next, a nucleophilic attack of Cys76 leads to the release of As(III) and the formation of a surface‐exposed Cys74‐Cys76 disulfide, ready for reduction by thioredoxin. [ABSTRACT FROM AUTHOR]

Substance P (SP), an important neuropeptide, has a crucial role in the progression of several cancers, including prostate cancer, through interacting with the neurokinin-1 receptor (NK1R). Oxidative stress is also involved in the onset and progression of prostate cancer. However, no studies have been performed on the cross-talk between the SP/NK1R system and cellular redox balance in prostate cancer, and how it is involved in tumorogenesis. We aimed to investigate the effect of the SP/NK1R system and the blockage of NK1R with its specific antagonist (aprepitant) on the cellular redox status of the prostate cancer cell line (PC3 and LNCaP). We performed the resazurin assay to evaluate the toxicity of the aprepitant on the PC3 and LNCaP cell lines. The intracellular reactive oxygen species (ROS) level was measured after SP and aprepitant treatment. The alterations of expression and activity of two crucial cellular oxidoreductases, glutaredoxin, and thioredoxin were evaluated by qRT-PCR and commercial kits (ZellBio GmbH), respectively. Our results revealed that SP increased ROS production and decreased the expression and activity of glutaredoxin and thioredoxin. On the other hand, treatment of cells with aprepitant showed reverse results. In conclusion, we found that the SP/NK1R system could promote prostate cancer progression by inducing oxidative stress. In addition, the inhibition of NK1R by aprepitant modulated the effect of the SP/NK1R system on the cellular redox system. Aprepitant might therefore be introduced as a candidate for the treatment of prostate cancer; however, more studies are required to confirm the validation of this hypothesis. [ABSTRACT FROM AUTHOR]

The interaction of chloroquine (CQ) with peptide-bonded-ethyl selenol and ethanethiol was experimentally and thermodynamically studied, indicating that CQ can bind to peptide-bonded active selenols through electrostatic interactions with a protonated triethylamine moiety, revealing a reasonable mechanism by which CQ inhibits thioredoxin reductase (TrxR) and significantly reduces oxidoreductase activity. [ABSTRACT FROM AUTHOR]

Unlike plants and animals, the phytoflagellate Euglena gracilis lacks catalase and contains a non-selenocysteine glutathione peroxidase-like protein (EgGPXL), two peroxiredoxins (EgPrx1 and EgPrx4), and one ascorbate peroxidase in the cytosol to maintain reactive oxygen species (ROS) homeostasis. In the present study, the full-length cDNA of three cytosolic EgGPXLs was obtained and further characterized biochemically and functionally. These EgGPXLs used thioredoxin instead of glutathione as an electron donor to reduce the levels of H2O2 and t-BOOH. The specific peroxidase activities of these enzymes for H2O2 and t-BOOH were 1.3 to 4.9 and 0.79 to 3.5 µmol/min/mg protein, respectively. Cytosolic EgGPXLs and EgPrx1/EgPrx4 were silenced simultaneously to investigate the synergistic effects of these genes on the physiological function of E. gracilis. The suppression of cytosolic EgGPXL genes was unable to induce any critical phenomena in Euglena under normal (100 μmol photons m−2 s−1) and high-light conditions (350 μmol photons m−2 s−1) at both autotrophic and heterotrophic states. Unexpectedly, the suppression of EgGPXL genes was able to rescue the EgPrx1/EgPrx4-silenced cell line from a critical situation. This study explored the potential resilience of Euglena to ROS, even with restriction of the cytosolic antioxidant system, indicating the involvement of some compensatory mechanisms. [ABSTRACT FROM AUTHOR]

Recently, we characterized the ferroptotic phenotype in the liver of diabetic mice and revealed nuclear factor (erythroid‐derived‐2)‐related factor 2 (Nrf2) inactivation as an integral part of hepatic injury. Here, we aim to investigate whether sulforaphane, an Nrf2 activator and antioxidant, prevents diabetes‐induced hepatic ferroptosis and the mechanisms involved. Male C57BL/6 mice were divided into four groups: control (vehicle‐treated), diabetic (streptozotocin‐induced; 40 mg/kg, from Days 1 to 5), diabetic sulforaphane‐treated (2.5 mg/kg from Days 1 to 42) and non‐diabetic sulforaphane‐treated group (2.5 mg/kg from Days 1 to 42). Results showed that diabetes‐induced inactivation of Nrf2 and decreased expression of its downstream antiferroptotic molecules critical for antioxidative defense (catalase, superoxide dismutases, thioredoxin reductase), iron metabolism (ferritin heavy chain (FTH1), ferroportin 1), glutathione (GSH) synthesis (cystine‐glutamate antiporter system, cystathionase, glutamate‐cysteine ligase catalitic subunit, glutamate‐cysteine ligase modifier subunit, glutathione synthetase), and GSH recycling ‐ glutathione reductase (GR) were reversed/increased by sulforaphane treatment. In addition, we found that the ferroptotic phenotype in diabetic liver is associated with increased ferritinophagy and decreased FTH1 immunopositivity. The antiferroptotic effect of sulforaphane was further evidenced through the increased level of GSH, decreased accumulation of labile iron and lipid peroxides (4‐hydroxy‐2‐nonenal, lipofuscin), decreased ferritinophagy and liver damage (decreased fibrosis, alanine aminotransferase, and aspartate aminotransferase). Finally, diabetes‐induced increase in serum glucose and triglyceride level was significantly reduced by sulforaphane. Regardless of the fact that this study is limited by the use of one model of experimentally induced diabetes, the results obtained demonstrate for the first time that sulforaphane prevents diabetes‐induced hepatic ferroptosis in vivo through the activation of Nrf2 signaling pathways. This nominates sulforaphane as a promising phytopharmaceutical for the prevention/alleviation of ferroptosis in diabetes‐related pathologies. [ABSTRACT FROM AUTHOR]

Citrus flavanones are recognized as promising bioactives within the concept of healthy aging. Thus, the present study investigated the effects of a nutritionally relevant dose of lemon extract (LE) on liver redox regulation and persulfidation levels in 24‐month‐old Wistar rats. LE (40 mg/kg b.m.) was administered orally once daily for 4 weeks. Control groups received either vehicle (sunflower oil) or remained intact. The applied methodology considered qPCR, Western blot, protein persulfidation levels evaluation, histochemistry in line with immunofluorescence, liver biochemical assays (glutathione, total ‐SH groups and malonaldehyde; MDA), liver enzymes in serum and in silico analysis to explore the potential interaction/binding between the proteins studied in the paper. Our results showed that LE increased glutathione peroxidase (GPx), reductase (GR), glutamate–cysteine ligase catalytic and modifier subunit, respectively, as well as Nrf2 gene expressions, but decreased the expression of superoxide dismutase 2 (SOD2). Upon LE application, protein expression showed upregulation of NRF2, SOD2, GPx, GR, and thioredoxin 1 (Trx1). LE significantly decreased the protein persulfidation levels and concentration of MDA, a marker of oxidative damage in the cell. Histological analysis showed a normal liver histoarchitecture without pathological changes, aligning with the normal serum level of hepatic enzymes. Obtained results showed that LE, by modulating hepatic redox regulators Nrf2 and Trx1, diminishes oxidative stress and alters the persulfidation levels, suggesting a considerable beneficial antioxidant potential of lemon flavanones in the old‐aged liver. [ABSTRACT FROM AUTHOR]

Selenium is an essential trace element in our diet, crucial for the composition of human selenoproteins, which include 25 genes such as glutathione peroxidases and thioredoxin reductases. The regulation of the selenoproteome primarily hinges on the bioavailability of selenium, either from dietary sources or cell culture media. This selenium-dependent control follows a specific hierarchy, with "housekeeping" selenoproteins maintaining constant expression while "stress-regulated" counterparts respond to selenium level fluctuations. This study investigates the variability in fetal bovine serum (FBS) selenium concentrations among commercial batches and its effects on the expression of specific stress-related cellular selenoproteins. Despite the limitations of our study, which exclusively used HEK293 cells and focused on a subset of selenoproteins, our findings highlight the substantial impact of serum selenium levels on selenoprotein expression, particularly for GPX1 and GPX4. The luciferase reporter assay emerged as a sensitive and precise method for evaluating selenium levels in cell culture environments. While not exhaustive, this analysis provides valuable insights into selenium-mediated selenoprotein regulation, emphasizing the importance of serum composition in cellular responses and offering guidance for researchers in the selenoprotein field. [ABSTRACT FROM AUTHOR]

In recent years, there has been an increasing tendency to create drugs based on certain commensal bacteria of the human microbiota and their ingredients, primarily focusing on live biotherapeutics (LBPs) and postbiotics. The creation of such drugs, termed pharmacobiotics, necessitates an understanding of their mechanisms of action and the identification of pharmacologically active ingredients that determine their target properties. Typically, these are complexes of biologically active substances synthesized by specific strains, promoted as LBPs or postbiotics (including vesicles): proteins, enzymes, low molecular weight metabolites, small RNAs, etc. This study employs omics technologies, including genomics, proteomics, and metabolomics, to explore the potential of Limosilactobacillus fermentum U-21 for innovative LBP and postbiotic formulations targeting neuroinflammatory processes. Proteomic techniques identified and quantified proteins expressed by L. fermentum U-21, highlighting their functional attributes and potential applications. Key identified proteins include ATP-dependent Clp protease (ClpL), chaperone protein DnaK, protein GrpE, thioredoxin reductase, LysM peptidoglycan-binding domaincontaining protein, and NlpC/P60 domain-containing protein, which have roles in disaggregase, antioxidant, and immunomodulatory activities. Metabolomic analysis provided insights into small-molecule metabolites produced during fermentation, revealing compounds with anti-neuroinflammatory activity. Significant metabolites produced by L. fermentum U-21 include GABA (γ-aminobutyric acid), niacin, aucubin, and scyllo-inositol. GABA was found to stabilize neuronal activity, potentially counteracting neurodegenerative processes. Niacin, essential for optimal nervous system function, was detected in vesicles and culture fluid, and it modulates cytokine production, maintaining immune homeostasis. Aucubin, an iridoid glycoside usually secreted by plants, was identified as having antioxidant properties, addressing issues of bioavailability for therapeutic use. Scyllo-inositol, identified in vesicles, acts as a chemical chaperone, reducing abnormal protein clumps linked to neurodegenerative diseases. These findings demonstrate the capability of L. fermentum U-21 to produce bioactive substances that could be harnessed in the development of pharmacobiotics for neurodegenerative diseases, contributing to their immunomodulatory, anti-neuroinflammatory, and neuromodulatory activities. Data of the HPLC-MS/MS analysis are available via ProteomeXchange with identifier PXD050857. [ABSTRACT FROM AUTHOR]

Ferroptosis is a recently discovered form of cell death that plays an important role in tumor growth and holds promise as a target for antitumor therapy. However, evidence in the regulation of ferroptosis in lung adenocarcinoma (LUAD) remains elusive. Here, we show that retinoic acid receptor alpha (RARA) is upregulated with the treatment of ferroptosis inducers (FINs). Pharmacological activation of RARA increases the resistance of LUAD to ferroptosis according to cell viability and lipid peroxidation assays, while RARA inhibitor or knockdown (KD) does the opposite. Through transcriptome sequencing in RARA-KD cells and chromatin immunoprecipitation (CHIP)-Seq data, we identify thioredoxin (TXN) and protein phosphatase 1 F (PPM1F) as downstream targets of RARA, both of which inhibit ferroptosis. We confirm that RARA binds to the promotor region of TXN and PPM1F and promotes their transcription by CHIP-qPCR and dual-luciferase assays. Overexpression of TXN and PPM1F reverses the effects of RARA knockdown on ferroptosis in vitro and vivo. Clinically, RARA knockdown or inhibitor increases cells' sensitivity to pemetrexed and cisplatin (CDDP). Immunohistochemistry (IHC) of LUAD from our cohort shows the same expression tendency of RARA and the downstream targets. Our study uncovers that RARA inhibits ferroptosis in LUAD by promoting TXN and PPM1F, and inhibiting RARA-TXN/PPM1F axis represents a promising strategy for improving the efficacy of FINs or chemotherapy in the treatment of LUAD patients. This study reveals RARA's role in inhibiting ferroptosis through the promotion of TXN and PPM1F, offering a potential therapeutic strategy for LUAD treatment by increasing efficacy of ferroptosis inducers or chemotherapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Thioredoxin (TRX) is a small protein with REDOX activity that plays a crucial role in a plant's growth, development, and stress resistance. The TRX family has been extensively studied in Arabidopsis, rice, and wheat, and so it is likely that its members have similar biological functions in Liriodendron that have not been reported in Liriodendron. In this study, we performed the genome-wide identification of the TRX gene family based on the Liriodendron chinense genome, leading to a total of 42 LcTRX gene members. A phylogenetic analysis categorized these 42 LcTRX proteins into 13 subfamilies. We further characterized their chromosome distributions, gene structures, conserved protein motifs, and cis-elements in the promoter regions. In addition, based on the publicly available transcriptome data for Liriodendron hybrid and following RT-qPCR experiments, we explored the expression patterns of LhTRXs to different abiotic stressors, i.e., drought, cold, and heat stress. Notably, we found that several LhTRXs, especially LhTRX-h3, were significantly upregulated in response to abiotic stress. In addition, the subcellular localization assay showed that LhTRX-h3 was mainly distributed in the cytoplasm. Subsequently, we obtained LhTRX-h3 overexpression (OE) and knockout (KO) callus lines in Liriodendron hybrid. Compared to the wild type (WT) and LhTRX-h3-KO callus proliferation of LhTRX-h3-OE lines was significantly enhanced with reduced reactive oxygen species (ROS) accumulation under drought stress. Our findings that LhTRX-h3 is sufficient to improve drought tolerance. and underscore the significance of the TRX gene family in environmental stress responses in Liriodendron. [ABSTRACT FROM AUTHOR]

Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in ferroptosis by regulating the cellular antioxidant response and maintaining redox balance. However, compounds that induce ferroptosis through dual antioxidant pathways based on Nrf2 have not been fully explored. In our study, we investigated the impact of Gambogic acid (GA) on MCF-7 cells and HepG2 cells in vitro. The cytotoxicity, colony formation assay and cell cycle assay demonstrated potent tumor-killing ability of GA, while its effect was rescued by ferroptosis inhibitors. Furthermore, RNA sequencing revealed the enrichment of ferroptosis pathway mediated by GA. In terms of ferroptosis indicators detection, evidences for GA were provided including reactive oxygen species (ROS) accumulation, alteration in mitochondrial membrane potential (MMP), disappearance of mitochondrial cristae, lipid peroxidation induction, malondialdehyde (MDA) accumulation promotion, iron ion accumulation as well as glutathione (GSH)/thioredoxin (Trx) depletion. Notably, Ferrostatin-1 (Fer-1) and Liproxstatin-1 (Lip-1) successfully rescued GA-induced MDA accumulation. In terms of mechanism, Nrf2 was found to play a pivotal role in GA-induced ferroptosis by inducing protein alterations through the iron metabolism pathway and GSH/Trx dual antioxidant pathway. Furthermore, GA exerted good antitumor activity in vivo through GSH/Trx dual antioxidant pathway, and Fer-1 significantly attenuated its efficacy. In conclusion, our findings first provided new evidence for GA as an inducer of ferroptosis, and Nrf2-mediated GSH/Trx dual antioxidant system played an important role in GA-induced ferroptosis. [Display omitted] • GA exhibited its anticancer activity by inducing ferroptosis in vitro and in vivo. • Nrf2 played an important role in GA-induced ferroptosis. • GSH/Thioredoxin dual antioxidant system was first found to induce ferroptosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Post-transcriptional regulation and RNA-binding proteins (RBPs) play vital roles in the occurrence of secondary injury after intracerebral hemorrhage (ICH). Therefore, we identified RBPs distinctively expressed after ICH by screening and determined thioredoxin1 (Txn1) as one of the most distinctive RBPs. We employed an ICH model and in vitro experiments to investigate the role of Txn1 in ICH. Firstly, we found that Txn1 was mainly expressed in microglia and neurons in the central nervous system, and its expression was significantly reduced in perihematomal tissue. Additionally, adeno-associated virus (AAV) carrying Txn1 was injected into the ICH rat model. Our results showed that overexpression of Txn1 reduced secondary injury and improved outcome in the ICH rat model. Moreover, to understand the therapeutic mechanism of Txn1 after ICH, we performed RNA immunoprecipitation combined with high-throughput sequencing. The results showed that Txn1 binds to inflammation- and apoptosis-related mRNAs and affects gene expression through RNA splicing and translation. Finally, RNA pull-down assays and in vitro experiments confirmed that Txn1 binds to metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), leading to reduced inflammation and apoptosis. Our study suggests that Txn1 is a potential therapeutic target for alleviating ICH-induced brain injury. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Gallinarum biovar Gallinarum (SG) causes fowl typhoid, a notifiable infectious disease in poultry. However, the pathogenic mechanism of SG-induced systemic infection in chickens remains unclear. Thioredoxin reductase (TrxB) is a redox protein crucial for regulating various enzyme activities in Salmonella serovar, but the role in SG-induced chicken systemic infection has yet to be determined. Here, we constructed a mutant SG strain lacking the trxB gene (trxB::Cm) and used chicken embryo inoculation and chicken oral infection to investigate the role of trxB gene in the pathogenicity of SG. Our results showed that trxB::Cm exhibited no apparent differences in colony morphology and growth conditions but exhibited reduced tolerance to H2O2 and increased resistance to bile acids. In the chicken embryo inoculation model, there was no significant difference in the pathogenicity of trxB::Cm and wild-type (WT) strains. In the chicken oral infection, the WT-infected group exhibited typical clinical symptoms of fowl typhoid, with complete mortality between days 6 and 9 post infection. In contrast, the trxB::Cm group showed a 100% survival rate, with no apparent clinical symptoms or pathological changes observed. The viable bacterial counts in the liver and spleen of the trxB::Cm-infected group were significantly reduced, accompanied by decreased expression of cytokines and chemokines (IL-1β, IL-6, IL-12, CXCLi1, TNF-α, and IFN-γ), which were significantly lower than those in the WT group. These results show that the pathogenicity of the trxB-deficient strain was significantly attenuated, indicating that the trxB gene is a crucial virulence factor in SG-induced systemic infection in chickens, suggesting that trxB may become a potentially effective target for controlling and preventing SG infection in chickens. [ABSTRACT FROM AUTHOR]

Saccharomyces boulardii (Sb) is a probiotic yeast for the treatment of gastrointestinal disorders, including inflammatory bowel disease (IBD). Little is known about the modulatory capacity of the Sb in IBD. Here, we found that oral gavage of Sb supernatant (SbS) alleviated gut inflammation, protected the intestinal barrier, and reversed DSS-induced down-regulated activation of epidermal growth factor receptor (EGFR) in colitis. Mass spectrum analysis showed that thioredoxin (Trx) is one of the critical secreted soluble proteins participating in EGFR activation detected in SbS. Trx exerted an array of significant effects on anti-inflammatory activity, including alleviating inflammation, protecting gut barrier, suppressing apoptosis, as well as reducing oxidative stress. Mechanistically, Trx promoted EGFR ligand gene expression and transactivated EGFR in a concentration-dependent manner. EGFR kinase inhibitor could block Trx-mediated preventive effects of intestinal epithelial injury. Our data suggested that Sb-derived soluble protein Trx could serve as a potential prophylactic, as a novel postbiotic against colitis, which provides a new strategy for the precision prevention and treatment of IBD.

Maintaining cellular redox homeostasis is critical for cell viability and growth, with disruptions implicated in cellular responses to chemicals and drugs. This study investigates the interactions between acylfulvenes (AFs), a class of DNA alkylating drugs, and thioredoxin (Trx), a key redox regulating enzyme. AFs are semi-synthetic derivatives of the natural product illudin S. While their cytotoxic properties are widely attributed to DNA alkylation, they also react with cellular thiols, such as Trx, and the implications of these interactions remain poorly understood. Through biochemical assays with isolated E. Coli Trx, and cellular experiments in a human cell line (HeLa), we elucidate AFs’ impact on Trx activity and cellular levels. AFs, particularly hydroxymethylacylfulvene (HMAF), inhibited Trx activity by covalently modifying its active site cysteines. Drug exposure also altered cellular Trx levels and nuclear accumulation. In contrast, illudin S, which has a less selective toxicity profile for cancer cells, minimally inhibited isolated Trx. These data underscore Trx as a potential target contributing to the chemotherapeutic potential of AFs and provide insights into molecular interactions governing their impact on cancer cells.

Cardiovascular disease (CVD) is one of the leading causes of mortality in humans, and oxidative stress plays a pivotal role in disease progression. This phenomenon typically arises from weakening of the cellular antioxidant system or excessive accumulation of peroxides. This review focuses on a specialized form of oxidative stress—disulfide stress—which is triggered by an imbalance in the glutaredoxin and thioredoxin antioxidant systems within the cell, leading to the accumulation of disulfide bonds. The genesis of disulfide stress is usually induced by extrinsic pathological factors that disrupt the thiol-dependent antioxidant system, manifesting as sustained glutathionylation of proteins, formation of abnormal intermolecular disulfide bonds between cysteine-rich proteins, or irreversible oxidation of thiol groups to sulfenic and sulfonic acids. Disulfide stress not only precipitates the collapse of the antioxidant system and the accumulation of reactive oxygen species, exacerbating oxidative stress, but may also initiate cellular inflammation, autophagy, and apoptosis through a cascade of signaling pathways. Furthermore, this review explores the detrimental effects of disulfide stress on the progression of various CVDs including atherosclerosis, hypertension, myocardial ischemia-reperfusion injury, diabetic cardiomyopathy, cardiac hypertrophy, and heart failure. This review also proposes several potential therapeutic avenues to improve the future treatment of CVDs.

Abstract Background To successfully replicate within the host cell, Toxoplasma gondii employs several mechanisms to overcome the host cell defenses and mitigate the harmful effects of the free radicals resulting from its own metabolic processes using effectors such as thioredoxin proteins. In this study, we characterize the location and functions of a newly identified thioredoxin in T. gondii, which was named Trx4. Methods We characterized the functional role of Trx4 in T. gondii Type I RH and Type II Pru strains by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The enzyme-catalyzed proximity labeling technique, the TurboID system, was employed to identify the proteins in proximity to Trx4. Results Trx4 was identified as a dense granule protein of T. gondii predominantly expressed in the parasitophorous vacuole (PV) and was partially co-localized with GRA1 and GRA5. Functional analysis showed that deletion of trx4 markedly influenced the parasite lytic cycle, resulting in impaired host cell invasion capacity in both RH and Pru strains. Mutation of Trx domains in Trx4 in RH strain revealed that two Trx domains were important for the parasite invasion. By utilizing the TurboID system to biotinylate proteins in proximity to Trx4, we identified a substantial number of proteins, some of which are novel, and others are previously characterized, predominantly distributed in the dense granules. In addition, we uncovered three novel proteins co-localized with Trx4. Intriguingly, deletion of trx4 did not affect the localization of these three proteins. Finally, a virulence assay demonstrated that knockout of trx4 resulted in a significant attenuation of virulence and a significant reduction in brain cyst loads in mice. Conclusions Trx4 plays an important role in T. gondii invasion and virulence in Type I RH strain and Type II Pru strain. Combining the TurboID system with CRISPR-Cas9 technique revealed many PV-localized proximity proteins associated with Trx4. These findings suggest a versatile role of Trx4 in mediating the processes that occur in this distinctive intracellular membrane-bound vacuolar compartment. Graphical Abstract

Lilium pumilum DC (L. pumilum DC) plays an important role in the rational utilization of salinized soil. To explore the molecular mechanism of salt-tolerant L. pumilum, the LpMYB4 was cloned. LpMYB4 close relationship with Bambusa emeiensis and Zea mays MYB4 throughout the phylogenetic tree construction. LpMYB4 protein was found to be localized in the nucleus. Prokaryotic and eukaryotic bacterial solution resistance experiments proved that the exogenous introduction of LpMYB4 made the overexpression strains obtain better survival ability under saline-alkaline stress. Compared with wild-type plants, tobacco plants overexpressing LpMYB4 had better growth and lower leaf wilting and lodging, the content of chlorophyll was higher, the content of hydrogen peroxide and superoxide anion was lower, the activity of peroxidase and superoxide dismutase was higher and the relative conductivity was lower under saline-alkaline stress. The analysis of seed germination and seedling resistance of transgenic plants under salt stress showed that LpMYB4 transgenic seeds were more tolerant to salt stress during germination and growth. Yeast two-hybrid and two-luciferase complementation experiments showed that LpMYB4 interacted with yeast two-hybrid and LpGPX6. The analysis of the role of LpMYB4 in improving plant saline-alkali resistance is helpful to the transformation of plant germplasm resources and has great significance for agriculture and sustainable development.

Understanding how plant pathogenic fungi adapt to their hosts is of critical importance to securing optimal crop productivity. In response to pathogenic attack, plants produce reactive oxygen species (ROS) as part of a multipronged defense response. Pathogens, in turn, have evolved ROS scavenging mechanisms to undermine host defense. Thioredoxins (Trx) are highly conserved oxidoreductase enzymes with a dithiol-disulfide active site, and function as antioxidants to protect cells against free radicals, such as ROS. However, the roles of thioredoxins in Verticillium dahliae, an important vascular pathogen, are not clear. Through proteomics analyses, we identified a putative thioredoxin (VdTrx1) lacking a signal peptide. VdTrx1 was present in the exoproteome of V. dahliae cultured in the presence of host tissues, a finding that suggested that it plays a role in host-pathogen interactions. We constructed a VdTrx1 deletion mutant ΔVdTrx1 that exhibited significantly higher sensitivity to ROS stress, H2O2, and tert-butyl hydroperoxide (t-BOOH). In vivo assays by live-cell imaging and in vitro assays by western blotting revealed that while VdTrx1 lacking the signal peptide can be localized within V. dahliae cells, VdTrx1 can also be secreted unconventionally depending on VdVps36, a member of the ESCRT-II protein complex. The ΔVdTrx1 strain was unable to scavenge host-generated extracellular ROS fully during host invasion. Deletion of VdTrx1 resulted in higher intracellular ROS levels of V. dahliae mycelium, displayed impaired conidial production, and showed significantly reduced virulence on Gossypium hirsutum, and model plants, Arabidopsis thaliana and Nicotiana benthamiana. Thus, we conclude that VdTrx1 acts as a virulence factor in V. dahliae.

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i's) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity. The clinical application of inhibitors targeting checkpoint kinase 1 (CHK1) is challenged by limited efficacy. Here, the authors identify that thioredoxin (Trx) system inhibition mediates sensitivity to CHK1 inhibitor via regulating the activity of ribonucleotide reductase, demonstrating the synergistic effect of CHK1 inhibitor and inhibitors targeting Trx system in lung cancer models. [ABSTRACT FROM AUTHOR]

During inflammation and tissue regeneration, the alarmin High Mobility Group Box 1 (HMGB1), in its reduced isoform, enhances the activity of the chemokine CXCL12, forming a heterocomplex that acts via the chemokine receptor CXCR4. Despite the established roles of both HMGB1 and CXCL12 in tumor progression and metastatic spread to distal sites, the role of the CXCL12/HMGB1 heterocomplex in cancer has never been investigated. By employing a newly established mass spectrometry protocol that allows an unambiguous distinction between reduced (red-HMGB1) and oxidized (ox-HMGB1) HMGB1 isoforms in cell lysates, we demonstrate that human epithelial cells derived from breast (MCF-7 and MDA-MB-231) and prostate (PC-3) cancer predominantly express red-HMGB1, while primary CD3+ T lymphocytes from peripheral blood express both HMGB1 isoforms. All these cancer cells release HMGB1 in the extracellular microenvironment together with varying concentrations of thioredoxin and thioredoxin reductase. The CXCL12/HMGB1 heterocomplex enhances, via CXCR4, the directional migration and invasiveness of cancer cells characterized by high metastatic potential that possess a fully active thioredoxin system, contributing to maintain red-HMGB1. On the contrary, cancer cells with low metastatic potential, lack thioredoxin reductase, promptly uptake CXCL12 and fail to respond to the heterocomplex. Our study demonstrates that the responsiveness of cancer cells to the CXCL12/HMGB1 heterocomplex, resulting in enhanced cell migration and invasiveness, depends on the maintenance of HMGB1 in its reduced isoform, and suggests disruption of the heterocomplex as a potential therapeutic target to inhibit invasion and metastatic spread in cancer therapies. [ABSTRACT FROM AUTHOR]

The reactivity of thioredoxin (Trx1) with the Au(I) drug auranofin (AF) and two therapeutic N-heterocyclic carbene (NHC)2-Au(I) complexes (bis [1-methyl-3-acridineimidazolin-2-ylidene]gold(I) tetrafluoroborate (Au3BC) and [1,3-diethyl-4,5-bis(4methoxyphenyl)imidazol-2-ylidene]gold(I) (Au4BC)) was investigated. Direct infusion (DI) electrospray ionization (ESI) mass spectrometry (MS) allowed information on the structure, stoichiometry, and kinetics of formation of Trx-Au adducts. The fragmentation of the formed adducts in the gas phase gave insights into the exact Au binding site within the protein, demonstrating the preference for Trx1 Cys32 or Cys35 of AF or the (NHC)2-Au(I) complex Au3BC, respectively. Reversed-phase HPLC suffered from the difficulty of elution of gold compounds, did not preserve the formed metal-protein adducts, and favored the loss of ligands (phosphine or NHC) from Au(I). These limitations were eliminated by capillary electrophoresis (CE) which enabled the separation of the gold compounds, Trx1, and the formed adducts. The ICP-MS/MS detection allowed the simultaneous quantitative monitoring of the gold and sulfur isotopes and the determination of the metallation extent of the protein. The hyphenation of the mentioned techniques was used for the analysis of Trx1-Au adducts for the first time. [ABSTRACT FROM AUTHOR]

HIV latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to-macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed protein kinase C signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing proinflammatory M1 macrophages suppressed HIV reactivation while anti-inflammatory M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive-oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and found that inhibitors of the thioredoxin (Trx) system acted as latency-promoting agents in T-cells and monocytes, but opposingly acted as latency-reversing agents in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase inhibition led to ROS induced NF-κB activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies. [ABSTRACT FROM AUTHOR]

Climate change, which causes periods with relatively high temperatures in winter in Poland, can lead to a shortening or interruption of the cold hardening of crops. Previous research indicates that cold acclimation is of key importance in the process of acquiring cereal tolerance to stress factors. The objective of this work was to verify the hypothesis that both natural temperature fluctuations and the plant genotype influence the content of metabolites as well as proteins, including antioxidant enzymes and photosystem proteins. The research material involved four winter triticale genotypes, differing in their tolerance to stress under controlled conditions. The values of chlorophyll a fluorescence parameters and antioxidant activity were measured in their seedlings. Subsequently, the contribution of selected proteins was verified using specific antibodies. In parallel, the profiling of the contents of chlorophylls, carotenoids, phenolic compounds, and proteins was carried out by Raman spectroscopy. The obtained results indicate that a better PSII performance along with a higher photosystem II proteins content and thioredoxin reductase abundance were accompanied by a higher antioxidant activity in the field-grown triticale seedlings. The Raman studies showed that the cold hardening led to a variation in photosynthetic dyes and an increase in the phenolic to carotenoids ratio in all DH lines. [ABSTRACT FROM AUTHOR]

Significance: Peroxiredoxins (Prdxs) with a single peroxidative cysteine (CP) in a conserved motif PXXX(T/S)XXCP within its thioredoxin fold, have been classified as the peroxiredoxin 6 (Prdx6) family. All Prdxs can reduce H2O2 and short chain hydroperoxides while Prdx6 in addition, can reduce phospholipid hydroperoxides (PLOOH) due to its ability to interact with peroxidized phospholipid substrate. The single CP of Prdx6 uses various external electron donors including glutathione thioredoxin, and ascorbic acid for resolution of its peroxidized state and, therefore, its peroxidase activity. Prdx6 proteins also exhibit Ca2+-independent phospholipase A2 (PLA2), lysophosphatidylcholine acyltransferase (LPCAT), and chaperone activities that depend on cellular localization and the oxidation and oligomerisation states of the protein. Thus, Prdx6 is a "moonlighting" enzyme. Recent Advance: Physiologically, Prdx6s have been reported to play an important role in protection against oxidative stress, repair of peroxidized cell membranes, mammalian lung surfactant turnover, activation of some NADPH oxidases, the regulation of seed germination in plants, as an indicator of cellular levels of reactive O2 species through Nrf-Klf9 activation, and possibly in male fertility, regulation of cell death through ferroptosis, cancer metastasis, and oxidative stress-related signalling pathways. Critical Issues: This review outlines Prdx6 enzyme unique structural features and explores its wide range of physiological functions. Yet, existing structural data falls short of fully revealing all of human Prdx6 multifunctional roles. Further endeavour is required to bridge this gap in its understanding. Although there are wide variations in both the structure and function of Prdx6 family members in various organisms, all Prdx6 proteins show the unique a long C-terminal extension that is also seen in Prdx1, but not in other Prdxs. Future Directions: As research data continues to accumulate, the potential for detailed insights into the role of C-terminal of Prdx6 in its oligomerisation and activities. There is a need for thorough exploration of structural characteristics of the various biological functions. Additionally, uncovering the interacting partners of Prdx6 and understanding its involvement in signalling pathways will significantly contribute to a more profound comprehension of its role. [ABSTRACT FROM AUTHOR]

Thioredoxin (Trx) is a small redox mediator protein involved in the regulation of various chloroplast functions by modulating the redox state of Trx target proteins in ever-changing light environments. Using reducing equivalents produced by the photosynthetic electron transport chain, Trx reduces the disulfide bonds on target proteins and generally turns on their activities. While the details of the protein-reduction mechanism by Trx have been well investigated, the oxidation mechanism that counteracts it has long been unclear. We have recently demonstrated that Trx-like proteins such as Trx-like2 and atypical Cys His-rich Trx (ACHT) can function as protein oxidation factors in chloroplasts. Our latest study on transgenic Arabidopsis plants indicated that the ACHT isoform ACHT2 is involved in regulating the thermal dissipation of light energy. To understand the role of ACHT2 in vivo, we characterized phenotypic changes specifically caused by ACHT2 overexpression in Arabidopsis. ACHT2-overexpressing plants showed growth defects, especially under high light conditions. This growth phenotype was accompanied with the impaired reductive activation of Calvin–Benson cycle enzymes, enhanced thermal dissipation of light energy, and decreased photosystem II activity. Overall, ACHT2 overexpression promoted protein oxidation that led to the inadequate activation of Calvin–Benson cycle enzymes in light and consequently induced negative feedback control of the photosynthetic electron transport chain. This study highlights the importance of the balance between protein reduction and oxidation in chloroplasts for optimal photosynthetic performance and plant growth. [ABSTRACT FROM AUTHOR]