Colorimetric and Electrochemical Dual-Mode Detection of Thioredoxin 1 Based on the Efficient Peroxidase-Mimicking and Electrocatalytic Property of Prussian Blue Nanoparticles.

As a potent detection method for cancer biomarkers in physiological fluid, a colorimetric and electrochemical dual-mode sensing platform for breast cancer biomarker thioredoxin 1 (TRX1) was developed based on the excellent peroxidase-mimicking and electrocatalytic property of Prussian blue nanoparticles (PBNPs). PBNPs were hydrothermally synthesized using K3[Fe(CN)6] as a precursor and polyvinylpyrrolidone (PVP) as a capping agent. The synthesized spherical PBNPs showed a significant peroxidase-like activity, having approximately 20 and 60% lower Km values for 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, respectively, compared to those of horseradish peroxidase (HRP). The PBNPs also enhanced the electron transfer on the electrode surface. Based on the beneficial features, PBNPs were used to detect target TRX1 via sandwich-type immunoassay procedures. Using the strategies, TRX1 was selectively and sensitively detected, yielding limit of detection (LOD) values as low as 9.0 and 6.5 ng mL-1 via colorimetric and electrochemical approaches, respectively, with a linear range of 10-50 ng mL-1 in both strategies. The PBNP-based TRX1 immunoassays also exhibited a high degree of precision when applied to real human serum samples, demonstrating significant potentials to replace conventional HRP-based immunoassay systems into rapid, robust, reliable, and convenient dual-mode assay systems which can be widely utilized for the identification of important target molecules including cancer biomarkers.

Trx4, a novel thioredoxin protein, is important for Toxoplasma gondii fitness.

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.

The cryo-EM structure of ASK1 reveals an asymmetric architecture allosterically modulated by TRX1.

Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.

Zn2+-dependent functional switching of ERp18, an ER-resident thioredoxin-like protein.

ERp18 is an endoplasmic reticulum (ER)-resident thioredoxin (Trx) family protein, similar to cytosolic Trx1. The Trx-like domain occupies a major portion of the whole ERp18 structure, which is postulated to be an ER paralog of cytosolic Trx1. Here, we elucidate that zinc ion (Zn2+) binds ERp18 through its catalytic motif, triggering oligomerization of ERp18 from a monomer to a trimer. While the monomeric ERp18 has disulfide oxidoreductase activity, the trimeric ERp18 acquires scavenger activity for hydrogen peroxide (H2O2) in the ER. Depletion of ERp18 thus causes the accumulation of H2O2, which is produced during the oxidative folding of nascent polypeptides in the ER. ERp18 knockdown in C. elegans without Prx4 and GPx7/8, both of which are also known to have H2O2 scavenging activity in the ER, shortened the lifespan, suggesting that ERp18 may form a primitive and essential H2O2 scavenging system for the maintenance of redox homeostasis in the ER.

Inhibition of the thioredoxin system for radiosensitization therapy of cancer.

Radiotherapy (RT) stands as a cornerstone in the clinical armamentarium against various cancers due to its proven efficacy. However, the intrinsic radiation resistance exhibited by cancer cells, coupled with the adverse effects of RT on normal tissues, often compromises its therapeutic potential and leads to unwanted side effects. This comprehensive review aims to consolidate our understanding of how radiosensitizers inhibit the thioredoxin (Trx) system in cellular contexts. Notable radiosensitizers, including gold nanoparticles (GNPs), gold triethylphosphine cyanide ([Au(SCN) (PEt3)]), auranofin, ceria nanoparticles (CONPs), curcumin and its derivatives, piperlongamide, indolequinone derivatives, micheliolide, motexafin gadolinium, and ethane selenide selenidazole derivatives (SeDs), are meticulously elucidated in terms of their applications in radiotherapy. In this review, the sensitization mechanisms and the current research progress of these radiosensitizers are discussed in detail, with the overall aim of providing valuable insights for the judicious application of Trx system inhibitors in the field of cancer radiosensitization therapy.

Komagataella phaffii Erp41 is a protein disulfide isomerase with unprecedented disulfide bond catalyzing activity when coupled to glutathione.

In the methylotrophic yeast Komagataella phaffii, we identified an endoplasmic reticulum-resident protein disulfide isomerase (PDI) family member, Erp41, with a peculiar combination of active site motifs. Like fungal ERp38, it has two thioredoxin-like domains which contain active site motifs (a and a'), followed by an alpha-helical ERp29c C-terminal domain (c domain). However, while the a domain has a typical PDI-like active site motif (CGHC), the a' domain instead has CGYC, a glutaredoxin-like motif which confers to the protein an exceptional affinity for GSH/GSSG. This combination of active site motifs has so far been unreported in PDI-family members. Homology searches revealed ERp41 is present in the genome of some plants, fungal parasites, and a few nonconventional yeasts, among which are Komagataella spp. and Yarrowia lipolytica. These yeasts are both used for the production of secreted recombinant proteins. Here, we analyzed the activity of K. phaffii Erp41. We report that it is nonessential in K. phaffii, and that it can catalyze disulfide bond formation in partnership with the sulfhydryl oxidase Ero1 in vitro with higher turnover rates than the canonical PDI from K. phaffii, Pdi1, but slower activation times. We show how Erp41 has unusually fast glutathione-coupled oxidation activity and relate it to its unusual combination of active sites in its thioredoxin-like domains. We further describe how this determines its unusually efficient catalysis of dithiol oxidation in peptide and protein substrates.

Exposure of human glioblastoma cells to thimerosal inhibits the thioredoxin system and decreases tumor growth-related factors.

Glioblastoma multiforme (GBM) is the most common, aggressive, and fatal primary malignant brain tumor in adults. The therapeutic efficacy of temozolomide (TMZ) is limited owing to frequent treatment resistance. The latter is in part related to the overexpression of redox systems such as the thioredoxin system. This system is fundamental for cell survival and proliferation, regulating hypoxia inducible factor-1alpha (HIF-1α) activity, in turn controlling vascular endothelial growth factor (VEGF), which is indispensable for tumor invasiveness, angiogenesis and microenvironment maintenance. HIF-1α can also be regulated by the signal transducer and activator of transcription 3 (STAT3), an oncogene stimulated by pro-inflammatory cytokines and growth factors. The thioredoxin system has several known inhibitors including mercury compounds such as Thimerosal (TmHg) which readily crosses the blood-brain barrier (BBB) and accumulates in the brain. Though previously used in various applications epidemiological evidence on TmHg's neurotoxicity is lacking. The objective of this study was to verify whether thimerosal is a suitable candidate for hard repurposing to control glioblastoma; therefore, the effects of this molecule were evaluated in human GBM (U87) cells. Our novel results show that TmHg decreased cellular viability (>50%) and migration (up to 90% decrease in wound closure), reduced thioredoxin reductase (TrxR/TXNRD1) and thioredoxin (Trx) activity, and increased reactive oxygen species (ROS) generation. Moreover, TmHg reduced HIF-1α expression (35%) as observed by immunofluorescence. Co-exposure of U87 cells to TmHg and TMZ reduced HIF-1α, VEGF, and phosphorylated STAT3. Consequently, TmHg alone or combined with chemotherapeutic drugs can reduce neoangiogenesis and ameliorate glioblastoma progression and treatment.

Cytoplasmic redox imbalance in the thioredoxin system activates Hsf1 and results in hyperaccumulation of the sequestrase Hsp42 with misfolded proteins.

Cells employ multiple systems to maintain homeostasis when experiencing environmental stress. For example, the folding of nascent polypeptides is exquisitely sensitive to proteotoxic stressors including heat, pH, and oxidative stress, and is safeguarded by a network of protein chaperones that concentrate potentially toxic misfolded proteins into transient assemblies to promote folding or degradation. The redox environment itself is buffered by both cytosolic and organellar thioredoxin and glutathione pathways. How these systems are linked is poorly understood. Here, we determine that specific disruption of the cytosolic thioredoxin system resulted in constitutive activation of the heat shock response in Saccharomyces cerevisiae and accumulation of the sequestrase Hsp42 into an exaggerated and persistent juxtanuclear quality control (JUNQ) compartment. Terminally misfolded proteins also accumulated in this compartment in thioredoxin reductase (TRR1)-deficient cells, despite apparently normal formation and dissolution of transient cytoplasmic quality control (CytoQ) bodies during heat shock. Notably, cells lacking TRR1 and HSP42 exhibited severe synthetic slow growth exacerbated by oxidative stress, signifying a critical role for Hsp42 under redox-challenged conditions. Finally, we demonstrated that Hsp42 localization patterns in trr1∆ cells mimic those observed in chronically aging and glucose-starved cells, linking nutrient depletion and redox imbalance with management of misfolded proteins via a process of long-term sequestration.

TXNIP-mediated crosstalk between oxidative stress and glucose metabolism.

Thioredoxin-interacting protein (TXNIP) has emerged as a key player in cancer and diabetes since it targets thioredoxin (TRX)-mediated redox regulation and glucose transporter (GLUT)-mediated metabolism. TXNIP consists of two arrestin (ARR, N-ARR and C-ARR) domains at its amino-terminus and two PPxY (PY) motifs and a di-leucine (LL) motif for endocytosis at its carboxyl-terminus. Here, we report that TXNIP shuffles between TRX and GLUTs to regulate homeostasis of intracellular oxidative stress and glucose metabolism. While TXNIP functions as a gatekeeper of TRX by default, it robustly interacted with class I GLUTs through its C-ARR domain upon increase of intracellular reactive oxygen species. This interaction prompted the surface expression downregulation and lysosomal degradation of GLUTs by its carboxyl-terminal LL endocytic signaling motif to attenuate glucose uptake. Consequently, TXNIP expression significantly limited glucose uptake, leading to the suppression of glycolysis, hexosamine biosynthesis, and the pentose phosphate pathway. Our findings establish a fundamental link between ROS and glucose metabolism through TXNIP and provide a promising target for the drug development against GLUT-related metabolic disorders.

Zbtb16 increases susceptibility of atrial fibrillation in type 2 diabetic mice via Txnip-Trx2 signaling.

Atrial fibrillation (AF) is the most prevalent sustained cardiac arrhythmia, and recent epidemiological studies suggested type 2 diabetes mellitus (T2DM) is an independent risk factor for the development of AF. Zinc finger and BTB (broad-complex, tram-track and bric-a-brac) domain containing 16 (Zbtb16) serve as transcriptional factors to regulate many biological processes. However, the potential effects of Zbtb16 in AF under T2DM condition remain unclear. Here, we reported that db/db mice displayed higher AF vulnerability and Zbtb16 was identified as the most significantly enriched gene by RNA sequencing (RNA-seq) analysis in atrium. In addition, thioredoxin interacting protein (Txnip) was distinguished as the key downstream gene of Zbtb16 by Cleavage Under Targets and Tagmentation (CUT&Tag) assay. Mechanistically, increased Txnip combined with thioredoxin 2 (Trx2) in mitochondrion induced excess reactive oxygen species (ROS) release, calcium/calmodulin-dependent protein kinase II (CaMKII) overactivation, and spontaneous Ca2+ waves (SCWs) occurrence, which could be inhibited through atrial-specific knockdown (KD) of Zbtb16 or Txnip by adeno-associated virus 9 (AAV9) or Mito-TEMPO treatment. High glucose (HG)-treated HL-1 cells were used to mimic the setting of diabetic in vitro. Zbtb16-Txnip-Trx2 signaling-induced excess ROS release and CaMKII activation were also verified in HL-1 cells under HG condition. Furthermore, atrial-specific Zbtb16 or Txnip-KD reduced incidence and duration of AF in db/db mice. Altogether, we demonstrated that interrupting Zbtb16-Txnip-Trx2 signaling in atrium could decrease AF susceptibility via reducing ROS release and CaMKII activation in the setting of T2DM.

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

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

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

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

TXM peptides inhibit SARS-CoV-2 infection, syncytia formation, and lower inflammatory consequences.

After three years of the SARS-CoV-2 pandemic, the search and availability of relatively low-cost benchtop therapeutics for people not at high risk for a severe disease are still ongoing. Although vaccines and new SARS-CoV-2 variants reduce the death toll, the long COVID-19 along with neurologic symptoms can develop and persist even after a mild initial infection. Reinfections, which further increase the risk of sequelae in multiple organ systems as well as the risk of death, continue to require caution. The spike protein of SARS-CoV-2 is an important target for both vaccines and therapeutics. The presence of disulfide bonds in the receptor binding domain (RBD) of the spike protein is essential for its binding to the human ACE2 receptor and cell entry. Here, we demonstrate that thiol-reducing peptides based on the active site of oxidoreductase thioredoxin 1, called thioredoxin mimetic (TXM) peptides, can prevent syncytia formation, SARS-CoV-2 entry into cells, and infection in a mouse model. We also show that TXM peptides inhibit the redox-sensitive HIV pseudotyped viral cell entry. These results support disulfide targeting as a common therapeutic strategy for treating infections caused by viruses using redox-sensitive fusion. Furthermore, TXM peptides exert anti-inflammatory properties by lowering the activation of NF-κB and IRF signaling pathways, mitogen-activated protein kinases (MAPKs) and lipopolysaccharide (LPS)-induced cytokines in mice. The antioxidant and anti-inflammatory effects of the TXM peptides, which also cross the blood-brain barrier, in combination with prevention of viral infections, may provide a beneficial clinical strategy to lower viral infections and mitigate severe consequences of COVID-19.

Suppression of pancreatic cancer proliferation through TXNIP-mediated inhibition of the MAPK signaling pathway.

Thioredoxin-interacting protein (TXNIP) is a crucial thioredoxin-binding protein that is recognized as a tumor suppressor in diverse malignancies, such as breast cancer, lung cancer, hepatocellular carcinoma, and thyroid cancer. However, the specific role and molecular mechanisms of TXNIP in the pathogenesis and progression of pancreatic cancer cells have not been determined. In this study, we investigate the relationship between TXNIP expression and overall survival prognosis in pancreatic cancer patients. Mechanistic studies are conducted to reveal the role of TXNIP in pancreatic cancer cell proliferation, migration, and regulation during malignancy. Our findings indicate that patients with high TXNIP expression have a more favorable prognosis. In vitro experiments with pancreatic cell lines show that overexpression of TXNIP suppresses the proliferation and migration of pancreatic cancer cells. Furthermore, we find that TXNIP inhibits the activation of the MAPK signaling pathway, thereby decreasing the malignant potential of pancreatic cancer. In conclusion, our study reveals TXNIP as a promising new predictive marker and therapeutic target for pancreatic cancer.

Thioredoxin domain containing 5 is involved in the hepatic storage of squalene into lipid droplets in a sex-specific way.

Hepatic thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family found associated with anti-steatotic properties of squalene and located in the endoplasmic reticulum and in lipid droplets. Considering that the latter are involved in hepatic squalene accumulation, the present research was aimed to investigate the role of TXNDC5 on hepatic squalene management in mice and in the AML12 hepatic cell line. Wild-type and TXNDC5-deficient (KO) mice were fed Western diets with or without 1% squalene supplementation for 6 weeks. In males, but not in females, absence of TXNDC5 blocked hepatic, but not duodenal, squalene accumulation. Hepatic lipid droplets were isolated and characterized using label-free LC-MS/MS analysis. TXNDC5 accumulated in this subcellular compartment of mice receiving squalene and was absent in TXNDC5-KO male mice. The latter mice were unable to store squalene in lipid droplets. CALR and APMAP were some of the proteins that responded to the squalene administration in all studied conditions. CALR and APMAP were positively associated with lipid droplets in the presence of squalene and they were decreased by the absence of TXNDC5. The increased squalene content was reproduced in vitro using AML12 cells incubated with squalene-loaded nanoparticles and this effect was not observed in an engineered cell line lacking TXNDC5. The phenomenon was also present when incubated in the presence of a squalene epoxidase inhibitor, suggesting a mechanism of squalene exocytosis involving CALR and APMAP. In conclusion, squalene accumulation in hepatic lipid droplets is sex-dependent on TXNDC5 that blocks its secretion.

Formalin-Fixed Paraffin-Embedded Proteomics of Malignant Mesothelioma and New Candidate Biomarkers Thioredoxin and Superoxide Dismutase 2 for Immunohistochemistry.

The pathogenesis of malignant mesothelioma (MM) has been extensively investigated, focusing on stress derived from reactive oxygen species. We aimed to identify diagnostic biomarkers of MM by analyzing proteins in formalin-fixed paraffin-embedded specimens using liquid chromatography-mass spectrometry. We extracted proteins from formalin-fixed paraffin-embedded sections of MM tissues (n = 7) and compared their profiles with those of benign mesothelial tissues (n = 4) and alveolar tissue (n = 1). Proteomic data were statistically assessed and profiled using principal component analysis. We were successful in the classification of MM and healthy tissue. The levels of superoxide dismutase 2 (SOD2), an enzyme that converts superoxide anion into oxygen and hydrogen peroxide, and thioredoxin (TXN), which plays a crucial role in reducing disulfide bonds in proteins, primarily contributed to the classification. Other redox-related proteins, such as pyruvate dehydrogenase subunit X, and ceruloplasmin also contributed to the classification. Protein-protein interaction analysis demonstrated that these proteins play essential roles in MM pathogenesis. Immunohistochemistry revealed that TXN levels were significantly lower, whereas SOD2 levels were significantly higher in MM and lung cancer tissues than in controls. Proteomic profiling suggested that MM tissues experienced increased exposure to hydrogen peroxide and other reactive oxygen species. Combining immunohistochemistry for TXN and SOD2 allows for differentiation among MM, lung cancer, and control tissues; hence, TXN and SOD2 may be promising MM biomarkers and therapeutic targets.

Kinetic and structural assessment of the reduction of human 2-Cys peroxiredoxins by thioredoxins.

We have studied the reduction reactions of two cytosolic human peroxiredoxins (Prx) in their disulfide form by three thioredoxins (Trx; two human and one bacterial), with the aim of better understanding the rate and mechanism of those reactions, and their relevance in the context of the catalytic cycle of Prx. We have developed a new methodology based on stopped-flow and intrinsic fluorescence to study the bimolecular reactions, and found rate constants in the range of 105 -106 m-1 s-1 in all cases, showing that there is no marked kinetic preference for the expected Trx partner. By combining experimental findings and molecular dynamics studies, we found that the reactivity of the nucleophilic cysteine (CN ) in the Trx is greatly affected by the formation of the Prx-Trx complex. The protein-protein interaction forces the CN thiolate into an unfavorable hydrophobic microenvironment that reduces its hydration and results in a remarkable acceleration of the thiol-disulfide exchange reactions by more than three orders of magnitude and also produces a measurable shift in the pKa of the CN . This mechanism of activation of the thiol disulfide exchange may help understand the reduction of Prx by alternative reductants involved in redox signaling.

Macrophage-expressed SRA ameliorates alcohol-induced liver injury by suppressing S-glutathionylation of Notch1 via recruiting thioredoxin.

Scavenger receptor A (SRA) is preferentially expressed in macrophages and implicated as a multifunctional pattern recognition receptor for innate immunity. Hepatic macrophages play a primary role in the pathogenesis of alcoholic liver disease. Herein, we observed that SRA expression was significantly increased in the liver tissues of mice with alcohol-related liver injury. SRA-deficient (SRA-/-) mice developed more severe alcohol-induced liver disease than wild-type mice. Enhanced liver inflammation existed in alcohol-challenged SRA-/- mice and was associated with increased Notch activation in hepatic macrophages compared with wild-type control animals. Mechanistically, SRA directly bound with Notch1 and suppressed its S-glutathionylation, thereby inhibiting Notch pathway activation. Further, we determined that the SRA interacted with thioredoxin-1 (Trx-1), a redox-active protein. SRA inhibited Trx-1 dimerization and facilitated the interaction of Trx-1 with Notch1. Application of a Trx-1-specific inhibitory agent during macrophage stimulation abolished SRA-mediated regulation of the Notch pathway and its downstream targets. In summary, our study revealed that SRA plays a critical role in macrophage inflammatory response by targeting Notch1 for its glutathionylation. SRA-mediated negative regulation of Notch activation might serve as a novel therapeutic strategy for alcohol-induced liver injury.

Redox Protein Thioredoxin Mediates Neurite Outgrowth in Primary Cultured Mouse Cerebral Cortical Neurons.

Thioredoxin system plays an important role in maintaining the cellular redox balance. Recent evidence suggests that thioredoxin (Trx) system may promote cell survival and neuroprotection. In this study, we explored the role of thioredoxin system in neuronal differentiation using a primary mouse cortical neuronal cell culture. First, Trx and Trx reductase (TrxR) protein levels were analyzed in cultured neurons from 1 to 32 days in vitro (DIV). The result showed that Trx and TrxR protein levels time-dependently increased in the neuron cell culture from 1 to 18 DIV. To establish the role of Trx in neuronal differentiation, Trx gene expression was knockdown in cultured neurons using Trx sgRNA CRISPR/Cas9 technology. Treatment with CRISPR/Cas9/Trx sgRNA decreased Trx protein levels and caused a reduction in dendritic outgrowth and branching of cultured neurons. Then, primary cortical neurons were treated with the Trx inhibitor PX12 to block Trx reducing activity. Treatment with PX12 also reduced dendritic outgrowth and branching. Furthermore, PX12 treatment reduced the ratio of phosphorylated cyclic AMP response element-binding protein (CREB)/total CREB protein levels. To investigate whether CREB phosphorylation is redox regulated, SH-SY5Y cells were treated with H2O2, which reduced phosphorylated CREB protein levels and increased CREB thiol oxidation. However, treatment with CB3, a Trx-mimetic tripeptide, rescued H2O2-decreased CREB phosphorylation. Our results suggest that Trx regulates neuronal differentiation and maturation of primary mouse cortical neurons by targeting CREB neurotrophic pathway. Trx may regulate CREB activation by maintaining the cellular redox balance.

Deficiency of thioredoxin-interacting protein results in age-related thrombocytopenia due to megakaryocyte oxidative stress.

BACKGROUND: Platelets are generated from megakaryocytes (MKs), mainly located in the bone marrow (BM). Megakaryopoiesis can be affected by genetic disorders, metabolic diseases, and aging. The molecular mechanisms underlying platelet count regulation have not been fully elucidated. OBJECTIVES: In the present study, we investigated the role of thioredoxin-interacting protein (TXNIP), a protein that regulates cellular metabolism in megakaryopoiesis, using a Txnip-/- mouse model. METHODS: Wild-type (WT) and Txnip-/- mice (2-27-month-old) were studied. BM-derived MKs were analyzed to investigate the role of TXNIP in megakaryopoiesis with age. The global transcriptome of BM-derived CD41+ megakaryocyte precursors (MkPs) of WT and Txnip-/- mice were compared. The CD34+ hematopoietic stem cells isolated from human cord blood were differentiated into MKs. RESULTS: Txnip-/- mice developed thrombocytopenia at 4 to 5 months that worsened with age. During ex vivo megakaryopoiesis, Txnip-/- MkPs remained small, with decreased levels of MK-specific markers. Critically, Txnip-/- MkPs exhibited reduced mitochondrial reactive oxygen species, which was related to AKT activity. Txnip-/- MkPs also showed elevated glycolysis alongside increased glucose uptake for ATP production. Total RNA sequencing revealed enrichment for oxidative stress- and apoptosis-related genes in differentially expressed genes between Txnip-/- and WT MkPs. The effects of TXNIP on MKs were recapitulated during the differentiation of human cord blood-derived CD34+ hematopoietic stem cells. CONCLUSION: We provide evidence that the megakaryopoiesis pathway becomes exhausted with age in Txnip-/- mice with a decrease in terminal, mature MKs that response to thrombocytopenic challenge. Overall, this study demonstrates the role of TXNIP in megakaryopoiesis, regulating mitochondrial metabolism.