Inhibition of the PERK/TXNIP/NLRP3 Axis by Baicalin Reduces NLRP3 Inflammasome-Mediated Pyroptosis in Macrophages Infected with Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) remains a significant threat to global health as it induces granuloma and systemic inflammatory responses during active tuberculosis. Mtb can induce macrophage pyroptosis, leading to the release of IL-1ß and tissue damage, promoting its spread. Here, we established an in vitro Mtb-infected macrophage model to seek an effective antipyroptosis agent. Baicalin, isolated from Radix Scutellariae, was found to reduce pyroptosis in Mtb-infected macrophages. Baicalin could inhibit activation of the PERK/eIF2α pathway and thus downregulates TXNIP expression and subsequently reduces activation of the NLRP3 inflammasome, resulting in reduced pyroptosis in Mtb-infected macrophages. In conclusion, baicalin reduced pyroptosis by inhibiting the PERK/TXNIP/NLRP3 axis and might thus be a new adjuvant host-directed therapy (HDT) drug.

Structure basis of non-structural protein pA151R from African Swine Fever Virus.

African Swine Fever Virus (ASFV) is an enveloped double-stranded DNA icosahedral virus that causes the devastating hemorrhagic fever of pigs. ASFV infections severely impact swine production and cause an enormous economic loss, but no effective vaccine and therapeutic regimen is available. pA151R is a non-structural protein of ASFV, which is expressed at both early and late stages of viral infection. Significantly, pA151R may play a key role in ASFV replication and virus assembly as suppressing pA151R expression can reduce virus replication. However, little is known about the functional and structural mechanisms of pA151R because it shares a very low sequence identity to known structures. It was proposed that pA151R might participate in the redox pathway owing to the presence of a thioredoxin active site feature, the WCTKC motif. In this study, we determined the crystal structure of pA151R. Based on the crystal structure, we found that pA151R comprises of a central five-stranded ß-sheet packing against two helices on one side and an incompact C-terminal region containing the WCTKC motif on the other side. Notably, two cysteines in the WCTKC motif, an additional cysteine C116 from the ß7-ß8 loop together with ND1 of H109 coordinate a Zn2+ ion to form a Zn-binding motif. These findings suggest that the structure of pA151R is significantly different from that of typical thioredoxins. Our structure should provide molecular insights into the understanding of functional and structural mechanisms of pA151R from ASFV and shall benefit the development of prophylactic and therapeutic anti-ASFV agents.

Salvianolic acid A attenuated myocardial infarction-induced apoptosis and inflammation by activating Trx.

Myocardial infarction (MI) is a leading cause of mortality worldwide and it is urgent to discover effective therapies. In this study, the protective effect of salvianolic acid A (SAL) on MI induced by left anterior descending coronary artery ligation surgery and H2O2-induced H9c2 damage was evaluated. Rats were intraperitoneally injected with SAL once a day for 2 days before MI. At 24-h post-MI, the SAL-treated group showed significantly decreased infarct rate and enhanced myocardial function. Meanwhile, myocardial injury enzymes such as aspartate transaminase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK) were significantly reduced by SAL treatment. Taking advantage of RNA-seq technology, 52 disease targets of MI were associated with differentially expressed genes after SAL treatment in MI, among which 21 inflammation-related genes and 16 MAPK cascade-related genes were found. Further experiment indicated that SAL treatment reduced inflammatory factors such as IL-1ß, IL-6, and TNF-α and decreased tunnel-positive cells and pro-apoptotic Bax after MI. Further investigation revealed that SAL treatment elevated thioredoxin (Trx) and inhibited the activation of c-jun N-terminal kinase (JNK) to attenuate apoptosis and inflammation after MI. Consistently, SAL protected cardiomyocytes against H2O2-induced H9c2 damage through increasing cell viability, decreasing cell apoptosis, and activating Trx and inhibiting JNK. Taken together, SAL inhibited cell apoptosis and inflammation through Trx/JNK signaling.

Thioredoxin-mediated redox signalling in plant immunity.

Activation of plant immune responses is associated with rapid production of vast amounts of reactive oxygen and nitrogen species (ROS/RNS) that dramatically alter cellular redox homeostasis. Even though excessive ROS/RNS accumulation can cause widespread cellular damage and thus constitute a major risk, plant cells have evolved to utilise these molecules as important signalling cues. Particularly their ability to modify redox-sensitive cysteine residues has emerged as a key mechanism to control the activity, conformation, protein-protein interaction and localisation of a growing number of immune signalling proteins. Regulated reversal of cysteine oxidation is dependent on activities of the conserved superfamily of Thioredoxin (TRX) enzymes that function as cysteine reductases. The plant immune system recruits specific TRX enzymes that have the potential to functionally regulate numerous immune signalling proteins. Although our knowledge of different TRX immune targets is now expanding, little remains known about how these enzymes select their substrates, what range of oxidized residues they target, and if they function selectively in different redox-mediated immune signalling pathways. In this review we discuss these questions by examining evidence showing TRX enzymes exhibit novel activities that play important roles in diverse aspects of plant immune signalling.

The Thioredoxin-Like Family of Selenoproteins: Implications in Aging and Age-Related Degeneration.

The thioredoxin-like (Rdx) family proteins contain four selenoproteins (selenoprotein H, SELENOH; selenoprotein T, SELENOT; selenoprotein V, SELENOV; selenoprotein W, SELENOW) and a nonselenoprotein Rdx12. They share a CxxU or a CxxC (C, cysteine; x, any amino acid; U, selenocysteine) motif and a stretch of eGxFEI(V) sequence. From the evolutionary perspective, SELENOW and SELENOV are clustered together and SELENOH and SELENOT are in another branch. Selenoproteins in the Rdx family exhibit tissue- and organelle-specific distribution and are differentially influenced in response to selenium deficiency. While SELENOH is nucleus-exclusive, SELENOT resides mainly in endoplasmic reticulum and SELENOW in cytosol. SELENOV is expressed essentially only in the testes with unknown cellular localization. SELENOH and SELENOW are more sensitive than SELENOT and SELENOV to selenium deficiency. While physiological functions of the Rdx family of selenoproteins are not fully understand, results from animal models demonstrated that (1) brain-specific SELENOT knockout mice are susceptible to 1-methyl-4-phenylpyridinium-induced Parkinson's disease in association with redox imbalance and (2) adult zebrafishes with heterozygous SELENOH knockout are prone to dimethylbenzanthracene-induced tumorigenesis together with increased DNA damage and oxidative stress. Further animal and human studies are needed to fully understand physiological roles of the Rdx family of selenoproteins in redox regulation, genome maintenance, aging, and age-related degeneration.

TXNDC9 promotes hepatocellular carcinoma progression by positive regulation of MYC-mediated transcriptional network.

The thioredoxin domain containing proteins are a group of proteins involved in redox regulation and have been recently reported to be associated with tumor progression. However, the role of thioredoxin proteins in hepatocellular carcinoma (HCC) remains largely unknown. Here in our study, we demonstrated that thioredoxin domain containing protein 9 (TXNDC9) was over-expressed in HCC and promoted HCC progression. We found that TXNDC9 expression was amplified in HCC tissues and associated with an advanced grade of HCC. And, we demonstrated that overexpression of TXNDC9 was correlated with poor prognosis of HCC. Furthermore, by using CRISPR-Cas9 mediated TXNDC9 knockout and RNA-seq analysis, we found that TXNDC9 accelerated HCC proliferation regulation. Moreover, we demonstrated that TXNDC9 directly interacted with MYC and knockout/knockdown of TXNDC9 decreased the protein levels of MYC and inhibited MYC-mediated transcriptional activation of its targets. Besides, we identified that TXNDC9 was trans-activated by FOXA1, JUND, and FOSL2 in HCC. Taken together, our study unveiled an oncogenic role of TXNDC9 in HCC and provided a mechanistic insight into the TXNDC9 mediated gene regulation network during HCC development.

Cellular stress and redox activity proteins are involved in gastric carcinogenesis associated with Helicobacter pylori infection expressing high levels of thioredoxin-1.

Helicobacter pylori infection is related to the development of gastric diseases. Our previous studies showed that high thioredoxin-1 (Trx1) expression in H. pylori can promote gastric carcinogenesis. To explore the underlying molecular mechanisms, we performed an isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis of stomach tissues from Mongolian gerbil infected with H. pylori expressing high and low Trx1. Differences in the profiles of the expressed proteins were analyzed by bioinformatics and verified using Western blot analysis. We found three candidate proteins, 14-3-3α/ß, glutathione-S-transferase (GST), and heat shock protein 70 (HSP70), in high Trx1 tissues compared with low Trx1 tissues and concluded that cellular stress and redox activity-related proteins were involved in the pathogenesis of gastric cancer associated with H. pylori Trx1.

[Do Myokines Have Potential as Exercise Mimetics?]

　Exercise is generally considered to have health benefits for the body, although its beneficial mechanisms have not been fully elucidated. Recent progressive research suggests that myokines, bioactive substances secreted from skeletal muscle, play an important role in mediating the benefits of exercise. There are three types of myokines in terms of the muscular secretion mechanism: those in which the secretion is promoted by stimulation, such as irisin, interleukin (IL)-6, and IL-15; those whose secretion is constitutive, such as thioredoxin, glutaredoxin, and peroxiredoxin; and those whose secretion is suppressed by stimulation, such as by a macrophage migration inhibitory factor. Although dozens of myokines have been reported, their physiological roles are not well understood. Therefore, there currently exists no advanced drug discovery research specifically targeting myokines, with the exception of Myostatin. Myostatin was discovered as a negative regulator of muscle growth. Myostatin is secreted from muscle cells as a myokine; it signals via an activin type IIB receptor in an autocrine manner, and regulates gene expressions involved in myogenesis. Given the studies to date that have been conducted on the utilization of myostatin inhibitors for the treatment of muscle weakness, including cachexia and sarcopenia, other myokines may also be new potential drug targets.

[Effects and related mechanism of overexpression of human thioredoxin on the inflammatory response in mice with viral myocarditis].

Objective: To observe the effects of recombinant adenovirus with human thioredoxin (hTRX) on the inflammatory response in mice with viral myocarditis and explore the related mechanism. Methods: Sixty Balb/c male mice were randomly divided into control group, myocarditis group, and hTRX group according to the random number table (n=20 each group). The myocarditis group and hTRX group were injected with 100 TCID(50) Coxackie virus B3 (0.1 ml) in the abdomen and control group were injected with saline. Two days before the viral injection, the hTRX group were injected with recombinant adenovirus vector coding the human thioredoxin gene by pericardial puncture and the control group and myocarditis group were injected with recombinant adenovirus vector without coding gene by pericardial puncture, all these mice were killed and hearts were removed 7 days later. The morphology of myocardial tissue in each group was detected by HE staining and the ultrastructure changes by electron microscope. The protein expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and NF-κB were detected by ELISA and Western blot. Immunohistochemical staining was performed to observe the protein expression levels of thioredoxin. Results: Necrosis of myocardial cells and a small amount of cell infiltration were found in the myocarditis group and necrosis and cell infiltration were significantly reduced in the hTRX group and no myocardial lesion was found in control group on HE stained sections. Electron microscope examination evidenced cell swelling and dissolved myofilament, vacuoles degeneration in mitochondria in the myocarditis group. These changes were significantly reduced in the hTRX group. There was no myocardial lesion in control group. The protein expression of TNF-α, IL-1ß and NF-κB were significantly upregulated in myocarditis group than in control group (all P<0.01). The protein expression of TNF-α, IL-1ß and NF-κB were significantly downregulated in hTRX group than in myocarditis group (all P<0.01). Immunohistochemical staining showed that protein expression of hTRX was higher in hTRX group than in myocarditis group (P<0.01). Conclusion: Recombinant adenovirus hTRX can attenuate cardiac injury in mice with acute myocarditis via inhibiting the inflammatory response and downregulating the expression of TNF-α, IL-1ß and NF-κB.

The thioredoxin-1 system is essential for fueling DNA synthesis during T-cell metabolic reprogramming and proliferation.

The thioredoxin-1 (Trx1) system is an important contributor to cellular redox balance and is a sensor of energy and glucose metabolism. Here we show critical c-Myc-dependent activation of the Trx1 system during thymocyte and peripheral T-cell proliferation, but repression during T-cell quiescence. Deletion of thioredoxin reductase-1 (Txnrd1) prevents expansion the CD4-CD8- thymocyte population, whereas Txnrd1 deletion in CD4+CD8+ thymocytes does not affect further maturation and peripheral homeostasis of αßT cells. However, Txnrd1 is critical for expansion of the activated T-cell population during viral and parasite infection. Metabolomics show that TrxR1 is essential for the last step of nucleotide biosynthesis by donating reducing equivalents to ribonucleotide reductase. Impaired availability of 2'-deoxyribonucleotides induces the DNA damage response and cell cycle arrest of Txnrd1-deficient T cells. These results uncover a pivotal function of the Trx1 system in metabolic reprogramming of thymic and peripheral T cells and provide a rationale for targeting Txnrd1 in T-cell leukemia.

Maintaining Cone Function in Rod-Cone Dystrophies.

Retinal degenerative diseases are a major cause of untreatable blindness due to a loss of photoreceptors. Recent advances in genetics and gene therapy for inherited retinal dystrophies (IRDs) showed that therapeutic gene transfer holds a great promise for vision restoration in people with currently incurable blinding diseases. Due to the huge genetic heterogeneity of IRDs that represents a major obstacle for gene therapy development, alternative therapeutic approaches are needed. This review focuses on the rescue of cone function as a therapeutic option for maintaining central vision in rod-cone dystrophies. It highlights recent developments in better understanding the mechanisms of action of the trophic factor RdCVF and its potential as a sight-saving therapeutic strategy.

Thioredoxin regulates G6PDH activity by changing redox states of OpcA in the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120.

Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first reaction in the oxidative pentose phosphate pathway. In green plant chloroplasts, G6PDH is a unique redox-regulated enzyme, since it is inactivated under the reducing conditions. This regulation is accomplished using a redox-active cysteine pair, which is conserved in plant G6PDH. The inactivation of this enzyme under conditions of light must be beneficial to prevent release of CO2 from the photosynthetic carbon fixation cycle. In the filamentous, heterocyst-forming, nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 (Anabaena 7120), G6PDH plays a pivotal role in providing reducing power for nitrogenase, and its activity is also reported to be suppressed by reduction, though Anabaena G6PDH does not conserve the critical cysteines for regulation. Based on the thorough analyses of the redox regulation mechanisms of G6PDH from Anabaena 7120 and its activator protein OpcA, we found that m-type thioredoxin regulates G6PDH activity by changing the redox states of OpcA. Mass spectrometric analysis and mutagenesis studies indicate that Cys393 and Cys399 of OpcA are responsible for the redox regulation property of this protein. Moreover, in vivo analyses of the redox states of OpcA showed that more than half of the OpcA is present as an oxidized form, even under conditions of light, when cells are cultured under the nitrogen-fixing conditions. This redox regulation of OpcA might be necessary to provide reducing power for nitrogenase by G6PDH in heterocysts even during the day.

New Clues in the Malignant Progression of Glioblastoma: Can the Thioredoxin System Play a Role?

AIM: To evaluate and compare the expression of thioredoxin reductase 1 (TrxR1) in primary and secondary glioblastoma samples. MATERIAL AND METHODS: Surgically resected human glioblastoma samples from 40 patients who underwent surgery at our institution were extracted from their histopathological specimens and divided into three groups. Ten histopathologically regular cerebral tissue samples, acquired from the non-neoplastic portion of the specimens, were assigned as the control group. Twenty specimens that included tumoral tissue from each type of glioblastoma (WHO grade IV, primary and secondary) were assigned as the primary and secondary glioblastoma groups. TrxR1 expression was analyzed by using both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Isocitrate dehydrogenase 1 (IDH1) mutation was analyzed by immunohistochemistry. Ki-67 proliferative index and apoptosis were also analyzed by immunohistochemistry. The differences between the groups were statistically compared and the correlation between these parameters was analyzed. RESULTS: The expressions of TrxR1 and Ki-67 values were significantly higher in primary glioblastoma. IDH1 mutation was significantly higher in secondary glioblastoma. TrxR1 expression was found to be highly correlated with the Ki-67 index. The apoptotic index was similar between primary and secondary glioblastoma. CONCLUSION: This study showed a high TrxR1 expression in primary glioblastoma which could indicate a role of the Trx system in promoting the malignant progression by some complex processes.

Thioredoxin-interacting protein regulates haematopoietic stem cell ageing and rejuvenation by inhibiting p38 kinase activity.

Ageing is a natural process in living organisms throughout their lifetime, and most elderly people suffer from ageing-associated diseases. One suggested way to tackle such diseases is to rejuvenate stem cells, which also undergo ageing. Here we report that the thioredoxin-interacting protein (TXNIP)-p38 mitogen-activated protein kinase (p38) axis regulates the ageing of haematopoietic stem cells (HSCs), by causing a higher frequency of long-term HSCs, lineage skewing, a decrease in engraftment, an increase in reactive oxygen species and loss of Cdc42 polarity. TXNIP inhibits p38 activity via direct interaction in HSCs. Furthermore, cell-penetrating peptide (CPP)-conjugated peptide derived from the TXNIP-p38 interaction motif inhibits p38 activity via this docking interaction. This peptide dramatically rejuvenates aged HSCs in vitro and in vivo. Our findings suggest that the TXNIP-p38 axis acts as a regulatory mechanism in HSC ageing and indicate the potent therapeutic potential of using CPP-conjugated peptide to rejuvenate aged HSCs.

Plastidic P2 glucose-6P dehydrogenase from poplar is modulated by thioredoxin m-type: Distinct roles of cysteine residues in redox regulation and NADPH inhibition.

A cDNA coding for a plastidic P2-type G6PDH isoform from poplar (Populus tremula x tremuloides) has been used to express and purify to homogeneity the mature recombinant protein with a N-terminus His-tag. The study of the kinetic properties of the recombinant enzyme showed an in vitro redox sensing modulation exerted by reduced DTT. The interaction with thioredoxins (TRXs) was then investigated. Five cysteine to serine variants (C145S - C175S - C183S - C195S - C242S) and a variant with a double substitution for Cys175 and Cys183 (C175S/C183S) have been generated, purified and biochemically characterized in order to investigate the specific role(s) of cysteines in terms of redox regulation and NADPH-dependent inhibition. Three cysteine residues (C145, C194, C242) are suggested to have a role in controlling the NADP+ access to the active site, and in stabilizing the NADPH regulatory binding site. Our results also indicate that the regulatory disulfide involves residues Cys175 and Cys183 in a position similar to those of chloroplastic P1-G6PDHs, but the modulation is exerted primarily by TRX m-type, in contrast to P1-G6PDH, which is regulated by TRX f. This unexpected specificity indicates differences in the mechanism of regulation, and redox sensing of plastidic P2-G6PDH compared to chloroplastic P1-G6PDH in higher plants.

Roles of the glutathione- and thioredoxin-dependent systems in the Escherichia coli responses to ciprofloxacin and ampicillin.

Recently, it was proposed that some antibiotics stimulate the production of reactive oxygen species (ROS), which contribute to cell death. Later, other research groups have provided arguments against ROS-mediated killing of bacteria by antibiotics. At present, there remain a number of unanswered questions in understanding of the role of ROS in killing by antibiotics. Mutants of Escherichia coli in components of the thioredoxin and glutaredoxin redox pathways used in this study possess a great variability in antioxidant activity, and they therefore are a useful model for the investigation of the role of oxidative stress in bactericidal effect of antibiotics. Statistical analysis did not reveal a significant correlation between the susceptibility of the mutants to ciprofloxacin and ampicillin and their resistance to peroxide stress. However, we found strong reverse correlations between the bactericidal activity of antibiotics and the specific growth rate of these mutants at the moment of drug addition. Supplements changing the level of intra- and extracellular glutathione considerably affected E. coli susceptibility to ciprofloxacin and ampicillin. The effect of GSH precursors on bactericidal activity of antibiotics was also observed in gshA mutants.

Human thioredoxin-1 attenuates the rate of lipopolysaccharide-induced preterm delivery in mice in association with its anti-inflammatory effect.

BACKGROUND: Maternal intrauterine infection/inflammation represents the major etiology of preterm delivery and the leading cause of neonatal mortality and morbidity. The aim of this study was to investigate the anti-inflammatory properties of thioredoxin-1 in vivo and its potential ability to attenuate the rate of inflammation-induced preterm delivery. METHODS: Two intraperitoneal injections of lipopolysaccharide from Escherichia coli were administered in pregnant mice on gestational day 15, with a 3-h interval between the injections. From either 1 h before or 1 h after the first lipopolysaccharide injection, mice received three intravenous injections of either recombinant human thioredoxin-1, ovalbumin, or vehicle, with a 3-h interval between injections. RESULTS: Intraperitoneal injection of lipopolysaccharide induced a rise of tumor necrosis factor-α, interferon-γ, monocyte chemotactic protein 1, and interleukin-6 in maternal serum levels and provoked preterm delivery. Recombinant human thoredoxin-1 prevented the rise in these proinflammatory cytokine levels. After the inflammatory challenge, placentas exhibited severe maternal vascular dilatation and congestion and a marked decidual neutrophil activation. These placental pathological findings were ameliorated by recombinant human thioredoxin-1, and the rate of inflammation-induced preterm delivery was attenuated. CONCLUSION: Thioredoxin-1 may thus represent a novel effective treatment to delay inflammation-induced preterm delivery.

Response of esophageal cancer cells to epigenetic inhibitors is mediated via altered thioredoxin activity.

We previously showed that histone deacetylase inhibitor (HDACi) and 5-azacytidine (AZA) treatment selectively induced cell death of esophageal cancer cells. The mechanisms of cancer selectivity, however, remained unclear. Here we examined whether the cancer selectivity of HDACi/AZA treatment is mediated by the thioredoxin (Trx) system and reactive oxygen species (ROS) in esophageal cancer cells. For this, we first analyzed human tissue specimens of 37 esophageal cancer patients by immunohistochemistry for Trx, Trx-interacting protein (TXNIP) and Trx reductase (TXNRD). This revealed a loss or at least reduction of nuclear Trx in esophageal cancer cells, compared with normal epithelial cells (P<0.001). Although no differences were observed for TXNIP, TXNRD was more frequently expressed in cancer cells (P<0.001). In the two main histotypes of esophageal squamous cell carcinomas (ESCCs, n=19) and esophageal adenomcarcinomas (EAC, n=16), similar Trx, TXNIP and TXNRD expression patterns were observed. Also in vitro, nuclear Trx was only detectable in non-neoplastic Het-1A cells, but not in OE21/ESCC or OE33/EAC cell lines. Moreover, the two cancer cell lines showed an increased Trx activity, being significant for OE21 (P=0.0237). After treatment with HDACi and/or AZA, ROS were exclusively increased in both cancer cell lines (P=0.048-0.017), with parallel decrease of Trx activity. This was variably accompanied by increased TXNIP levels upon AZA, MS-275 or MS-275/AZA treatment for 6 or 24 h in OE21, but not in Het-1A or OE33 cells. In summary, this study evaluated Trx and its associated proteins TXNIP and TXNRD for the first time in esophageal cancers. The analyses revealed an altered subcellular localization of Trx and strong upregulation of TXNRD in esophageal cancer cells. Moreover, HDACi and AZA disrupted Trx function and induced accumulation of ROS with subsequent apoptosis in esophageal cancer cells exclusively. Trx function is hence an important cellular mediator conferring non-neoplastic cell resistance for HDACi and/or AZA.