Thioredoxin Family Research Articles

Functional characterization of peroxiredoxin 5 from yellowtail clownfish (Amphiprion clarkii): Immunological expression assessment, antioxidant activities, heavy metal detoxification, and nitrosative stress mitigation

Peroxiredoxin 5 (Prdx5) is the last recognized member of Prdx family. It is a unique, atypical, 2-Cys antioxidant enzyme, protecting cells from death caused by reactive oxygen species (ROS). In this study, the Prdx5 ortholog of Amphiprion clarkii (AcPrdx5) was identified and characterized to explore its specific structural features and functional properties. The open reading frame of AcPrdx5 is 573 bp long and encodes 190 amino acids containing a mitochondrial targeting sequence, thioredoxin domain, and two conserved cysteine residues responsible for antioxidant function. The predicted molecular weight and theoretical isoelectric point of AcPrdx5 are 20.3 kDa and 9.01, respectively. AcPrdx5 sequences were found to be highly conserved across the other orthologs from various organisms and it distinctively clustered within the fish Prdx5 subclade of the phylogenetic tree. The expression of AcPrdx5 was ubiquitously detected among twelve tested tissues, with the highest level in the brain. Furthermore, the mRNA levels of AcPrdx5 in the blood and head-kidney tissues were significantly (p < 0.05) upregulated following polyinosinic-polycytidylic acid (Poly I:C), lipopolysaccharide (LPS), and Vibrio harveyi immune challenge. A concentration-dependent antioxidant potential of recombinant AcPrdx5 was observed in insulin disulfide bond reduction, heavy metal detoxification, free radical and hydrogen peroxide (H2O2) scavenging assays. Additionally, AcPrdx5 overexpression in fathead minnow (FHM) cells upregulated the antioxidant-associated gene (Rrm1, MAPK, SOD2, and PRDX1) expression after H2O2 treatment, and promoted cell viability upon arsenic (As) exposure. In macrophages, AcPrdx5 overexpression effectively suppressed substantial nitric oxide production under lipopolysaccharide treatment. Collectively, our results suggest that AcPrdx5 may play roles in both antioxidant defense system and innate immune response against pathogenic invasions in A. clarkii.

Nme8 is essential for protection against chemotherapy drug cisplatin-induced male reproductive toxicity in mice.

Cisplatin (CP), a chemotherapy drug commonly used in cancers treatment, causes serious reproductive toxicity. With younger cancer patients and increasing survival rates, it is important to preserve their reproductive capacity. NME8 is highly expressed in testis and contains thioredoxin and NDPK domains, suggesting it may be a target against the CP-induced reproductive toxicity. We deleted exons 6-7 of the Nme8 in mice based on human mutation sites and observed impaired transcript splicing. In mice, Nme8 was not essential for spermatogenesis, possibly due to functional compensation by its paralog, Nme5. Nme8 expression was elevated and translocated to the nucleus in response to two weeks of CP treatment. Under CP treatment, Nme8 deficiency further impaired antioxidant capacity, induced lipid peroxidation and increased ROS level, and failed to activate autophagy, resulting in aggravated DNA damage in testes and sperm. Consequently, the proliferation and differentiation of spermatogonia and the meiosis of spermatocyte were almost completely halted, and sperm motility was impaired. Our research indicates that NME8 protects against CP-induced testis and sperm damage. This may provide new insights into the physiological functions of the Nme family and potential targets for preserving fertility in young male cancer patients.

Protein folding dependence on selenoprotein M contributes to steady cartilage extracellular matrix repressing ferroptosis via PERK/ATF4/CHAC1 axis

ObjectiveInitiation of endoplasmic reticulum (ER) stress is pivotal to the advancement of osteoarthritis (OA). We aimed to explore the function of ER-resident selenoprotein M (SELM) in cartilage-forming chondrocytes, investigating how SELM participates in cartilage extracellular matrix (ECM) metabolism and ER stress modulation. MethodsArticular cartilage samples with knee OA undergoing total knee arthroplasty were categorized into OA-smooth and OA-damaged groups, with primary chondrocytes extracted from smooth areas. Destabilization of the medial meniscus was induced in male C57BL6/J mice, with sham operations on the left knee as controls. After 8 weeks, knee joint tissues were collected for analysis. Histology and immunohistochemistry examined cartilage damage. Molecular biology techniques investigated how SELM affects ECM metabolism and ER stress regulation. RNA sequencing revealed the pathway changes after SELM intervention. AlphaFold demonstrated how SELM interacts with other molecules. Cultured cartilage explants helped determine the effects of SELM supplementation. ResultsSELM expression was reduced in the damaged cartilage. Increasing SELM levels positively impacted ECM equilibrium. Decreasing SELM expression activated genes linked to degenerative ailments and impaired the cellular response to misfolded proteins, initiating the PERK/P-EIF2A/ATF4 pathway and exacerbating GSH/GSSG imbalance via the ATF4/CHAC1 axis. SELM likely participated in protein folding and modification by leveraging its thioredoxin domains. In vitro SELM supplementation mitigated IL-1β effects on damaged cartilage explants and suppressed beneficial chondrocyte phenotypes. ConclusionsOur results confirm the involvement of SELM in ER stress-induced cartilage damage as well as protein folding, pointing to new directions in molecular therapy for degenerative diseases.

Parecoxib Enhances Resveratrol against Human Colorectal Cancer Cells through Akt and TXNDC5 Inhibition and MAPK Regulation.

In this study, we discovered the mechanisms underlying parecoxib and resveratrol combination's anti-cancer characteristics against human colorectal cancer DLD-1 cells. We studied its anti-proliferation and apoptosis-provoking effect by utilizing cell viability 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, fluorescence microscope, gene overexpression, Western blot, and flow cytometry analyses. Parecoxib enhanced the ability of resveratrol to inhibit cell viability and increase apoptosis. Parecoxib in combination with resveratrol strongly enhanced apoptosis by inhibiting the expression of thioredoxin domain containing 5 (TXNDC5) and Akt phosphorylation. Parecoxib enhanced resveratrol-provoked c-Jun N-terminal kinase (JNK) and p38 phosphorylation. Overexpression of TXNDC5 and repression of JNK and p38 pathways significantly reversed the inhibition of cell viability and stimulation of apoptosis by the parecoxib/resveratrol combination. This study presents evidence that parecoxib enhances the anti-cancer power of resveratrol in DLD-1 colorectal cancer cells via the inhibition of TXNDC5 and Akt signaling and enhancement of JNK/p38 MAPK pathways. Parecoxib may be provided as an efficient drug to sensitize colorectal cancer by resveratrol.

The Thioredoxin Fold Protein (TFP2) from Extreme Acidophilic Leptospirillum sp. CF-1 Is a Chaperedoxin-like Protein That Prevents the Aggregation of Proteins under Oxidative Stress.

Extreme acidophilic bacteria like Leptospirillum sp. require an efficient enzyme system to counteract strong oxygen stress conditions in their natural habitat. The genome of Leptospirillum sp. CF-1 encodes the thioredoxin-fold protein TFP2, which exhibits a high structural similarity to the thioredoxin domain of E. coli CnoX. CnoX from Escherichia coli is a chaperedoxin that protects protein substrates from oxidative stress conditions using its holdase function and a subsequent transfer to foldase chaperones for refolding. Recombinantly produced and purified Leptospirillum sp. TFP2 possesses both thioredoxin and chaperone holdase activities in vitro. It can be reduced by thioredoxin reductase (TrxR). The tfp2 gene co-locates with genes for the chaperone foldase GroES/EL on the chromosome. The "tfp2 cluster" (ctpA-groES-groEL-hyp-tfp2-recN) was found between 1.9 and 8.8-fold transcriptionally up-regulated in response to 1 mM hydrogen peroxide (H2O2). Leptospirillum sp. tfp2 heterologously expressed in E. coli wild type and cnoX mutant strains lead to an increased tolerance of these E. coli strains to H2O2 and significantly reduced intracellular protein aggregates. Finally, a proteomic analysis of protein aggregates produced in E. coli upon exposition to oxidative stress with 4 mM H2O2, showed that Leptospirillum sp. tfp2 expression caused a significant decrease in the aggregation of 124 proteins belonging to fifteen different metabolic categories. These included several known substrates of DnaK and GroEL/ES. These findings demonstrate that Leptospirillum sp. TFP2 is a chaperedoxin-like protein, acting as a key player in the control of cellular proteostasis under highly oxidative conditions that prevail in extreme acidic environments.

Unraveling the Role of the Liriodendron Thioredoxin (TRX) Gene Family in an Abiotic Stress Response.

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.

Hydrogen peroxide positively regulates ABA signaling via oxidative modification of the C2H2-type zinc finger protein ZFP36 in rice

The rice zinc finger protein ZFP36 serves as a pivotal regulator of the hydrogen peroxide (H2O2) signaling pathway in response to abscisic acid (ABA). Its role is crucial for integrating H2O2 signals with the plant defense mechanisms against water deficit and oxidative stress. However, it remains unclear whether ZFP36 directly modulates ABA-induced H2O2 signaling. This study explored the effects of oxidative post-translational modifications (OxiPTMs) on ZFP36 in rice, with an emphasis on the H2O2-induced oxidation through its cysteine (Cys) residues. We found that ZFP36 undergoes oxidative modification as a target of H2O2 in the presence of ABA, specifically at Cys32. Employing quantitative detection and fluorescence assays, we observed that ZFP36 oxidation enhances the expression and activity of genes encoding protective antioxidant enzymes. Moreover, our investigation into the thioredoxin (Trx) and glutaredoxin (Grx) families revealed that OsTrxh1 facilitates the reduction of oxidized ZFP36. Genetic evidence indicates that ZFP36 positively influences rice resilience to oxidative and water stress, while OsTrxh1 exerts an opposing effect. These insights reveal a distinctive pathway for plant cells to perceive ABA-induced H2O2 signaling, advance our comprehension of H2O2 signaling dynamics, and ABA-related plant responses, and lay a vital groundwork for enhancing crop stress tolerance.

Sodium-glucose cotransporter 2 inhibitors attenuate vascular calcification by suppressing endoplasmic reticulum protein thioredoxin domain containing 5 dependent osteogenic reprogramming

AimsVascular calcification is strongly linked to the development of major adverse cardiovascular events, but effective treatments are lacking. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are an emerging category of oral hypoglycemic drugs that have displayed marked effects on metabolic and cardiovascular diseases, including recently reported vascular medial calcification. However, the roles and underlying mechanisms of SGLT2 inhibitors in vascular calcification have not been fully elucidated. Thus, we aimed to further determine whether SGLT2 inhibitors protect against vascular calcification and to investigate the mechanisms involved. Methods and resultsA computed tomography angiography investigation of coronary arteries from 1554 patients with type 2 diabetes revealed that SGLT2 inhibitor use was correlated with a lower Agatston calcification score. In the vitamin D3 overdose, 5/6 nephrectomy chronic kidney disease-induced medial calcification and Western diet-induced atherosclerotic intimal calcification models, dapagliflozin (DAPA) substantially alleviated vascular calcification in the aorta. Furthermore, we showed that DAPA reduced vascular calcification via Runx2-dependent osteogenic transdifferentiation in vascular smooth muscle cells (VSMCs). Transcriptome profiling revealed that thioredoxin domain containing 5 (TXNDC5) was involved in the attenuation of vascular calcification by DAPA. Rescue experiments showed that DAPA-induced TXNDC5 downregulation in VSMCs blocked the protective effect on vascular calcification. Furthermore, TXNDC5 downregulation disrupted protein folding-dependent Runx2 stability and promoted subsequent proteasomal degradation. Moreover, DAPA downregulated TXNDC5 expression via amelioration of oxidative stress and ATF6-dependent endoplasmic reticulum stress. Consistently, the class effects of SGLT2 inhibitors on vascular calcification were validated with empagliflozin in intimal and medial calcification models. ConclusionsSGLT2 inhibitors ameliorate vascular calcification through blocking endoplasmic reticulum stress-dependent TXNDC5 upregulation and promoting subsequent Runx2 proteasomal degradation, suggesting that SGLT2 inhibitors are potentially beneficial for vascular calcification treatment and prevention.

Analysis of thioredoxin 1 levels in a mouse model of unilateral renal ischemia-reperfusion injury

Kidney disease, acute and chronic, is associated with high morbidity and mortality. Currently, a major obstacle in the field of nephrology is the lack of novel and more sensitive biomarkers that would allow an early diagnose and a proper monitoring of disease progression. Oxidative stress and the increased production of reactive oxygen species (ROS) have been implicated in the pathophysiology of a variety of renal diseases. Proteins of the thioredoxin (Trx) family, among others thioredoxins (Trxs) and glutaredoxins (Grxs), represent one of the most important protective systems in the defense against ROS as well as in the repair of their intracellular effects. Thioredoxin 1 (Trx1) is a cytosolic redox protein that catalyzes the reduction of protein disulfides using the thiol groups of two cysteine residues on its CGPC active site. Trx1 is abundantly expressed in different segments of the nephron and has been shown to be released from cells under conditions of oxidative stress in vitro and in vivo. We hypothesized that renal IRI induces the release of Trx1 from cells into extracellular fluids such as serum and tubular fluid/urine and that Trx1 increase may correlate with the degree of damage in the kidney. We established an effective mouse model of unilateral renal ischemia-reperfusion injury (IRI) and studied Trx1 levels in the serum, urine, as well as in various nephron segments of mice following IRI or sham surgery. We compared different reperfusion times (4h, 8h, 16h, 24h, 48h, 96h, and 168h) to track Trx1 levels fluctuation over time. Serum levels of Trx1 increased 4h after renal IRI, while other markers such as blood urea nitrogen and creatinine remained low. Serum Trx1 showed a positive correlation with tubular injury score helping predict intrarenal damage over time. Additionally, immunohistochemistry studies revealed an increase in Trx1 signals in the proximal tubules and collecting ducts as well as a strong nuclear localization in the same nephron segments that became more intense by 96h, a time point characterized by an intense mitotic activity in the tubules. Between 16 and 24h, Trx1 appeared in the lumen of proximal tubules, however urinary levels of Trx1 did not significantly increase. Based on these findings we conclude that serum Trx1 may represent a novel biomarker for assessing renal damage. In addition to this, we hypothesize that intrarenal changes in Trx1 expression and localization might be related to the activation of regeneration mechanisms in the renal tubular epithelium. Further studies are needed to understand the role of Trx1 in the regeneration process after renal IRI. This work was funded by the Center of Integrative Mammalian Research of Ross University School of Veterinary Medicine and by a Startup funding from Long Island University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Estrogen deficiency in rats alters the expression and localization of redox proteins in the bone and bone marrow

Imbalances in bone formation/bone resorption ratio can occur under diverse conditions and diseases leading to an alteration of bone mineral density and strength. One of the most dramatic changes altering bone’s microarchitecture occurs under estrogen deficient conditions in humans and in other mammals. For instance, estrogen deficiency during menopause in women induces the formation and accumulation of reactive oxygen species (ROS) and a dysregulation of redox signaling. The bone and bone marrow are especially susceptible to the effects of oxidative stress and ROS. In tissues like the bone, cells are equipped with important defense systems to maintain homeostasis and to survive. Redox proteins of the thioredoxin family represent the most important protective system in cells that act against ROS and repair their intracellular effects. Redox proteins of the thioredoxin family comprise thioredoxins (Trxs), glutaredoxins (Grxs), and peroxiredoxins (Prxs). From all these protein classes, Prxs are the only ones that work as classical ROS scavengers being able to reduce hydrogen peroxide (H2O2), peroxynitrites and organic peroxides. Prxs are localized in different cellular compartments such as mitochondria (Prx3), cytosol and nucleus (Prx1 and Prx2), endoplasmic reticulum (Prx4), and peroxisomes (Prx5). We hypothesized that changes in estrogen levels may alter the expression and localization of Prxs in the bone and bone marrow. In this study, we analyzed the cellular localization and expression patterns of Prxs in the bone and bone marrow of rats as well as the effects of estrogens on their expression levels. We compared sham, ovariectomized, as well as ovariectomized/estrogen-replaced female rats of the same age. Western blot and immunohistochemistry revealed that all peroxiredoxins were abundantly expressed in different cell types of the bone and bone marrow such as osteocytes, chondrocytes, osteoclasts, bone lining cells, osteoblasts, and hematopoietic precursor cells. After ovariectomy, redox protein levels were significantly reduced in most cell types of the bone and bone marrow compartment. In addition to this, estrogen deficient animals showed increased levels of Prx1 in bone marrow supernatant suggesting a major redox dysregulation in this compartment. Estrogen replacement restored the levels of most redox proteins in the bone and bone marrow compartment and reduced the amount of extracellular Prx1. Based on these results, we conclude that estrogen deficiency negatively affects the ability of bone and bone marrow cells to maintain redox proteins at physiological levels. These results also reveal the widespread effects that are mediated by estrogen affecting different protein systems and tissues in the body. This work was funded by a Seed Discovery Grant (grant #3670920201102 to JG) from Long Island University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Thioredoxin Domain Containing 5 (TXNDC5): Friend or Foe?

This review focuses on the thioredoxin domain containing 5 (TXNDC5), also known as endoplasmic reticulum protein 46 (ERp46), a member of the protein disulfide isomerase (PDI) family with a dual role in multiple diseases. TXNDC5 is highly expressed in endothelial cells, fibroblasts, pancreatic β-cells, liver cells, and hypoxic tissues, such as cancer endothelial cells and atherosclerotic plaques. TXNDC5 plays a crucial role in regulating cell proliferation, apoptosis, migration, and antioxidative stress. Its potential significance in cancer warrants further investigation, given the altered and highly adaptable metabolism of tumor cells. It has been reported that both high and low levels of TXNDC5 expression are associated with multiple diseases, such as arthritis, cancer, diabetes, brain diseases, and infections, as well as worse prognoses. TXNDC5 has been attributed to both oncogenic and tumor-suppressive features. It has been concluded that in cancer, TXNDC5 acts as a foe and responds to metabolic and cellular stress signals to promote the survival of tumor cells against apoptosis. Conversely, in normal cells, TXNDC5 acts as a friend to safeguard cells against oxidative and endoplasmic reticulum stress. Therefore, TXNDC5 could serve as a viable biomarker or even a potential pharmacological target.

The role and mechanism of TXNDC5 in disease progression.

Thioredoxin domain containing protein-5 (TXNDC5), also known as endothelial protein-disulfide isomerase (Endo-PDI), is confined to the endoplasmic reticulum through the structural endoplasmic reticulum retention signal (KDEL), is a member of the PDI protein family and is highly expressed in the hypoxic state. TXNDC5 can regulate the rate of disulfide bond formation, isomerization and degradation of target proteins through its function as a protein disulfide isomerase (PDI), thereby altering protein conformation, activity and improving protein stability. Several studies have shown that there is a significant correlation between TXNDC5 gene polymorphisms and genetic susceptibility to inflammatory diseases such as rheumatoid, fibrosis and tumors. In this paper, we detail the expression characteristics of TXNDC5 in a variety of diseases, summarize the mechanisms by which TXNDC5 promotes malignant disease progression, and summarize potential therapeutic strategies to target TXNDC5 for disease treatment.

Functional diversity of YbbN/CnoX proteins: Insights from a comparative analysis of three thioredoxin-like oxidoreductases from Pseudomonas aeruginosa, Xylella fastidiosa and Escherichia coli

YbbN/CnoX are proteins that display a Thioredoxin (Trx) domain linked to a tetratricopeptide domain. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl. Here, we compared EcYbbN with YbbN proteins from Xylella fastidiosa (XfYbbN) and from Pseudomonas aeruginosa (PaYbbN). EcYbbN presents a redox active Cys residue at Trx domain (Cys63), 24 residues away from SQHC motif (SQHC[N24]C) that can form mixed disulfides with target proteins. In contrast, XfYbbN and PaYbbN present two Cys residues in the CXXC (CAPC) motif, while only PaYbbN shows the Cys residue equivalent to Cys63 of EcYbbN. Our phylogenetic analysis revealed that most of the YbbN proteins are in the bacteria domain of life and that their members can be divided into four groups according to the conserved Cys residues. EcYbbN (SQHC[N24]C), XfYbbN (CAPC[N24]V) and PaYbbN (CAPC[N24]C) are representatives of three sub-families. In contrast to EcYbbN, both XfYbbN and PaYbbN: (1) reduced an artificial disulfide (DTNB) and (2) supported the peroxidase activity of Peroxiredoxin Q from X. fastidiosa, suggesting that these proteins might function similarly to the canonical Trx enzymes. Indeed, XfYbbN was reduced by XfTrx reductase with a high catalytic efficiency (kcat/Km = 1.27 x 107 M−1 s−1), similar to the canonical XfTrx (XfTsnC). Furthermore, EcYbbN and XfYbbN, but not PaYbbN displayed HOCl-induced holdase activity. Remarkably, EcYbbN gained disulfide reductase activity while lost the HOCl-activated chaperone function, when the SQHC was replaced by CQHC. In contrast, the XfYbbN CAPA mutant lost the disulfide reductase activity, while kept its HOCl-induced chaperone function. In all cases, the induction of the holdase activity was accompanied by YbbN oligomerization. Finally, we showed that deletion of ybbN gene did not render in P. aeruginosa more sensitive stressful treatments. Therefore, YbbN/CnoX proteins display distinct properties, depending on the presence of the three conserved Cys residues.

Genome-wide analysis and expression divergence of protein disulfide isomerase (PDI) gene family members in chickpea (Cicer arietinum) under salt stress.

Chickpea (Cicer arietinum ) is a grain crop that is an important source of protein, vitamins, carbohydrates and minerals. It is highly sensitive to salt stress, and salt damage to cellular homeostasis and protein folding affects production. Plants have several mechanisms to prevent cellular damages under abiotic stresses, such as proteins in the endoplasmic reticulum (protein isulfide somerases (PDIs) and PDI-like proteins), which help prevent the build-up of mis-folded proteins that are damaged under abiotic stresses. In this study, we completed initial comprehensive genome-wide analysis of the chickpea PDI gene family. We found eight PDI genes are distributed on six out of eight chromosomes. Two pairs of paralogous genes were found to have segmental duplications. The phylogenetic analysis showed that the PDI s have a high degree of homology in C. arietinum, Cicer reticulatum, Lens culinaris, Phaseolus acutifolius, Pisum sativum and Oryza sativa . The gene structure analysis displayed that CaPDI1-CaPDI8 have 9-12 exons except for CaPDI5 , which has 25 exons. Subcellular localisation indicated accumulation of CaPDIs in endoplasmic reticulum. Protein-conserved motifs and domain analysis demonstrated that thioredoxin domains of PDI family is present in all CaPDIs. CaPDI proteins have strong protein-protein interaction. In silico expression analysis showed that four out of eight PDI genes (CPDI2, CaPDI6, CaPDI7 and CaPDI8 ) were expressed under salt stress. Of these, expression of CaPDI2 and CaPDI8 was the highest. This work indicated that PDI genes are involved in salt stress tolerance in chickpea and the CaPDIs may be further studied for their role of inducing salt tolerance.

Systematic analysis of the thioredoxin gene family in Citrus sinensis: identification, phylogenetic analysis, and gene expression patterns

ABSTRACT Thioredoxin (TRX) proteins play essential roles in reactive oxygen species scavenging in plants. We executed an exhaustive analysis of the TRX gene family in Citrus sinensis (CsTRXs), encompassing identification, phylogenetic analysis, detection of conserved motifs and domains, gene structure, cis-acting elements, gene expression trends, and subcellular localization analysis. Our findings established that a total of 22 CsTRXs with thioredoxin domains were identified in the genome of C. sinensis. Phylogenetic analysis indicated that CsTRXs were divided into six subclusters. Conserved motifs analysis of CsTRXs indicated a wide range of conserved motifs. A significant number of cis-acting elements associated with both abiotic and biotic stress responses, inclusive of numerous phytohormone-related elements, were detected in the promoter regions of CsTRXs. The expression levels of CsTRXs including CsTRXf1, CsTRXh1, CsTRXm1, CsTRXo3, CsTRXx2 and CsTRXy1 were observed to be reduced upon pathogen infection. Subcellular localization analysis found that CsTRXf1, CsTRXm1, CsTRXo3, CsTRXx2 and CsTRXy1 were predominantly localized in chloroplasts, whereas CsTRXh1 was distributed indiscriminately. This research yields integral data on CsTRXs, facilitating future efforts to decipher the gene functions of CsTRXs.

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.

The chloroplast protein HCF164 is predicted to be associated with Coffea SH9 resistance factor against Hemileia vastatrix

To explore the connection between chloroplast and coffee resistance factors, designated as SH1 to SH9, whole genomic DNA of 42 coffee genotypes was sequenced, and entire chloroplast genomes were de novo assembled. The chloroplast phylogenetic haplotype network clustered individuals per species instead of SH factors. However, for the first time, it allowed the molecular validation of Coffea arabica as the maternal parent of the spontaneous hybrid “Híbrido de Timor”. Individual reads were also aligned on the C. arabica reference genome to relate SH factors with chloroplast metabolism, and an in-silico analysis of selected nuclear-encoded chloroplast proteins (132 proteins) was performed. The nuclear-encoded thioredoxin-like membrane protein HCF164 enabled the discrimination of individuals with and without the SH9 factor, due to specific DNA variants linked to chromosome 7c (from C. canephora-derived sub-genome). The absence of both the thioredoxin domain and redox-active disulphide center in the HCF164 protein, observed in SH9 individuals, raises the possibility of potential implications on redox regulation. For the first time, the identification of specific DNA variants of chloroplast proteins allows discriminating individuals according to the SH profile. This study introduces an unexplored strategy for identifying protein/genes associated with SH factors and candidate targets of H. vastatrix effectors, thereby creating new perspectives for coffee breeding programs.

Breakdown of Arabidopsis thaliana thioredoxins and glutaredoxins based on electrostatic similarity-Leads to common and unique interaction partners and functions.

The reversible reduction and oxidation of protein thiols was first described as mechanism to control light/dark-dependent metabolic regulation in photosynthetic organisms. Today, it is recognized as an essential mechanism of regulation and signal transduction in all kingdoms of life. Proteins of the thioredoxin (Trx) family, Trxs and glutaredoxins (Grxs) in particular, catalyze thiol-disulfide exchange reactions and are vital players in the operation of thiol switches. Various Trx and Grx isoforms are present in all compartments of the cell. These proteins have a rather broad but at the same time distinct substrate specificity. Understanding the molecular basis of their target specificity is central to the understanding of physiological and pathological redox signaling. Electrostatic complementarity of the redoxins with their target proteins has been proposed as a major reason. Here, we analyzed the electrostatic similarity of all Arabidopsis thaliana Trxs, Grxs, and proteins containing such domains. Clustering of the redoxins based on this comparison suggests overlapping and also distant target specificities and thus functions of the different sub-classes including all Trx isoforms as well as the three classes of Grxs, i.e. CxxC-, CGFS-, and CC-type Grxs. Our analysis also provides a rationale for the tuned substrate specificities of both the ferredoxin- and NADPH-dependent Trx reductases.

Early plasma proteomic biomarkers and prediction model of acute respiratory distress syndrome after cardiopulmonary bypass: a prospective nested cohort study.

Early recognition of the risk of acute respiratory distress syndrome (ARDS) after cardiopulmonary bypass (CPB) may improve clinical outcomes. The main objective of this study was to identify proteomic biomarkers and develop an early prediction model for CPB-ARDS. We conducted three prospective nested cohort studies of all consecutive patients undergoing cardiac surgery with CPB at XXX Hospital. Plasma proteomic profiling was performed in ARDS patients and matched controls (Cohort 1, April, 2021 to July, 2021) at multiple timepoints: before CPB (T1), at end of CPB (T2), and 24 h after CPB (T3). Then, for Cohort 2 (August, 2021 to July, 2022), biomarker expression was measured and verified in the plasma. Furthermore, lung ischemia/reperfusion injury (LIRI) models and sham-operation were established in 50 rats to explore the tissue-level expression of biomarkers identified in the aforementioned clinical cohort. Subsequently, a machine learning-based prediction model incorporating protein and clinical predictors from Cohort 2 for CPB-ARDS was developed and internally validated. Model performance was externally validated on Cohort 3 (January, 2023 to March, 2023). A total of 709 proteins were identified, with 9, 29, and 35 altered proteins between ARDS cases and controls at T1, T2, and T3, respectively, in Cohort 1. Following quantitative verification of several predictive proteins in Cohort 2, higher levels of thioredoxin domain containing 5 (TXNDC5), cathepsin L (CTSL), and NPC intracellular cholesterol transporter 2 (NPC2) at T2 were observed in CPB-ARDS patients. A dynamic online predictive nomogram was developed based on three proteins (TXNDC5, CTSL, and NPC2) and two clinical risk factors (CPB time, massive blood transfusion), with excellent performance (precision: 83.33%, sensitivity: 93.33%, specificity: 61.16%, F1 score: 85.05%). Mean area under the receiver operating characteristics curve (AUC) of the model after 10-fold cross-validation was 0.839 (95% confidence interval (CI): 0.824 to 0.855). Model discrimination and calibration were maintained during external validation dataset testing, with AUC of 0.820 (95% CI: 0.685 to 0.955) and Brier Score of 0.177 (95% CI: 0.147 to 0.206). Moreover, the considerably overexpressed TXNDC5 and CTSL proteins identified in the plasma of patients with CPB-ARDS, exhibited a significant upregulation in the lung tissue of LIRI rats. This study identified several novel predictive biomarkers, developed and validated a practical prediction tool using biomarker and clinical factor combinations for individual prediction of CPB-ARDS risk. Assessing the plasma TXNDC5, CTSL, and NPC2 levels might identify patients who warrant closer follow-up and intensified therapy for ARDS prevention following major surgery.

Molecular features, antioxidant potential, and immunological expression assessment of thioredoxin-like protein 1 (TXNL1) in yellowtail clownfish (Amphiprion clarkii)

Thioredoxin-like protein 1 (TXNL1) is a redox-active protein belonging to the thioredoxin family, which mainly controls the redox status of cells. The TXNL1 gene from Amphiprion clarkii (AcTXNL1) was obtained from a pre-established transcriptome database. The AcTXNL1 is encoded with 289 amino acids and is predominantly localized in the cytoplasm and nucleus. The TXN domain of AcTXNL1 comprises a34CGPC37 motif with redox-reactive thiol (SH-) groups. The spatial distribution pattern of AcTXNL1 mRNA was examined in different tissues, and the muscle was identified as the highest expressed tissue. AcTXNL1 mRNA levels in the blood and gills were significantly increased in response to different immunostimulants. In vitro antioxidant capacity of the recombinant AcTXNL1 protein (rACTXNL1) was evaluated using the ABTS free radical-scavenging activity assay, cupric ion reducing antioxidant capacity assay, turbidimetric disulfide reduction assay, and DNA nicking protection assay. The potent antioxidant activity of rAcTXNL1 exhibited a concentration-dependent manner in all assays. Furthermore, in the cellular environment, overexpression of AcTXNL1 increased cell viability under H2O2 stress and reduced nitric oxide (NO) production induced by lipopolysaccharides (LPS). Collectively, the experimental results revealed that AcTXNL1 is an antioxidant and immunologically important gene in A. clarkii.