Thioredoxin Domain-containing Protein Research Articles

Single-cell RNA sequencing analysis reveals the role of TXNDC5 in keloid formation

Objective: Thioredoxin domain-containing protein 5 (TXNDC5) is associated with fibrosis in a variety of organs, but its mechanism of action in keloid is unclear. In this study, we aimed to investigate the mechanism of TXNDC5 in keloid. Material and Methods: Single-cell RNA sequencing data of keloid and normal scar samples obtained from public databases were normalized and clustered using the Seurat package. Pathway enrich analysis was conducted using biological process enrichment analysis and Gene Set Enrichment Analysis (GSEA). In addition, TXNDC5 expression and its effects on migration and invasion of keloid fibroblasts (KFs) were validated based on cell function experiments. Results: A total of five cell types were obtained. The KF clusters were further clustered into two fibroblast subtypes (Fibroblast cells 1 and Fibroblast cells 2). Biological process enrichment analysis showed that transforming growth factor beta (TGF-β) signaling pathway was enriched in the two fibroblast subtypes. GSEA analysis demonstrated that genes in TGF-β signaling pathway were mainly enriched in Fibroblast cells 1, and that genes involved in cell proliferation, migration, and the TGF-β signaling pathway were all high-expressed in fibroblast cells 1. TXNDC5 was positively correlated with fibroblast proliferation, migration and TGF-β signaling pathway, and AUCell score. The cellular experiment confirmed that the messenger RNA and protein levels of TXNDC5 and TGF-β1 were high-expressed in KFs cells (P<0.001), and that knockdown of TXNDC5 downregulated TGF-β1 expression and inhibited migration and invasion of KFs (P<0.0001). Conclusion: Our study indicated that TGF-β signaling pathway was enriched in fibroblast cells, and TXNDC5 was positively correlated with proliferation, migration, and TGF-β signaling pathway. Cellular experiment demonstrated that knocking down TXNDC5 downregulated TGF-β1 expression, and suppressed migration and invasion of KFs. The current discoveries provided a new therapeutic strategy for the treatment of keloid.

Proteo-genomic analyses in relatively lean Chinese adults identify proteins and pathways that affect general and central adiposity levels

Adiposity is an established risk factor for multiple diseases, but the causal relationships of different adiposity types with circulating protein biomarkers have not been systematically investigated. We examine the causal associations of general and central adiposity with 2923 plasma proteins among 3977 Chinese adults (mean BMI = 23.9 kg/m²). Genetically-predicted body mass index (BMI), body fat percentage (BF%), waist circumference (WC), and waist-to-hip ratio (WHR) are significantly (FDR < 0.05) associated with 399, 239, 436, and 283 proteins, respectively, with 80 proteins associated with all four and 275 with only one adiposity trait. WHR is associated with the most proteins (n = 90) after adjusting for other adiposity traits. These associations are largely replicated in Europeans (mean BMI = 27.4 kg/m²). Two-sample Mendelian randomisation (MR) analyses in East Asians using cis-protein quantitative trait locus (cis-pQTLs) identified in GWAS find 30/2 proteins significantly affect levels of BMI/WC, respectively, with 10 showing evidence of colocalisation, and seven (inter-alpha-trypsin inhibitor heavy chain H3, complement factor B, EGF-containing fibulin-like extracellular matrix protein 1, thioredoxin domain-containing protein 15, alpha-2-antiplasmin, fibronectin, mimecan) are replicated in separate MR using different cis-pQTLs identified in Europeans. These findings identified potential novel mechanisms and targets, to our knowledge, for improved treatment and prevention of obesity and associated diseases.

Insights into the functional properties of thioredoxin domain-containing protein 12 (TXNDC12): Antioxidant activity, immunological expression, and wound-healing effect in yellowtail clownfish (Amphiprion clarkii)

Thioredoxin domain-containing protein 12 (TXNDC12) is a member of the thioredoxin-like superfamily that contributes to various thiol-dependent metabolic activities in all living organisms. In this research, the TXNDC12 gene from yellowtail clownfish (Amphiprion clarkii) was structurally characterized using in silico tools, assessed for immunological expression, and evaluated for biological activity using recombinant protein and cellular overexpression. The deduced coding sequence of AcTXNDC12 comprised a 522-bp nucleotide, encoding 173 amino acids with a predicted molecular mass of 19.198 kDa. The AcTXNDC12 protein consists of a66WCGAC70 active motif and a170GDEL173 signature. The highest tissue-specific expression of AcTXNDC12 was observed in the brain tissue, with significant modulation observed in the blood and gill tissues following stimulation of polyinosinic: polycytidylic acid, lipopolysaccharides (LPS), and Vibrio harveyi. In functional assays, recombinant AcTXNDC12 protein (rAcTXNDC12) showed insulin disulfide reduction activity, 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) decolorization antioxidant capacity, and ferric (Fe3+) reducing antioxidant potential. Additionally, a significant reduction in nitric oxide production was observed in AcTXNDC12-overexpressed RAW 264.7 cells upon LPS stimulation. Furthermore, genes associated with the regulation of oxidative stress, including nuclear factor erythroid 2-related factor 2 (Nrf2), catalase (Cat), peroxiredoxin 1 (Prx1), and ribonucleotide reductase catalytic subunit M1 (Rrm1) were significantly upregulated in fathead minnow cells overexpressing AcTXNDC12 in response to H2O2 treatment. The scratch wound healing assay demonstrated tissue regeneration and cell proliferation ability upon AcTXNDC12 overexpression. Altogether, the current study elucidated the antioxidant activity, immunological importance, and wound-healing effect of the AcTXNDC12 gene in yellowtail clownfish, providing valuable insights for advancing the aquaculture of A. clarkii fish.

Proteome-wide Ligand and Target Discovery by Using Strain-Enabled Cyclopropane Electrophiles.

The evolving use of covalent ligands as chemical probes and therapeutic agents could greatly benefit from an expanded array of cysteine-reactive electrophiles for efficient and versatile proteome profiling. Herein, to expand the current repertoire of cysteine-reactive electrophiles, we developed a new class of strain-enabled electrophiles based on cyclopropanes. Proteome profiling has unveiled that C163 of lactate dehydrogenase A (LDHA) and C88 of adhesion regulating molecule 1 (ADRM1) are ligandable residues to modulate the protein functions. Moreover, fragment-based ligand discovery (FBLD) has revealed that one fragment (Y-35) shows strong reactivity toward C66 of thioredoxin domain-containing protein 12 (TXD12), and its covalent binding has been demonstrated to impact its downstream signal pathways. TXD12 plays a pivotal role in enabling Y-35 to exhibit its antisurvival and antiproliferative effects. Finally, dicarbonitrile-cyclopropane has been demonstrated to be an electrophilic warhead in the development of GSTO1-involved dual covalent inhibitors, which is promising to alleviate drug resistance.

Redox Enzymes P4HB and PDIA3 Interact with STIM1 to Fine-Tune Its Calcium Sensitivity and Activation.

Sensing the lowering of endoplasmic reticulum (ER) calcium (Ca2+), STIM1 mediates a ubiquitous Ca2+ influx process called the store-operated Ca2+ entry (SOCE). Dysregulated STIM1 function or abnormal SOCE is strongly associated with autoimmune disorders, atherosclerosis, and various forms of cancers. Therefore, uncovering the molecular intricacies of post-translational modifications, such as oxidation, on STIM1 function is of paramount importance. In a recent proteomic screening, we identified three protein disulfide isomerases (PDIs)-Prolyl 4-hydroxylase subunit beta (P4HB), protein disulfide-isomerase A3 (PDIA3), and thioredoxin domain-containing protein 5 (TXNDC5)-as the ER-luminal interactors of STIM1. Here, we demonstrated that these PDIs dynamically associate with STIM1 and STIM2. The mutation of the two conserved cysteine residues of STIM1 (STIM1-2CA) decreased its Ca2+ affinity both in cellulo and in situ. Knockdown of PDIA3 or P4HB increased the Ca2+ affinity of wild-type STIM1 while showing no impact on the STIM1-2CA mutant, indicating that PDIA3 and P4HB regulate STIM1's Ca2+ affinity by acting on ER-luminal cysteine residues. This modulation of STIM1's Ca2+ sensitivity was further confirmed by Ca2+ imaging experiments, which showed that knockdown of these two PDIs does not affect STIM1-mediated SOCE upon full store depletion but leads to enhanced SOCE amplitudes upon partial store depletion. Thus, P4HB and PDIA3 dynamically modulate STIM1 activation by fine-tuning its Ca2+ binding affinity, adjusting the level of activated STIM1 in response to physiological cues. The coordination between STIM1-mediated Ca2+ signaling and redox responses reported herein may have implications for cell physiology and pathology.

Modeling alcohol-associated liver disease in humans using adipose stromal or stem cell-derived organoids

Alcohol-associated liver disease (ALD) is a prevalent liver disease, yet research is hampered by the lack of suitable and reliable human ALD models. Herein, we generated human adipose stromal/stem cell (hASC)-derived hepatocellular organoids (hAHOs) and hASC-derived liver organoids (hALOs) in a three-dimensional system using hASC-derived hepatocyte-like cells and endodermal progenitor cells, respectively. The hAHOs were composed of major hepatocytes and cholangiocytes. The hALOs contained hepatocytes and nonparenchymal cells and possessed a more mature liver function than hAHOs. Upon ethanol treatment, both steatosis and inflammation were present in hAHOs and hALOs. The incubation of hALOs with ethanol resulted in increases in the levels of oxidative stress, the endoplasmic reticulum protein thioredoxin domain-containing protein 5 (TXNDC5), the alcohol-metabolizing enzymes ADH1B and ALDH1B1, and extracellular matrix accumulation, similar to those of liver tissues from patients with ALD. These results present a useful approach for understanding the pathogenesis of ALD in humans, thus facilitating the discovery of effective treatments.

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.

Hepatitis E virus ORF3 protein hijacking thioredoxin domain-containing protein 5 (TXNDC5) for its stability to promote viral particle release.

Hepatitis E virus (HEV) infection is the leading cause of acute viral hepatitis worldwide. After the synthesis and modification in the cells, the mature ORF3 protein is essential for HEV release. However, the host protein involved in this process has yet to be determined. Here, we reported a novel host protein, thioredoxin domain-containing protein 5 (TXNDC5), as a chaperone, contributing to HEV release by facilitating ORF3 protein stability in the endoplasmic reticulum through interacting with non-palmitoylated ORF3 protein. However, we also found that in the knockout-TXNDC5 stable cell lines, the HEV ORF3 protein may hijack other proteins for its stabilization. For the first time, our study demonstrated the involvement of TXNDC5 in viral particle release. These findings provide some new insights into the process of the HEV life cycle, the interaction between HEV and host factors, and a new direction for antiviral design.

Abstract 285: Targeting TXNDC5 in stromal fibroblasts resolves desmoplasia and resistance to immune checkpoint blockade in mesenchymal-type colorectal cancer

Abstract Objectives: Mesenchymal-type colorectal cancer (CRC), characterized by strong stromal infiltration and immune tolerance, resists immune checkpoint blockade and has poor outcomes. Cancer-associated fibroblasts (CAFs), abundant in tumor stroma, actively remodel the extracellular matrix (ECM), modulate immune evasion, and drive tumor progression. We have recently identified thioredoxin domain-containing protein 5 (TXNDC5), a protein disulfide isomerase (PDI), as a critical mediator of fibroblast activation and ECM remodeling in organ fibrosis. We hypothesized that TXNDC5 could contribute to fibroblast activation, stroma formation and disease progression in cancer, especially in the stroma-enriched fibrogenic mesenchymal-type CRC. Methods: Transcriptome databases of CRC were re-analyzed to determine the clinical relevance of TXNDC5. Experimentally, CRC was induced in mouse lines by azoxymethane (AOM) and dextran sulfate sodium (DSS) stimuli, a model sharing multiple characteristics with human mesenchymal-type CRC. Human colonic fibroblast line CCD-18co was used to investigate the molecular mechanisms by which TXNDC5 regulates colonic fibroblast activities. Fibroblast-specific TXNDC5 knockout (Col1a2-Cre/ERT2*TXNDC5fl/fl) mice were generated, combining with single-cell RNA sequencing analyses on AOM/DSS-induced CRC tumors in these animals, to clarify how fibroblast TXNDC5 impact tumor microenvironment, CRC progression and response to immune checkpoint blockade. Findings: TXNDC5 was predominantly expressed in stromal fibroblasts of human and mouse CRC. Fibroblast-specific deletion of Txndc5 lessened CAF activation, attenuated tumor fibrosis and reduced tumor burden in AOM/DSS-induced CRC. Mechanistically, increased TXNDC5 levels augments TGFβ signaling in CAF by post-translational stabilization of TGFBR1 through its PDI activity. In addition, deletion of Txndc5 in CAFs led to less tumor desmoplasia, decompressed tumor vessels and attenuated intra-tumoral hypoxia, thereby easing immune tolerance and increasing cytotoxic T cell infiltration in CRC. Single-cell transcriptome analysis revealed a marked change of intra-tumoral immune cell populations upon fibroblast-specific deletion of TXNDC5, shifting from myeloid-derived suppressive cells to cytotoxic tumor-infiltrating lymphocytes. Importantly, depletion of TXNDC5 in CAFs potentiated the anti-tumor effects of immune checkpoint blockade with anti-PD1 therapy in CRC. Conclusions: Our data suggest an important yet previously unrecognized role of fibroblast TXNDC5 in CRC progression, through enhancing CAF activation, stroma formation and immune escape. Combining immune checkpoint blockade with TXNDC5 deletion synergistically improved anti-tumor effects in CRC. Targeting TXNDC5, therefore, can be a novel therapeutic approach for CRC patients. Citation Format: Kai-Lin Cheng, Chih-I Chen, Shu-Han Yu, Huai-Wen Liang, Yi-Wei Tsai, Chen-Ting Hung, Yu-Shan Lin, Yi-Shiuan Tzeng, Sung-Jan Lin, Yueh-Feng Wu, Jen-Kuang Lee, Chia-Hui Yu, Shuei-Liong Lin, Shih-Yu Chen, Tzu-Tang Wei, Yun-Ju Huang, Ruey-Hwa Chen, Ching-Chow Chen, Kai-Chien Yang. Targeting TXNDC5 in stromal fibroblasts resolves desmoplasia and resistance to immune checkpoint blockade in mesenchymal-type colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 285.

TXNDC12 inhibits pancreatic tumor cells ferroptosis by regulating GSH/GGT7 and promotes its growth and metastasis.

Background: Thioredoxin domain-containing protein 12 (TXNDC12) is upregulated in a variety of tumours, including pancreatic cancer (PAAD), and its high expression is closely associated with poor prognosis. However, the regulatory mechanism of TXNDC12 in PAAD has not been reported. The aim of this study is to reveal the precise mechanism of TXNDC12 in regulating PAAD progression. Methods: The expression of TXNDC12 in pan-cancer as well as PAAD was verified by TCGA and GTEx databases, Western blot and RT-qPCR. CCK8 assay, clone formation assay and cell cycle assay were used to observe the effect of TXNDC12 on the proliferation of PAAD cells, the migration and invasion capacities were verified by wound healing assay and Transwell assay. The effect of TXNDC12 on apoptosis of MIA PaCa-2 and PANC-1 cells was detected using Hochest and flow cytometry. Finally, the interaction of TXNDC12 with GGT7 was predicted by STRING database and confirmed by CO-IP assay, the effect of TXNDC12 on ferroptosis through GGT7 was evaluated by GSH assay, MDA assay, ROS assay and Western blot. Results: TXNDC12 is upregulated in PAAD tissues, and patients with high TXNDC12 levels generally have shorter survival times. Knockdown of TXNDC12 significantly inhibited the proliferation, migration and invasion and promoted apoptosis of MIA PaCa-2 and PANC-1 cells. Mechanistically, knockdown of TXNDC12 resulted in a decrease in intracellular GSH content and an increase in GSSG content, as well as elevated levels of pro-ferroptosis factors, such as MDA and ROS. STRING database predicted that TXNDC12 interacts with GGT7, and CO-IP assay was used to validate this result. Finally, the effect of knocking down TXNDC12 on pancreatic cancer cell functions was able to be reversed by overexpression of GGT7. Conclusion: TXNDC12 inhibits ferroptosis in PAAD cells through the GSH/GGT7 axis thereby promoting their development.

TXNDC12 inhibits lipid peroxidation and ferroptosis

Ferroptosis is a type of regulated cell death characterized by lipid peroxidation and subsequent damage to the plasma membrane. Here, we report a ferroptosis resistance mechanism involving the upregulation of TXNDC12, a thioredoxin domain-containing protein located in the endoplasmic reticulum. The inducible expression of TXNDC12 during ferroptosis in leukemia cells is inhibited by the knockdown of the transcription factor ATF4, rather than NFE2L2. Mechanistically, TXNDC12 acts to inhibit lipid peroxidation without affecting iron accumulation during ferroptosis. When TXNDC12 is overexpressed, it restores the sensitivity of ATF4-knockdown cells to ferroptosis. Moreover, TXNDC12 plays a GPX4-independent role in inhibiting lipid peroxidation. The absence of TXNDC12 enhances the tumor-suppressive effects of ferroptosis induction in both cell culture and animal models. Collectively, these findings demonstrate anendoplasmic reticulum-based anti-ferroptosis pathway in cancer cells with potential translational applications.

High Thioredoxin Domain-Containing Protein 11 Expression Is Associated with Tumour Progression in Glioma.

Glioblastoma (GBM) is the most common primary brain malignancy in adults. Despite multimodal treatment that involves maximal safe resection, concurrent chemoradiotherapy, and tumour treatment for supratentorial lesions, the prognosis remains poor. The current median overall survival is only <2 years, and the 5-year survival is only 7.2%. Thioredoxin domain-containing protein 11 (TXNDC11), also known as EF-hand binding protein 1, was reported as an endoplasmic reticulum stress-induced protein. The present study aimed to elucidate the prognostic role of TXNDC11 in GBM. We evaluated the clinical parameters and TXNDC11 scores in gliomas from hospitals. Additionally, proliferation, invasion, migration assays, apoptosis, and temozolomide (TMZ)-sensitivity assays of GBM cells were conducted to evaluate the effects of short interfering RNA (siRNA) on these processes. In addition, these cells were subjected to Western blotting to detect the expression levels of N-cadherin, E-cadherin, and Cyclin D1. High levels of TXNDC11 protein expression were significantly associated with World Health Organization (WHO) high-grade tumour classification and poor prognosis. Multivariate analysis revealed that in addition to the WHO grade, TXNDC11 protein expression was also an independent prognostic factor of glioma. In addition, TXNDC11 silencing inhibited proliferation, migration, and invasion and led to apoptosis of GBM cells. However, over-expression of TXNDC11 enhanced proliferation, migration, and invasion. Further, TXNDC11 knockdown downregulated N-cadherin and cyclin D1 expression and upregulated E-cadherin expression in GBM cells. Knock-in TXNDC11 return these. Finally, in vivo, orthotopic xenotransplantation of TXNDC11-silenced GBM cells into nude rats promoted slower tumour growth and prolonged survival time. TXNDC11 is a potential oncogene in GBMs and may be an emerging therapeutic target.

Mitochondria of Live Mizuhopecten yessoensis Scallops Can Sensitively Respond to Quality Changes during Dry/Reimmersed Storage as Determined by TMT-Labeled Proteomic Analysis.

Dry/reimmersed storage is often used in the transportation of live scallops. In this study, tandem mass tag (TMT)-labeled protein omics were used to quantitatively analyze the protein changes in scallops during dry/reimmersed stress. The results showed that during dry storage, scallops maintained cellular redox homeostasis through the upregulation of SCO1-like protein and thioredoxin domain-containing protein and reduced organic acids from the ATP synthetic process by the downregulation of NADH dehydrogenase, thereby reducing the damage caused during dry storage. During reimmersed storage, mitochondrial proteins underwent very sensitive changes. By upregulating aerobic respiration-related proteins (including proteins involved in glucose phosphate metabolism, glyceraldehyde 3-phosphate metabolism, etc.), the ATP synthesis ability was improved. However, the damage to the mitochondrial structure by dry storage could not be completely recovered, even by reimmersion. This included some apoptosis-related proteins that were obviously upregulated. In summary, compared with ATP-related indexes, mitochondria can respond more sensitively to dry storage stress.

Thioredoxin domain-containing protein 9 protects cells against UV-B-provoked apoptosis via NF-κB/p65 activation in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC), a type of non-melanoma skin cancer (NMSC), is the most common malignancy worldwide. Thioredoxin (TXN) domain-containing protein 9 (TXNDC9) is a member of the TXN family that is important in cell differentiation. However, the biological function of this protein in cancer, particularly cSCC, is still unknown. In the present study, our experiments revealed the protective effects of TXNDC9 on UV-B-irritated cSCC cells. The initial findings showed that TXNDC9 is significantly upregulated in cSCC tissue and cells compared to normal skin tissue and keratinocytes. UV-B radiation robustly induces the expression of TXNDC9, and UV-B-induced cSCC cell death is boosted by TXNDC9 deficiency. Moreover, cSCC cells lacking TXNDC9 displayed attenuated activation of the NF-κB pathway. Additional studies by inhibiting TXNDC9 confirmed this finding, as TXNDC9 deficiency attenuated UV-B radiation-induced translocation of NF-κB p65 from the cytoplasm to the nucleus of cSCC. In conclusion, our work demonstrates the biological roles of TXNDC9 in cSCC progression and may provide a novel therapeutic target to treat cSCC in the future.

Thioredoxin domain-containing protein 12 (TXNDC12) in red spotted grouper (Epinephelus akaara): Molecular characteristics, disulfide reductase activities, and immune responses

Thioredoxins are small ubiquitous redox proteins that are involved in many biological processes. Proteins with thiol-disulfide bonds are essential regulators of cellular redox homeostasis and diagnostic markers for redox-dependent diseases. Here, we identified and characterized the thioredoxin domain-containing protein 12 (EaTXNDC12) gene in red spotted grouper (Epinephelus akaara), evaluated transcriptional responses, and investigated the activity of the recombinant protein using functional assays. EaTXNDC12 is a 19.22-kDa endoplasmic reticulum (ER)-resident protein with a 522-bp open reading frame and 173 amino acids, including a signal peptide. We identified a conserved active motif (66WCGAC70) and ER retention motif (170GDEL173) in the EaTXNDC12 amino acid sequence. Relative EaTXNDC12 mRNA expression was analyzed using 12 different tissues, with the highest expression seen in brain tissue, while skin tissue showed the lowest expression level. Furthermore, mRNA expression in response to immune challenges was analyzed in the head kidney, blood, and gill tissues. EaTXNDC12 was significantly modulated in response to bacterial endotoxin lipopolysaccharide (LPS), nervous necrosis virus (NNV), and polyinosinic:polycytidylic acid (poly(I:C)) challenges in all of the tested tissues. Recombinant EaTXNDC12 (rEaTXNDC12) displayed antioxidant ability in an insulin reductase assay, and a capacity for free radical inhibition in a 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay. In addition, a DNA nicking assay revealed that purified rEaTXNDC12 exhibited concentration-dependent DNA protection activity, while results from 2-hydroxyethyl disulfide and L-dehydroascorbic assays indicated that rEaTXNDC12a possesses reducing ability. Furthermore, fathead minnow (FHM) cells transfected with EaTXNDC12-pcDNA demonstrated significantly upregulated cell survival against H2O2-induced apoptosis. Collectively, the results of this study strengthen our knowledge of EaTXNDC12 with respect to cellular redox hemostasis and immune regulation in Epinephelus akaara.

Expression analysis and functional characterization of thioredoxin domain-containing protein 11.

The endoplasmic reticulum (ER) is a crucial organelle that regulates both the folding, modification and transport of many proteins and senses certain stimuli inside and outside of cells. ER-associated degradation (ERAD), including SEL1L is a crucial mechanism to maintain homeostasis. In this study, we performed comparative proteome analysis in wild-type (wt) and SEL1L-deficient cells. We found constitutively high expression of thioredoxin domain-containing protein 11 (TXNDC11) mRNA and protein in our SEL1L-deficient HEK293 cells by RT-PCR and Western blot analysis. The TXNDC11 gene possesses a well-conserved unfolded protein response element (UPRE) around its transcription start site, and ER stress increased TXNDC11 mRNA and luciferase reporter activity via this putative UPRE in HEK293 cells. The amounts of TXNDC11 protein in wild-type and SEL1L-deficient cells with or without thapsigargin (Tg) treatment were parallel to their mRNAs in these cells, which was almost proportional to spliced XBP1 (sXBP1) mRNA expression. The establishment and characterization of TXNDC11-deficient HEK293 cells revealed that the expression of three different ER resident stress sensors, ATF6α, CREB3 and CREB3L2, is regulated by TXNDC11. The rate of disappearance of the three proteins by CHX treatment in wt cells was remarkably different, and the full-length CREB3L2 protein was almost completely degraded within 15min after CHX treatment. TXNDC11 deficiency increased the expression of each full-length form under resting conditions and delayed their disappearance by CHX treatment. Interestingly, the degree of increase in full-length CREB3/CREB3L2 by TXNDC11 deficiency was apparently higher than that in full-length ATF6α. The increase in these proteins by TXNDC11 deficiency was hardly correlated with the expression of each mRNA. Treatment with ER stress inducers influenced each full-length mature form, and the difference in each full-length form observed in wt and TXNDC11-deficient cells was smaller. This study demonstrated that TXNDC11 is an ER stress-inducible gene regulated by the IRE1-sXBP1 pathway. In addition, TXNDC11 is involved in the regulation of ATF6α, CREB3 and CREB3L2 protein expression, although the contribution to the stability of these proteins is quite variable. Therefore, its further characterization will provide new insights for understanding protein homeostasis in ER physiology and pathology.

M6A Methyltransferase METTL3 Promotes the Progression of Primary Acral Melanoma via Mediating TXNDC5 Methylation.

m6A modification is one of the most important post-transcriptional modifications in RNA and plays an important role in promoting translation or decay of RNAs. The role of m6A modifications has been highlighted by increasing evidence in various cancers, which, however, is rarely explored in acral melanoma. Here, we demonstrated that m6A level was highly elevated in acral melanoma tissues, along with the expression of METTL3, one of the most important m6A methyltransferase. Besides, higher expression of METTL3 messenger RNA (mRNA) correlated with a higher stage in primary acral melanoma patients. Knockdown of METTL3 decreased global m6A level in melanoma cells. Furthermore, METTL3 knockdown suppressed the proliferation, migration, and invasion of melanoma cells. In METTL3 knockdown xenograft mouse models, we observed decreased volumes and weights of melanoma tissues. Mechanistically, we found that METTL3 regulates certain m6A-methylated transcripts, thioredoxin domain containing protein 5 (TXNDC5), with the confirmation of RNA-seq, MeRIP-seq, and Western blot. These data suggest that METTL3 may play a key role in the progression of acral melanoma, and targeting the m6A dependent-METTL3 signaling pathway may serve as a promising therapeutic strategy for management of patients of acral melanomas.

Selenium Deficiency Induces Pathological Cardiac Lipid Metabolic Remodeling and Inflammation.

Selenium (Se) disequilibrium is closely involved in many cardiac diseases, although its in vivo mechanism remains uncertain. Therefore, a pig model is created in order to generate a comprehensive picture of cardiac response to dietary Se deficiency. A total of 24 pigs are divided into two equal groups, which were fed a diet with either 0.007mg kg-1 Se or 0.3mg kg-1 Se for 16 weeks. Se deficiency causes cardiac oxidative stress by blocking glutathione and thioredoxin systems and increases thioredoxin domain-containing protein S-nitrosylation. Energy production is disordered, as free fatty acids are overloaded, the tricarboxylic acid cycle is strengthened, and three respiratory chain proteins enhance S-nitrosylation. Excess free fatty acids lead to increased synthesis of diacylglycerol, phosphatidylcholine, and phosphatidylethanolamine, where the latter two are vulnerable to oxidation and causes an increase in malondialdehyde. Moreover, increased palmitic acid enhances de novo ceramide synthesis and accumulation. Additionally, Se deficiency initiates inflammation via cytosolic DNA-sensing pathways, which activates downstream interferon regulatory factor 7 and nuclear factor kappaB. The present study identifies a lipid metabolic vulnerability and inflammation initiation pathway via Se deficiency, which may provide targets for human redox imbalance-induced cardiac disease treatment.

Impact of the Glycemic Level on the Salivary Proteome of Middle-Aged and Elderly People With Type 2 Diabetes Mellitus: An Observational Study.

Type 2 diabetes mellitus (T2DM) is an increasing global public health concern, but its impact on the salivary proteome is still unclear. To evaluate the effect of glycemic levels in middle-aged and elderly individuals with T2DM on salivary proteomics, we compared the differences by liquid chromatography tandem mass spectrometry (LC–MS/MS). Unstimulated whole saliva samples from 8 T2DM patients with good glycemic control (G group, HbA1c <6.5%) and 16 patients with poor control (P group, HbA1c ≥6.5%) were analyzed by LC–MS/MS in the data-independent acquisition mode (Clinical register number: ChiCTR1900023582.). After functional annotation, cluster analysis and receiver operating characteristic (ROC) curve analysis were carried out to screen and evaluate candidate proteins. A total of 5,721 proteins were quantified, while 40 proteins differed significantly. In the P group, proteins involved in oxidative stress-related processes were upregulated, whereas proteins related to salivary secretion were downregulated. The combination of thioredoxin domain-containing protein 17, zymogen granule protein 16B, and FAM3 metabolism regulating signaling molecule D yielded an area under the curve of 0.917 which showed a robust ability to distinguish the P and G groups. In conclusion, poorly controlled hyperglycemia may affect salivary proteins through various pathways, including oxidative stress and glandular secretion. Furthermore, the differentially expressed proteins, especially the three proteins with the best differentiation, might serve as an anchor point for the further study of hyperglycemia and oral diseases.

High level of TXNDC9 predicts poor prognosis and contributes to the NF-κB-regulated metastatic potential in gastric cancer.

Gastric cancer (GC) is the most frequent malignant tumor in the digestive system, with high metastasis potential and poor prognosis. This study aimed to investigate the prognostic value and biological function of thioredoxin domain-containing protein 9 (TXNDC9) in GC. The expression of TXNDC9 was analyzed based on The Cancer Genome Atlas (TCGA) database. The prognostic value of TXNDC9 was evaluated by Kaplan-Meier curves and Cox regression analysis. The mRNA and protein expression of TXNDC9 were analyzed using quantitative real-time PCR and western blot analysis. The effects of TXNDC9 on GC cell invasion and EMT were assessed in vitro, and its effects on tumorigenesis were confirmed using animal experiments. The activity of the NF-κB signaling pathway was examined by both in vitro and in vivo experiments. TXNDC9 was highly expressed in GC tissues and cell lines. A high level of TXNDC9 was associated with poor overall survival and served as an independent prognostic biomarker in GC patients. The knockdown of TXNDC9 led to restrained GC cell invasion, microtubule formation, and EMT in vitro, and suppressed tumorigenesis in vivo. In addition, the NF-κB signaling pathway was demonstrated to mediate the functional role of TXNDC9 in GC. In conclusion, this study found that high TXNDC9 predicted poor prognosis in GC, and served as an oncogene by enhancing tumor cell invasion and EMT through the NF-κB signaling pathway.