TXNRD1 Antibody

What is TXNRD1 and what cellular functions does it regulate?

TXNRD1 is a homodimeric flavoprotein that primarily functions to reduce the disulfide protein thioredoxin (Trx) to its dithiol-containing form. It contains a selenocysteine residue at the C-terminal active site that is essential for catalysis. TXNRD1 participates in regulating multiple cellular processes including DNA synthesis and repair, signal transduction, and antioxidant defense. Within the antioxidant defense pathway, TXNRD1 works alongside thioredoxin to provide resistance to oxidative stress . Additionally, TXNRD1 exhibits reductase activity on hydrogen peroxide (H₂O₂), further contributing to its antioxidant functions. Certain isoforms of TXNRD1 have been found to induce actin and tubulin polymerization, enhance transcriptional activity of estrogen receptors, and mediate cell death in specific contexts .

Which applications and sample types have been validated for TXNRD1 antibodies?

TXNRD1 antibodies have been validated for multiple experimental applications including Western blotting (WB), immunohistochemistry on paraffin-embedded samples (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF). According to validation studies, TXNRD1 antibodies successfully detect the protein in human, mouse, and rat samples . Specifically, positive Western blot detection has been confirmed in multiple cell lines (HeLa, MCF-7, Jurkat) and tissue samples (mouse and rat heart tissue). For immunohistochemistry, TXNRD1 antibodies have demonstrated positive detection in human testis tissue, human breast cancer tissue, and mouse testis tissue. Optimal results for IHC may require antigen retrieval with TE buffer at pH 9.0 or alternatively with citrate buffer at pH 6.0 .

How can researchers accurately assess TXNRD1 expression in tissue samples?

For accurate assessment of TXNRD1 expression in tissue samples, researchers should employ a combination of techniques. Immunohistochemistry can reveal the spatial distribution of TXNRD1, which has been shown to be predominantly expressed in the medial layer of vasculature in lung tissue . Immunofluorescence staining provides enhanced specificity and can be used to co-localize TXNRD1 with other markers. For quantitative assessment, Western blotting of tissue homogenates provides reliable results when normalized to appropriate housekeeping proteins. When analyzing TXNRD1 in clinical samples, serum concentration can be measured and compared against healthy controls. In pulmonary hypertension research, TXNRD1 expression has been shown to be significantly downregulated in lung homogenates from monocrotaline-treated rats compared to controls, which can be confirmed by both Western blot and immunofluorescence staining .

What are the standardized assays for measuring TXNRD1 activity?

Two standardized assays are widely used to measure TXNRD1 activity: the insulin-coupled TXN1 reduction assay and the DTNB reduction assay.

The insulin-coupled TXN1 reduction assay measures TXNRD1 activity by monitoring absorbance at 340 nm. The assay proceeds in two stages: first measuring NADPH oxidation, and then monitoring insulin precipitation. During this process, NADPH-reduced TXNRD1 reduces the active sites of TXN1, which subsequently cleaves the disulfide bonds of insulin alpha and beta chains, resulting in the formation of turbid precipitates in the reaction solution .

The DTNB reduction assay monitors the formation of TNB anionic radicals spectrophotometrically at 412 nm. A decrease in the rate of TNB formation indicates inhibition of TXNRD1 activity. For this assay, 10 μL of treated TXNRD1 is typically added to each well of a 96-well plate, followed by 190 μL DTNB master mix, and absorbance is monitored at 412 nm for 5 minutes with measurements taken at 5-second intervals .

What protocol should be followed to evaluate potential inhibitors of TXNRD1?

The evaluation of potential TXNRD1 inhibitors follows a systematic protocol with several critical steps. First, TXNRD1 (0.2 μM) should be reduced by incubation with 100 μM NADPH in TE buffer at room temperature for 10 minutes. The reduced TXNRD1 is then incubated with various concentrations of the inhibitor in TE buffer for 1 hour at room temperature. It is crucial to ensure that the volume of DMSO or other organic solvents in the buffer system remains below 2% to avoid non-specific effects .

Following inhibitor treatment, TXNRD1 activity can be assessed using either the insulin-coupled TXN1 reduction assay or the DTNB reduction assay as described previously. For comprehensive characterization of inhibitors, researchers should perform four complementary assays: cellular TXNRD activity measurement, recombinant enzyme-based activity determination, differential scanning fluorimetry (DSF), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This multi-assay approach facilitates thorough screening and development of potential small-molecule inhibitors of TXNRD1 .

How can researchers isolate and culture pulmonary artery smooth muscle cells (PASMCs) for TXNRD1 studies?

For TXNRD1 studies in pulmonary arterial hypertension research, proper isolation and culture of PASMCs is essential. After isolation, PASMCs should be confirmed by immunofluorescence with alpha-smooth muscle actin (alpha-sma) to verify cell identity. When studying TXNRD1 regulation in these cells, PDGF-BB (platelet-derived growth factor-BB) stimulation serves as an effective model, as it has been identified as a key mediator of PASMC proliferation in PAH progression. Following PDGF-BB stimulation, researchers can observe suppression of TXNRD1 expression .

For knockdown studies, siRNA targeting TXNRD1 has been shown to exacerbate proliferative disorder, migration, and apoptosis resistance in PASMCs, suggesting TXNRD1's protective role against pathological vascular remodeling. These cellular models provide valuable platforms for investigating TXNRD1's role in pulmonary vascular pathophysiology and for screening potential therapeutic compounds targeting this pathway .

What evidence supports TXNRD1 as a biomarker for idiopathic pulmonary arterial hypertension (IPAH)?

Integrated bioinformatic analysis of multiple datasets has identified TXNRD1 as a promising biomarker for IPAH diagnosis. Validation studies using the GSE113439 dataset confirmed that TXNRD1 is consistently downregulated in IPAH patients. Clinical validation with patient samples demonstrated that serum TXNRD1 concentration was significantly lower in IPAH patients compared to healthy controls .

ROC curve analysis has shown that TXNRD1 has excellent predictive efficiency as a diagnostic biomarker with an AUC value of 0.795. At the optimal expression cutoff value of 0.60, sensitivity and specificity were 92.3% and 66.7%, respectively. Furthermore, TXNRD1 levels exhibit significant negative correlations with critical clinical parameters including mean pulmonary arterial pressure (mPAP) and pulmonary vascular resistance (PVR), suggesting its potential utility as a marker of disease severity .

What clinical parameters correlate with TXNRD1 expression in IPAH patients?

Analysis of the relationship between serum TXNRD1 levels and clinical characteristics of IPAH patients has revealed significant correlations with multiple parameters. Most notably, TXNRD1 levels demonstrate strong negative correlations with mean pulmonary arterial pressure (mPAP) and pulmonary vascular resistance (PVR), suggesting that lower TXNRD1 levels are associated with more severe hemodynamic impairment .

The following table summarizes the clinical characteristics of IPAH patients and controls in a validation study:

Interestingly, TXNRD1 levels did not show significant correlations with 6-minute walk distance (6MWD) or N-terminal pro-brain natriuretic peptide (NT-proBNP) levels, suggesting that TXNRD1 may reflect specific aspects of pulmonary vascular pathophysiology rather than general cardiac function or exercise capacity .

Which signaling pathways interact with TXNRD1 in pathological contexts?

Gene Set Enrichment Analysis (GSEA) has identified several signaling pathways that consistently correlate with TXNRD1 across multiple datasets. The three most prominently enriched pathways are the mTORC1 signaling pathway, MYC targets, and the unfolded protein response .

The mTORC1 pathway is a central regulator of cell growth and metabolism, suggesting TXNRD1 may influence vascular remodeling through modulation of this pathway. The association with MYC targets implies potential roles in cell cycle regulation and proliferation, which are key processes in vascular pathology. The unfolded protein response connection indicates possible involvement in endoplasmic reticulum stress, which has been implicated in various cardiovascular diseases including pulmonary hypertension .

These pathway associations provide valuable insights into the molecular mechanisms through which TXNRD1 may influence disease pathogenesis and suggest potential therapeutic approaches targeting these interactions.

How can researchers effectively validate TXNRD1 findings across multiple experimental models?

Comprehensive validation of TXNRD1 findings requires integration of data from multiple experimental models. In IPAH research, a robust validation approach includes four complementary levels: bioinformatic analysis of gene expression datasets, serum protein measurements in patient samples, animal model experiments, and in vitro cellular studies .

For bioinformatic validation, researchers should utilize the Robust rank aggregation (RRA) method to screen differentially expressed genes across multiple datasets, followed by validation in independent cohorts. At the clinical level, serum TXNRD1 concentrations should be compared between patients and healthy controls, with ROC curve analysis to assess diagnostic potential .

In animal studies, models such as monocrotaline-treated rats provide valuable platforms for validating TXNRD1 expression changes. Researchers should measure TXNRD1 expression in lung homogenates using Western blotting and assess tissue localization through immunofluorescence staining. At the cellular level, isolated PASMCs stimulated with PDGF-BB serve as an effective model for studying TXNRD1 regulation in a controlled environment .

What methodological challenges should researchers anticipate when studying TXNRD1?

Several methodological challenges require consideration when studying TXNRD1. The selenocysteine residue at the C-terminal active site is essential for catalysis but can complicate protein expression and purification. Additionally, TXNRD1 exists in multiple isoforms with distinct functions – isoform 1 induces actin and tubulin polymerization, isoform 4 enhances transcriptional activity of both estrogen receptors (ESR1 and ESR2), and isoform 5 specifically enhances ESR2 activity .

When measuring TXNRD1 activity, maintaining proper concentrations of NADPH and ensuring that organic solvent concentrations remain below 2% are critical considerations. For clinical studies, a significant limitation is often the availability of sufficient patient samples, as noted in research on IPAH .

Additionally, while animal models provide valuable insights, researchers should be aware of their limitations. The monocrotaline-treated rat model, though widely used, may not capture all aspects of human disease. Future studies may benefit from utilizing additional models such as hypoxia + Sugen5416 treated mice to provide more comprehensive validation .

How should researchers integrate bioinformatic analysis with experimental TXNRD1 research?

Effective integration of bioinformatics with experimental TXNRD1 research requires a systematic approach. Researchers should begin with comprehensive database mining, such as utilizing GEO datasets, followed by robust statistical methods like the RRA approach to identify consistently differentially expressed genes across multiple datasets .

Functional annotation through GO and KEGG enrichment analysis should be performed to understand the biological context of expression changes. Construction of protein-protein interaction networks using tools like MCODE and CytoHubba can help identify hub genes and potential interaction partners of TXNRD1 .

GSEA analysis provides valuable insights into the pathways and biological processes associated with TXNRD1. Researchers should validate bioinformatic predictions through experimental approaches, including measurement of protein expression, activity assays, and functional studies in relevant cell types. This integrated approach enables identification of TXNRD1 as both a biomarker and a potential therapeutic target, as demonstrated in IPAH research .

What are promising therapeutic approaches targeting TXNRD1?

Several inhibitors have been reported to target TXNRD1 activity, with potential applications as anti-tumor medications and possibly for other conditions like pulmonary hypertension. Future research should focus on developing selective TXNRD1 inhibitors with improved pharmacokinetic properties and reduced off-target effects . Additionally, given that TXNRD1 appears to be downregulated in IPAH, approaches to enhance or restore TXNRD1 function might represent a novel therapeutic strategy for this condition .

The connection between TXNRD1 and pathways such as mTORC1 signaling, MYC targets, and the unfolded protein response suggests that combination therapies targeting TXNRD1 alongside these pathways might provide synergistic effects. Furthermore, the negative correlation between TXNRD1 levels and clinical parameters like mPAP and PVR indicates that TXNRD1-targeted therapies might directly impact disease severity in conditions like PAH .

What unresolved questions remain in TXNRD1 research?

Despite significant advances, several important questions in TXNRD1 research remain unresolved. The precise mechanisms by which TXNRD1 downregulation contributes to pulmonary vascular remodeling in PAH are not fully understood. Additionally, while TXNRD1 has been identified as a potential biomarker for IPAH, larger clinical studies are needed to validate its diagnostic and prognostic utility .

The relationship between TXNRD1 and established PAH treatments has not been thoroughly investigated. Understanding whether current therapies modulate TXNRD1 expression or activity could provide insights into their mechanisms of action and guide the development of more effective treatment approaches. Furthermore, while TXNRD1 knockdown exacerbates proliferation, migration, and apoptosis resistance in PASMCs, the detailed molecular mechanisms underlying these effects require further exploration .