TNFRSF10A Human Fc

What is TNFRSF10A and what role does it play in cellular signaling pathways?

TNFRSF10A (tumor necrosis factor receptor superfamily member 10A), also known as DR4, CD261, or TRAILR1, is a cell surface receptor protein involved in apoptotic, necroptotic, and inflammatory pathways . It functions as a receptor for the cytokine TNF-related apoptosis-inducing ligand (TRAIL/TNFSF10) in death receptor signaling pathways . When TRAIL binds to TNFRSF10A, it activates the receptor by exposing its cytoplasmic death domain, which then recruits the adapter molecule FADD and activates caspases to initiate apoptosis . Beyond apoptosis, TNFRSF10A is also involved in the activation of NFκβ-mediated inflammatory signaling pathways .

Studies with FADD-deficient mice have demonstrated that FADD is required for TNFRSF10A-mediated apoptosis . TNFRSF10A is closely related to TRAIL R2/DR5 (TNFRSF10B), sharing approximately 55% amino acid sequence identity .

What is a TNFRSF10A Human Fc chimera and how does it function differently from native TNFRSF10A?

A TNFRSF10A Human Fc chimera (TRAIL R1/Fc chimera) is a recombinant fusion protein that combines the extracellular domain of human TNFRSF10A (typically Ala24-Asn239) with an immunoglobulin Fc region . Unlike native TNFRSF10A, which is a transmembrane protein that transduces apoptotic signals into cells, the TNFRSF10A-Fc chimera functions as a soluble decoy receptor that can bind to and neutralize TRAIL in solution.

The human TRAIL R1/Fc chimera effectively neutralizes the ability of TRAIL to induce apoptosis . This makes it particularly useful for studying TRAIL-mediated biological processes by allowing researchers to selectively block TRAIL signaling. The Fc portion of the chimera provides several advantages, including:

• Extended half-life in experimental systems

• Enhanced stability

• Easier detection and purification

• Potential for dimerization, increasing avidity for TRAIL

What are the primary functions of TNFRSF10A in normal physiology versus disease states?

TNFRSF10A plays diverse roles in both normal physiology and pathological conditions:

In normal physiology:

• Mediates immune surveillance by facilitating elimination of virus-infected and transformed cells

• Participates in the regulation of inflammatory responses through NFκβ pathway activation

• Contributes to the maintenance of immune homeostasis

In disease states:

• Dysregulation of TNFRSF10A has been implicated in sensitization to apoptosis

• May contribute to development of multiple diseases when expression or function is altered

• Overexpression of TNFRSF10A can lead to decreased cell viability (demonstrating a 1.60% ± 0.33% increase in TUNEL-positive cells compared to control)

• In cancer cells, TNFRSF10A expression may be altered, affecting sensitivity to TRAIL-induced apoptosis

• TNFRSF10A signaling may affect immune cell proliferation, particularly M2 macrophages

Expression analyses have shown that TNFRSF10A is detected in various tissues, including human brain neurons, where it is primarily localized to the cytoplasm .

How should researchers design experiments using TNFRSF10A Human Fc to study TRAIL-mediated apoptosis?

When designing experiments with TNFRSF10A Human Fc for studying TRAIL-mediated apoptosis, researchers should consider the following methodological approaches:

Experimental design considerations:

• Dose optimization: Titrate TNFRSF10A-Fc concentrations (typically 10-100 ng/mL) against a fixed TRAIL concentration to determine optimal neutralization ratios

• Pre-incubation protocol: For maximum neutralization efficacy, pre-incubate TNFRSF10A-Fc with TRAIL for 30-60 minutes before adding to cells

• Appropriate controls: Include TRAIL alone (positive control), buffer only (negative control), and an irrelevant Fc protein (specificity control)

• Cell-specific considerations: Test multiple cell lines, as TRAIL sensitivity varies between cell types

Methodological approach for TRAIL neutralization assay:

Research has shown that inhibition of TRAIL-induced cytotoxicity by TNFRSF10A-Fc is dose-dependent, with the ND50 (neutralization dose 50%) typically in the range of 0.02-0.055 μg/mL .

What are the critical parameters for validating TNFRSF10A Human Fc biological activity in cell-based assays?

Validating the biological activity of TNFRSF10A Human Fc is essential for experimental reliability. Key validation strategies include:

Functional validation approaches:

• Neutralization assay: Test the ability of TNFRSF10A-Fc to inhibit TRAIL-induced cytotoxicity in susceptible cell lines (e.g., L-929 mouse fibroblasts in the presence of actinomycin D) . A functional protein should demonstrate dose-dependent protection against TRAIL-mediated cell death.

• Flow cytometry validation: Confirm binding specificity by analyzing TNFRSF10A-Fc interaction with target cells expressing TRAIL. For example, research has used this approach to validate antibodies against TNFRSF10A in HeLa cells .

• Western blot analysis: Detect TNFRSF10A-Fc using anti-TNFRSF10A or anti-Fc antibodies. Western blotting can detect TRAIL R1/TNFRSF10A at approximately 50 kDa under reducing conditions, as demonstrated in TF-1 human erythroleukemic cells .

• Inhibition of downstream signaling: Measure the effect of TNFRSF10A-Fc on TRAIL-induced caspase activation, PARP cleavage, or other apoptotic markers.

Important validation controls:

• Positive control: Anti-TNFRSF10A neutralizing antibody (to confirm TRAIL neutralization by an alternative method)

• Specificity control: Test neutralization of alternative death ligands (e.g., FasL) to confirm TRAIL specificity

• Dose-response analysis: Use multiple concentrations to establish the relationship between TNFRSF10A-Fc concentration and neutralization capacity

How does cellular stress affect TNFRSF10A signaling and how should this be accounted for in experimental design?

Cellular stress significantly impacts TNFRSF10A signaling, creating important considerations for experimental design:

Effects of cellular stress on TNFRSF10A signaling:

Research has demonstrated that cellular stress conditions, such as treatment with tunicamycin (a chemical stressor), can alter TNFRSF10A-mediated cell death responses . For example:

These data demonstrate that knockdown of TNFRSF10A increases cell death specifically under stress conditions, while TNFRSF10A overexpression increases cell death regardless of stress .

Recommendations for experimental design:

• Include stress and non-stress conditions: Test TNFRSF10A-Fc under both baseline and stress conditions to capture context-dependent effects

• Monitor stress markers: Include measurements of cellular stress indicators (e.g., ER stress markers, ROS levels)

• Time-course analysis: Examine the kinetics of TRAIL sensitivity following stress induction

• Consider dose-dependent effects: Test a range of stressor concentrations to establish dose-response relationships

• Cell type considerations: Different cell types may respond differently to combined stress and TRAIL stimulation

What is the relationship between TNFRSF10A and the lncRNA AC100861.1, and how might this impact experimental design?

Recent research has uncovered a complex relationship between TNFRSF10A and the long non-coding RNA AC100861.1 that has important implications for experimental design:

Key findings about AC100861.1 and TNFRSF10A:

• Genomic proximity: AC100861.1 is a lncRNA that lies head-to-head with TNFRSF10A in the genome

• Independent expression regulation: Manipulation of one gene does not substantially affect expression of the other:

  • AC100861.1 overexpression (295.71 ± 12.96 fold increase) did not significantly affect TNFRSF10A levels (1.07 ± 0.16 fold change)

  • TNFRSF10A overexpression (4.12 ± 0.36 fold increase) did not substantially alter AC100861.1 expression (1.55 ± 0.30 fold change)

• Functional similarities but distinct mechanisms: Both genes affect cell viability:

  • AC100861.1 overexpression led to decreased cell viability, with further decreases observed under cellular stress conditions

  • AC100861.1 appears to regulate necroptotic and inflammatory signaling pathways independently of TNFRSF10A

• Subcellular localization: RNA fluorescence in situ hybridization (RNA-FISH) has shown that AC100861.1 transcript is primarily localized to the cytoplasm

Experimental design implications:

• Genetic manipulation considerations: When overexpressing or knocking down TNFRSF10A, confirm that AC100861.1 expression remains unchanged, and vice versa

• Pathway analysis expansion: Include markers of both apoptotic and necroptotic/inflammatory pathways when studying either gene

• Subcellular fractionation: Consider separate analysis of cytoplasmic and membrane fractions to distinguish effects related to membrane-bound TNFRSF10A versus cytoplasmic AC100861.1

• Combined approaches: For comprehensive understanding, consider manipulating both genes simultaneously using combinatorial approaches

How can TNFRSF10A Human Fc be utilized in studying necroptotic and inflammatory pathways beyond apoptosis?

While TNFRSF10A is primarily known for its role in apoptosis, emerging research indicates it also participates in necroptotic and inflammatory signaling, expanding the utility of TNFRSF10A Human Fc:

Applications for studying necroptotic pathways:

• Differential pathway analysis: Use TNFRSF10A-Fc in combination with:

  • Caspase inhibitors (e.g., z-VAD-fmk) to block apoptosis

  • RIPK1 inhibitors (e.g., necrostatin-1) to block necroptosis

  • MLKL inhibitors (e.g., necrosulfonamide) to block necroptotic execution

• Molecular markers: After TRAIL treatment with/without TNFRSF10A-Fc neutralization, monitor:

  • RIPK1/RIPK3 phosphorylation and complex formation

  • MLKL phosphorylation and oligomerization

  • Membrane permeabilization and release of DAMPs

Applications for studying inflammatory signaling:

• NFκβ pathway analysis: Use TNFRSF10A-Fc to determine TRAIL-dependency of:

  • IκB degradation and p65 nuclear translocation

  • NFκβ-driven gene transcription (using reporter assays)

  • Pro-inflammatory cytokine production

• Experimental approach for dissecting inflammatory vs. apoptotic outcomes:

This approach helps distinguish between TRAIL's direct effects on inflammatory signaling versus inflammatory responses secondary to apoptosis.

What are the methodological considerations for using TNFRSF10A Human Fc in comparative studies with other TRAIL receptors?

TNFRSF10A is one of several TRAIL receptors, requiring careful methodological considerations for comparative studies:

Key considerations for comparative receptor studies:

• Receptor specificity: TNFRSF10A (TRAIL R1) is most closely related to TNFRSF10B (TRAIL R2/DR5), sharing 55% amino acid sequence identity . Additionally, two TRAIL decoy receptors exist which antagonize TRAIL-induced apoptosis .

• Receptor expression profiling: Before comparative studies, quantify expression levels of all TRAIL receptors in the experimental system using:

  • Flow cytometry for surface expression

  • Western blotting for total protein levels

  • qRT-PCR for mRNA expression

• Selective receptor targeting: Use receptor-specific tools in parallel experiments:

  • TNFRSF10A-Fc for TRAIL R1 blockade

  • TNFRSF10B-Fc for TRAIL R2 blockade

  • Receptor-specific neutralizing antibodies

• Cross-reactivity controls: Test for potential cross-reactivity between closely related receptors, particularly at high concentrations of blocking agents

Methodological approach for receptor contribution analysis:

• Stepwise neutralization strategy:

  • TNFRSF10A-Fc alone

  • TNFRSF10B-Fc alone

  • Both receptor-Fc proteins combined

  • Compare results to determine relative receptor contributions

• Genetic validation:

  • Complement neutralization studies with receptor-specific siRNA or CRISPR knockouts

  • Compare phenotypes between neutralization and genetic approaches

• Cell type considerations:

  • Compare receptor function across multiple cell types with different receptor expression profiles

  • Consider tissue context when interpreting results

What are the most common technical issues when working with TNFRSF10A Human Fc and how can they be addressed?

Researchers working with TNFRSF10A Human Fc may encounter several technical challenges that require specific troubleshooting approaches:

Common issues and solutions:

• Limited neutralization efficacy:

  • Problem: Insufficient blocking of TRAIL activity

  • Potential causes: Inadequate concentration, protein degradation, improper storage

  • Solutions:

    • Perform fresh titration experiments

    • Prepare new protein aliquots

    • Confirm TRAIL concentration is not excessive

    • Extend pre-incubation time between TRAIL and TNFRSF10A-Fc

• Non-specific effects:

  • Problem: Changes in cellular responses not attributable to TRAIL neutralization

  • Potential causes: Fc-mediated effects, contaminants in preparation

  • Solutions:

    • Include irrelevant Fc protein control

    • Test heat-inactivated TNFRSF10A-Fc

    • Filter protein solution before use

    • Use alternative protein batches

• Inconsistent results across experiments:

  • Problem: Variable outcomes between replicate experiments

  • Potential causes: Cell passage effects, variable receptor expression, inconsistent handling

  • Solutions:

    • Standardize cell culture conditions

    • Verify receptor expression levels periodically

    • Develop detailed protocols with precise timing

    • Use internal controls for normalization

• Detection limitations:

  • Problem: Difficulty measuring TNFRSF10A-Fc activity or binding

  • Potential causes: Insufficient sensitivity, interference from sample components

  • Solutions:

    • Try alternative detection methods (ELISA, flow cytometry, functional assays)

    • Optimize sample preparation to reduce interference

    • Include concentration standards for quantitative comparison

How should researchers interpret contradictory results when studying TNFRSF10A signaling pathways?

Contradictory results are common in complex signaling systems like TNFRSF10A pathways. A systematic approach to resolving these contradictions includes:

Methodological approach to resolving contradictions:

• Comprehensive experimental characterization:

  • Test multiple cell lines to identify cell type-specific effects

  • Perform time-course analyses to capture temporal dynamics

  • Use concentration gradients to identify threshold effects

• Pathway integration assessment:

  • Consider the activation state of parallel pathways (e.g., NFκβ signaling)

  • Investigate potential roles of the lncRNA AC100861.1

  • Evaluate cellular stress conditions that might alter TRAIL sensitivity

• Technical validation:

  • Confirm findings using multiple detection methods

  • Verify antibody and reagent specificity

  • Perform genetic validation (siRNA, CRISPR) alongside pharmacological approaches

• Contextual factors to consider:

  • Proliferation status (dividing vs. quiescent cells)

  • Metabolic state (glycolytic vs. oxidative)

  • Differentiation status (stem/progenitor vs. terminally differentiated)

Interpretation framework for contradictory findings:

What controls are essential when designing conclusive experiments with TNFRSF10A Human Fc?

Well-designed controls are critical for generating conclusive data with TNFRSF10A Human Fc. Key controls should include:

Essential experimental controls:

• Negative controls:

  • Untreated cells (baseline viability/function)

  • Irrelevant Fc fusion protein (control for non-specific Fc effects)

  • Heat-inactivated TNFRSF10A Human Fc (control for specific binding activity)

  • Isotype control antibodies for detection methods

• Positive controls:

  • TRAIL alone (confirm TRAIL activity)

  • Known TRAIL-sensitive cell line (system validation)

  • Alternative TRAIL neutralizing agent (e.g., anti-TRAIL antibody)

• Specificity controls:

  • TRAIL R2/DR5 Fc chimera (compare receptor specificity)

  • Titration of TNFRSF10A Human Fc (establish dose-dependency)

  • Pre-incubation vs. co-incubation of TRAIL and TNFRSF10A Human Fc

• Pathway validation controls:

  • Caspase inhibitors (confirm apoptotic mechanism)

  • Necroptosis inhibitors (distinguish death pathways)

  • NFκβ pathway inhibitors (assess inflammatory signaling)

Control implementation strategy:

For maximum rigor, controls should be implemented in a systematic matrix that enables clear isolation of TNFRSF10A-specific effects from technical and biological confounders. This approach facilitates identification of specific molecular mechanisms and helps resolve seemingly contradictory results.

Western blot analysis can be used to validate TNFRSF10A detection, with TF-1 human erythroleukemic cell line lysates showing a specific band for TRAIL R1/TNFRSF10A at approximately 50 kDa under reducing conditions .

How does understanding the AC100861.1 lncRNA expand our knowledge of TNFRSF10A biology?

Recent research on the AC100861.1 lncRNA represents a significant advance in understanding TNFRSF10A biology:

The discovery that AC100861.1 lies head-to-head with TNFRSF10A in the genome but functions independently has revealed new complexity in the regulation of death receptor signaling . While both genes can influence cell viability, they appear to do so through different mechanisms - TNFRSF10A primarily through death receptor signaling and AC100861.1 through effects on necroptotic and inflammatory pathways .

This finding challenges previous assumptions about the TNFRSF10A genomic locus and suggests that comprehensive understanding of TRAIL signaling requires consideration of both protein-coding and non-coding elements. Future research should explore whether therapeutic strategies targeting this locus should consider both TNFRSF10A and AC100861.1 to achieve optimal outcomes.

What are the emerging applications of TNFRSF10A Human Fc in studying cancer and immune interactions?

TNFRSF10A Human Fc is finding new applications in studying the complex interactions between cancer cells and immune components:

Recent work has revealed that glycosylation patterns can significantly impact TRAIL receptor function on tumor cells, affecting their interactions with immune cells . TNFRSF10A Human Fc can be used to investigate how such modifications alter TRAIL sensitivity and immune recognition.

Additionally, TNFRSF10A signaling appears to influence immune cell proliferation, particularly M2 macrophages , suggesting potential applications in studying tumor-associated macrophage biology. By selectively blocking TRAIL-TNFRSF10A interactions, researchers can dissect the contribution of this pathway to tumor immune evasion and immunotherapy responses.

Future directions include investigating how TNFRSF10A Human Fc might be combined with immune checkpoint inhibitors to enhance anti-tumor immune responses or used as a tool to identify patients likely to benefit from TRAIL-targeted therapies.