Recombinant Human Tumor necrosis factor receptor superfamily member 10B (TNFRSF10B), partial (Active)

What is TNFRSF10B and what are its primary functions in cellular systems?

TNFRSF10B (also known as DR5, TRAIL-R2, CD262, KILLER, TRICK2) is a type 1 membrane protein belonging to the TNF receptor superfamily. It contains extracellular cysteine-rich domains, a transmembrane domain, and a cytoplasmic death domain . The protein is located at chromosome 8p21.3 and is one of five known receptors for TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) .

TNFRSF10B primarily functions as a death receptor that, upon binding trimeric TRAIL, triggers apoptosis through the extrinsic death pathway. When activated, it forms a death-inducing signaling complex (DISC) containing pro-caspase-8, ultimately leading to programmed cell death . Most notably, TRAIL specifically targets fully transformed cells without significantly affecting normal ones, making TNFRSF10B a promising target for cancer therapeutics .

How does TNFRSF10B differ from other TRAIL receptors in the TNF receptor family?

• TNFRSF10B contains a functional intracellular death domain capable of transducing apoptotic signals

• The decoy receptors compete for TRAIL binding but cannot initiate apoptosis

• Unlike some receptors, there are no known rat or mouse homologs to TNFRSF10B

The ratio of functional death receptors to decoy receptors often determines cellular sensitivity to TRAIL-induced apoptosis.

What genomic alterations of TNFRSF10B are commonly observed in cancer tissues?

Genomic and expression alterations of TNFRSF10B vary significantly across cancer types and subtypes:

In head and neck squamous cell carcinoma (HNSCC), the TRAIL receptor family members (TNFRSF10A/B/C/D) exhibit significant differences in copy number variations (CNV) between tumors with different HPV status . These genomic alterations may contribute to differential sensitivity to TRAIL-based therapies and agents targeting TRAILRs.

What are the optimal reconstitution and storage conditions for recombinant TNFRSF10B proteins?

For optimal activity of recombinant TNFRSF10B proteins, researchers should follow these guidelines:

Reconstitution:

• Lyophilized forms should be reconstituted at 500 μg/mL in PBS

• Allow complete solubilization before using in experiments

Storage:

• Use manual defrost freezers and avoid repeated freeze-thaw cycles

• Store reconstituted protein at temperatures recommended by manufacturers (typically -20°C to -80°C)

• Ship at ambient temperature but store immediately upon receipt

These conditions are critical for maintaining protein stability and functional activity in experimental settings.

Why is crosslinking necessary for some recombinant TNFRSF10B formats?

Crosslinking is essential for His-tagged TNFRSF10B formats because:

• TNFRSF10B requires receptor trimerization for optimal biological activity

• In natural systems, trimeric TRAIL induces oligomerization of the receptor, which is necessary for DISC formation and downstream signaling

• Recombinant forms may not spontaneously form the optimal configuration without additional crosslinking

The crosslinking antibody (e.g., anti-6x histidine) enhances biological activity by promoting receptor clustering similar to what occurs with endogenous TRAIL binding . Without proper crosslinking, the recombinant protein may not achieve the same level of specific activity described in product datasheets.

What bioassays are most appropriate for evaluating TNFRSF10B functional activity?

Several bioassays can effectively measure TNFRSF10B functional activity:

• Cytotoxicity Inhibition Assay:

  • Recombinant TRAIL R2/TNFRSF10B can inhibit TRAIL-mediated cytotoxicity in L-929 mouse fibroblast cells

  • The ED50 for this inhibitory effect is typically 0.75-6 ng/mL

• Apoptosis Detection:

  • Flow cytometry with Annexin V/PI staining to quantify early and late apoptotic cells

  • Caspase-8 and caspase-3 activation assays to measure downstream effector activation

  • PARP cleavage detection by Western blot

• Receptor Binding Assays:

  • Competitive binding assays with labeled TRAIL to measure receptor-ligand interactions

  • Surface plasmon resonance to determine binding kinetics and affinity

When designing these assays, it's crucial to include appropriate positive and negative controls to ensure the specificity of TNFRSF10B-mediated effects.

How does the HPV status affect TNFRSF10B expression and function in head and neck cancers?

HPV status significantly influences TNFRSF10B genomic alterations and potentially its function in head and neck squamous cell carcinoma (HNSCC):

HPV(+) HNSCC:

• More frequently display neutral or one-copy gain of TNFRSF10B

• Harbor less frequent FADD amplification and overexpression

• Show more frequent shallow deletion of BIRC2/3 loci

• Are generally less sensitive to SMAC mimetic birinapant

HPV(-) HNSCC:

• Have higher percentage of one-copy loss of TNFRSF10B

• Show different patterns of copy number variations of death pathway molecules

These distinct genomic profiles suggest that HPV status may be an important consideration when developing TRAIL-based therapeutic strategies for HNSCC. Oropharyngeal tumors, which are often HPV(+), showed the highest percentage of one-copy loss of FADD and BIRC2/3, with more frequent gain in TRAIL and its receptors .

What molecular mechanisms link cellular senescence to TNFRSF10B expression?

Cellular senescence involves a biphasic program that affects TNFRSF10B expression:

• Early Phase:

  • Activation of senescence-associated secretory (SAS) factors

  • Upregulation of inflammatory and anti-apoptogenic genes

• Late Phase:

  • Repression of DNA and chromatin components

  • Downregulation of CFLAR (which encodes c-FLIP, an inhibitor of apoptosis)

  • Increased expression of TNFRSF10B/DR5

This temporal regulation creates a window of vulnerability where senescent cells or their neighboring premalignant cells may become sensitized to TRAIL-induced apoptosis. The senescence secretome contains factors that lead to activation of the TRAIL pathway in premalignant cells, suggesting potential therapeutic opportunities for intervention at benign tumor stages .

How do genomic co-alterations of TNFRSF10B and other death pathway molecules affect therapeutic responses?

The therapeutic response to TRAIL-based treatments is influenced by the complex interplay of genomic alterations across multiple death pathway molecules:

These co-alterations create distinct molecular profiles that can predict sensitivity to various therapeutic approaches. For example, tumors with TNFRSF10B gain but FADD loss might respond differently to TRAIL than tumors with both components intact. Similarly, tumors with BIRC2/3 amplification may require combination therapy with SMAC mimetics to overcome resistance .

What factors contribute to variable TRAIL sensitivity despite TNFRSF10B expression?

Several factors can explain discrepancies between TNFRSF10B expression and TRAIL sensitivity:

• Receptor Dynamics:

  • Post-translational modifications affecting receptor function

  • Differential receptor localization in membrane vs. cytoplasm

  • Variations in receptor clustering efficiency

• Downstream Pathway Status:

  • Expression levels of anti-apoptotic proteins (c-FLIP, IAPs, Bcl-2 family)

  • Caspase-8 activation efficiency

  • Presence of downstream inhibitors

• Experimental Considerations:

  • TRAIL preparation quality and concentration

  • Duration of treatment

  • Cell density and culture conditions

When encountering unexpected resistance, researchers should systematically evaluate each of these factors to identify the specific mechanism operating in their model system.

How can researchers optimize experimental design when studying TNFRSF10B-mediated apoptosis?

A robust experimental design for studying TNFRSF10B-mediated apoptosis should include:

• Appropriate Controls:

  • Positive control (known TRAIL-sensitive cell line)

  • Negative control (either TRAIL-resistant cell line or TRAIL + pan-caspase inhibitor)

  • TNFRSF10B blocking antibody control

• Multiple Readouts:

  • Early apoptosis markers (phosphatidylserine externalization)

  • Executioner caspase activation (caspase-3/7)

  • Late apoptosis markers (DNA fragmentation)

• Validation Approaches:

  • Confirm TNFRSF10B expression by flow cytometry and Western blot

  • Verify the absence of mutations in the death domain

  • Assess expression of other TRAIL receptors (TNFRSF10A/C/D)

• Optimized Reagents:

  • Properly reconstituted and stored recombinant proteins

  • Validated crosslinking antibodies when using His-tagged formats

  • Fresh preparations of all reagents for each experiment

This comprehensive approach minimizes variability and increases confidence in experimental results.

How might TNFRSF10B-targeted therapeutics overcome resistance mechanisms in cancer?

Innovative approaches to overcome TRAIL resistance include:

• Receptor-Specific Targeting:

  • DR5-selective TRAIL variants with enhanced affinity

  • DR5-B functionalized with tumor-penetrating peptides like iRGD

  • Antibody-drug conjugates specifically targeting TNFRSF10B

• Combination Strategies:

  • Sensitization with agents that upregulate TNFRSF10B expression

  • Co-targeting anti-apoptotic proteins (IAPs, c-FLIP)

  • Exploiting senescence-induced vulnerability periods

• Novel Delivery Systems:

  • Nanoparticle formulations to enhance delivery and stability

  • Multimeric TRAIL formats to promote more effective receptor clustering

  • Tumor microenvironment-responsive activation systems

These approaches take advantage of the tumor-selective nature of TRAIL-induced apoptosis while addressing common resistance mechanisms .

What is the significance of TNFRSF10B in non-apoptotic signaling pathways?

Beyond its canonical role in apoptosis, TNFRSF10B can activate several non-apoptotic pathways:

• Inflammatory Signaling:

  • NF-κB activation leading to pro-inflammatory cytokine production

  • MAPK pathway activation affecting cell survival and proliferation

• Cell Migration and Invasion:

  • Potential role in activating Src family kinases

  • Effects on matrix metalloproteinase expression

• Metabolism:

  • Emerging evidence suggesting links to metabolic pathways

  • Potential crosstalk with glutamine utilization pathways

Understanding these non-canonical functions is essential for predicting potential off-target effects of TNFRSF10B-targeted therapies and may reveal new therapeutic opportunities.

How does the TNFRSF10B genomic landscape vary across different cancer types?

TNFRSF10B genomic alterations show distinct patterns across cancer types:

These cancer-specific patterns suggest that TNFRSF10B may play different roles in the pathogenesis of various malignancies . Researchers should consider cancer type-specific genomic contexts when designing experiments and interpreting results related to TNFRSF10B function.