Recombinant Human Tumor necrosis factor receptor superfamily member 1A protein (TNFRSF1A), partial (Active)

What is TNFRSF1A and what are its primary functions in normal physiology?

TNFRSF1A, also known as TNF Receptor Type I or TNFR1, is a 55 kDa type I transmembrane protein member of the TNF receptor superfamily . In humans, the full TNFRSF1A protein contains 455 amino acids . Physiologically, it functions as a primary receptor for tumor necrosis factor alpha (TNFα), mediating multiple cellular responses including inflammation, apoptosis, and cell survival signaling.

The receptor contains an extracellular domain that binds TNFα, a transmembrane domain, and an intracellular domain that initiates downstream signaling cascades . When TNFα binds to TNFRSF1A, it triggers activation of nuclear factor-kappa B (NF-κB) signaling pathway, leading to transcription of inflammatory and cell survival genes . This interaction plays a crucial role in host defense against pathogens and in tissue homeostasis through regulation of inflammation and cell death.

TNFRSF1A signaling is tightly regulated, and dysregulation can contribute to various pathological conditions, including autoimmune disorders, inflammatory diseases, and cancer development .

What genetic variations of TNFRSF1A have been identified and what are their clinical implications?

Multiple clinically significant mutations in the TNFRSF1A gene have been identified, with the most notable being those associated with Tumor Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS) . TRAPS is characterized by recurrent fevers lasting approximately 3 weeks to a few months, occurring at variable intervals from every 6 weeks to years apart .

The T79M mutation is a well-established TRAPS-causing mutation, while G87V is another confirmed pathogenic variant . The T90I variant has been identified but is currently classified as a variant of unknown significance . These mutations primarily affect the extracellular domain of the receptor, particularly in exon 3 of the coding sequence .

Clinical manifestations of TRAPS include abdominal pain, muscle pain, joint pain, periorbital edema, skin rash, and inflammation affecting various body systems including the eyes, heart muscle, joints, throat, and mucous membranes . Approximately 15-20% of TRAPS patients develop amyloidosis in adulthood, which can lead to kidney failure due to amyloid protein accumulation .

Beyond TRAPS, TNFRSF1A variants have implications in cancer biology, particularly in renal cell carcinoma, where altered TNFRSF1A signaling contributes to tumor progression .

What transcript variants of TNFRSF1A exist and how do they differ functionally?

The TNFRSF1A gene generates 18 distinct transcript variants through alternative splicing . These variants differ significantly in their structure, stability, and functional properties. The primary transcript (ENST00000162749.7, TNFRSF1A-201) is 2,171 base pairs in length and encodes a 455 amino acid protein . This variant is designated as the MANE Select and Ensembl Canonical transcript, representing the most biologically relevant form .

Other significant protein-coding variants include:

• TNFRSF1A-204 (2,379 bp): Encodes a longer 528 amino acid protein

• TNFRSF1A-212 (1,527 bp): Encodes a 412 amino acid protein

• TNFRSF1A-211 (1,063 bp): Encodes a 298 amino acid protein with an incomplete CDS at the 3' end

Several transcripts undergo nonsense-mediated decay, including variants that encode truncated proteins ranging from 65 to 228 amino acids . These include TNFRSF1A-217, TNFRSF1A-218, and TNFRSF1A-203, which encode 218 amino acid proteins, and TNFRSF1A-207, which encodes a 65 amino acid protein .

Additionally, there are non-protein coding variants classified as retained intron transcripts (TNFRSF1A-202, TNFRSF1A-216, TNFRSF1A-215, TNFRSF1A-206, and TNFRSF1A-210) . These transcript variations contribute to the complex regulation of TNFRSF1A expression and function across different tissues and cellular contexts.

How do TNFRSF1A mutations affect TNFα signaling in experimental models?

TNFRSF1A mutations associated with TRAPS have been extensively studied using both in vitro and in vivo experimental models. Contrary to initial expectations, research with TRAPS mutant mice (T79M, G87V, and T90I) has revealed that these mutations do not enhance inflammatory responses but instead suppress TNFα signaling .

In murine models, TRAPS mutations (T79M and G87V) demonstrated reduced mortality rates following administration of lipopolysaccharide (LPS) and D-galactosamine, which typically induce TNFα-dependent lethal hepatitis . Similarly, when crossed with human TNFα transgenic mice, these mutations strongly suppressed the development of TNFα-mediated arthritis .

At the cellular level, primary bone marrow-derived macrophages from T79M and G87V mutant mice showed attenuated inflammatory responses to TNFα compared to wild-type cells . This reduced responsiveness appears to be specific to TNFα, as these mutations did not alter the cells' response to LPS stimulation .

The molecular mechanism behind this unexpected dampening of TNFα signaling involves altered TNFR1 trafficking. TRAPS mutant macrophages exhibited increased levels of TNFR1 in whole-cell lysates but significantly decreased cell surface expression . This suggests that the mutations cause retention of the receptor intracellularly, reducing its availability for TNFα binding at the cell surface.

These findings challenge earlier hypotheses about TRAPS pathogenesis and suggest that inflammation in TRAPS may be driven by as-yet unidentified disease-specific proinflammatory factors rather than enhanced TNFα signaling .

What is the role of TNFRSF1A in cancer progression, particularly in renal cell carcinoma?

TNFRSF1A has emerged as a significant cancer marker, particularly in renal cell carcinoma (RCC). Research has identified a critical role for the TNF signaling pathway in the development of clear cell RCC (ccRCC), the most common histological subtype comprising 70-80% of all kidney cancer cases .

Single-cell analysis has revealed tumor microenvironment (TME) heterogeneity in RCC, with specific crosstalk identified between TNF and TNFRSF1A . This communication within the TME appears to be a key factor in tumor progression. Functional validation through in vitro experiments has demonstrated that TNFRSF1A promotes proliferation, migration, and invasion of ccRCC cells .

Multiple experimental techniques have been employed to investigate TNFRSF1A's cancer-promoting characteristics:

• 5-ethynyl-2'-deoxyuridine incorporation assays to measure cell proliferation

• Cell Counting Kit-8 assays for cell viability assessment

• Colony formation assays to evaluate clonogenic potential

• Transwell assays to quantify cell migration and invasion capabilities

• Cell cycle and apoptosis assays to determine effects on cellular turnover

These findings suggest that TNFRSF1A could serve as a potential therapeutic target in RCC. Disrupting TNFRSF1A function or expression might impede tumor progression, offering a novel strategy for precision medicine approaches to improve prognosis in RCC patients .

What methodologies are optimal for studying TNFRSF1A-mediated cell signaling pathways?

Multiple methodological approaches have proven effective for investigating TNFRSF1A-mediated signaling. Western blotting is widely used to analyze downstream signaling events, particularly focusing on NF-κB pathway activation through phosphorylation of key components such as p65 and p105 .

For inhibition studies, TNFRSF1A antibodies at concentrations of 4-10 μg/mL have been shown to effectively block TNFα-induced signaling, with maximum inhibition observed at 10 μg/mL . The inhibitory effect can be measured by assessing phosphorylation of NF-κB components at early time points (5 minutes post-stimulation) following TNFα treatment (typically 10 ng/mL) .

Cell-based assays to evaluate TNFRSF1A function include:

• Cytotoxicity assays using L-929 mouse fibroblast cells, where recombinant TNFRSF1A inhibits TNFα-induced cell death in a dose-dependent manner

• Flow cytometry to analyze surface expression markers such as SSEA-4 in response to TNFα and TNFR1 blockade

• Gene expression analysis via qPCR to measure transcriptional changes in target genes like OCT-4 and NANOG

When performing these assays, optimal conditions include using recombinant human TNF RI/TNFRSF1A at 0.3 μg/mL, recombinant human TNFα at 0.25 ng/mL, and the metabolic inhibitor actinomycin D at 1 μg/mL when appropriate . The typical ND50 (neutralization dose) for anti-TNFRSF1A antibodies ranges from 1-6 μg/mL under these conditions .

How can CRISPR/Cas9 be utilized to generate TNFRSF1A mutant models?

CRISPR/Cas9 technology has been successfully employed to generate TNFRSF1A mutant mouse models for studying TRAPS mutations. The genome editing by electroporation of Cas9 protein (GEEP) method has proven particularly effective for introducing specific point mutations in the Tnfrsf1a gene .

To generate TRAPS mutant mice (T79M, G87V, or T90I), CRISPR RNAs (crRNAs) are designed to target exon 3 of the TNFR1 coding sequence . The crRNA design should include:

• The target sequence (20 nucleotides)

• The protospacer adjacent motif (PAM) sequence (NGG for Cas9 from Streptococcus pyogenes)

• Modifications to introduce the desired mutation

Examples of successful crRNA designs include:

• T79M: 5′-CAGGGCGGGATACAGTCTGCAGG-3′ (PAM sequence in bold)

• G87V: 5′-GTCTGCAGGGAGTGTGAAAAAAA-3′

• T90I: 5′-GTAATTCTGGGAAGCCGTAAAGG-3′

The CRISPR/Cas9 components are delivered via electroporation to fertilized eggs, which are then implanted into pseudopregnant females. Resulting offspring are genotyped to confirm successful introduction of the desired mutation. This approach ensures physiological expression levels of the mutant protein, avoiding artifacts associated with overexpression systems .

Using this methodology, researchers have successfully generated T79M and G87V mutant mice (C57BL/6N background) at Setsuro Tech (Tokushima, Japan) and T90I mutant mice at Kawasaki Medical School (Kurashiki, Japan) . These models provide valuable tools for investigating the in vivo effects of TRAPS mutations on inflammation, immune responses, and disease pathogenesis.

What considerations should be made when using recombinant TNFRSF1A proteins in experimental assays?

When utilizing recombinant human TNF RI/TNFRSF1A in experimental settings, several important considerations should guide experimental design and interpretation:

• Protein stability and carrier proteins: Recombinant TNFRSF1A is typically formulated with carrier proteins such as bovine serum albumin (BSA) to enhance stability and increase shelf-life . This addition should be considered when designing experiments, particularly immunological assays where BSA might interfere.

• Protein format: Various forms of recombinant TNFRSF1A are available, including:

  • The extracellular domain alone

  • Fc chimera proteins that combine the TNFRSF1A extracellular domain with an Fc region

  The choice between these formats depends on the experimental objectives, with Fc chimeras offering advantages in purification, detection, and increased half-life.

• Antibody selection and validation: For detection of TNFRSF1A or inhibition studies, monoclonal antibodies like clone #16805 have been validated for specific applications . When using these antibodies, it's critical to note that some may recognize TNFRSF1A only under non-reducing conditions, highlighting the importance of proper sample preparation .

• Dosage optimization: In inhibition assays, the concentration of recombinant TNFRSF1A significantly impacts results. Typical effective concentrations are 0.3 μg/mL for recombinant TNFRSF1A when used with 0.25 ng/mL recombinant human TNFα . Dose-response curves should be generated to determine optimal concentrations for specific experimental systems.

• Complementary assays: To comprehensively assess TNFRSF1A function, multiple assay types should be employed, including:

  • Binding assays to confirm interaction with TNFα

  • Cell viability/cytotoxicity assays to measure functional outcomes

  • Signaling assays to detect downstream pathway activation

  • Gene expression analysis to evaluate transcriptional responses

How can researchers effectively measure TNFRSF1A-mediated NF-κB activation in cellular models?

Measuring NF-κB activation downstream of TNFRSF1A is critical for understanding its signaling mechanisms. Western blotting provides a robust method for analyzing NF-κB pathway components, particularly the phosphorylation status of p65 and p105 subunits .

When designing experiments to measure TNFRSF1A-mediated NF-κB activation, consider the following protocol elements:

• Cell preparation: Culture appropriate cell types (e.g., dental pulp stem cells, macrophages, or cancer cell lines) in serum-containing media until 70-80% confluent .

• TNFR1 blockade: For inhibition studies, pre-incubate cells with anti-TNFR1 antibody at varying concentrations (4, 6, and 10 μg/mL) for 1 hour prior to TNFα stimulation .

• TNFα stimulation: Treat cells with recombinant human TNFα at 10 ng/mL .

• Timing: Harvest cells at early time points (5 minutes post-stimulation) for analysis of immediate signaling events .

• Protein extraction and analysis: Prepare whole cell lysates and analyze by Western blotting using phospho-specific antibodies against NF-κB components (p-p65, p-p105) .

This approach allows for dose-dependent analysis of TNFR1 inhibition on NF-κB activation. Research has shown that anti-TNFR1 antibody inhibits TNFα-induced phosphorylation of p105 and p65 in a dose-dependent manner, with maximum effect observed at 10 μg/mL .

Complementary approaches include flow cytometric analysis of cell surface markers like SSEA-4, which can be partially inhibited by TNFR1 blockade , and qPCR analysis of downstream gene expression (e.g., OCT-4 and NANOG), which shows variable responses to TNFR1 inhibition .

What therapeutic approaches target TNFRSF1A in TRAPS and other inflammatory disorders?

While there is no cure for TRAPS, several therapeutic approaches target TNFRSF1A-mediated inflammation. Current treatment strategies focus on managing symptoms and preventing long-term complications such as amyloidosis .

First-line treatments include:

More targeted approaches include:

• Interleukin-1 inhibitors: Medications that block interleukin-1, a protein involved in inflammation, have shown significant benefit in TRAPS management . These biologics target the downstream inflammatory cascade rather than directly affecting TNFRSF1A function.

Interestingly, TNF inhibitors, which might seem logical given TNFRSF1A's role in TNF signaling, have shown mixed results in TRAPS patients. This paradoxical response aligns with research findings showing that TRAPS mutations actually decrease responsiveness to TNFα rather than enhance it .

Research models suggest that TRAPS-associated inflammation may be driven by unidentified disease-specific proinflammatory factors rather than enhanced TNFα signaling . This insight opens new avenues for therapeutic development targeting these yet-unidentified factors.

For targeting TNFRSF1A in cancer contexts, particularly renal cell carcinoma, disrupting TNFRSF1A function shows promise as a potential therapeutic strategy to impede tumor progression . This approach may represent an advance in precision medicine for improving patient prognosis.