TNFRSF12A Human, sf9

What is the molecular structure of human TNFRSF12A and how does it function in cellular signaling?

TNFRSF12A (Tumor necrosis factor receptor superfamily member 12A), also known as FN14, CD266 antigen, or TweakR, is the smallest member of the TNF receptor superfamily. It is a type I transmembrane protein consisting of 129 amino acids with a distinct domain structure: a 27 amino acid signal peptide, a 53 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 28 amino acid cytoplasmic domain . The protein contains only one cysteine-rich region in its extracellular domain, unlike other TNF receptor family members that typically have multiple such regions .

Functionally, TNFRSF12A acts as a receptor for TNFSF12/TWEAK and contains a TRAF binding motif in its cytoplasmic domain that interacts with TRAFs 1, 2, and 3 . This interaction initiates a signal transduction cascade that, depending on the cellular context, can lead to diverse biological responses including cell death, cell proliferation, and angiogenesis . The receptor is involved in positive regulation of extrinsic apoptotic signaling pathways and regulation of wound healing . It also promotes angiogenesis, proliferation of endothelial cells, and may modulate cellular adhesion to matrix proteins .

How is TNFRSF12A gene expression regulated in normal versus disease states?

TNFRSF12A expression appears to be tightly regulated by epigenetic mechanisms, particularly DNA methylation. In hepatocellular carcinoma (HCC), the methylation status of TNFRSF12A is significantly associated with patient survival outcomes . Notably, hypermethylation of TNFRSF12A correlates with longer survival (58.0%; 95% CI: 48.7–69.0%) compared to hypomethylation (39.6%; 95% CI: 30.9–50.9%) .

Research has revealed an interesting pattern where TNFRSF12A promoter methylation frequency is significantly higher in healthy controls (77.4%) than in HCC patients (53.4%) . This suggests that hypomethylation of the TNFRSF12A promoter may contribute to cancer development and progression. Additionally, TNFRSF12A mRNA levels were found to be lower in HCC patients with methylated TNFRSF12A promoters compared to those without methylation, confirming the functional impact of this epigenetic regulation .

The Human Protein Atlas data indicates that TNFRSF12A is expressed across various human tissues, with expression patterns varying significantly between normal and disease states . TNFRSF12A has been specifically associated with pathological processes in diseases such as glioblastoma and multiple sclerosis .

What are the comparative advantages of Sf9 insect cells versus E. coli for expressing human TNFRSF12A?

When selecting an expression system for human TNFRSF12A, researchers must consider several key factors that affect protein quality and functionality:

Sf9 Insect Cell Advantages:

• Ability to perform eukaryotic post-translational modifications, including glycosylation, phosphorylation, and proper disulfide bond formation, which may be critical for TNFRSF12A functionality

• More suitable environment for proper folding of complex human proteins like TNFRSF12A

• Better capacity for expressing transmembrane proteins in their native conformation

• Scalable suspension culture capabilities for larger-scale protein production

Sf9 Insect Cell Limitations:

• Glycosylation patterns differ from mammalian cells, potentially affecting protein function

• Longer production timeline (typically 3-5 days post-infection)

• More complex and expensive system to maintain and optimize

• Requires specialized expertise in baculovirus expression technology

E. coli Advantages:

• Simpler, more cost-effective expression system

• Faster growth and potentially higher protein yields

• Well-established protocols and optimization strategies

• Demonstrated success with the extracellular domain of TNFRSF12A (as seen with the 53 amino acid non-glycosylated polypeptide)

E. coli Limitations:

• Cannot perform glycosylation or other complex post-translational modifications

• Tendency to form inclusion bodies requiring refolding

• Endotoxin contamination concerns for certain applications

• Challenges with full-length membrane protein expression

The optimal choice depends on research objectives: E. coli may be sufficient for structural studies of the extracellular domain alone, while Sf9 cells would be preferred for functional studies requiring the full-length protein in a near-native state.

What experimental protocols yield optimal recombinant TNFRSF12A when expressed in Sf9 cells?

Based on established baculovirus expression system methodologies, the following optimized protocol is recommended for high-quality TNFRSF12A production in Sf9 cells:

Vector Design Considerations:

• Construct selection: Based on experimental needs, choose between:

  • Full-length TNFRSF12A (all domains)

  • Extracellular domain only (similar to the 53 aa fragment described)

  • Fusion constructs with tags for detection and purification

• Promoter selection: The polyhedrin (polh) promoter provides strong expression in the late phase of baculovirus infection

• Sequence optimization:

  • Codon optimization for insect cell expression

  • Signal sequence optimization for proper membrane targeting

  • Addition of affinity tags (His6, FLAG, etc.) with protease cleavage sites

Sf9 Cell Culture Optimization:

• Maintain cells in Sf-900 medium containing 3% FBS and 1% antibiotic-antimycotic at 27°C

• Infect cells at density of 1.5-2.0 × 10^6 cells/ml for optimal protein production

• Determine optimal multiplicity of infection (MOI) through empirical testing

• Monitor expression using appropriate detection methods (Western blot, fluorescence if using GFP fusion)

• Harvest cells 48-72 hours post-infection, based on expression kinetics testing

Purification Strategy:

• Cell lysis and membrane fraction isolation

• Detergent screening to identify optimal solubilization conditions

• Affinity chromatography using engineered tags

• Size exclusion and/or ion exchange chromatography for further purification

• Quality control via SDS-PAGE, Western blotting, and functional assays

Storage Considerations:

• Formulate in stabilizing buffer (similar to PBS, pH 7.4 recommended for TNFRSF12A)

• Consider lyophilization for long-term storage

• Store desiccated below -18°C for maximum stability

This comprehensive approach addresses the critical parameters for obtaining functional TNFRSF12A protein suitable for downstream applications.

How can methylation status of TNFRSF12A be accurately assessed in experimental and clinical samples?

Accurate assessment of TNFRSF12A methylation status is critical for understanding its regulation and potential as a biomarker. Based on established methodologies, the following comprehensive approach is recommended:

Methylation-Specific PCR (MSP) Protocol:

• DNA extraction from clinical samples or cultured cells using standardized protocols

• Bisulfite conversion: Treatment of DNA with sodium bisulfite to convert unmethylated cytosines to uracils while leaving methylated cytosines unchanged

• Primer design: Development of primer pairs specific to either methylated or unmethylated sequences in the TNFRSF12A promoter region

• PCR amplification: Using optimized conditions for the specific primers

• Analysis: Gel electrophoresis to visualize methylation status qualitatively

Quantitative Methylation Analysis:

• Quantitative methylation-specific PCR (qMSP) for more precise methylation level determination

• Pyrosequencing or next-generation sequencing approaches for site-specific methylation quantification

• Integration with The Cancer Genome Atlas (TCGA) methylation data for comparative analysis

Correlation with Expression:

• Parallel RNA extraction from matching samples

• RT-qPCR analysis of TNFRSF12A expression levels

• Statistical correlation between methylation status and expression

• Western blot analysis to confirm changes at the protein level

Clinical Correlation Analysis:

• Collection of comprehensive clinical data including survival information

• Kaplan-Meier survival analysis stratified by methylation status

• Multivariate analysis to identify independent prognostic value

When implemented in clinical studies of HCC, this approach revealed that patients with methylated TNFRSF12A promoters had mean survival time of 553.89 days (SE 40.235, 95% CI: 475.03–632.75) compared to 503.497 days (SE 39.080, 95% CI: 426.90–580.09) for those without methylation . This demonstrates the biomarker potential of TNFRSF12A methylation status.

What experimental approaches are most effective for investigating TNFRSF12A's role in cellular signaling pathways?

To comprehensively elucidate TNFRSF12A's role in cellular signaling, a multi-faceted experimental approach is recommended:

Genetic Manipulation Strategies:

• CRISPR/Cas9 gene editing:

  • Complete knockout of TNFRSF12A to assess loss-of-function effects

  • Domain-specific mutations, particularly in the TRAF binding motif

  • Knock-in of tagged versions for tracking endogenous protein

• Inducible expression systems:

  • Tetracycline-regulated expression for dose-controlled studies

  • Cell type-specific promoters for tissue-relevant expression

Stimulation and Pathway Analysis:

• TWEAK ligand stimulation experiments:

  • Dose-response (0.1-100 ng/mL) and time-course (5 min to 24 h) studies

  • Analysis of downstream signaling nodes:

    • Western blotting for phosphorylated proteins in Akt and TNF signaling pathways

    • Immunoprecipitation of TRAF1, TRAF2, and TRAF3 to confirm recruitment

    • Transcriptional profiling via RNA-seq to identify regulated genes

• Pathway inhibition approach:

  • Small molecule inhibitors targeting key nodes

  • Combinatorial inhibition to map pathway interactions

  • siRNA knockdown of pathway components to confirm specificity

Functional Outcome Assessment:

• Apoptosis assays:

  • Annexin V/PI flow cytometry

  • Caspase activity measurements

  • TUNEL assay for DNA fragmentation

• Angiogenesis and proliferation assays:

  • Endothelial tube formation

  • BrdU or EdU incorporation for proliferation

  • Scratch wound healing assay

• Matrix adhesion studies:

  • Cell adhesion assays to different extracellular matrix components

  • Focal adhesion complex analysis by immunofluorescence

Systems Biology Integration:

• Protein interaction network analysis:

  • BioID or proximity labeling approaches

  • Co-immunoprecipitation followed by mass spectrometry

  • Placement of TNFRSF12A in the context of Akt Signaling and TNF Superfamily pathways

• Multi-omics integration:

  • Correlation of transcriptomics, proteomics, and epigenomics data

  • Pathway enrichment analysis using tools like ConsensusPathDB

This methodological framework enables comprehensive characterization of TNFRSF12A's role in cellular signaling across different biological contexts.

How can contradictory data about TNFRSF12A function in different experimental systems be reconciled?

Contradictory findings regarding TNFRSF12A function across different experimental systems are common and require systematic approaches to reconciliation:

Cell Type-Specific Effects Analysis:

• Comparative expression studies across cell lines:

  • Baseline TNFRSF12A expression quantification by qPCR and Western blot

  • Analysis of co-expressed signaling components (especially TRAF proteins)

  • Assessment of endogenous TWEAK levels that might affect receptor signaling

• Systematic pathway mapping in multiple cell types:

  • Side-by-side stimulation experiments with standardized protocols

  • Broad phosphoproteomic analysis to identify differentially activated pathways

  • Transcriptional response profiling to identify cell type-specific gene regulation

Technical Variation Mitigation:

• Protocol standardization:

  • Consistent recombinant protein sources and concentrations

  • Standardized stimulation timeframes

  • Uniform lysis and analysis methods

• Multiple methodological approaches:

  • Verification of key findings using orthogonal techniques

  • Positive and negative controls for each experimental system

  • Detailed reporting of experimental conditions to facilitate reproduction

Reconciliation of Methylation Data:
As highlighted in the hepatocellular carcinoma studies, TNFRSF12A promoter methylation shows distinct patterns between healthy and diseased tissues . To reconcile potentially contradictory methylation data:

• Site-specific methylation analysis:

  • Map specific CpG sites affected in different contexts

  • Correlate site-specific methylation with expression changes

  • Consider regional versus single CpG effects

• Integrative analysis:

  • Combine methylation data with histone modification patterns

  • Assess chromatin accessibility in different cell types

  • Evaluate transcription factor binding sites affected by methylation

• Functional validation:

  • Site-directed mutagenesis of specific CpG sites

  • Reporter assays to directly test promoter activity

  • CRISPR-based epigenetic editing to manipulate methylation status

When faced with contradictory data, researchers should consider that TNFRSF12A may have genuinely different functions in different cellular contexts, reflecting its evolved role in complex regulatory networks rather than a simple binary function.

What quality control parameters are essential when working with recombinant TNFRSF12A proteins?

Ensuring consistent quality of recombinant TNFRSF12A is critical for reliable experimental outcomes. The following comprehensive quality control framework is recommended:

Structural Integrity Assessment:

• SDS-PAGE analysis:

  • Purity assessment (>95% recommended for most applications)

  • Molecular weight verification (5.6 kDa for E.coli-produced extracellular domain)

  • Aggregation state evaluation under reducing and non-reducing conditions

• Mass spectrometry characterization:

  • Sequence confirmation through peptide mapping

  • Post-translational modification analysis

  • Disulfide bond mapping for proper folding assessment

• Secondary/tertiary structure analysis:

  • Circular dichroism spectroscopy

  • Thermal stability testing

  • Limited proteolysis to assess folded domains

Functional Quality Controls:

• Ligand binding assays:

  • ELISA-based binding assays with recombinant TWEAK

  • Surface plasmon resonance for binding kinetics determination

  • Competition assays with known TWEAK antibodies

• Cellular activity testing:

  • Induction of established TNFRSF12A-dependent signaling pathways

  • Comparison to commercially validated standards

  • Batch-to-batch consistency verification

Stability Evaluation:

• Accelerated stability testing:

  • Temperature sensitivity analysis

  • Freeze-thaw stability (limit to <3 cycles)

  • Long-term storage testing at recommended conditions (-18°C, desiccated)

• Formulation optimization:

  • Solubility testing in different buffers (PBS, pH 7.4 recommended)

  • Excipient screening for stability enhancement

  • Concentration-dependent aggregation assessment

Production Process Controls:

• Endotoxin testing:

  • Limulus Amebocyte Lysate (LAL) assay for E. coli-produced protein

  • Specification: <1.0 EU/mg protein for most research applications

• Host cell protein analysis:

  • ELISA or mass spectrometry-based detection

  • Western blot with anti-host cell protein antibodies

• Production documentation:

  • Detailed batch records

  • Cell growth and induction parameters

  • Purification chromatograms and yields

For TNFRSF12A produced in Sf9 cells, additional quality controls should include glycosylation pattern analysis and baculovirus clearance verification. Implementing this comprehensive quality control framework ensures that experimental variations can be attributed to biological factors rather than protein quality inconsistencies.

What emerging technologies could advance our understanding of TNFRSF12A biology?

Several cutting-edge technologies offer promising avenues for deepening our understanding of TNFRSF12A biology:

Advanced Structural Biology Approaches:

• Cryo-electron microscopy:

  • Determination of the full-length TNFRSF12A structure in membrane environment

  • Visualization of TNFRSF12A-TWEAK complexes

  • Analysis of receptor clustering and oligomerization states

• Single-molecule techniques:

  • FRET-based studies of conformational changes upon ligand binding

  • Single-particle tracking to monitor receptor diffusion and clustering

  • Force spectroscopy to measure TWEAK-TNFRSF12A binding strength

Spatial Transcriptomics and Proteomics:

• In situ sequencing and imaging:

  • Spatial mapping of TNFRSF12A expression in tissues

  • Correlation with methylation patterns and disease markers

  • Integration with expression data from The Human Protein Atlas

• Proximity labeling proteomics:

  • TurboID or APEX2 fusions to map the TNFRSF12A proximal proteome

  • Domain-specific interaction partners identification

  • Dynamic changes in the interactome upon TWEAK stimulation

Advanced Genome Editing Technologies:

• Base editing and prime editing:

  • Precise modification of TNFRSF12A regulatory elements

  • Introduction of clinically relevant variants

  • Targeted demethylation of promoter regions to modulate expression

• CRISPR activation/interference:

  • Targeted modulation of TNFRSF12A expression without genetic modification

  • Epigenetic editing of specific CpG sites in the promoter

  • High-throughput screening of regulatory elements

Organoid and Advanced Cell Culture Systems:

• Patient-derived organoids:

  • Testing TNFRSF12A function in disease-relevant contexts

  • Personalized medicine approaches for cancer types showing TNFRSF12A dysregulation

  • Drug screening in physiologically relevant systems

• Microfluidic systems:

  • Cell migration and invasion studies in TNFRSF12A-modulated cells

  • Analysis of angiogenesis and wound healing in controlled gradients

  • High-throughput screening of TNFRSF12A-targeted therapeutics

The integration of these emerging technologies promises to significantly advance our understanding of TNFRSF12A biology and potentially reveal new therapeutic opportunities for diseases associated with this receptor.

How might TNFRSF12A methylation status be leveraged for clinical applications?

The promising findings regarding TNFRSF12A methylation, particularly in hepatocellular carcinoma, suggest several potential clinical applications:

Prognostic Biomarker Development:

• Multi-center validation studies:

  • Expansion of methylation analysis across larger patient cohorts

  • Standardization of methylation detection methods for clinical implementation

  • Integration with existing prognostic models for enhanced prediction accuracy

• Liquid biopsy applications:

  • Detection of TNFRSF12A methylation in circulating tumor DNA

  • Longitudinal monitoring of methylation changes during treatment

  • Early detection of disease recurrence based on methylation patterns

Therapeutic Target Identification:

• Methylation-based patient stratification:

  • Selection of patients most likely to benefit from specific therapies

  • Identification of synergistic treatment combinations based on methylation status

  • Development of companion diagnostics for TNFRSF12A-targeted therapies

• Epigenetic therapy approaches:

  • Evaluation of demethylating agents in hypermethylated contexts

  • Development of targeted methylation modifiers for TNFRSF12A

  • Combination strategies with existing treatments

Clinical Implementation Framework:

• Diagnostic assay development:

  • PCR-based methylation detection kits for clinical laboratories

  • Quality control standards and reference materials

  • Integration into existing molecular pathology workflows

• Clinical decision support systems:

  • Algorithms incorporating TNFRSF12A methylation with other clinical parameters

  • Risk stratification tools for patient management

  • Treatment decision flowcharts based on methylation status