Recombinant Human Protein FAM176A (FAM176A)

What is FAM176A and what are its primary functions in cellular processes?

FAM176A (family with sequence similarity 176 member A), also known as TMEM166 (transmembrane protein 166), is a novel molecule involved in programmed cell death. It functions as a regulator of both autophagy and apoptosis, essentially facilitating two distinct cellular death mechanisms. The protein is expressed broadly in most human normal tissues and organs in a cell- and tissue-type-specific manner .

Functionally, FAM176A appears to mediate cell death through multiple pathways:

• Induction of autophagy (demonstrated by autophagosome formation)

• Activation of apoptotic cascades via caspase-9 and caspase-3, but not caspase-8

• Cell cycle regulation, particularly G2/M phase arrest

• Interference with DNA damage repair processes

These multiple functions position FAM176A as a potential tumor suppressor, with its decreased expression documented in various human cancers including gastric, esophageal, adrenal cortical carcinoma, pituitary adenoma, pancreatic islet cell tumors, and lung cancers .

How is FAM176A expression regulated in normal versus cancerous tissues?

Research has revealed:

• Significant downregulation in gastric cancer, esophagus cancer, adrenal cortical carcinoma, pituitary adenoma samples, pancreatic islet cell tumor, and parathyroid adenoma

• Absence of expression in certain lung cancer cell lines (H1299 and H520), while maintained in others (A549)

• The restoration of FAM176A expression in cancer cells typically induces strong anti-tumor effects through multiple cell death pathways

This differential expression pattern suggests epigenetic or transcriptional regulation mechanisms that are disrupted during carcinogenesis, making FAM176A restoration a potential therapeutic strategy.

What are the key considerations when designing experiments to study FAM176A-induced cell death?

When designing experiments to investigate FAM176A-induced cell death, researchers should incorporate multiple approaches to capture the dual autophagy and apoptosis mechanisms. Based on published methodologies, a comprehensive experimental design should include:

• Cell line selection: Choose appropriate cell lines that either lack endogenous FAM176A expression (like H1299 cells) for overexpression studies or lines with normal expression for knockdown experiments

• Delivery method determination: Consider adenovirus vectors for efficient transfection, as demonstrated in key studies (e.g., Ad5-FAM176A) with appropriate controls (Ad5-Null)

• Time-course and dose-dependency analysis: Design experiments with multiple MOI (multiplicity of infection) values (100, 200, 400, 800) and time points (24h, 48h, 72h) to fully characterize the dose and time-dependent effects

• Multiple cell death assessments:

  • Autophagy markers (LC3-I to LC3-II conversion, autophagosome formation)

  • Apoptosis assays (Annexin V staining, PI exclusion)

  • Caspase activation (measuring caspase-3, caspase-9, and caspase-8 activities)

  • Cell cycle analysis (flow cytometry)

  • Morphological changes (microscopy)

• Pathway inhibitor studies: Include specific inhibitors (e.g., z-VAD-FMK for caspase inhibition) to determine pathway dependencies

• Biological replicates: Ensure at least three independent experiments for statistical validation

This multi-parameter approach allows for comprehensive characterization of the complex cell death mechanisms activated by FAM176A.

How should researchers approach experimental controls when working with recombinant FAM176A?

Proper experimental controls are critical for generating reliable and interpretable data when studying FAM176A. Based on published methodologies, researchers should implement the following control strategies:

• Vector controls: When using adenovirus vectors for FAM176A expression, include the same vector without the FAM176A gene (e.g., Ad5-Null compared to Ad5-FAM176A) to account for any vector-specific effects

• Cell line controls:

  • Include both FAM176A-expressing and non-expressing cell lines (e.g., A549 versus H1299) for comparative analysis

  • When possible, use isogenic cell lines that differ only in FAM176A expression

• Treatment controls for mechanistic studies:

  • Include autophagy inhibitors (e.g., 3-methyladenine)

  • Use caspase inhibitors (e.g., z-VAD-FMK) to differentiate apoptotic from non-apoptotic death

  • Apply pathway-specific inhibitors to isolate particular signaling cascades

• Dose and time controls:

  • Include multiple concentration points to establish dose-response curves

  • Incorporate various time points to characterize temporal dynamics of responses

• Technical controls:

  • For protein expression analysis, include housekeeping genes/proteins (β-actin, GAPDH)

  • For PCR, include no-template controls and reference genes (like GUSB)

  • For recombinant protein studies, include tag-only proteins to control for tag effects

By implementing these comprehensive controls, researchers can distinguish FAM176A-specific effects from experimental artifacts and establish causality in the observed phenomena.

What methodologies are most effective for studying FAM176A's role in both autophagy and apoptosis simultaneously?

Studying the dual role of FAM176A in both autophagy and apoptosis requires sophisticated methodological approaches. Based on published research, the most effective integrated methodology includes:

• Temporal analysis with dual fluorescent reporters:

  • GFP-LC3 puncta formation to track autophagosome formation

  • RFP-Annexin V for real-time apoptosis detection

  • Time-lapse confocal microscopy to determine the sequence and potential relationship between these processes

• Biochemical pathway dissection:

  • Western blotting for autophagy markers (LC3-I/II conversion, p62/SQSTM1 degradation)

  • Caspase activity assays (particularly caspase-3 and caspase-9)

  • PARP cleavage detection

  • Mitochondrial membrane potential analysis

• Genetic manipulation approaches:

  • Knockdown of key autophagy genes (ATG5, ATG7, Beclin-1) to assess dependence of cell death on autophagy

  • Caspase inhibition (both chemical and genetic) to assess apoptosis contribution

  • Domain-specific mutations in FAM176A to map functional regions responsible for each process

• Flow cytometry-based multiplexed analysis:

  • Simultaneous detection of autophagy (using autophagy dyes) and apoptosis markers (Annexin V/PI)

  • Cell cycle analysis to correlate with G2/M arrest

• Electron microscopy:

  • Ultrastructural analysis to distinguish and quantify autophagic vacuoles and apoptotic bodies in the same cells

This integrated approach enables researchers to determine the relationship between FAM176A-induced autophagy and apoptosis—whether they occur sequentially, in parallel, or interdependently.

How can researchers investigate the potential role of FAM176A in neurodegenerative diseases?

Research has implicated FAM176A in neurodegenerative disease processes through its genetic association with CSF progranulin (PGRN) levels. To investigate this connection, researchers should consider these methodological approaches:

• Genetic association studies:

  • Genotype the rs708384 SNP in the FAM171A2 gene, which has been associated with CSF PGRN levels and increased risk of Alzheimer's disease, Parkinson's disease, and frontotemporal dementia

  • Perform linear regression models accounting for age, sex, educational level, and APOE ε4 status to analyze associations

• In vitro functional studies:

  • Develop cellular models with rs708384 mutations using CRISPR/Cas9 genome editing

  • Measure the effects on FAM171A2 expression and PGRN production

  • Investigate cellular consequences in neuron-like cells and microglia

• Protein-protein interaction studies:

  • Investigate potential interactions between FAM176A and neurodegenerative disease-related proteins

  • Use co-immunoprecipitation, proximity ligation assays, or FRET approaches

  • Focus on proteins involved in autophagy pathways that are relevant to neurodegeneration

• Biomarker development:

  • Assess correlations between FAM176A expression/activity and established neurodegenerative disease biomarkers

  • Develop assays to measure FAM176A in patient samples (CSF, blood)

• Animal model studies:

  • Generate transgenic mouse models with altered FAM176A expression

  • Assess cognitive and motor function

  • Analyze tissue for pathological hallmarks of neurodegenerative diseases

  • Measure PGRN levels in brain tissue and CSF

This research direction is particularly promising given the identification of FAM171A2 as a key regulator of progranulin expression, which has established roles in neurodegenerative pathologies.

What are the optimal storage and handling conditions for recombinant FAM176A protein?

Proper storage and handling of recombinant FAM176A protein is critical for maintaining its activity and stability. Based on product information provided by suppliers, researchers should follow these guidelines:

Storage conditions:

• Store lyophilized protein at -20°C to -80°C for up to 12 months

• For reconstituted protein:

  • Short-term storage (1-2 weeks): 2-8°C

  • Long-term storage (up to 3 months): -20°C to -80°C in aliquots with added glycerol (1:1 volume ratio)

Reconstitution protocol:

• Reconstitute at 0.25 µg/μl in 200 μl sterile water for short-term use

• For long-term storage, add an equal volume of glycerol after reconstitution

• Centrifuge vial before opening to collect all material

• When reconstituting, gently pipet and wash down the sides of the vial to ensure full recovery

Handling precautions:

• Avoid repeated freeze-thaw cycles which significantly reduce activity

• Prepare working aliquots after first thaw to minimize freeze-thaw damage

• Protect from light during storage and handling

• Maintain sterile conditions during reconstitution and aliquoting

Formulation considerations:

• Typical commercial formulations include 5% trehalose and 5% mannitol as protectants

• Buffer systems may include PBS (58mM Na₂HPO₄, 17mM NaH₂PO₄, 68mM NaCl, pH 8.0)

• Some preparations may include GSH (100mM) in the elution buffer

Following these guidelines will help ensure maximum stability and activity of recombinant FAM176A for experimental applications.

What methods are recommended for validating the activity of recombinant FAM176A protein in experimental systems?

Validating the activity of recombinant FAM176A protein is essential before using it in experimental systems. Based on published methodologies, researchers should employ a multi-faceted validation approach:

• Structural and purity validation:

  • SDS-PAGE with Coomassie Brilliant Blue staining to confirm molecular weight (expected 40-60 kDa) and purity (>85%)

  • Western blot with anti-FAM176A or anti-tag antibodies

  • Mass spectrometry to confirm protein identity and integrity

• Functional activity assays:

  • Cell-based assays measuring cell viability after treatment (MTT assay)

  • Assessment of autophagy induction (LC3-I to LC3-II conversion)

  • Apoptosis assays (Annexin V/PI staining)

  • Caspase activation assays (particularly caspase-3 and caspase-9)

• Dose-response validation:

  • Test multiple concentrations to establish dose-response relationships

  • Compare effects to literature reports on FAM176A activity

  • Document time-dependency of observed effects

• Cell line selection for validation:

  • Primary validation in H1299 cells (well-established model)

  • Secondary validation in additional relevant cell lines

  • Comparison to adenovirus-mediated FAM176A expression effects

• Confirmation of protein-protein interactions:

  • Co-immunoprecipitation assays to confirm known interaction partners

  • Proximity ligation assays to validate interactions in cellular contexts

The validation should be comparative, ideally using previously characterized FAM176A preparations or adenovirus-expressed FAM176A as reference standards to ensure consistency across experiments.

How should researchers interpret conflicting results in FAM176A functional studies?

When faced with conflicting results in FAM176A studies, researchers should implement a systematic analytical approach:

• Context-dependent effects analysis:

  • Cell type differences: FAM176A may produce different effects in different cell types due to varying expression of interaction partners or downstream effectors. Compare results between cell types systematically (e.g., H1299 vs. A549)

  • Expression level variations: Different expression levels may trigger different cellular responses. Quantify protein levels across experiments and correlate with observed effects

• Methodological differences assessment:

  • Delivery method impact: Compare results between adenoviral vector-mediated expression and recombinant protein treatment

  • Assay sensitivity differences: Some assays may detect early events while others capture late-stage processes

• Temporal dynamics consideration:

  • Time-course analysis: FAM176A may induce autophagy first, followed by apoptosis. Establish comprehensive time-course experiments with multiple readouts

  • Cell cycle effects: G2/M arrest may precede or follow other cellular responses

• Statistical approach to contradictions:

  • Meta-analysis of multiple experiments

  • Increased biological replicates (n≥5) to improve statistical power

  • Use of multiple statistical tests to verify results

• Technical validation:

  • Protein tag effects: Different tags (His, GST) may affect protein function

  • Endotoxin contamination: Verify endotoxin levels are below threshold that could influence results

  • Batch variation: Compare results across multiple batches of recombinant protein

When publishing, transparently report contradictory results and provide possible explanations based on the above analysis framework.

What statistical approaches are most appropriate for analyzing FAM176A experimental data?

When analyzing experimental data related to FAM176A studies, researchers should employ appropriate statistical methods based on the type of experiments conducted:

• For cell viability and proliferation studies:

  • Two-way ANOVA for analyzing dose and time effects simultaneously

  • Post-hoc tests (e.g., Tukey's or Bonferroni) for multiple comparisons

  • Graphical representation showing both dose-response and time-course data with error bars representing standard deviation

• For apoptosis and autophagy quantification:

  • Student's t-test for comparing two conditions (e.g., Ad5-FAM176A vs. Ad5-Null)

  • One-way ANOVA for comparing multiple conditions

  • Present data as mean ± SD from at least three independent experiments

  • Include p-value significance indicators (*P < 0.05, **P < 0.001, ***P < 0.0001)

• For gene expression analysis:

  • Normalization to housekeeping genes for RT-qPCR data

  • ΔΔCt method for relative quantification

  • Non-parametric tests if data does not follow normal distribution

• For biomarker potential analysis:

  • ROC curve analysis to determine diagnostic value

  • AUC (Area Under Curve) calculation to assess accuracy

  • Sensitivity and specificity calculations at optimal cut-off values

  • Youden index determination

Example of biomarker statistical analysis for reference:

*Example shown is for EVA1A, another cell death regulator protein. Similar statistical approaches would apply to FAM176A analysis .

• For genetic association studies:

  • Linear regression models adjusting for covariates (age, sex, education)

  • Calculation of hazard ratios with 95% confidence intervals

  • Permutation-based empiric corrections for multiple testing

What approaches should researchers consider for exploring FAM176A's potential as a cancer therapeutic target?

Based on FAM176A's demonstrated anti-tumor activities, researchers exploring its therapeutic potential should consider these methodological approaches:

• Advanced delivery system development:

  • Optimize adenoviral vectors beyond Ad5 for improved targeting and reduced immunogenicity

  • Develop non-viral delivery methods such as lipid nanoparticles or cell-penetrating peptide conjugates

  • Design tissue-specific promoters to restrict FAM176A expression to cancer cells

• Combination therapy investigation:

  • Test FAM176A expression in combination with:

    • Conventional chemotherapeutics (especially those inducing G2/M arrest)

    • Radiation therapy (potentially enhancing DNA damage effects)

    • Immunotherapy (exploring potential immunogenic cell death induction)

  • Use factorial experimental designs to identify synergistic combinations

• FAM176A domain engineering:

  • Identify the minimal functional domains required for anti-cancer effects

  • Design truncated versions with enhanced stability or activity

  • Create chimeric proteins combining FAM176A domains with cancer-targeting modules

• In vivo efficacy and safety studies:

  • Develop xenograft models using FAM176A-negative cancer cell lines

  • Test both local and systemic delivery methods

  • Conduct comprehensive toxicology assessment in multiple tissues

  • Evaluate immune response to FAM176A-based therapeutics

• Biomarker development for patient stratification:

  • Identify molecular signatures predicting sensitivity to FAM176A restoration

  • Develop companion diagnostics to measure endogenous FAM176A levels

  • Correlate FAM176A expression with response to conventional therapies

This multi-faceted approach acknowledges both the promising anti-cancer activities of FAM176A and the challenges of developing it into a viable therapeutic strategy.

How can researchers effectively design studies to explore the molecular interactions between FAM176A and other cell death regulators?

To elucidate the complex interactions between FAM176A and other cell death regulatory proteins, researchers should implement these advanced methodological approaches:

• Comprehensive protein-protein interaction mapping:

  • Unbiased approaches:

    • Proximity-dependent biotin labeling (BioID or APEX)

    • Co-immunoprecipitation followed by mass spectrometry

    • Yeast two-hybrid screening

  • Targeted approaches:

    • Co-immunoprecipitation with known autophagy/apoptosis regulators

    • FRET or BRET assays for direct interaction detection

    • Mammalian two-hybrid assays

• Domain-specific interaction studies:

  • Generate truncation mutants to map interaction domains

  • Create point mutations in conserved residues

  • Use peptide arrays to identify specific binding motifs

  • Employ structural biology approaches (X-ray crystallography, cryo-EM) for interaction interfaces

• Pathway perturbation analysis:

  • Conduct CRISPR/Cas9 knockout or knockdown of potential interaction partners

  • Use small molecule inhibitors of specific pathway components

  • Perform epistasis analysis by manipulating multiple genes simultaneously

  • Employ phosphoproteomics to identify signaling cascades

• Dynamic interaction studies:

  • Use live-cell imaging with fluorescently tagged proteins

  • Implement real-time interaction biosensors

  • Conduct temporal analysis of complex formation following stimulation

  • Apply mathematical modeling to determine interaction kinetics

• Integration with transcriptomics and proteomics:

  • Perform RNA-seq and proteomics in FAM176A-expressing versus control cells

  • Use pathway enrichment analysis to identify affected networks

  • Apply systems biology approaches to model the cell death interactome

  • Validate key nodes through targeted experiments

This systematic approach will help elucidate the position of FAM176A within the broader cell death regulatory network and potentially identify novel therapeutic targets or combination strategies.