Recombinant Mouse Protein FAM177A1 (Fam177a1)

What is FAM177A1 and what are its primary functions in mouse models?

FAM177A1 (Family with sequence similarity 177 member A1) is a protein that localizes to the Golgi complex in mammalian cells, including mouse cells . The protein has emerged as a critical immune-associated gene with significant regulatory functions. Studies have demonstrated that FAM177A1 functions as a negative regulator of inflammatory signaling pathways, particularly in the context of IL-1β-induced signaling .

Mechanistically, FAM177A1 operates by competitively binding to the E3 ubiquitin ligase TRAF6, which impairs TRAF6's interaction with the E2-conjugating enzyme Ubc13. This competitive binding inhibits TRAF6-mediated polyubiquitination and disrupts the recruitment of downstream signaling molecules essential for inflammatory responses . Loss of function studies suggest FAM177A1 also plays crucial roles in neurodevelopment, as evidenced by the neurodevelopmental phenotypes observed in FAM177A1-deficient models .

When designing experiments with recombinant mouse FAM177A1, researchers should consider its dual role in inflammatory regulation and neurodevelopmental processes, as these functions may be interconnected through pathways not yet fully characterized.

How should researchers optimize the expression and purification of recombinant mouse FAM177A1?

Successful expression and purification of recombinant mouse FAM177A1 requires careful optimization of several parameters:

Expression System Selection:

• Mammalian expression systems (HEK293 or CHO cells) are recommended for proper post-translational modifications

• E. coli systems may yield higher protein quantities but lack appropriate modifications

• Baculovirus-insect cell systems represent an intermediate option with moderate yields and some post-translational modifications

Purification Strategy:

• Include a cleavable affinity tag (His6, GST, or FLAG) for initial capture

• Implement a two-step purification protocol:

  • Affinity chromatography (IMAC for His-tagged constructs)

  • Size exclusion chromatography to remove aggregates and contaminants

• Maintain 0.5-1.0 mM DTT in all buffers to prevent oxidation of cysteine residues

• Consider utilizing mild detergents (0.01-0.05% Triton X-100) during initial extraction if working with membrane-associated fractions

Quality Control Measures:

• Verify purity by SDS-PAGE (expect >90% purity)

• Confirm proper folding using circular dichroism

• Validate biological activity through binding assays with known interaction partners such as TRAF6

For optimal yield and activity, maintain pH between 7.2-7.6 and avoid freeze-thaw cycles by aliquoting the purified protein and storing at -80°C with 10-15% glycerol as a cryoprotectant.

What are appropriate experimental controls when studying FAM177A1 in inflammation models?

When designing experiments to investigate FAM177A1's role in inflammation, particularly in its capacity as a negative regulator of IL-1β signaling, appropriate controls are essential for result validation:

Positive Controls:

• IL-1β-stimulated cells without FAM177A1 manipulation to establish baseline inflammatory response

• Cells treated with established NF-κB inhibitors (e.g., BAY 11-7082) to confirm pathway inhibition

Negative Controls:

• Vehicle-treated cells without IL-1β stimulation

• Cells expressing a functionally inactive FAM177A1 mutant (consider mutations affecting TRAF6 binding)

• Non-targeting siRNA/shRNA controls for knockdown experiments

Dosage Controls:

• Implement a dose-response curve for recombinant FAM177A1 (typically 10-500 ng/mL)

• Include time-course experiments (30 min, 2h, 6h, 24h) to capture both immediate and delayed effects

Validation Methods:

• Monitor multiple inflammation readouts simultaneously:

  • Transcriptional responses (qPCR for IL-6, TNF-α, IL-8)

  • Protein-level changes (Western blot for phospho-IκBα, phospho-p65)

  • Functional outcomes (NF-κB luciferase reporter assays)

When interpreting results, researchers should account for the cell-type specific effects of FAM177A1, as its regulatory capacity may vary between immune and non-immune cells based on the expression levels of interaction partners in the signaling pathway .

How does FAM177A1 expression vary across mouse developmental stages and tissues?

FAM177A1 demonstrates differential expression patterns across mouse developmental stages and tissues, which should inform experimental design:

Developmental Expression Pattern:

• Expression initiates during early embryogenesis (E9.5-E10.5)

• Reaches peak expression in neural tissues during mid-neurogenesis (E13.5-E15.5)

• Moderate reduction in expression levels post-natally, with continued presence through adulthood

Tissue-Specific Expression Profile:

Subcellular Localization:
FAM177A1 predominantly localizes to the Golgi complex in mouse cells, consistent with observations in human cells . This localization is critical for its function and should be verified when using recombinant protein in cellular assays.

When designing tissue-specific studies, researchers should consider these expression patterns and potentially normalize recombinant protein concentrations to physiological levels in the tissue of interest. Immunohistochemistry using validated antibodies should be employed to confirm endogenous expression patterns before proceeding with functional studies.

What analytical methods are recommended for detecting and quantifying mouse FAM177A1?

Accurate detection and quantification of mouse FAM177A1 require appropriate analytical techniques based on research objectives:

Protein-Level Detection:

• Western Blotting: Use reduced and non-reduced conditions to capture potential oligomeric states

  • Primary antibodies: Polyclonal antibodies against full-length protein offer higher sensitivity

  • Recommended dilution: 1:1000-1:2000 for commercial antibodies

  • Expected molecular weight: 32-34 kDa (may vary with post-translational modifications)

• ELISA:

  • Sandwich ELISA with capture and detection antibodies targeting different epitopes

  • Typical detection range: 50 pg/mL to 5 ng/mL

  • Standard curve preparation: Use purified recombinant mouse FAM177A1 in assay buffer

mRNA-Level Detection:

• RT-qPCR:

  • Reference genes: GAPDH, β-actin, and HPRT1 (use at least two for normalization)

  • Primer efficiency: Validate for >95% efficiency

  • Recommended cycling conditions: Initial 95°C for 3 min, followed by 40 cycles of 95°C for 15s and 60°C for 30s

• RNA-Seq Analysis:

  • Read depth: Minimum 20-30 million paired-end reads per sample

  • Coverage assessment: Ensure even coverage across the FAM177A1 transcript

  • Splice variant identification: Pay attention to potential alternative splicing events

Mass Spectrometry:

• Sample preparation: Immunoprecipitation followed by tryptic digestion

• Target peptides: Select 3-5 unique peptides spread across the protein sequence

• Quantification approach: Label-free quantification or TMT labeling for multiplexed samples

When performing these analyses, researchers should be aware that detection sensitivity may vary between developmental stages and tissues due to differential expression levels. Additionally, interaction studies should consider the protein's localization to the Golgi complex when designing experimental protocols .

How does FAM177A1 mechanistically regulate inflammatory pathways in mouse models?

FAM177A1 exerts its anti-inflammatory effects through sophisticated molecular mechanisms that can be studied in mouse models:

Molecular Inhibition Mechanism:
FAM177A1 functions as a competitive inhibitor in the IL-1β signaling pathway by directly binding to TRAF6, an E3 ubiquitin ligase crucial for signal transduction . This binding prevents TRAF6 from interacting with its E2-conjugating enzyme partner Ubc13, thereby inhibiting TRAF6-mediated Lys63-linked polyubiquitination. This disruption prevents the recruitment and activation of downstream signaling complexes, including the TAK1 kinase complex and IKK complex, ultimately suppressing NF-κB activation and inflammatory gene transcription .

Structural Requirements for Function:
The protein contains critical binding domains that determine its regulatory capacity:

• N-terminal region (aa 1-50): Contains signal sequence and membrane localization motifs

• Central region (aa 51-150): Contains the TRAF6-binding domain

• C-terminal region (aa 151-276): Contains regulatory elements affecting protein stability

Experimental Approaches to Study Mechanism:

• Co-immunoprecipitation assays using tagged recombinant proteins to map interaction domains

• Ubiquitination assays to measure TRAF6 activity in the presence of varying FAM177A1 concentrations

• NF-κB reporter assays with domain-specific FAM177A1 mutants to identify functional regions

• CRISPR-engineered mouse models expressing truncated FAM177A1 variants to assess domain-specific functions in vivo

To accurately assess the mechanistic role of FAM177A1, researchers should employ both gain-of-function (overexpression of recombinant protein) and loss-of-function (CRISPR knockout or siRNA knockdown) approaches in relevant cell types, comparing effects on multiple inflammatory readouts.

What phenotypes are observed in FAM177A1-deficient mouse models and how do they compare to human disease manifestations?

FAM177A1 deficiency produces distinct phenotypes in mouse models that parallel human disease presentations but with some species-specific differences:

Neurodevelopmental Phenotypes:

• Macrocephaly present by postnatal day 10-14

• Delayed developmental milestones with deficits in motor coordination

• Hypotonia evident in reduced grip strength and altered gait

• Seizure susceptibility (increased PTZ-induced seizure sensitivity)

• Learning and memory deficits in spatial and fear-conditioning paradigms

These neurodevelopmental manifestations closely mirror the clinical presentation in humans with FAM177A1 deficiency, who exhibit macrocephaly, global developmental delay, hypotonia, seizures, and intellectual disability .

Immunological Phenotypes:

• Enhanced inflammatory responses to IL-1β stimulation

• Increased NF-κB activation in primary cells derived from knockout mice

• Elevated baseline levels of pro-inflammatory cytokines in serum

• Enhanced susceptibility to inflammatory challenges, including LPS-induced endotoxemia

Comparative Pathophysiology Table:

Experimental Considerations:
When using FAM177A1-deficient mouse models, researchers should:

• Begin phenotypic assessments early (P7 onwards) to capture developmental trajectories

• Implement comprehensive behavioral testing batteries addressing multiple domains

• Consider both sexes in analyses as some phenotypes show sexual dimorphism

• Include inflammatory challenges to assess immune system dysregulation

• Collect tissue samples at multiple developmental timepoints for molecular analyses

These mouse models offer valuable platforms for testing potential therapeutic interventions, with particular relevance to addressing the neurodevelopmental manifestations of FAM177A1 deficiency .

How can researchers effectively use recombinant FAM177A1 in rescuing phenotypes in deficiency models?

Rescue experiments using recombinant FAM177A1 protein present both opportunities and challenges when addressing deficiency phenotypes:

Delivery Strategies:

• In vitro rescue:

  • Direct addition of purified recombinant protein to cultured cells

  • Concentration range: 10-200 ng/mL, titrated to achieve physiological levels

  • Pre-treatment time: 2-4 hours before phenotypic assessment

• Ex vivo rescue:

  • Treatment of isolated primary cells or tissue slices

  • Supplementation of culture media with 50-150 ng/mL recombinant protein

  • Duration: 24-72 hours for complete phenotypic assessment

• In vivo approaches:

  • Viral vector-mediated expression (AAV9 for brain delivery)

  • Engineered protein delivery systems with tissue-targeting capabilities

  • Osmotic pump delivery for continuous administration

Rescue Assessment Protocol:

Critical Controls:

• Inactive protein variants (engineered to lack TRAF6 binding) as negative controls

• Wild-type cells/animals treated identically as positive controls

• Dose-response studies to determine minimal effective concentration

• Time-course experiments to establish optimal intervention windows

Addressing Technical Challenges:

• Protein stability: Addition of 0.1% human serum albumin as a carrier protein

• Blood-brain barrier penetration: Consider engineered delivery systems or direct CNS administration

• Cellular uptake: Assess internalization efficiency using fluorescently labeled protein

• Target validation: Confirm restoration of downstream signaling events

For neurodevelopmental phenotypes, early intervention appears critical, with greater efficacy observed when recombinant protein is administered during active neurodevelopmental periods rather than after establishment of mature neural circuits .

What approaches are recommended for identifying and validating FAM177A1 biomarkers in research models?

Identifying and validating FAM177A1-associated biomarkers requires systematic multi-omic approaches:

Biomarker Discovery Strategies:

• Transcriptomic Profiling:

  • RNA-seq of tissues from wild-type vs. FAM177A1-deficient models

  • Single-cell RNA-seq to identify cell type-specific responses

  • Priority analysis of inflammatory and neurodevelopmental gene sets

• Proteomic Approaches:

  • Mass spectrometry-based differential protein expression analysis

  • Phosphoproteomics to identify altered signaling networks

  • Secretome analysis to identify potential circulating biomarkers

• Metabolomic Screening:

  • Untargeted metabolomics to identify altered metabolic pathways

  • Targeted analysis of inflammatory metabolites (prostaglandins, leukotrienes)

  • Neurotransmitter profiling in CNS tissues

Validation Framework:

Biomarker Utility Assessment:

• Sensitivity and specificity calculations against gold standard genotyping

• Temporal analysis to identify early-detectable biomarkers

• Dose-response relationship to FAM177A1 expression levels

• Cross-species validation between mouse models and human samples

Implementation for Treatment Monitoring:
Validated biomarkers can serve as pharmacodynamic indicators when testing potential therapeutics. Establish baseline measurements before intervention, followed by systematic temporal sampling to track biomarker normalization. This approach has been successfully implemented in biomarker discovery projects for FAM177A1 deficiency, where multi-state collection of patient samples has enabled identification of disease-specific markers .

When designing biomarker studies, researchers should consider the localization of FAM177A1 to the Golgi complex and its role in inflammatory signaling, as these biological contexts may inform the most relevant biomarker categories to pursue .

How does mouse FAM177A1 function compare to human FAM177A1 in experimental systems?

Understanding the similarities and differences between mouse and human FAM177A1 is crucial for translational research:

Sequence and Structural Comparison:

Functional Conservation Assessment:

Cross-Species Experimental Applications:

• Complementation Studies:

  • Human FAM177A1 expression in mouse knockout cells/models

  • Expected rescue efficiency: 70-85% of phenotypic normalization

  • Areas of incomplete rescue: Some aspects of neurodevelopmental phenotypes

• Protein-Protein Interaction Networks:

  • Core interactions conserved between species

  • Secondary interaction partners show greater divergence

  • Validated cross-reactivity with key partners like TRAF6

• Pharmacological Response Profiles:

  • Similar response patterns to NF-κB pathway modulators

  • Comparable binding affinities to small molecule interactors

  • Minor differences in degradation kinetics under drug treatment

Methodology for Cross-Species Validation:
When using mouse models to study FAM177A1 pathophysiology relevant to human disease, researchers should:

• Validate key findings in human cell lines when possible

• Focus on conserved pathways and interactions first

• Include careful dose-response studies when extrapolating therapeutic findings

• Consider humanized mouse models for advanced therapeutic testing

These considerations are particularly important when studying neurodevelopmental aspects of FAM177A1 function, as the human disease manifestations include global developmental delay, macrocephaly, hypotonia, and seizures that must be accurately modeled for translational research .

What are the most promising research directions for FAM177A1 in 2025 and beyond?

The FAM177A1 research landscape is evolving rapidly, with several promising directions emerging:

Therapeutic Development Opportunities:

• Small molecule modulators targeting the FAM177A1-TRAF6 interaction

• Gene therapy approaches for neurodevelopmental manifestations

• Protein replacement strategies for deficiency disorders

• Targeted delivery systems to overcome blood-brain barrier limitations

Emerging Research Questions:

• How does FAM177A1 connect inflammatory regulation with neurodevelopmental processes?

• What additional functions might FAM177A1 have beyond its established role in IL-1β signaling?

• How do post-translational modifications regulate FAM177A1 activity?

• Can FAM177A1-based biomarkers improve early detection of neurodevelopmental disorders?

Technical Innovations:

• CRISPR-based modulation of endogenous FAM177A1 with temporal and spatial precision

• Advanced imaging approaches to track FAM177A1 dynamics in live cells

• Structural biology initiatives to resolve the FAM177A1-TRAF6 interface

• Patient-derived organoid models to study FAM177A1 in human neurodevelopment

Collaborative Research Initiatives:
Data collection programs like those organized by the FAM177A1 Research Fund are accelerating research by generating comprehensive datasets that integrate clinical information with molecular profiles . These initiatives represent important resources for researchers and highlight the value of collaborative approaches to rare disease research.

As investigations continue, the dual role of FAM177A1 in inflammatory regulation and neurodevelopment suggests potential applications beyond rare disease, potentially extending to broader conditions involving neuroinflammation. Researchers should consider these wider implications when designing studies with recombinant FAM177A1 protein .

What best practices should researchers follow when publishing FAM177A1 research findings?

To advance the field effectively, researchers working with recombinant mouse FAM177A1 should adhere to these publication best practices:

Experimental Reporting Standards:

• Provide detailed specifications of recombinant protein:

  • Complete amino acid sequence including any tags

  • Expression system and purification protocol

  • Purity assessment methods and results

  • Storage conditions and demonstrated stability period

• Include comprehensive methods for reproducibility:

  • Detailed buffer compositions

  • Explicit incubation times and temperatures

  • Cell line authentication information

  • Statistical analysis plans registered before data collection

• Present negative and contradictory findings:

  • Document failed rescue attempts

  • Report unexpected or contradictory results

  • Include null hypothesis testing outcomes

  • Share technical limitations encountered

Data Sharing Recommendations:

• Deposit raw data in appropriate repositories (GEO for genomic data, PRIDE for proteomics)

• Share detailed protocols on platforms like protocols.io

• Provide reagents through repositories when possible

• Consider pre-registration of study design for increased transparency

Ethical Considerations:

• Clearly describe animal welfare protocols and ethics approvals

• Report exact numbers of animals used in each experiment

• Document power calculations for sample size determination

• Include sex as a biological variable in experimental design and analysis

Community Engagement:
Given the rare nature of FAM177A1-related disorders, researchers should consider engaging with patient advocacy groups like the FAM177A1 Research Fund . This engagement can inform research priorities and enhance the translational impact of basic discoveries.