Recombinant Human Protein FAM8A1 (FAM8A1)

What is FAM8A1 and what cellular pathways is it involved in?

FAM8A1 (Family with Sequence Similarity 8 Member A1, also named AHCP) is a 413 amino acid protein that plays a significant role in the ubiquitin-dependent Endoplasmic Reticulum-Associated Degradation (ERAD) pathway. It appears to be part of the Hrd1p ubiquitin ligase complex, which is critical for the degradation of misfolded proteins in the endoplasmic reticulum . Protein interaction studies suggest FAM8A1 functions within the endoplasmic reticulum quality control (ERQC) system .

How does FAM8A1 differ from FAM81A?

While similar in nomenclature, FAM8A1 and FAM81A are distinct proteins with different functions. FAM8A1 is associated with the ERAD pathway , whereas FAM81A has been characterized as a component of the postsynaptic density in neurons . FAM81A is selectively expressed in brain tissue and shows a post-natal gradual increase in expression levels that parallels PSD-95, a key synaptic protein . Recent research from Kobe University has identified FAM81A as a crucial protein for postsynaptic density formation .

What protein interactions govern FAM8A1 function?

According to STRING database analysis, FAM8A1 interacts with several proteins involved in ER quality control and degradation pathways, with high confidence scores:

These interactions suggest FAM8A1 functions within a protein complex that identifies and processes misfolded proteins for degradation .

What expression systems are most effective for producing recombinant FAM8A1?

For optimal expression of recombinant human FAM8A1, mammalian expression systems such as HEK-293 cells have proven effective . This approach helps ensure proper folding and post-translational modifications that may be critical for protein function. The recombinant protein can be designed with fusion tags (e.g., His-tag) to facilitate purification while maintaining structural integrity . When designing experiments requiring recombinant FAM8A1, researchers should consider:

• Expression in mammalian cells for proper folding

• Inclusion of purification tags (e.g., His-tag)

• One-step affinity chromatography for purification

• Verification of full-length expression (all 413 amino acids)

How can I validate the functionality of purified recombinant FAM8A1?

Functional validation of recombinant FAM8A1 should focus on its known interactions and biological role. A multi-step approach includes:

• Protein interaction analysis: Co-immunoprecipitation or pull-down assays with known binding partners (OS9, SYVN1, SEL1L)

• In vitro binding assays: Using purified components to verify direct protein-protein interactions

• Cell-based functional assays: Complementation studies in FAM8A1-depleted cells to rescue ERAD function

• Structural integrity verification: Circular dichroism or thermal shift assays to confirm proper folding

Researchers should verify that the recombinant protein reproduces the known interactions with components of the ERAD machinery before using it in more complex experimental systems .

What controls should be included when studying FAM8A1 in ERAD pathways?

When investigating FAM8A1's role in ERAD pathways, include these essential controls:

• Positive controls: Well-characterized ERAD substrates (e.g., misfolded glycoproteins)

• Negative controls: Non-ERAD substrates that should not be affected by FAM8A1 manipulation

• Specificity controls: Rescue experiments with wild-type FAM8A1 following knockdown

• Pathway positioning controls: Parallel manipulation of other ERAD components (OS9, SYVN1) to determine epistatic relationships

• Subcellular localization verification: Confirming proper ER localization of FAM8A1 in experimental systems

These controls help distinguish direct effects of FAM8A1 from indirect consequences or experimental artifacts .

How can researchers study the evolutionary conservation of FAM8A1?

The evolutionary conservation of FAM8A1 can be investigated using both computational and experimental approaches:

• Comparative sequence analysis: Align FAM8A1 sequences from different species to identify conserved domains and motifs

• Functional complementation studies: Test whether FAM8A1 from different species (human, mouse, rat, etc.) can functionally substitute in cellular models

• Expression pattern comparison: Compare tissue expression profiles across species

• Structural conservation analysis: Compare predicted or resolved protein structures

Current research indicates FAM8A1 is conserved across multiple species including human, cynomolgus/rhesus macaque, rat, mouse, feline, canine, bovine, and equine, suggesting evolutionary importance of its function .

What methodologies are appropriate for investigating FAM8A1's role in protein quality control?

To investigate FAM8A1's role in protein quality control, consider these methodological approaches:

• CRISPR/Cas9-mediated knockout or knockdown: To assess the impact of FAM8A1 depletion on ERAD efficiency

• Inducible expression systems: To study gain-of-function effects without adaptation

• Protein interaction mapping: Using proximity labeling (BioID, APEX) to identify the complete FAM8A1 interactome

• Live cell imaging: To monitor dynamics of FAM8A1 localization during ER stress

• Misfolded protein reporter systems: To quantitatively measure ERAD efficiency in the presence/absence of FAM8A1

These approaches help position FAM8A1 within the complex network of protein quality control mechanisms and identify its specific contributions .

How might FAM8A1 function be altered in disease states involving ER stress?

Based on its role in ERAD, FAM8A1 function may be particularly relevant in diseases characterized by ER stress and protein misfolding:

• Neurodegenerative disorders: Conditions like Alzheimer's or Parkinson's disease involve protein aggregation that may be influenced by ERAD efficiency

• Metabolic disorders: Conditions affecting the liver or pancreas where protein secretion and ER stress are prominent

• Cancer: Many cancers exhibit elevated ER stress due to high protein synthesis demands

Research approaches should include:

• Analysis of FAM8A1 expression in disease tissues

• Correlation of FAM8A1 levels with disease progression markers

• Assessment of whether FAM8A1 modulation affects disease phenotypes in model systems

• Identification of disease-associated variants in FAM8A1 that may alter function

What are the challenges in distinguishing between FAM8A1 and similar proteins in experimental systems?

Due to nomenclature similarities with proteins like FAM81A, researchers must be vigilant about specificity:

• Antibody validation: Thoroughly validate antibodies against recombinant standards and knockdown controls

• Primer design for qPCR: Design primers in unique regions to avoid cross-amplification

• Mass spectrometry verification: Use peptide fingerprinting to confirm protein identity

• Expression pattern comparison: FAM8A1 appears to be expressed in multiple tissues, while FAM81A shows brain-specific expression

The case of FAM8A1 vs. FAM81A illustrates a common challenge in protein research where similar nomenclature can lead to confusion in literature and experimental interpretation .

How can researchers address challenges in studying membrane-associated proteins like FAM8A1?

FAM8A1 contains hydrophobic regions suggesting membrane association, which presents specific experimental challenges:

• Protein extraction optimization: Use appropriate detergents for solubilization without denaturation

• Native conformation preservation: Consider native membrane mimetics (nanodiscs, liposomes) for structural studies

• Interaction studies: Employ techniques suitable for membrane protein interactions (e.g., MYTH, FRET)

• Localization studies: Use appropriate fixation methods that preserve membrane structures when performing immunofluorescence

These technical considerations are crucial for accurate characterization of FAM8A1's cellular functions and interactions within membrane-associated complexes .

What approaches can overcome variability in FAM8A1 expression studies?

When studying FAM8A1 expression patterns, researchers encounter several sources of variability:

• Reference gene selection: Carefully validate multiple reference genes for normalization

• Sample preparation consistency: Standardize tissue collection and processing protocols

• Quantification methods: Use absolute quantification with standard curves when possible

• Single-cell techniques: Consider single-cell RNA-seq to address cellular heterogeneity

• Protein half-life considerations: Account for potential post-transcriptional regulation

These approaches help ensure reproducible and reliable expression data that can be compared across studies and experimental conditions .

What are emerging opportunities for therapeutic targeting of FAM8A1?

Given its role in protein quality control, FAM8A1 presents several potential therapeutic opportunities:

• Modulation of ERAD efficiency: Small molecules targeting FAM8A1 could potentially enhance clearance of disease-associated misfolded proteins

• Pathway-specific intervention: Targeting specific FAM8A1 interactions might allow precise modulation of protein degradation

• Biomarker development: FAM8A1 levels or modifications might serve as biomarkers for ER stress states

• Personalized medicine approaches: Genetic variations in FAM8A1 might predict response to therapies targeting protein quality control

Research should focus on developing high-throughput screening assays to identify compounds that modulate FAM8A1 activity or its protein interactions .

How might multi-omics approaches advance understanding of FAM8A1 function?

Integrating multiple omics technologies can provide comprehensive insights into FAM8A1 biology:

• Proteomics + transcriptomics: Correlate FAM8A1 protein levels with gene expression profiles

• Interactomics + structural biology: Map the complete interaction network and structural basis

• Genomics + phenomics: Connect genetic variations with functional outcomes

• Metabolomics integration: Identify metabolic consequences of FAM8A1 dysfunction

These integrated approaches help position FAM8A1 within broader cellular networks and identify unexpected connections to other pathways and functions .

What new technologies might enhance study of FAM8A1 in cellular contexts?

Emerging technologies offer new opportunities for studying FAM8A1 dynamics and functions:

• Cryo-electron tomography: Visualize FAM8A1 in its native cellular environment

• Optogenetic control: Develop light-inducible FAM8A1 variants to control activity with spatiotemporal precision

• Protein engineering approaches: Create biosensors to monitor FAM8A1 activity in real-time

• Organoid systems: Study FAM8A1 function in more physiologically relevant 3D tissue models

• AI-driven protein structure prediction: Generate improved structural models to guide functional studies

These technologies may help overcome current limitations in understanding the dynamic aspects of FAM8A1 function in complex cellular environments .