Recombinant Mouse Interferon-induced transmembrane protein 2 (Ifitm2)

What is Recombinant Mouse IFITM2 and what are its primary functions?

Recombinant Mouse IFITM2 (also known as Dispanin subfamily A member 2c or Interferon-inducible protein 1-8D) is a membrane-associated protein with a molecular mass of approximately 19.4 kDa (predicted) or 19 kDa (measured) . It belongs to the interferon-induced transmembrane protein family, which are interferon-stimulated genes (ISGs) involved in antiviral defense.

The primary functions of IFITM2 include:

• Mediating broad-spectrum antiviral responses against multiple viruses

• Enhancing type I interferon signaling pathways

• Interacting with pattern recognition receptors like MDA5 to promote IFN-β production

• Regulating endocytic processes in specific cell types, particularly in neural development

Recent studies have revealed that IFITM2 creates a positive feed-forward loop with type I IFN, establishing a critical role in enforcing innate immune responses . Furthermore, IFITM2 has been shown to modulate endocytosis in radial glial cells during brain development, indicating its importance extends beyond purely antiviral functions .

How does IFITM2 differ from other IFITM family members?

While IFITM proteins share structural similarities, they exhibit functional distinctions:

IFITM2 shows the strongest antiviral effect and ability to activate IFN-β among the three IFITM proteins, making it a particularly important subject for antiviral research . Unlike IFITM1, both IFITM2 and IFITM3 can restrict Rift Valley Fever Virus replication . The proteins also show differential activities in SARS-CoV-2 infection, where IFITM2 and IFITM3, but not IFITM1, demonstrated inhibitory effects against SARS-CoV-2 Spike-pseudotyped vesicular stomatitis virus .

What are the structural characteristics of Recombinant Mouse IFITM2?

Recombinant Mouse IFITM2 protein typically consists of 144 amino acid residues (Met1~Phe144) . Key structural characteristics include:

• Predicted molecular mass: 19.4 kDa

• Actual measured molecular mass: 19 kDa

• Isoelectric point: 6.8

• Membrane protein with hydrophobic domains

• Contains functional motifs important for endocytosis, including a YXXø motif that influences subcellular localization

The recombinant version used in research is often expressed with an N-terminal His Tag to facilitate purification and experimental applications . The protein is typically formulated in PBS (pH 7.4) containing 0.01% SKL and 5% Trehalose for stability .

How should researchers handle and reconstitute Recombinant IFITM2 for experiments?

Proper handling of Recombinant Mouse IFITM2 is crucial for maintaining its biological activity:

• Reconstitution Protocol:

  • Reconstitute in 10mM PBS (pH 7.4) to a concentration of 0.1-1.0 mg/mL

  • Avoid vortexing, which can damage the protein structure

  • Ensure complete solubilization before proceeding with experiments

• Storage Recommendations:

  • For short-term storage (up to one month): 2-8°C

  • For long-term storage (up to 12 months): Aliquot and store at -80°C

  • Avoid repeated freeze/thaw cycles as this reduces activity

• Stability Considerations:

  • Thermal stability should be monitored through accelerated thermal degradation testing

  • Maintain cold chain during handling

  • Consider adding stabilizing agents for dilute solutions

The protein's purity (>90%) makes it suitable for use as a positive control, immunogen, or in analytical techniques like SDS-PAGE and Western blotting .

What experimental models are most suitable for studying IFITM2 function?

Based on current research, several experimental models have proven effective for studying IFITM2:

• Cell Culture Systems:

  • HEK293 cells: Demonstrated to be suitable for IFITM2 overexpression and viral infection studies

  • Neural stem cell models: Appropriate for studying IFITM2's role in endocytosis and brain development

  • Multiple cell lines for viral restriction studies: Vero, SW13, HepG2, and CER cells

• Genetic Manipulation Approaches:

  • siRNA knockdown: Effective for studying loss-of-function

    • Validated siRNA: human IFITM-2 (catalog no. L-020103-00; ON-TARGET plus)

  • Overexpression systems: Useful for gain-of-function studies

  • CRISPR/Cas9 gene editing: For generating stable knockout or knock-in cell lines

• Virus Infection Models:

  • Encephalomyocarditis virus (EMCV): Used to demonstrate IFITM2's antiviral activity

  • Rift Valley Fever Virus: Shown to be restricted by IFITM2 and IFITM3 but not IFITM1

  • SARS-CoV-2 pseudovirus systems: For studying coronavirus restriction

What methods are recommended for detecting IFITM2 expression and localization?

Several validated methods can be used to detect and localize IFITM2:

• Protein Detection:

  • Western blotting: Using specific anti-IFITM2 antibodies

  • ELISA: For quantitative measurement in complex samples

  • Immunoprecipitation: Especially useful for studying protein interactions

• Localization Studies:

  • Immunofluorescence microscopy: For visualization of subcellular localization

  • Subcellular fractionation: To separate membrane compartments

  • Live-cell imaging with fluorescently tagged IFITM2: For dynamic studies

• Expression Analysis:

  • RT-qPCR: For mRNA expression levels

  • Flow cytometry: For cell surface expression analysis

  • Single-cell RNA sequencing: For expression patterns in heterogeneous populations

Researchers should note that IFITM2 is primarily localized to endosomal compartments, which should inform experimental design when studying its localization and interactions .

How does IFITM2 modulate the type I interferon signaling pathway?

IFITM2 has been demonstrated to enhance type I interferon signaling through multiple mechanisms:

• Interaction with Pattern Recognition Receptors:

  • IFITM2 directly binds to melanoma differentiation-associated protein 5 (MDA5), as confirmed by co-immunoprecipitation assays

  • This interaction enhances MDA5's ability to detect viral RNA and trigger downstream signaling

• Feed-Forward Amplification:

  • IFITM2 expression is induced by type I interferons

  • Once expressed, IFITM2 further enhances IFN-β production, creating a positive feedback loop

  • This amplification is critical for robust antiviral responses

• Pathway-Specific Effects:

  • IFITM2 specifically enhances MDA5-triggered IFN-β activation

  • Interestingly, IFITM2 does not affect IFN-β activation induced by downstream components including MAVS, TBK1, and IRF3 (5D)

  • This specificity suggests IFITM2 functions primarily at the level of pattern recognition

The N-terminal domain of IFITM2 plays a particularly important role in its ability to activate the IFN-β signaling pathway, highlighting structure-function relationships that could be exploited for therapeutic development .

What is the role of IFITM2 in neural development and how does it differ from its antiviral functions?

Recent research has revealed a previously unrecognized role for IFITM2 in neural development that appears distinct from its antiviral functions:

• Endocytic Regulation in Neural Stem Cells:

  • IFITM2 is highly expressed near the ventricular surface in the developing brain

  • It modulates endocytosis in radial glial cells (RGCs), which are neural stem cells

  • Loss of IFITM2 impairs endosome formation and disrupts RGC maintenance

• Molecular Mechanisms:

  • The YXXø endocytic motif on IFITM2 is essential for its subcellular localization

  • Mutations in this motif reduce endocytic vesicle formation

  • K82 and K87 residues interact with phosphoinositides to promote endocytic vesicle formation

  • Polarized localization of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) on the ventricular side suggests its involvement in vesicle formation

• Signaling Pathway Interactions:

  • IFITM2 deficiency leads to reduced phosphorylation of AKT and GSK3β

  • This alteration in signaling affects neural stem cell maintenance and fate decisions

  • These effects appear to be independent of interferon signaling

This dual role highlights IFITM2 as a multifunctional protein that links innate immunity and developmental processes, suggesting evolutionary conservation of cellular machinery across different biological contexts.

How do researchers reconcile contradictory findings regarding IFITM2's role in viral infections?

The scientific literature contains several contradictory findings regarding IFITM2's role in viral infections, particularly for SARS-CoV-2. Researchers can address these contradictions through:

• Methodological Considerations:

  • Different experimental systems: Studies use various cell types, viral strains, and expression levels

  • Pseudovirus vs. authentic virus: Results may differ between these systems

  • Timing of measurements: Early vs. late infection stages may show different effects

• Specific Case of SARS-CoV-2:

  • Some studies show IFITM2 inhibits SARS-CoV-2 Spike-pseudotyped vesicular stomatitis virus infection

  • Other studies report no inhibition by IFITM3 in similar pseudovirus systems

  • Conflicting results exist regarding IFITM2's effect on Spike-mediated cell fusion

• Reconciliation Approaches:

  • Side-by-side comparison of different systems under identical conditions

  • Detailed analysis of protein localization in different cell types

  • Consideration of concentration-dependent effects

  • Assessment of interactions with other host factors that may modify IFITM2 activity

• Proposed Resolution Framework:

  Aspect	Approach to Reconciliation
  Cell Type Differences	Test multiple relevant cell lines in parallel
  Viral System	Compare pseudovirus and authentic virus in the same study
  Expression Levels	Use inducible systems to test dose-dependent effects
  Temporal Dynamics	Perform time-course experiments
  Cofactor Requirements	Analyze proteomic interactions in different contexts

These contradictions highlight the complexity of virus-host interactions and the context-dependent nature of restriction factors like IFITM2 .

What are the critical functional domains of IFITM2 and how do they contribute to its diverse activities?

IFITM2 contains several functional domains that contribute to its various biological activities:

• N-Terminal Domain:

  • Critical for antiviral activity

  • Plays an active role in IFN-β activation

  • Contributes to protein-protein interactions, including binding to MDA5

• Endocytic Motif (YXXø):

  • Essential for subcellular localization of IFITM2

  • Mutations in this motif reduce endocytic vesicle formation

  • Critical for IFITM2's function in neural stem cell maintenance

• Lysine Residues (K82 and K87):

  • Interact with phosphoinositides

  • Promote endocytic vesicle formation

  • Essential for proper function in radial glial cells

• Transmembrane Domains:

  • Contribute to membrane association and protein stability

  • May influence the protein's ability to restrict viral entry

  • Affect interaction with other membrane components

• Post-Translational Modification Sites:

  • Potential sites for ubiquitination, palmitoylation, and phosphorylation

  • Regulate protein turnover and functional activity

  • May be differentially modified in response to various stimuli

Understanding these domains provides insights into how IFITM2 performs its diverse functions and offers potential targets for therapeutic interventions or experimental manipulations.

What are common challenges when working with Recombinant IFITM2 and how can they be addressed?

Researchers often encounter several challenges when working with Recombinant IFITM2:

• Protein Stability Issues:

  • Challenge: IFITM2 can lose activity during storage or handling

  • Solution: Store at -80°C in small aliquots to avoid freeze-thaw cycles; include stabilizing agents in buffers; monitor thermal stability through accelerated degradation tests

• Solubility Problems:

  • Challenge: As a membrane protein, IFITM2 may aggregate or precipitate

  • Solution: Reconstitute in appropriate buffers (10mM PBS, pH 7.4); avoid vortexing; consider adding mild detergents for certain applications

• Detection Difficulties:

  • Challenge: Low expression levels or antibody cross-reactivity with other IFITM proteins

  • Solution: Use tagged versions for easier detection; validate antibodies for specificity; employ multiple detection methods in parallel

• Functional Assay Variability:

  • Challenge: Inconsistent results in antiviral or signaling assays

  • Solution: Standardize cell culture conditions; control for endogenous IFITM expression; include appropriate positive and negative controls

• Expression System Limitations:

  • Challenge: Difficulty expressing functional protein in certain systems

  • Solution: Optimize codon usage for the expression system; consider eukaryotic expression for proper post-translational modifications; test multiple tags and fusion partners

How can researchers optimize knockdown or overexpression studies of IFITM2?

Effective manipulation of IFITM2 expression is critical for functional studies:

• siRNA Knockdown Optimization:

  • Use validated siRNA sequences (e.g., human IFITM-2 catalog no. L-020103-00; ON-TARGET plus)

  • Include nontargeting siRNA controls (e.g., catalog no. D-001810-04; ON-TARGET plus)

  • Assess knockdown efficiency by RT-qPCR and Western blot

  • Optimize transfection conditions for each cell type

  • Consider the timing of knockdown relative to experimental readouts

• Overexpression Strategies:

  • Select appropriate vectors based on experimental requirements

  • Consider inducible expression systems for temporal control

  • Validate expression levels and subcellular localization

  • Be aware that very high expression levels may cause artifacts

  • Include empty vector controls and consider dose-response relationships

• CRISPR/Cas9 Gene Editing:

  • Design specific guide RNAs to minimize off-target effects

  • Validate edited clones by sequencing and functional assays

  • Consider potential compensatory mechanisms from other IFITM family members

  • Generate rescue cell lines to confirm specificity of observed phenotypes

• Assessment Framework:

  Parameter	Method	Considerations
  Expression Level	RT-qPCR, Western blot	Analyze both mRNA and protein levels
  Subcellular Localization	Immunofluorescence, fractionation	Confirm proper targeting
  Functional Impact	Viral infection, signaling assays	Include appropriate controls
  Off-Target Effects	RNA-seq, proteomics	Monitor for unexpected changes
  Compensation	Family member expression analysis	Check for upregulation of related proteins

What experimental designs best elucidate IFITM2's interactions with other proteins?

To effectively study IFITM2's protein-protein interactions:

• Co-Immunoprecipitation Approaches:

  • Tag IFITM2 with epitopes such as Myc for easier pulldown

  • Use bidirectional co-IP (e.g., pull down with anti-FLAG for MDA5 and anti-Myc for IFITM2)

  • Include appropriate controls (e.g., IgG control, non-interacting protein)

  • Validate interactions in multiple cell types and conditions

• Proximity Labeling Methods:

  • BioID or TurboID fusion proteins can identify proximal interactors

  • APEX2-based approaches work well for membrane proteins like IFITM2

  • These methods can capture transient or weak interactions missed by co-IP

• Fluorescence-Based Interaction Assays:

  • Förster Resonance Energy Transfer (FRET) for direct interaction studies

  • Bimolecular Fluorescence Complementation (BiFC) to visualize interactions in living cells

  • Fluorescence correlation spectroscopy for quantitative interaction measurements

• Structural Biology Approaches:

  • Cryo-EM for larger complexes

  • NMR for structural determination of protein domains

  • X-ray crystallography for high-resolution interaction details

• Domain Mapping Studies:

  • Generate deletion mutants to identify interaction domains

  • Use peptide arrays to pinpoint specific binding motifs

  • Alanine scanning mutagenesis to identify critical residues

The interaction between IFITM2 and MDA5 has been successfully demonstrated using co-IP approaches, providing a validated method for studying this particular interaction .

What emerging applications of IFITM2 research hold the most promise?

Several emerging areas of IFITM2 research show significant potential:

• Therapeutic Applications:

  • Development of IFITM2-inspired antiviral peptides or mimetics

  • Targeted enhancement of IFITM2 activity to boost innate immunity

  • Potential applications in viral diseases where IFITM2 shows strong restriction activity

• Neurological Development:

  • Further exploration of IFITM2's role in brain development

  • Potential implications for neurodevelopmental disorders

  • Study of IFITM2 in adult neurogenesis and brain repair mechanisms

• Broad-Spectrum Antiviral Strategies:

  • Leveraging IFITM2's ability to restrict multiple viruses for pandemic preparedness

  • Identifying common mechanisms that could be therapeutically targeted

  • Understanding resistance mechanisms that viruses develop against IFITM2

• Pathway Integration Analysis:

  • Systems biology approaches to map IFITM2's position in cellular networks

  • Study of crosstalk between antiviral and developmental pathways

  • Identification of novel regulatory mechanisms controlling IFITM2 expression and function

How might comparative studies across species advance our understanding of IFITM2 function?

Comparative analysis of IFITM2 across species offers valuable insights:

• Evolutionary Conservation and Divergence:

  • Compare human, mouse, and other mammalian IFITM2 homologs

  • Identify conserved domains that likely serve critical functions

  • Analyze species-specific variations that might reflect adaptation to different viral threats

• Host-Pathogen Co-evolution:

  • Study IFITM2 variations in species with different susceptibilities to viral infections

  • Identify potential signatures of positive selection in IFITM genes

  • Correlate IFITM2 sequence variations with species-specific viral resistance patterns

• Functional Conservation Across Systems:

  • Compare IFITM2's role in neural development across vertebrates

  • Examine whether dual functionality (antiviral and developmental) is conserved

  • Identify species-specific regulatory mechanisms controlling IFITM2 expression

• Translational Relevance:

  • Determine how findings in mouse models translate to human IFITM2 function

  • Develop improved animal models that better recapitulate human IFITM2 biology

  • Leverage comparative insights for therapeutic development

Existing research already demonstrates the value of this approach, with studies examining both mouse and human models showing conservation of IFITM2's role in neurogenesis .