Recombinant Human Interferon-induced transmembrane protein 10 (IFITM10)

What is IFITM10 and how does it differ from other IFITM family proteins?

IFITM10 is one of five functional IFITM homologs identified in humans (along with IFITM1, IFITM2, IFITM3, and IFITM5). Unlike the immune-related IFITM1, IFITM2, and IFITM3 which share high sequence similarity (>90% identity over 70% of coding sequence), IFITM10 has significantly different gene and protein sequences. A key distinguishing characteristic is that IFITM10 is insensitive to interferon stimulation due to the absence of interferon-stimulated response elements (ISREs) and IFN gamma-activated sites (GAS) in its promoter region, which are present in IFITM1, IFITM2, and IFITM3 . Additionally, while most IFITMs demonstrate strong antiviral properties, IFITM10 has been observed to exert only weak antiviral effects in experimental settings .

Is IFITM10 expression induced by interferons like other IFITM family members?

Unlike IFITM1, IFITM2, and IFITM3, which are strongly upregulated in response to interferons, IFITM10 expression is not enhanced by interferon stimulation. Experimental evidence shows that interferon-α stimulation of embryonic fibroblast cells does not enhance the expression of IFITM10 . This lack of interferon responsiveness is attributed to the absence of interferon-stimulated response elements (ISREs) and IFN gamma-activated sites (GAS) in the promoter region of the IFITM10 gene, which are present in the promoters of interferon-responsive IFITM genes . This fundamental difference in regulation suggests that IFITM10 likely serves biological functions distinct from the classical interferon-mediated antiviral responses associated with other IFITM proteins.

What functional assays have been used to characterize IFITM10's antiviral activity?

Several experimental approaches have been employed to assess IFITM10's potential antiviral activity, though studies are more limited compared to other IFITM proteins. Research with chicken IFITM10 has utilized:

• Viral infection assays: Forced expression of IFITM10 in cell culture systems showed slight inhibition of VSV-G-pseudotyped lentiviral vector infectivity .

• Cell fusion assays: IFITM10 inhibited cell fusion when HeLa cells transfected with VSV-G expression vector were treated with low pH buffer, suggesting potential interference with membrane fusion events critical for viral entry .

• Comparative expression analysis: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) has been used to measure expression levels of IFITM10 relative to other IFITM proteins across different tissues and developmental stages .

These methodological approaches provide preliminary evidence for weak antiviral properties, though the physiological relevance remains uncertain.

How does IFITM10's antiviral activity compare to other IFITM proteins?

IFITM10 demonstrates significantly weaker antiviral activity compared to IFITM1, IFITM2, and IFITM3. While IFITM1-3 are well-established restriction factors that block viral replication by preventing the fusion of virus and host-cell membranes (thereby restricting numerous pathogenic viruses including influenza A virus and Ebola virus), IFITM10 exhibits only slight inhibition of viral infectivity in experimental settings . This difference in antiviral potency correlates with IFITM10's lack of interferon responsiveness. The mechanism underlying IFITM10's weak antiviral activity may differ from other IFITMs, which typically function by altering membrane properties to prevent viral fusion. Further comparative studies are needed to elucidate whether IFITM10 utilizes similar or distinct mechanisms for its observed antiviral effects.

Through what mechanisms might IFITM10 restrict viral infection?

The precise mechanisms through which IFITM10 might restrict viral infection remain largely unexplored. Based on limited experimental evidence and knowledge of other IFITM proteins, potential mechanisms include:

• Membrane modification: Like other IFITM proteins, IFITM10 may alter membrane properties, albeit less efficiently, to inhibit virus-host membrane fusion events.

• VSV-G-mediated fusion inhibition: Experiments show IFITM10 can inhibit cell fusion mediated by VSV-G under low pH conditions, suggesting interference with pH-dependent fusion mechanisms .

• Alternative non-IFN dependent pathways: Given its IFN-independence, IFITM10 may utilize distinct cellular pathways to exert its weak antiviral effects.

Methodological approaches to explore these mechanisms could include membrane fluidity assays, lipid composition analysis, and targeted mutagenesis of putative functional domains to identify residues critical for the observed antiviral activity.

What are the recommended methods for studying IFITM10 expression patterns?

For comprehensive characterization of IFITM10 expression patterns, researchers should consider multiple complementary approaches:

• Quantitative RT-PCR: This method has been successfully employed to examine IFITM10 expression across different tissues and developmental stages, allowing comparison with other IFITM family members .

• RNA-seq analysis: For genome-wide expression profiling and identification of co-regulated genes.

• In situ hybridization: To localize IFITM10 expression in specific tissue regions, particularly during development.

• Immunohistochemistry/immunofluorescence: Using validated antibodies to detect protein-level expression and subcellular localization.

• Reporter gene assays: To study promoter activity and regulatory mechanisms controlling IFITM10 expression.

When designing experiments, researchers should consider developmental stage, tissue specificity, and appropriate controls, including comparison with other IFITM family members, especially in contexts where differential expression has been observed, such as embryonic versus adult tissues and primordial germ cells.

What strategies can be used to study IFITM10 function in viral infection models?

To investigate IFITM10's potential role in viral infections, several experimental strategies are recommended:

• Overexpression systems: Transfect cells with IFITM10 expression constructs and challenge with various viral vectors or live viruses, as demonstrated with VSV-G-pseudotyped lentiviral vectors .

• CRISPR/Cas9-mediated knockout: Generate IFITM10-deficient cell lines or animal models to assess the impact on viral susceptibility.

• Domain swapping: Create chimeric proteins between IFITM10 and other IFITM proteins to identify functional domains responsible for antiviral activity.

• Microscopy-based fusion assays: Employ cell-cell fusion assays using viral fusion proteins (like VSV-G) to assess IFITM10's impact on membrane fusion events .

• Comparative studies: Perform side-by-side comparisons with other IFITM proteins under identical conditions to quantify relative antiviral potency.

These approaches should be implemented with appropriate controls and across multiple viral systems to comprehensively characterize IFITM10's functional properties.

How has IFITM10 evolved across different species?

IFITM10 exhibits interesting evolutionary patterns that distinguish it from other IFITM family members. A notable evolutionary characteristic is that vertebrate IFITM10 genes are divided into two distinct groups: aquatic and terrestrial types . Aquatic vertebrate IFITM10 genes have undergone positive selection and several gene duplications during evolution, suggesting adaptation to aquatic environments . This evolutionary divergence between aquatic and terrestrial IFITM10 implies environment-specific selective pressures that may have shaped IFITM10 function. In contrast to IFITM10, IFITM5 has remained highly conserved with little evidence of gene duplication or positive selection, consistent with its specialized role in bone development . The evolutionary history of IFITM10 suggests it may serve functions beyond antiviral activity, potentially related to environmental adaptation.

What do comparative genomics analyses reveal about IFITM10 conservation and adaptation?

Comparative genomics approaches provide insights into IFITM10's evolutionary history and potential functional divergence:

• Sequence conservation analysis: Unlike highly conserved IFITM5, IFITM10 shows evidence of adaptive evolution, particularly in aquatic vertebrates.

• Selective pressure analysis: The detection of positive selection in aquatic IFITM10 variants suggests environment-specific adaptation .

• Gene duplication patterns: Multiple duplication events in aquatic vertebrate IFITM10 genes indicate potential functional diversification.

• Promoter element analysis: The absence of interferon response elements in IFITM10 across species represents a conserved feature distinguishing it from immune-related IFITMs .

These evolutionary patterns suggest IFITM10 may have evolved functions distinct from the classical antiviral roles of other IFITM proteins, potentially related to environmental adaptation or developmental processes.

How does IFITM10 compare to other IFITMs in terms of protein sequence and domain organization?

IFITM10 differs substantially from other IFITM family members in sequence and domain organization:

• Sequence similarity: IFITM10 shares significantly less sequence homology with IFITM1-3, which maintain high mutual similarity (>90% identity in coding regions). In contrast, IFITM5 and IFITM10 are markedly different from the immune-related IFITMs and from each other .

• Domain structure: While all IFITMs share a general topology with intramembrane and transmembrane domains, specific functional motifs may differ. The presence and conservation of crucial motifs like the oligomerization-mediating GXXXG motif in IFITM10 remain to be fully characterized .

• Post-translational modification sites: IFITM1-3 function is regulated by various post-translational modifications including palmitoylation, ubiquitination, and phosphorylation. The conservation of these regulatory sites in IFITM10 requires further investigation.

These structural differences likely underpin the distinct functional properties of IFITM10 compared to other family members.

How do the cellular localization and trafficking of IFITM10 compare to other IFITM proteins?

The cellular localization and trafficking patterns of IFITM10 remain less characterized compared to other IFITM proteins:

• IFITM1-3 localization: IFITM1 predominantly localizes to the plasma membrane, while IFITM2 and IFITM3 are found primarily in endosomal and lysosomal compartments .

• Trafficking signals: IFITM2 and IFITM3 contain an N-terminal endocytic signal (YxxΦ motif) that mediates their internalization from the cell surface .

• IFITM10 localization: Detailed studies of IFITM10's subcellular distribution are lacking in the current literature. Determining whether IFITM10 contains sorting signals and identifying its predominant cellular compartments represent important research questions.

• Developmental changes: Given IFITM10's differential expression during development, particularly in primordial germ cells , its localization may vary with developmental stage and cell type.

Methodological approaches to address these questions include fluorescent protein tagging, immunofluorescence microscopy, subcellular fractionation, and comparison of trafficking in different cell types and developmental stages.