Recombinant Mouse Transmembrane protein 239 (Tmem239)

What is the evolutionary conservation pattern of TMEM239?

TMEM239 orthologs are distributed among eutheria and metatheria mammalian lineages but are notably absent in prototheria. No human paralogs for TMEM239 have been identified, suggesting it has a specialized function that evolved after the divergence of monotremes from other mammals . When designing comparative studies, researchers should be aware of this evolutionary distribution pattern, particularly when selecting appropriate animal models for functional studies.

What is the tissue expression profile of TMEM239?

TMEM239 demonstrates a restricted expression pattern, with primary expression in the testis and brain based on human expressed sequence tag (EST) profiles. According to protein abundance database (PaxDb) analyses, TMEM239 falls within the bottom 10% relative to all other proteins in both mice and humans, indicating it is not abundantly expressed . Moderate expression levels are observed in testes, with lower expression in various tissues including brain and submaxillary gland . This limited expression profile suggests tissue-specific functions and should inform experimental design when studying this protein.

What are the recommended storage conditions for maintaining recombinant TMEM239 stability?

Recombinant TMEM239 protein is typically supplied as a lyophilized powder and should be stored at -20°C/-80°C upon receipt. Aliquoting is necessary to avoid repeated freeze-thaw cycles which can lead to protein degradation. For working stocks, storage at 4°C for up to one week is recommended . Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol (final concentration) for long-term storage. This glycerol concentration helps prevent protein aggregation during freeze-thaw cycles.

How can researchers effectively knockout TMEM239 for functional studies?

Researchers have successfully employed CRISPR/Cas9 technology to generate TMEM239 knockout cell lines and animals. For cellular models, designing specific single guide RNAs (sgRNAs) targeting the TMEM239 coding region followed by electroporation of ribonucleoprotein complexes (RNPs) has proven effective . For animal models, somatic cell nuclear transfer (SCNT) technology has been successfully used to generate TMEM239 knockout piglets . Alternative approaches include siRNA knockdown, which has been validated in primary alveolar macrophages (PAMs) with specific TMEM239-targeted siRNAs achieving significant reduction in expression levels .

What are the known protein interaction partners of TMEM239?

TMEM239 has been demonstrated to interact with several proteins involved in cellular trafficking and signaling pathways:

These interactions suggest TMEM239 may function at the interface of cellular trafficking, particularly in endosomal pathways, and immune responses.

What role does TMEM239 play in viral infection processes?

TMEM239 has been identified as an important host factor facilitating African swine fever virus (ASFV) entry into early endosomes . Genome-wide CRISPR knockout screening revealed that TMEM239 deletion significantly reduced ASFV replication by impeding viral entry into early endosomes . Mechanistically, TMEM239 interacts with Rab5A, an early endosomal marker, and TMEM239 knockout affects the co-localization of viral capsid protein p72 with Rab5A shortly after viral infection . This interaction appears critical for the early stages of viral infection, as knockout of TMEM239 significantly reduced ASFV replication in both cell culture and ex vivo studies using peripheral blood mononuclear cells from TMEM239 knockout piglets .

How does TMEM239 contribute to endosomal trafficking pathways?

TMEM239 functions in endosomal trafficking through its interaction with Rab5A, a key regulator of early endosome formation and function . Co-immunoprecipitation assays and colocalization studies using confocal microscopy have confirmed this interaction . The functional significance of this interaction is evident in viral infection models, where TMEM239 knockout disrupts the normal trafficking of viral particles into early endosomes . Researchers investigating endosomal trafficking should consider TMEM239 as a potential regulator, particularly in contexts involving pathogen entry or cellular internalization processes.

What neurological disorders have been associated with TMEM239?

A genome-wide association study identified SNP rs7360412, located in the 3'UTR of TMEM239, as a top marker for fractional anisotropy in bipolar disorder . Fractional anisotropy, detected by diffusion tensor imaging, is used to assess white matter integrity, which is highly heritable and reduced in both bipolar patients and their unaffected relatives . This suggests TMEM239 may play a role in neurological function, particularly in white matter development or maintenance. Researchers studying neurological disorders should consider TMEM239 as a candidate gene, particularly in bipolar disorder studies focusing on white matter abnormalities.

What infectious disease processes involve TMEM239?

Beyond its role in ASFV infection, TMEM239 has been implicated in Leishmaniasis progression. RNA-seq analysis of human and Leishmania primary cutaneous lesions revealed that decreased expression of TMEM239 in primary cutaneous lesions indicates a higher probability of Mucosal Leishmaniasis (ML) development from Localized Cutaneous Leishmaniasis (LCL) . Additionally, TMEM239's interaction with viral proteins like Human T-cell leukemia virus type-1 Protein TAX-1 suggests potential roles in other viral infections . This multifaceted involvement in infectious disease processes makes TMEM239 a valuable target for researchers studying host-pathogen interactions.

How can TMEM239 be targeted for development of antiviral strategies?

Given TMEM239's essential role in ASFV infection, several approaches could be explored for antiviral development:

• Small molecule inhibitors: Compounds disrupting the TMEM239-Rab5A interaction could potentially block viral entry. High-throughput screening assays using fluorescence resonance energy transfer (FRET) or split-luciferase complementation between tagged TMEM239 and Rab5A could identify candidate molecules.

• Genetic modification: The creation of TMEM239 knockout pigs has demonstrated reduced viral replication in ex vivo studies , suggesting gene editing approaches as a potential strategy for developing ASF-resistant pig lineages.

• Peptide inhibitors: Synthetic peptides mimicking the interaction domains between TMEM239 and its viral binding partners could competitively inhibit these interactions, potentially blocking viral entry.

Researchers should consider the tissue-specific expression of TMEM239 when designing targeting strategies to minimize potential side effects.

What methodological approaches can be used to study the subcellular localization and trafficking of TMEM239?

Several complementary techniques can effectively characterize TMEM239 localization and trafficking:

• Immunofluorescence microscopy: Using antibodies against endogenous TMEM239 or epitope-tagged recombinant versions, coupled with markers of subcellular compartments (e.g., Rab5A for early endosomes) .

• Live-cell imaging: Expressing TMEM239 fused to fluorescent proteins allows real-time tracking of its movement through cellular compartments, particularly valuable for studying dynamic trafficking events.

• Subcellular fractionation: Physical separation of cellular compartments followed by Western blotting can biochemically confirm TMEM239 localization to specific membrane fractions.

• Proximity labeling: Techniques such as BioID or APEX2 fused to TMEM239 can identify proximal proteins in living cells, providing insights into its local interaction network.

How can researchers investigate the potential role of TMEM239 in glioblastoma?

While current research on TMEM239 in glioblastoma is limited, methodological approaches to investigate its potential involvement could include:

• Expression analysis: Examining TMEM230 (a related transmembrane protein) has been performed in glioblastoma multiforme (GBM) and low-grade gliomas using The Cancer Genome Atlas (TCGA) datasets . Similar approaches could be applied to TMEM239, analyzing its expression across different grades of gliomas and correlating with clinical outcomes.

• Functional studies: CRISPR/Cas9-mediated knockout or overexpression of TMEM239 in glioblastoma cell lines followed by assessment of proliferation, migration, invasion, and tumor microenvironment modulation.

• Patient-derived xenografts: Examining TMEM239 expression in patient-derived glioblastoma samples and testing targeted interventions in xenograft models.

Researchers should employ differential gene expression analysis using tools like DESEQ2 with appropriate p-value cut-offs (<0.0001) and log2 fold change thresholds (>0.58) to identify statistically significant associations .