Recombinant Human Transmembrane protein 239 (TMEM239)

What is TMEM239 and what is its cellular localization?

TMEM239 (Transmembrane Protein 239) is a membrane-integrated protein that plays a significant role in endosomal trafficking. It is primarily localized to early endosomal compartments where it interacts with Rab5A, an early endosomal marker protein. Multiple experimental approaches have demonstrated that TMEM239 is an integral component of membrane structures involved in cellular trafficking pathways . The protein consists of 195 amino acids in humans with several predicted transmembrane domains that anchor it within cellular membranes .

What is the expression pattern of TMEM239 across different tissues?

Based on human expressed sequence tag (EST) profiles, TMEM239 demonstrates a tissue-specific expression pattern:

According to protein abundance database (PaxDb) analyses, TMEM239 expression falls within the bottom 10% relative to all other proteins in both mice and humans, indicating it is not abundantly expressed in most tissues . This restricted expression pattern suggests specialized functions in specific cellular contexts.

What are the known protein interaction partners of TMEM239?

TMEM239 engages in several important protein-protein interactions that suggest roles in cellular signaling, membrane transport, and immune function:

These interactions indicate TMEM239 may function at the intersection of membrane trafficking, immune responses, and cell signaling pathways.

What methods have proven effective for generating TMEM239 knockout models?

Several approaches have been successfully employed to generate TMEM239 knockout models:

CRISPR/Cas9 Genome Editing:

• For cellular models, researchers have used lentiviral delivery of sgRNAs targeting TMEM239 in WSL-Cas9 cells. The protocol involves:

  • Cloning sgRNA sequences into lentiGuide-puro plasmid

  • Transfecting plasmids with helper vectors into HEK293T cells

  • Harvesting lentivirus and transducing target cells at MOI of 0.3

  • Selecting with puromycin to establish knockout cell lines

  • Confirming knockout efficiency via RT-qPCR and Sanger sequencing

• For animal models, CRISPR/Cas9 has been used to generate TMEM239 knockout pigs, though with significant health impacts on the animals, suggesting careful consideration of developmental effects .

RNA Interference Approaches:
For transient knockdown experiments, siRNA delivery has been effective:

• Synthetic siRNAs targeting TMEM239 transfected at 30 nM concentration using Lipofectamine RNAiMAX

• Knockdown efficiency evaluated 36 hours post-transfection by RT-qPCR

• Multiple siRNAs should be used as controls for off-target effects

What are recommended protocols for producing recombinant TMEM239 protein?

While direct expression protocols were not fully detailed in the search results, the following approaches have been used successfully:

Expression Systems:

• Cell-free protein synthesis (CFPS) from Nicotiana tabacum has proven effective for producing human TMEM239 (AA 1-195) with Strep Tag .

• Alternative expression systems include mammalian, bacterial, and insect cell systems, each with specific advantages for membrane protein production.

Purification Strategy:

• Affinity purification using Strep-tag technology

• For membrane proteins like TMEM239, proper detergent selection is crucial for maintaining native structure

• Quality assessment via SDS-PAGE, Western Blot, and analytical SEC (HPLC) should confirm >70-80% purity

Applications:
The recombinant TMEM239 protein has been successfully used in:

• ELISA

• SDS-PAGE

• Western Blotting

• Functional studies

How can researchers effectively detect TMEM239 localization in cells?

To study TMEM239 localization:

• Immunofluorescence microscopy:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.2% Triton X-100

  • Block with 5% BSA

  • Incubate with anti-TMEM239 antibodies (available commercially)

  • Co-stain with markers like anti-Rab5A for early endosomes

  • Analyze co-localization using confocal microscopy

• Subcellular fractionation:

  • Separate cellular components through differential centrifugation

  • Isolate membrane fractions enriched for early endosomes

  • Confirm TMEM239 presence via Western blotting

• Co-immunoprecipitation:

  • Use anti-TMEM239 antibodies to pull down protein complexes

  • Identify interaction partners like Rab5A through mass spectrometry or Western blotting

How was TMEM239 identified as a crucial host factor for ASFV infection?

TMEM239 was identified through a comprehensive genome-wide CRISPR knockout (GeCKO) screen:

• Experimental design:

  • A GeCKO library containing 186,510 specific sgRNAs targeting 20,580 pig genes was constructed

  • Wild boar lung (WSL) cells expressing Cas9 were transduced with the lentiviral GeCKO library

  • Cells were challenged with genotype II ASFV in three successive rounds of infection

  • Surviving cells were harvested and deep sequencing was performed to identify enriched sgRNAs

• Validation approach:

  • Individual TMEM239 knockout cell lines were generated using three different sgRNAs

  • Knockout efficiency was confirmed at the mRNA level using RT-qPCR

  • ASFV replication was assessed in these knockout cells compared to wildtype controls

  • Multiple ASFV strains were tested to confirm consistency of the phenotype

• Key findings:

  • TMEM239 knockout cells showed significantly reduced ASFV replication

  • The effect was observed across multiple ASFV strains

  • The phenotype was confirmed in both cell lines and primary cells

This systematic approach provided strong evidence that TMEM239 is a critical host factor for ASFV infection.

What is the mechanism by which TMEM239 facilitates ASFV entry?

TMEM239 plays a crucial role in facilitating ASFV entry into early endosomes through several mechanisms:

• Interaction with early endosomal machinery:

  • TMEM239 directly interacts with Rab5A, a key marker and regulator of early endosomes

  • This interaction may facilitate viral trafficking to appropriate endosomal compartments

• Effect on viral capsid localization:

  • In wild-type cells, viral capsid protein p72 co-localizes with Rab5A shortly after infection

  • In TMEM239 knockout cells, this co-localization is significantly disrupted

  • This suggests TMEM239 is required for proper routing of incoming virions

• Temporal dynamics:

  • The effect is observed shortly after viral infection

  • This indicates TMEM239 functions during the early stages of the viral life cycle

  • The protein likely acts at the entry/trafficking stage rather than during replication or assembly

The molecular details of how TMEM239 coordinates with Rab5A to facilitate viral entry require further investigation, but the current evidence strongly supports its role in early endosomal entry of ASFV.

How do TMEM239 knockout experiments affect ASFV replication in different experimental systems?

The effect of TMEM239 knockout on ASFV replication has been demonstrated across multiple experimental systems:

Key observations from these studies:

• Cellular models:

  • TMEM239 knockout consistently reduced ASFV replication

  • Multiple viral strains were tested (ad-7GD, ad-HRB1, WT-SD) with similar results

  • Quantification was performed using both viral genome copies (p72) and viral titers

• Ex vivo studies:

  • PBMCs isolated from TMEM239 knockout piglets showed significant resistance to ASFV infection

  • This validates the in vitro findings in a more physiologically relevant context

• Methodological considerations:

  • Some reviewers noted concerns about the use of different virus strains across experiments

  • The high MOI used in some experiments with gene-deleted virus strains rather than wild-type virus may affect interpretation

  • Proper controls are essential to rule out indirect effects on virus replication

What are the implications of TMEM239 research for ASF control strategies?

Research on TMEM239 opens several promising avenues for ASF control:

What potential roles might TMEM239 play in other viral infections?

While the current research has focused on ASFV, the endosomal localization and interaction with Rab5A suggest TMEM239 could be relevant for other viruses that utilize endosomal entry pathways:

• Potential relevance to other DNA viruses:

  • Many large DNA viruses utilize endosomal entry mechanisms

  • The interaction with early endosomal machinery suggests TMEM239 could potentially affect other viruses that require trafficking through these compartments

• Host-pathogen interaction data:

  • TMEM239 interaction with the Human T-cell leukemia virus type-1 Protein TAX-1 has been documented

  • This suggests potential roles in other viral infections beyond ASFV

• Research approaches to explore broader viral relevance:

  • Screen TMEM239 knockout cells against a panel of viruses with different entry mechanisms

  • Assess TMEM239 expression changes during various viral infections

  • Evaluate TMEM239 interaction with proteins from different viral families

How does the structure-function relationship of TMEM239 contribute to its role in endosomal trafficking?

Understanding the structure-function relationship of TMEM239 requires investigation of:

• Predicted structural features:

  • TMEM239 is a 195 amino acid protein with multiple transmembrane domains

  • Functional domains that mediate interaction with Rab5A need to be identified

  • Post-translational modifications may regulate its function

• Key regions for investigation:

  • The cytoplasmic domains likely mediate interaction with trafficking machinery

  • Transmembrane regions anchor the protein in the appropriate cellular compartments

  • Structure prediction tools suggest potential protein-protein interaction motifs

• Experimental approaches:

  • Site-directed mutagenesis of key residues/domains followed by functional assays

  • Protein-fragment complementation assays to map interaction domains

  • Structural studies using cryo-EM or X-ray crystallography (challenging for membrane proteins)

What are the best approaches for analyzing contradictory data in TMEM239 research?

When confronted with contradictory data on TMEM239 function:

• Methodological considerations:

  • Evaluate differences in experimental systems (cell types, knockout methods)

  • Consider virus strain variations and their potential impact

  • Assess MOI differences across studies that may affect outcomes

• Controls and validation:

  • Use multiple siRNAs/sgRNAs to rule out off-target effects

  • Include reconstitution experiments (re-expression of TMEM239 in knockout cells)

  • Validate findings across multiple experimental systems

• Statistical approaches:

  • Perform meta-analysis of multiple independent studies

  • Use appropriate statistical tests with correction for multiple comparisons

  • Report effect sizes rather than just p-values to better assess biological significance

• Experimental design recommendations:

  • Include time-course experiments to capture kinetic differences

  • Use both high and low MOI conditions to assess dose-dependent effects

  • Employ multiple quantification methods (viral genome copies, titers, reporter assays)

What clinical associations have been identified for TMEM239?

Several clinical associations with TMEM239 have been documented:

• Neuropsychiatric conditions:

  • SNP rs7360412, located in the 3'UTR of TMEM239, was identified in a genome-wide association study of bipolar disorder

  • This SNP was associated with fractional anisotropy, a measure of white matter integrity

  • White matter integrity is reduced in both bipolar patients and their unaffected relatives

• Infectious disease:

  • Decreased expression of TMEM239 in primary cutaneous lesions has been associated with a higher probability of Mucosal Leishmaniasis (ML) development

  • This suggests potential roles in the host response to Leishmania infection

• Research directions:

  • Investigate TMEM239 expression in neuropsychiatric disorders

  • Explore potential roles in other infectious diseases beyond ASFV and Leishmaniasis

  • Consider TMEM239 variants as potential biomarkers for disease susceptibility

What are the technical challenges in developing TMEM239-based interventions?

Developing interventions targeting TMEM239 faces several challenges:

• Genetic modification approaches:

  • TMEM239 knockout piglets showed developmental issues, suggesting complete knockout may not be viable

  • Partial knockdown or tissue-specific modifications may be more appropriate

  • CRISPR/Cas9 gene editing in livestock remains technically challenging and regulatory approval is complex

• Small molecule development:

  • Targeting protein-protein interactions (like TMEM239-Rab5A) is generally more difficult than targeting enzymes

  • The limited structural information on TMEM239 complicates structure-based drug design

  • Specificity is critical to avoid disrupting essential cellular functions

• Delivery systems:

  • Targeting endosomal proteins may require specialized delivery systems

  • Tissue-specific delivery would help minimize off-target effects

  • Large animal models are needed to validate efficacy and safety

What are the most promising research directions for TMEM239?

Based on current knowledge, several research directions appear particularly promising:

• Structural studies:

  • Determine the three-dimensional structure of TMEM239

  • Map the interaction interface with Rab5A and other binding partners

  • Identify druggable pockets for therapeutic development

• Functional genomics:

  • Create conditional or inducible knockout models to bypass developmental issues

  • Perform domain-specific mutagenesis to identify functional regions

  • Investigate tissue-specific roles of TMEM239

• Comparative virology:

  • Test TMEM239's role in the entry of other viruses that utilize endocytic pathways

  • Compare mechanisms across different viral families

  • Identify common principles that could inform broad-spectrum interventions

• Translational research:

  • Develop and test small molecule inhibitors of TMEM239-Rab5A interaction

  • Explore gene editing approaches that modify rather than eliminate TMEM239

  • Investigate naturally occurring TMEM239 variants that might confer ASFV resistance

The identification of TMEM239 as a critical host factor for ASFV entry opens significant new avenues for both basic research and applied interventions against this economically devastating disease.