Recombinant Human Transmembrane and coiled-coil domain-containing protein 4 (TMCO4)

What is the genomic location and structure of the TMCO4 gene?

TMCO4 (Transmembrane and coiled-coil domains 4) is a protein-coding gene located on the minus strand of chromosome 1 at position 1p36.13. The gene spans 118,172 base pairs, extending from position 19,682,213 to 19,800,385. It comprises 16 exons that can generate twenty different mRNA transcript variants (X1-X20) through alternative splicing. TMCO4 lacks common aliases and is flanked by genes CAPZB and LOC105376823 on chromosome 1 .

What is the predicted protein structure of TMCO4?

TMCO4 is predicted to be a transmembrane protein that traverses the endoplasmic reticulum membrane three times. Based on structural similarities with other TMCC family proteins, TMCO4 likely contains coiled-coil domains that facilitate protein-protein interactions and potential oligomerization. The coiled-coil regions are critical for mediating specific interactions with partner proteins and may contribute to the formation of higher-order structures .

How does TMCO4 compare to other TMCC family members?

While TMCO4 shares structural similarities with TMCC family proteins (TMCC1, TMCC2, and TMCC3), it has distinct features and potential functions. Like other TMCC proteins, TMCO4 contains transmembrane regions that anchor it to the endoplasmic reticulum. Studies on TMCC3 have shown that the transmembrane domains contribute to localization in the ER, while coiled-coil regions mediate oligomerization. TMCC3 forms trimers and possibly hexamers, whereas TMCC1 was reported to primarily form dimers through its second coiled-coil domain. By comparison, the specific oligomerization patterns of TMCO4 remain to be characterized in detail .

What cell line models are available for studying TMCO4?

The TMCO4 Knockout HEK293 cell line represents a valuable model system for investigating TMCO4 function. This genetically modified line, derived from the well-characterized HEK293 background, offers several advantages for TMCO4 research, including:

• Complete elimination of TMCO4 protein expression

• Robust growth conditions and high transfectability

• Compatibility with established protocols for transfection and protein expression

• Ideal for comparing wild-type versus knockout phenotypes

This cell line enables researchers to assess the effects of TMCO4 loss on cellular behavior, including ion channel activity, metabolic pathways, and signaling cascades in a controlled environment .

How can recombinant TMCO4 protein be expressed and purified?

Based on methodologies used for related TMCC proteins, recombinant TMCO4 expression can be achieved using the following approach:

• Clone the full-length TMCO4 cDNA into an expression vector (such as pcDNA-3.1/Myc-His)

• Design appropriate primers based on the TMCO4 sequence (similar to the approach used for TMCC3 where primers targeted specific domains)

• Amplify the TMCO4 cDNA by RT-PCR from human tissue cDNA libraries

• Create deletion mutants as needed for domain analysis by designing primers that exclude specific regions

• Transform expression vectors into suitable host cells (mammalian cells preferred for proper post-translational modifications)

• Express and purify using affinity chromatography via engineered tags

For domain-specific studies, deletion mutants lacking specific coiled-coil regions can be generated to assess their contribution to protein function and interaction .

What techniques are most effective for studying TMCO4 localization?

The subcellular localization of TMCO4 can be effectively studied using multiple complementary approaches:

• Immunofluorescence microscopy: Using specific antibodies against TMCO4 or epitope tags in recombinant constructs, combined with organelle markers (particularly ER markers).

• Confocal microscopy: For high-resolution co-localization studies with ER markers and potential interacting partners.

• Subcellular fractionation: To biochemically separate cellular compartments and detect TMCO4 distribution by western blotting.

• Domain deletion analysis: Creating transmembrane domain deletion mutants to assess their contribution to proper localization, as demonstrated with TMCC3 where transmembrane domains were shown to be essential for ER localization .

What proteins interact with TMCO4?

While specific TMCO4 interaction partners have not been comprehensively characterized, insights can be drawn from studies of related proteins:

To identify TMCO4-specific interacting partners, immunoprecipitation of tagged TMCO4 followed by mass spectrometry analysis would be an effective approach, similar to the method used to identify TMCC3 interactions .

How can researchers investigate TMCO4's potential role in cellular signaling?

To elucidate TMCO4's involvement in cellular signaling pathways, researchers can employ the following methodological approaches:

• Phosphorylation analysis: Investigate potential phosphorylation sites on TMCO4 and determine kinases responsible, particularly given the interaction between TMCC3 and 14-3-3 proteins, which often bind phosphorylated targets.

• Calcium signaling assays: Given the potential involvement of TMCO4 in ion transport, calcium imaging techniques and electrophysiology can reveal its role in calcium homeostasis.

• Signal transduction pathway analysis: Examine changes in signaling pathway activation (e.g., MAPK, PI3K/Akt) in TMCO4 knockout versus wild-type cells using phospho-specific antibodies.

• Transcriptional profiling: RNA-seq analysis comparing wild-type and TMCO4 knockout cells to identify downstream gene expression changes.

• Proteomic profiling: Using approaches similar to the independent component analysis (ICA) method described for breast cancer proteogenomics to extract pathway-level signatures associated with TMCO4 function .

How can TMCO4 knockout models be utilized to understand its physiological role?

TMCO4 knockout models provide powerful tools for elucidating physiological functions through multiple experimental approaches:

• Phenotypic characterization: Systematic analysis of cellular phenotypes in TMCO4 knockout HEK293 cells, including:

  • Growth rate and viability assessments

  • Morphological changes using microscopy

  • Stress response to various stimuli (oxidative stress, ER stress)

• Electrophysiological studies: Given TMCO4's potential role in ion channel activity, patch-clamp recordings can identify changes in membrane potential and ion currents.

• Rescue experiments: Re-introducing wild-type or mutant TMCO4 into knockout cells to determine which domains are critical for function.

• Omics integration: Combining transcriptomics, proteomics, and metabolomics data from knockout cells to construct a comprehensive view of TMCO4's role in cellular homeostasis.

• Drug response profiling: Screening TMCO4 knockout cells against compound libraries to identify synthetic lethality relationships or altered drug sensitivity profiles .

What evidence suggests TMCO4's involvement in disease processes?

Current evidence for TMCO4's role in disease processes remains preliminary but suggests several potential associations:

• Cancer biology: TMCO4 reportedly interacts with proteins implicated in cancer development, suggesting a possible role in oncogenic signaling or tumor progression. This warrants investigation in cancer cell lines and patient samples .

• Ion transport disorders: Given TMCO4's predicted role in cellular homeostasis and ion transport, it may contribute to disorders characterized by ion imbalance or membrane potential dysregulation .

• Neurological conditions: By analogy to TMCC3, which shows high expression in human brain and potential involvement in neuronal development, TMCO4 might have neurological implications that could be explored through expression analysis in neural tissues .

How can proteogenomic approaches advance TMCO4 research?

Proteogenomic integration provides powerful insights into TMCO4 function through:

• Independent Component Analysis (ICA): This statistical approach, as applied to breast cancer proteogenomics, can extract mechanistic information by identifying co-regulated protein networks involving TMCO4. The method has been shown to extract more specific information about biological processes compared to traditional clustering approaches .

• Activity signature correlation: By establishing associations between TMCO4 expression patterns and clinical features in patient cohorts, researchers can uncover potential functional relevance. The approach used in breast cancer research identified significant associations between protein expression signatures and clinical variables .

• Multi-omics data integration: Combining genomic, transcriptomic, and proteomic data to understand TMCO4 regulation across multiple biological levels, though previous research has highlighted potential redundancy in information content across different omics layers .

What are common challenges in TMCO4 protein expression and purification?

Researchers working with TMCO4 may encounter several technical challenges:

• Protein solubility issues: As a transmembrane protein, TMCO4 may present solubility challenges during purification. Consider:

  • Using mild detergents optimized for membrane proteins

  • Expressing truncated versions lacking transmembrane domains for soluble protein studies

  • Employing fusion tags that enhance solubility

• Preserving native conformation: Ensuring that recombinant TMCO4 maintains its native structure, particularly for functional studies:

  • Optimize buffer conditions to maintain protein stability

  • Use gentle purification methods to preserve oligomeric states

  • Consider co-expression with interacting partners to stabilize complexes

• Verifying expression levels: For both overexpression and knockout validation:

  • Use multiple detection methods (western blot, qPCR, immunofluorescence)

  • Include appropriate controls to confirm specificity

  • Validate antibodies thoroughly to ensure detection of the correct protein

How can researchers differentiate between direct and indirect effects in TMCO4 knockout studies?

To distinguish direct TMCO4-dependent phenotypes from secondary effects:

• Rescue experiments: Re-introduce wild-type TMCO4 into knockout cells to confirm that phenotypes are directly due to TMCO4 absence.

• Domain-specific mutants: Introduce TMCO4 variants with specific domain deletions or mutations to identify critical functional regions.

• Temporal control: Use inducible TMCO4 knockout systems to distinguish immediate versus long-term adaptive responses.

• Dose-dependent effects: Employ partial knockdown approaches using siRNA or shRNA with varying efficiencies to establish dose-response relationships.

• Acute inhibition: Where available, use specific inhibitors of TMCO4 function to compare acute versus genetic inhibition phenotypes.

What parameters should be considered when designing experiments with recombinant TMCO4?

When working with recombinant TMCO4, researchers should consider:

• Expression system selection: Choose between:

  • Prokaryotic systems (E. coli) for high yield but potential folding issues

  • Insect cells for improved folding of complex proteins

  • Mammalian cells (like HEK293) for native post-translational modifications

• Tag selection and position: Consider:

  • N-terminal versus C-terminal tags based on predicted topology

  • Tag size and potential interference with function

  • Cleavable tags for downstream applications requiring native protein

• Codon optimization: For expression in heterologous systems, codon optimization can significantly improve yield.

• Deletion mutant design: When creating truncation variants:

  • Carefully analyze domain boundaries to avoid disrupting critical structural elements

  • Consider the design approach used for TMCC3, where deletion mutants targeting specific coiled-coil regions revealed their contribution to oligomerization .