Recombinant Mouse CKLF-like MARVEL transmembrane domain-containing protein 6 (Cmtm6)

What is CKLF-like MARVEL transmembrane domain-containing protein 6 (Cmtm6)?

Cmtm6 belongs to the chemokine-like factor gene superfamily, a novel family that shares similarities with both the chemokine and transmembrane 4 superfamilies of signaling molecules. The protein contains 4 transmembrane segments and consists of 183 amino acids. Mouse Cmtm6 shares approximately 88.5% sequence similarity with human CMTM6, indicating high evolutionary conservation of this protein . The gene is part of a cluster of chemokine-like factor genes located on a specific chromosome, and while it is widely expressed across many tissues, its precise physiological function is still being elucidated through ongoing research.

What is the expression pattern of Cmtm6 in normal mouse tissues?

Mouse Cmtm6, similar to its human counterpart, demonstrates widespread tissue expression with particular abundance in leukocytes, placenta, and testis. Northern blot analyses have detected transcripts of approximately 3.88 kb in these tissues, with smaller transcript variants also observed in leukocytes . When conducting expression studies, it's methodologically important to establish baseline expression levels across multiple tissue types using qRT-PCR with appropriate housekeeping genes for normalization. This comparative approach allows researchers to identify tissues with physiologically relevant expression levels before proceeding with functional studies.

What are the most reliable methods for detecting and quantifying recombinant mouse Cmtm6 in experimental samples?

For quantitative detection of mouse Cmtm6, sandwich ELISA represents a highly specific and sensitive method. Based on human CMTM6 detection parameters, which can serve as a reference point, typical detection ranges fall between 61.7-5000 pg/mL with sensitivity around 2.9 pg/mL . When implementing this method:

• Sample preparation is critical - serum, plasma, and other biological fluids require appropriate dilution series to ensure measurements fall within the linear range of detection

• Include both positive and negative controls in each assay to validate results

• Consider cross-reactivity potential, especially when analyzing complex biological samples

• For tissue samples, optimize protein extraction protocols with appropriate detergents that preserve transmembrane protein integrity

Western blotting provides qualitative confirmation but requires careful optimization of lysis conditions to effectively solubilize this multi-pass transmembrane protein.

How should researchers design knockdown or overexpression experiments for mouse Cmtm6?

When manipulating Cmtm6 expression levels for functional studies, consider these methodological approaches:

• For knockdown studies:

  • Use at least 2-3 different shRNA sequences targeting distinct regions of Cmtm6 mRNA to control for off-target effects

  • Validate knockdown efficiency at both mRNA (qRT-PCR) and protein levels (western blot)

  • Include appropriate non-targeting shRNA controls

• For overexpression studies:

  • Consider both constitutive and inducible expression systems

  • Include epitope tags that don't interfere with protein trafficking

  • Validate expression levels and proper subcellular localization through immunofluorescence

The effectiveness of Cmtm6 manipulation should be carefully validated before proceeding with functional assays. Research has shown that shRNA-mediated knockdown of CMTM6 can effectively alter phenotypes in cancer models, suggesting similar approaches would be valid for mouse Cmtm6 studies .

What cellular pathways and processes is mouse Cmtm6 known to regulate?

Mouse Cmtm6, like its human counterpart, appears to function in several key cellular processes:

• Protein trafficking and stability:

  • Regulates the intracellular trafficking of cell surface proteins

  • Maintains expression of specific membrane proteins by preventing their degradation

• Metabolic regulation:

  • Influences glucose uptake through regulation of glucose transporters

  • Impacts cellular glycolysis and the Warburg effect in cancer cells

• Cell cycle regulation:

  • Interacts with cell cycle regulatory proteins such as p21

  • Can protect p21 from ubiquitination mediated by various E3 ligase complexes (SCF SKP2, CRL4 CDT2, APC/C CDC20), thereby influencing cell cycle progression in a cell-cycle-independent manner

• Immune regulation:

  • Maintains expression levels of immune checkpoint proteins

  • Influences tumor immune microenvironment through regulation of cytokine/chemokine secretion

When investigating these pathways, researchers should design experiments that can distinguish direct from indirect effects of Cmtm6 through appropriate controls and time-course analyses.

How does mouse Cmtm6 compare with human CMTM6 in terms of function and experimental applications?

Despite the high sequence similarity (88.5%) between mouse Cmtm6 and human CMTM6 , researchers should be aware of potential functional differences when translating findings between species:

• Conserved functions:

  • Both are involved in protein trafficking mechanisms

  • Both influence cell proliferation pathways

  • Both interact with the immune regulatory system

• Experimental considerations:

  • When using mouse models to study human disease relevance, validate key findings in human cell systems

  • Consider species-specific interaction partners through comparative proteomic approaches

  • For antibody-based detection, carefully validate cross-reactivity between species

Studies have demonstrated that manipulation of CMTM6 in both human and mouse cancer cell lines produces similar phenotypic outcomes, suggesting substantial functional conservation .

How can researchers effectively investigate Cmtm6's role in protein trafficking and membrane localization?

To elucidate Cmtm6's function in protein trafficking, implement these methodological approaches:

• Subcellular fractionation and co-localization studies:

  • Use confocal microscopy with markers for endosomes, lysosomes, and plasma membrane

  • Employ synchronized pulse-chase experiments with fluorescently tagged target proteins

  • Quantify co-localization coefficients for statistical analysis

• Protein trafficking pathway analysis:

  • Investigate specific Rab-dependent transport mechanisms, particularly focusing on Rab11-dependent recycling pathways

  • Utilize dominant-negative Rab protein mutants to dissect specific trafficking steps

  • Employ super-resolution microscopy for detailed visualization of trafficking events

• Protein stability assessment:

  • Implement cycloheximide (CHX) chase assays (10 μM) to block protein synthesis and track degradation kinetics

  • Use proteasome inhibitors (e.g., MG132 at 20 μM for 6 hours) to determine degradation pathways

  • Perform ubiquitination assays with immunoprecipitation to directly assess ubiquitin conjugation

These approaches have successfully revealed that CMTM6 maintains Rab11 mRNA levels, Rab11 activity, and Rab11-dependent transport of membrane proteins such as Glut1 to the plasma membrane .

What experimental approaches should be used to investigate Cmtm6's role in tumor growth and immune regulation?

For studying Cmtm6's functions in tumor progression and immune regulation:

• In vitro tumor cell analysis:

  • Compare 2D and 3D culture systems (e.g., ultra-low attachment cell culture plates)

  • Utilize Live-Dead Cell Staining Kits to assess cell viability in 3D tumoroids

  • Perform BrdU incorporation assays to distinguish between proliferation and apoptosis effects

• Immune interaction studies:

  • Analyze secreted cytokine/chemokine profiles using targeted proteomics

  • Co-culture systems with immune cells to assess direct interaction effects

  • Flow cytometry to characterize immune cell activation states

• In vivo tumor models:

  • Use both immunocompromised models (to assess direct tumor effects) and immunocompetent models (to assess immune interaction)

  • Implement tissue-specific inducible knockout/overexpression systems

  • Perform detailed characterization of tumor microenvironment through multi-parameter immunohistochemistry or single-cell sequencing approaches

Research has demonstrated that CMTM6 knockdown inhibits CRC tumor growth in immunocompromised mice and CRC liver metastasis in immunocompetent mice, suggesting both direct tumor cell effects and immune regulatory functions .

How should researchers interpret conflicting data regarding Cmtm6's role in cancer progression?

The literature contains seemingly contradictory findings regarding CMTM6's role in cancer, with evidence supporting both tumor-promoting and tumor-suppressing functions. When encountering conflicting data:

These apparent contradictions highlight the context-dependent nature of CMTM6 function and underscore the importance of comprehensive experimental design when studying this protein.

What are the key considerations when interpreting gene expression data for Cmtm6 across different experimental systems?

When analyzing Cmtm6 expression data:

• Technical considerations:

  • Different detection methods (qRT-PCR, RNA-seq, microarray) may yield varying results

  • Verify expression changes using multiple methodologies

  • Consider transcript variants and appropriate primer design

• Analytical approaches:

  • Implement Gene Set Enrichment Analysis (GSEA) with appropriate statistical thresholds (p < 0.05, normalized enrichment score > 1)

  • Use coexpression analysis to identify functionally related genes

  • Apply Gene Ontology (GO) and pathway analysis (KEGG) to contextualize expression changes

• Integration with public databases:

  • Compare findings with TCGA, GTEx, and Oncomine data

  • Utilize resources like GEPIA2 for cross-validation

  • Consider both mRNA and protein expression using resources like The Human Protein Atlas

Researchers have successfully employed these approaches to reveal that CMTM6 expression correlates with specific immune cell infiltration patterns and chemokine expression profiles, providing context for functional studies .

What are the most promising avenues for future research on mouse Cmtm6?

Based on current knowledge gaps, these research directions hold significant potential:

• Structural biology approaches:

  • Determine the three-dimensional structure of Cmtm6 to understand its transmembrane organization

  • Characterize the MARVEL domain's contribution to protein-protein interactions

  • Identify critical residues for functional interactions through site-directed mutagenesis

• Systems biology integration:

  • Develop comprehensive protein-protein interaction networks using proximity labeling approaches

  • Implement CRISPR-Cas9 screens to identify synthetic lethal interactions

  • Apply multi-omics approaches to understand Cmtm6's position in cellular regulatory networks

• Translational applications:

  • Investigate Cmtm6 as a potential target for modulating immune checkpoint pathways

  • Explore the relationship between Cmtm6 expression and treatment response

  • Develop conditional knockout mouse models to assess tissue-specific functions

These directions build upon established findings while addressing fundamental gaps in our understanding of Cmtm6 biology and potential therapeutic applications.

How can researchers effectively study the interaction between Cmtm6 and the immune system in mouse models?

To investigate immune regulatory functions of Cmtm6:

• Experimental model considerations:

  • Compare results between immunocompetent and immunodeficient mouse models

  • Utilize tissue-specific and inducible Cmtm6 knockout/overexpression systems

  • Consider humanized mouse models for translational relevance

• Analytical approaches:

  • Implement multi-parameter flow cytometry to characterize immune cell populations

  • Use single-cell RNA sequencing to identify cell-type specific responses

  • Analyze cytokine/chemokine profiles in tumor microenvironment

• Functional validation:

  • Perform immune cell depletion studies to identify critical immune populations

  • Combine with immune checkpoint blockade to assess potential synergistic effects

  • Utilize ex vivo immune cell assays to validate in vivo findings

Research has shown that CMTM6 expression positively correlates with markers of T helper cells (Th1, Th2, Tfh, Th17, and Tregs) and macrophages (M1 and M2), suggesting it plays a role in recruiting and regulating these immune cell populations .