Recombinant Mouse Transmembrane protein 106C (Tmem106c)

What is Tmem106c and how does it relate to other TMEM106 family proteins?

Tmem106c is a transmembrane protein belonging to the TMEM106 family, which includes the better-characterized TMEM106A and TMEM106B proteins. While specific information on Tmem106c is more limited, bioinformatic analysis predicts it to be a type II transmembrane protein with a conserved transmembrane domain, similar to other family members . TMEM106B has been established as a risk factor for frontotemporal dementia (FTD) caused by GRN mutation, with research showing that elevated levels increase FTD risk . TMEM106A has been characterized for its immunobiological functions on mouse macrophages, where it can trigger activation and polarization toward an M1-like phenotype . Recent research has also investigated TMEM106C expression across multiple cancer types, particularly in liver hepatocellular carcinoma (LIHC) .

What expression patterns have been documented for Tmem106c in mouse tissues?

While the search results don't provide specific information about Tmem106c expression patterns, related family member Tmem106a shows variable expression across mouse tissues. High expression levels of TMEM106C have been investigated across 34 different cancer types using normalized pan-cancer RNA-sequencing methods . In contrast to Tmem106a, which shows high expression on mouse macrophage surfaces and increases with inflammatory stimulation , researchers investigating Tmem106c should conduct tissue-specific expression analysis using techniques such as RT-PCR, immunohistochemistry, or RNA-sequencing to establish expression patterns.

How is recombinant mouse Tmem106c typically produced for research applications?

Based on production methods for related proteins, recombinant mouse Tmem106c would likely be expressed in mammalian expression systems such as HEK293 cells to ensure proper post-translational modifications, particularly glycosylation. For instance, recombinant mouse TMEM106B is expressed from HEK293 with an hFc tag at the C-terminus, containing amino acid range Pro119-Gln275 . A similar approach may be applicable for Tmem106c production, potentially using a variety of tags (His, Fc, FLAG) depending on experimental needs. The protein would typically be purified using affinity chromatography followed by additional purification steps to achieve >95% purity, and quality control would include techniques such as Bis-Tris PAGE and HPLC .

What are the recommended storage and reconstitution protocols for recombinant mouse Tmem106c?

While specific information for Tmem106c is not provided, standard protocols for similar recombinant proteins suggest storage at -20°C to -80°C for 12 months from the date of receipt in lyophilized form . After reconstitution, storage at -80°C for up to 3 months is typically recommended. Reconstitution should begin with centrifuging the tube before opening, then dissolving the lyophilized protein in distilled water to a concentration exceeding 100 μg/ml . To optimize storage stability, it's advisable to aliquot the reconstituted protein into smaller quantities to minimize freeze-thaw cycles, which can degrade protein structure and function .

What are the most effective methods for studying Tmem106c function in vitro?

Based on approaches used for related proteins and TMEM106C cancer research, effective methods include:

• Gene knockout or knockdown studies using CRISPR-Cas9 or siRNA technologies

• Overexpression systems using appropriate vectors

• Protein-protein interaction studies (co-immunoprecipitation, proximity ligation assays)

• Functional assays relevant to suspected biological roles

For TMEM106C specifically, CRISPR-Cas9 gene editing has been successfully employed to construct knockout cells targeting specific exons. The guide RNA sequences for TMEM106C targeting have included: second exon (5'-CACCGTTCTTGCTTTCGCCTGCAGG-3' and 5'-AAACCCTGCAGGCGAAAGCAAGAA-3') and fourth exon (5'-CACCGTCAGTCCTTGTGGATGATGA-3' and 5'-AAACTCATCATCCACAAGGACTGA-3') . Knockout efficiency verification can be performed using qPCR with primers such as 5'-AGGGGACAGGCTACATTCCA-3' (upstream) and 5'-ACCACCAAACCAGATGCCAG-3' (downstream) .

How can researchers effectively validate antibodies for Tmem106c detection?

Proper antibody validation for Tmem106c should include multiple complementary approaches:

• Western blot analysis using recombinant Tmem106c protein as a positive control

• Analysis of specificity using knockout/knockdown cell lysates as negative controls

• Immunoprecipitation followed by mass spectrometry to confirm target specificity

• Cross-reactivity testing against other TMEM106 family members

• Validation across multiple techniques (flow cytometry, immunofluorescence, ELISA)

When developing flow cytometry protocols, researchers should follow approaches similar to those used for Tmem106a, where cells were stained with fluorophore-coupled antibodies for 1 hour at 4°C before analysis . For confocal microscopy, similar staining protocols can be employed to visualize subcellular localization, as has been done to confirm the surface expression of Tmem106a on peritoneal macrophages .

What cellular assays are recommended for investigating Tmem106c's biological functions?

Based on methodologies employed in TMEM106C cancer research, recommended assays include:

• Proliferation assays using cell counting kit-8 with measurements taken every six hours over 5 days

• Migration and invasion assays using 24-well transwell chambers

• Cell cycle analysis using flow cytometry with appropriate DNA staining

• Apoptosis detection assays using standard flow cytometry protocols

• Secretion assays measuring cytokines/chemokines if Tmem106c is suspected to have immune functions similar to Tmem106a

For signaling pathway analyses, researchers should consider examining phosphorylation of potential downstream mediators including MAPKs (ERK-1/2, JNK, p38) and NF-κB pathway components as observed with Tmem106a activation .

How can structural predictions and molecular docking be applied to study Tmem106c interactions?

Advanced structural biology approaches can enhance understanding of Tmem106c function:

• Protein structure prediction using tools like AlphaFold to generate structural models of Tmem106c

• Molecular docking simulations to investigate potential ligand interactions

• Identification of critical binding domains through in silico analysis

• Structure-guided mutational studies to validate predicted interaction sites

This approach has been successfully applied to TMEM106C research, where AlphaFold was used to predict the crystal structure of the TMEM106C protein receptor, followed by molecular docking simulations with nitidine chloride to investigate binding interactions . The interactions were visualized using discovery studio visualizer, revealing conventional hydrogen bonds, pi-donor hydrogen bonds, and pi-pi T-shaped molecular interactions .

What approaches should be considered when studying potential roles of Tmem106c in disease models?

For disease model studies involving Tmem106c, consider:

• Creation of transgenic mouse models (knockout, conditional knockout, or overexpression)

• Analysis of Tmem106c expression in existing disease models

• Therapeutic targeting approaches (similar to research on TMEM106B in FTD models)

• Correlation of Tmem106c expression with disease severity or progression

In cancer research specifically, xenograft models have been established by subcutaneously inoculating cancer cells into nude mice. For example, LIHC xenograft models were created by inoculating 10^7 SMMC7721 cells into the right axilla of six-week-old nude mice to study TMEM106C's role and potential therapeutic targeting .

How can researchers investigate potential interactions between Tmem106c and other TMEM106 family members?

To explore functional relationships between TMEM106 family proteins:

• Co-expression analysis in various tissues and cell types

• Co-immunoprecipitation studies to detect physical interactions

• Double knockout/knockdown experiments to identify compensatory mechanisms

• Comparative signaling pathway analysis

Research on TMEM106B has shown that reducing its levels has therapeutic potential in mouse models of FTD, with crossing of Grn-deficient mice to Tmem106b knockout mice resulting in rescue of behavioral defects and lysosomal dysfunction . Similar approaches could be considered for investigating functional relationships between Tmem106c and other family members.

What bioinformatic approaches can be used to predict Tmem106c function and interactions?

Comprehensive bioinformatic strategies include:

• Expression correlation analysis across tissues and disease states

• Pathway enrichment analysis using tools like Gene Set Enrichment Analysis (GSEA)

• Protein-protein interaction network construction

• Single-cell RNA sequencing data analysis to identify co-expressed genes

For TMEM106C, researchers have successfully employed correlation analysis to identify LIHC overexpressed genes (OEGs) and genes correlated with TMEM106C (using Spearman correlation coefficients ≥ 0.3, P < 0.05) . Functional annotation of co-expressed genes was performed using clusterProfiler, and single-cell RNA-seq data was analyzed to confirm co-expression patterns with potential upstream transcription factors .

How do mouse and human Tmem106c/TMEM106C compare in structure and function?

While detailed comparative data is not provided in the search results, researchers should consider:

• Sequence homology analysis between mouse and human orthologs

• Conservation of key functional domains and motifs

• Comparative expression patterns across tissues

• Species-specific differences in post-translational modifications

For related family member TMEM106B, research has established it as a risk factor for frontotemporal dementia in humans, with elevated levels increasing disease risk . Comparison of mouse and human orthologs would help determine the translational relevance of findings from mouse models to human disease applications.

What are the key experimental controls that should be included when working with recombinant Tmem106c?

Critical controls include:

• Protein purity validation (>95% by techniques such as Bis-Tris PAGE and HPLC)

• Endotoxin testing (<1EU per μg by LAL method)

• Functional validation compared to native protein

• Tag-only controls to rule out tag-mediated effects

• Denatured protein controls for structural specificity

Additionally, when conducting functional studies using CRISPR-Cas9 knockout systems, verification of knockout efficiency using qPCR is essential, as demonstrated in TMEM106C research . For cellular assays, appropriate positive controls (such as LPS for immune activation studies, as used in Tmem106a research) should be included.

How can researchers distinguish between effects of Tmem106c and other TMEM106

family members in experimental systems?

To ensure specificity:

• Use highly specific antibodies validated against all family members

• Design knockout/knockdown strategies that target unique regions

• Consider compensatory mechanisms through simultaneous measurement of all family members

• Validate findings using multiple independent methodologies

Research on Tmem106a demonstrated macrophage activation and polarization toward M1 phenotype, with upregulation of markers like CD80, CD86, CD69, and MHC II, plus induction of TNF-α, IL-1β, IL-6, CCL2, and NO release . Researchers should determine whether Tmem106c has similar immunomodulatory effects or distinct functions to properly differentiate between family members.

What evidence exists for Tmem106c's role in cancer biology and potential therapeutic applications?

Recent research has investigated TMEM106C across 34 cancer types, with particular focus on liver hepatocellular carcinoma (LIHC):

• Expression analysis showed differential expression patterns across cancer types

• Functional studies using TMEM106C knockout in LIHC cell lines revealed effects on:

  • Cell proliferation

  • Migration and invasion capabilities

  • Cell cycle progression and apoptosis

Molecular docking studies have identified potential interactions between TMEM106C and anti-cancer compounds like nitidine chloride, suggesting possible therapeutic applications . Correlation analysis in CellMiner has been employed to predict additional candidate compounds that might target TMEM106C, providing avenues for future drug development .

How might single-cell sequencing technologies advance our understanding of Tmem106c function?

Single-cell technologies offer several advantages for Tmem106c research:

• Cell-type specific expression profiling across tissues

• Identification of rare cell populations with unique Tmem106c expression patterns

• Trajectory analysis to understand dynamic regulation during differentiation or disease progression

• Co-expression network analysis at single-cell resolution

TMEM106C researchers have already begun utilizing single-cell RNA-seq data (GSE112271) to confirm co-expression patterns between TMEM106C and its upstream transcriptional factors , demonstrating the value of this approach for understanding regulatory mechanisms.

What are the known interactions between Tmem106c and the tumor microenvironment?

Research has begun exploring TMEM106C's relationship with the tumor microenvironment:

• Infiltration levels of immune and stromal cells in the tumor microenvironment were calculated across 33 cancer types using the microenvironment cell populations counter algorithm

• Spearman correlation coefficients were employed to evaluate associations between TMEM106C expression and tumor microenvironment components

Given that related family member Tmem106a activates macrophages and polarizes them toward an M1-like phenotype through MAPKs and NF-κB pathway activation , researchers should investigate whether Tmem106c similarly influences immune cell populations within the tumor microenvironment, potentially affecting anti-tumor immunity.