Recombinant Mouse Transmembrane protein 106A (Tmem106a)

What is the basic structure and cellular localization of mouse Tmem106a?

Mouse Tmem106a is a type II transmembrane protein with 261 amino acids and an isoelectric point of 7.04. It features a conserved transmembrane domain located at amino acids 93-115. The protein contains an intracellular N-terminal domain, a single transmembrane domain, and an extracellular C-terminal domain . The gene is located on chromosome 11 and encompasses nine exons and eight introns. Comparative analysis demonstrates that Tmem106a is highly conserved across various animal species .

Experimentally, Tmem106a's transmembrane nature can be verified through subcellular fractionation followed by Western blotting or through immunofluorescence microscopy with membrane-specific markers. Additionally, domain-specific antibodies can help determine the orientation of the protein within the membrane.

What is the tissue expression pattern of mouse Tmem106a?

Tmem106a exhibits a distinct expression pattern across mouse tissues. High mRNA expression levels are observed in lung, kidney, intestine, and lymphoid nodes as determined by semi-quantitative RT-PCR . At the cellular level, Tmem106a is abundantly expressed in myeloid cells, particularly macrophages . Within the immune system, Tmem106a protein is detected on the surface of mouse macrophages and is highly expressed in PMA-stimulated THP-1 macrophages .

For comprehensive expression profiling, researchers should employ a combination of qRT-PCR for transcript quantification and Western blotting or immunohistochemistry for protein detection across various tissues and cell types.

How is Tmem106a expression regulated during inflammatory responses?

Tmem106a expression demonstrates dynamic regulation during inflammatory responses. Upon lipopolysaccharide (LPS) stimulation, Tmem106a levels significantly increase in mouse bone marrow-derived macrophages (mBMDMs) at both mRNA and protein levels . This upregulation suggests Tmem106a plays a role in the inflammatory response cascade. Flow cytometry analysis has shown that treatment with LPS (100 ng/ml) enhances TMEM106A protein expression in PMA-stimulated THP-1 cells .

To study this regulation experimentally, researchers can use real-time qPCR for temporal expression analysis following inflammatory stimuli and examine various signaling pathway inhibitors to identify the regulatory mechanisms controlling Tmem106a expression during inflammation.

What role does Tmem106a play in macrophage activation and polarization?

Tmem106a serves as a trigger for macrophage activation with influence toward M1 polarization through activation of the MAPKs and NF-κB pathways . Activation of Tmem106a by anti-Tmem106a antibody stimulation upregulates surface expression of activation markers including CD80, CD86, CD69, and MHC II on macrophages . This activation induces the release of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, CCL2, and nitric oxide (NO), but not the anti-inflammatory cytokine IL-10 .

Mechanistically, Tmem106a activation significantly increases inducible nitric oxide synthase (iNOS) production and STAT1 phosphorylation, while having no effect on ARGINASE-1 or STAT6 phosphorylation, indicating polarization toward an M1-like phenotype . Further investigation has shown that anti-Tmem106a stimulation increases phosphorylation of ERK-1/2, JNK, p38 MAPK, NF-κB p65, and IKKα/β, and promotes nuclear translocation of the cytosolic NF-κB p65 subunit .

How does Tmem106a affect the inflammatory response during sepsis?

Tmem106a plays a protective role during inflammatory responses and sepsis. Tmem106a knockout (KO) mice demonstrate increased sensitivity to LPS-induced septic shock compared to wild-type mice . Following LPS challenge, Tmem106a KO mice display significantly higher serum levels of pro-inflammatory cytokines TNF-α, IL-6, and IFN-β . Histopathological examination reveals increased inflammatory cell infiltration, hemorrhage, and interstitial pneumonitis in the lungs of Tmem106a KO mice after LPS challenge .

Bone marrow transplantation experiments indicate that macrophages are key mediators of Tmem106a's anti-inflammatory effects. Chimeric mice reconstituted with Tmem106a-deficient bone marrow produce higher levels of inflammatory cytokines when challenged with LPS compared to those reconstituted with wild-type bone marrow .

What is the role of Tmem106a in viral infection?

TMEM106A functions as a host antiviral factor that inhibits the release of multiple enveloped viruses from the cell surface . The transmembrane domain (TM) and extracellular C-terminal domain (CD) of TMEM106A are critical for its antiviral activity . Truncation mutant analysis demonstrated that the TMC mutant (composed of TM and CD) exhibits antiviral activity comparable to the full-length protein, while other mutants lacking the membrane localization capability show little effect on viral production .

This suggests that membrane localization is essential for TMEM106A's antiviral function. The protein appears to specifically target the release process of enveloped viruses, providing a potential cellular defense mechanism against viral infection.

How is Tmem106a involved in cancer progression?

Tmem106a functions as a tumor suppressor in several types of cancer. Expression of TMEM106A is markedly downregulated in hepatocellular carcinoma (HCC) compared to normal liver tissue . Similarly, decreased expression has been observed in gastric cancer, renal cancer, and non-small-cell lung carcinoma . Restoration of TMEM106A expression significantly inhibits tumor cell proliferation and induces cell death .

In HCC specifically, TMEM106A overexpression suppresses malignant behavior of HCC cells in vitro and decreases tumorigenicity and lung metastasis in vivo . Mechanistically, TMEM106A inhibits epithelial-mesenchymal transition (EMT) of HCC cells through inactivation of the Erk1/2/Slug signaling pathway .

What is the relationship between TMEM106A methylation and cancer prognosis?

Tumor-specific DNA methylation of TMEM106A is frequently observed in tumor tissues from HCC patients . Pyrosequencing analysis reveals a significant relationship between TMEM106A methylation and downregulation of protein expression . Receiver operating characteristic (ROC) curve analysis demonstrates that TMEM106A methylation in tumor samples differs significantly from that in non-malignant adjacent tissues of HCC patients .

How can researchers generate and validate Tmem106a knockout models?

Researchers can generate Tmem106a knockout mice using CRISPR/Cas9 genome editing. A specific approach documented in the literature involves using guide RNA (5′-GCTCACCTCTCGGAAGGATG-3′) targeting close to the start codon in exon 3 of mouse Tmem106a . Mouse embryos (C57BL/6J × FVB/N) can be injected with gRNAs and Cas9 mRNA. Editing should be confirmed by sequencing PCR products from genomic DNA.

For verification, genotyping can be performed by PCR using specific oligonucleotides (5′-TTCACTTGCAGAAATCCCTTAAA-3′ and 5′-GCCAGCCTGAGACTGCATAC-3′), which will yield products of different sizes for wild-type (577 bp) and mutant (429 bp) alleles . Knockout efficiency should be validated at protein level using Western blot and immunohistochemistry.

What experimental approaches can be used to study Tmem106a in macrophage activation?

Several complementary approaches can be employed to study Tmem106a's role in macrophage activation:

• Gene expression analysis: qRT-PCR to quantify Tmem106a mRNA levels and expression of M1 markers (TNF-α, IL-6, IL-1β, iNOS) and M2 markers (Arginase-1, IL-10) following various stimuli.

• Protein expression and localization: Flow cytometry to assess cell surface expression of Tmem106a and activation markers (CD80, CD86, MHC II). Immunofluorescence microscopy to visualize subcellular localization.

• Functional assays: ELISA to measure cytokine production (TNF-α, IL-6, IL-1β, IL-10), and Griess assay to quantify nitric oxide production as indicators of macrophage activation.

• Signaling pathway analysis: Western blotting to detect phosphorylation of key signaling molecules including MAPKs (ERK1/2, JNK, p38), NF-κB, STAT1, and STAT6 .

• Loss and gain of function: siRNA knockdown or CRISPR-mediated knockout of Tmem106a, followed by stimulation with LPS or other activators to assess the functional impact on macrophage responses .

How can Tmem106a methylation be analyzed in tumor samples?

Analysis of Tmem106a methylation in tumor samples can be performed using several techniques:

• Methylation-Specific PCR (MSP): This technique uses primers designed to specifically amplify either methylated or unmethylated DNA after bisulfite conversion. It provides a qualitative assessment of methylation status at specific CpG sites .

• Pyrosequencing: This quantitative method allows for precise measurement of the degree of methylation at individual CpG sites. It provides percentage values of methylation at each analyzed position, enabling more detailed analysis than MSP .

• Correlation analysis: Combined analysis of methylation data with expression data (qRT-PCR, immunohistochemistry) to establish relationships between methylation and gene/protein expression.

• Demethylation experiments: Treatment of hypermethylated cell lines with demethylating agents (e.g., 5-Aza-2′-deoxycytidine) to confirm that methylation is responsible for reduced Tmem106a expression .

• Clinical correlation: Analysis of methylation status in relation to clinical parameters (tumor size, survival) to establish prognostic significance .

What is the potential of Tmem106a as a therapeutic target for inflammatory diseases?

Given Tmem106a's role in regulating inflammatory responses, it presents a potential therapeutic target for inflammatory diseases. Tmem106a knockout mice show enhanced susceptibility to LPS-induced septic shock and elevated production of pro-inflammatory cytokines . This suggests that enhancing Tmem106a function might mitigate excessive inflammatory responses in conditions like sepsis.

Therapeutic approaches could include:

• Development of agonists to enhance Tmem106a function during excessive inflammation

• Gene therapy approaches to restore or increase Tmem106a expression

• Small molecule modulators of the Tmem106a signaling pathway

Research methodologies to explore this potential should combine in vitro studies with primary macrophages and in vivo studies using relevant disease models, such as LPS-induced endotoxemia or cecal ligation and puncture (CLP) for sepsis .

How might Tmem106a be exploited in cancer diagnostics and therapeutics?

Tmem106a's tumor suppressor function and its epigenetic regulation through methylation offer potential applications in cancer diagnostics and therapeutics:

Diagnostic applications:

• Development of methylation-specific assays for TMEM106A as a biomarker for cancer detection and prognosis, particularly in HCC

• Use of TMEM106A expression levels as a prognostic indicator in various cancers

Therapeutic approaches:

• Demethylating agents to restore TMEM106A expression in tumors with hypermethylation

• Gene therapy to reintroduce TMEM106A in cancers with reduced expression

• Small molecule activators of pathways downstream of TMEM106A, particularly targeting the Erk1/2/Slug signaling pathway in HCC

Research should focus on validating TMEM106A methylation as a biomarker in large patient cohorts and developing targeted approaches to restore its tumor suppressor function in specific cancer types.

What are the optimal methods for producing recombinant mouse Tmem106a?

Production of high-quality recombinant mouse Tmem106a requires careful consideration of expression systems and purification strategies:

• Expression system selection: Mammalian expression systems (HEK293, CHO cells) are recommended for proper folding and post-translational modifications of membrane proteins. For studies requiring only specific domains, bacterial systems may be sufficient.

• Expression constructs: Design constructs with appropriate tags (His, FLAG, myc) for detection and purification. Consider using truncated versions (such as the extracellular domain) for easier expression and purification.

• Purification strategy: For full-length protein, detergent solubilization followed by affinity chromatography is typically required. For secreted domains, standard protein purification techniques can be employed.

• Quality control: Verify protein integrity by SDS-PAGE, Western blot, and mass spectrometry. Functional validation through binding assays or cell-based activity assays is essential.

• Storage conditions: Optimize buffer composition and storage conditions to maintain protein stability and functionality.

What are the key considerations for developing antibodies against Tmem106a?

Development of specific and effective antibodies against Tmem106a requires:

• Antigen selection: Choose unique epitopes with high antigenicity, preferably in the extracellular domain for applications requiring detection of the native protein on cell surfaces. Peptide antibodies can be generated using boxed sequences identified in the literature .

• Antibody type: Consider the application when choosing between polyclonal and monoclonal antibodies. Polyclonal antibodies provide broader epitope recognition, while monoclonal antibodies offer higher specificity.

• Validation methods:

  • Western blot using tissue samples known to express Tmem106a (lung, kidney, lymphoid tissue)

  • Immunohistochemistry on tissue sections with known expression patterns

  • Flow cytometry on cells with confirmed Tmem106a expression

  • Knockout or knockdown controls to confirm specificity

  • Blocking experiments to validate functional antibodies

• Cross-reactivity: Test for cross-reactivity with related proteins, particularly other TMEM family members, to ensure specificity.

• Applications optimization: Optimize conditions (dilutions, blocking agents, incubation times) for each specific application (Western blot, immunohistochemistry, flow cytometry, functional studies).