Recombinant Human Transmembrane protein 106A (TMEM106A)

Where is TMEM106A primarily expressed in human and mouse tissues?

TMEM106A is constitutively expressed on the plasma membrane of macrophages and shows high expression in myeloid cells . Expression analysis by qRT-PCR has confirmed that TMEM106A is highly expressed in mouse macrophages compared to other immune cells . In PMA-stimulated THP-1 human macrophage cells, high mRNA levels of TMEM106A have been detected . The expression varies across different cell types, with some cell lines showing inducible expression upon interferon treatment. For example, 293A, 293A-SCARB2, and Vero cells all show increased TMEM106A expression following type I interferon (IFN-α2b) treatment .

How is TMEM106A expression regulated?

TMEM106A expression is significantly upregulated by type I interferon stimulation, classifying it as an interferon-stimulated gene (ISG) . Time-course experiments have shown that TMEM106A mRNA levels increase progressively following IFN-α2b treatment in both 293A-SCARB2 and Vero cell lines . Additionally, lipopolysaccharide (LPS) stimulation enhances TMEM106A expression in macrophages. In mouse bone marrow-derived macrophages (BMDMs), LPS treatment significantly increases Tmem106a mRNA levels . Similarly, in PMA-stimulated THP-1 cells, LPS (100 ng/ml) treatment further enhances TMEM106A protein expression as confirmed by flow cytometry analysis . This dual regulation by both type I interferons and LPS suggests that TMEM106A plays important roles in both antiviral and antibacterial immune responses.

What methods can be used to measure TMEM106A expression?

Several complementary techniques can be employed to quantify TMEM106A expression at both mRNA and protein levels:

RNA level detection:

• RT-qPCR is the most commonly used method for measuring TMEM106A mRNA levels. RNA extraction is typically performed using TRIzol reagent, followed by DNase treatment to remove genomic DNA contamination . cDNA synthesis using reverse transcriptase precedes qPCR analysis.

• For normalization, GAPDH mRNA is commonly used as an internal control .

Protein level detection:

• Western blotting can be used to assess TMEM106A protein levels, with appropriate antibodies against human or mouse TMEM106A.

• Flow cytometry provides a quantitative measure of TMEM106A protein expression at the single-cell level, particularly useful for analyzing expression in specific cell populations .

• Immunofluorescence microscopy can visualize the subcellular localization of TMEM106A and its colocalization with interacting partners .

How can TMEM106A function be manipulated in experimental settings?

Overexpression systems:

• Plasmid-based transient transfection: TMEM106A cDNA can be cloned into expression vectors (e.g., pcDNA4) and transfected into cells using Lipofectamine 2000 or other transfection reagents .

• Inducible expression systems: Doxycycline-inducible TMEM106A expression has been established in THP-1 cells to study its antiviral activity .

Knockdown/knockout approaches:

• RNA interference: shRNA targeting TMEM106A can be delivered using vectors like pSUPER-GFP-106A-shRNA, with knockdown efficiency verified by RT-qPCR and western blotting .

• CRISPR-Cas9 gene editing: TMEM106A knockout cells have been generated using CRISPR-Cas9 technology with specific sgRNAs targeting the TMEM106A gene .

• Knockout mice: Tmem106a knockout mice have been created to study its function in vivo, particularly in the context of inflammatory responses .

Domain mapping and mutational analysis:

• Truncated forms of TMEM106A can be generated to map functional domains. For example, studies have analyzed the antiviral activity of the extracellular region versus the cytoplasmic domain .

What experimental approaches can be used to study TMEM106A interactions with other proteins?

Co-immunoprecipitation (Co-IP):

• TMEM106A protein interactions can be studied by co-expressing tagged versions of TMEM106A (e.g., myc-tagged) with potential binding partners, followed by immunoprecipitation and detection of co-precipitated proteins .

• For example, interaction between TMEM106A and HIV-1 gp120 has been demonstrated by co-expressing myc-tagged TMEM106A with FLAG-tagged gp120 or gp160 in 293T cells, followed by immunoprecipitation and western blotting .

Confocal microscopy for colocalization:

• Fluorescently tagged TMEM106A can be co-expressed with potential interacting partners to visualize their colocalization in cells .

• This approach has been used to demonstrate colocalization of TMEM106A with SCARB2, providing evidence for their physical association .

Structural analysis:

• Nuclear magnetic resonance (NMR) spectroscopy has been employed to characterize the conformational properties of the TMEM106A cytoplasmic domain, revealing its intrinsically disordered nature with weakly populated secondary structure elements .

• Three-dimensional NMR spectra including CCC(CO)NH, HN(CO)CACB, HSQC-TOCSY, and HSQC-NOESY have been used for detailed structural characterization .

What is the role of TMEM106A in antiviral immunity?

TMEM106A functions as an interferon-induced antiviral factor with activity against both enveloped and non-enveloped viruses:

Against enveloped viruses:

• TMEM106A inhibits human immunodeficiency virus type-1 (HIV-1) by trapping viral particles and preventing their release from the cell surface .

• The mechanism appears similar to that of the HIV-1-releasing inhibitory protein BST-2, depending on interactions between the plasma membrane and virion membrane .

• Doxycycline-inducible expression of TMEM106A in THP-1 cells inhibits the replication of HIV-1 strain NL4-3-R3A .

Against non-enveloped viruses:

• TMEM106A restricts enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) infections .

• It specifically blocks SCARB2-mediated viral infection by interfering with virus binding to host cells .

• TMEM106A associates with SCARB2 (scavenger receptor class B member 2), a known receptor for EV-A71, and prevents viral attachment .

How does TMEM106A inhibit EV-A71 infection?

TMEM106A employs a specific mechanism to block EV-A71 infection through interaction with the viral receptor SCARB2:

• Association with SCARB2: TMEM106A physically associates with SCARB2, a type-III membrane protein with a 400 amino acid luminal domain that serves as a receptor for EV-A71 .

• Blocking antibody accessibility: Colocalization of TMEM106A with SCARB2 blocks the accessibility of antibodies targeting the luminal domain of SCARB2, particularly antibodies directed against regions of helices 2, 5, and 14, suggesting these regions are occupied by TMEM106A .

• Interference with viral binding: This association specifically interferes with EV-A71 binding to host cells via SCARB2, thereby preventing viral entry .

• Membrane anchoring requirement: The antiviral activity depends on correct membrane anchoring of the extracellular region of TMEM106A, highlighting the importance of proper positioning for the TMEM106A-SCARB2 interaction .

• Specificity for SCARB2-mediated infection: TMEM106A blocks SCARB2-mediated viral infection but does not affect infections mediated by other receptors like PSGL1 .

How is the antiviral function of TMEM106A against HIV-1 antagonized?

HIV-1 has evolved a mechanism to counteract TMEM106A restriction:

• Envelope glycoprotein antagonism: HIV-1 envelope glycoprotein (Env) serves as an antagonist of TMEM106A-mediated restriction .

• Direct interaction with gp120: The HIV-1 surface glycoprotein gp120 interacts with the extracellular domain of TMEM106A .

• Interference with TMEM106A intermolecular interactions: This interaction may disrupt the intermolecular association of TMEM106A molecules required for viral restriction .

• Co-IP confirmation: Co-immunoprecipitation assays have demonstrated that TMEM106A interacts with both gp120 and unprocessed gp160 .

This antagonism represents a viral countermeasure to overcome host restriction factors, highlighting the evolutionary arms race between viruses and host immunity.

How does TMEM106A affect macrophage polarization and function?

TMEM106A plays a critical role in regulating macrophage activation and inflammatory responses:

• Regulation of M1 polarization: TMEM106A negatively regulates the polarization of macrophages toward the pro-inflammatory M1 phenotype .

• Impact on activation markers: Deletion of Tmem106a in mouse macrophages enhances the expression of activation markers including CD80, CD86, and MHC-II upon LPS stimulation .

• Regulation of inflammatory cytokines: Tmem106a ablation leads to upregulation of pro-inflammatory cytokines and mediators including TNF-α, IL-6, IFN-β, and inducible nitric oxide synthase (iNOS) in response to LPS .

• Signaling pathway modulation: TMEM106A negatively regulates MAPK and NF-κB signaling pathways, which are critical for inflammatory gene expression .

• In vivo relevance: Tmem106a knockout mice show increased sensitivity to LPS-induced septic shock compared to wild-type mice, indicating that TMEM106A serves as a brake on excessive inflammatory responses .

What experimental models are available to study TMEM106A in inflammation?

In vitro models:

• Primary cell cultures: Bone marrow-derived macrophages (BMDMs) from wild-type and Tmem106a knockout mice can be compared for inflammatory responses to stimuli like LPS .

• Cell lines: PMA-stimulated THP-1 human macrophage cells express TMEM106A and can be used to study its function through overexpression or knockdown approaches .

In vivo models:

• Tmem106a knockout mice: These mice display enhanced sensitivity to LPS-induced septic shock and can be used to study the role of TMEM106A in systemic inflammatory conditions .

• Macrophage-specific deletion: Models with macrophage-specific deletion of Tmem106a help distinguish its role in macrophages from effects in other cell types .

Clinical relevance:

• Peripheral monocytes from patients with sepsis show altered TMEM106A levels, suggesting its involvement in human inflammatory diseases .

What is known about TMEM106A's role in cancer?

TMEM106A has been identified as a tumor suppressor gene with altered expression in several cancer types:

• Downregulation in multiple cancers: TMEM106A expression is decreased in gastric cancer, renal cancer, and non-small-cell lung carcinoma .

• Antiproliferative effects: Restoration of TMEM106A expression can significantly inhibit tumor cell proliferation and induce cell death .

• Potential mechanisms: While the exact mechanisms remain to be fully elucidated, TMEM106A's role in modulating inflammatory signaling pathways such as MAPK and NF-κB may contribute to its tumor-suppressive functions .

Is there a connection between TMEM106A and neurodegenerative diseases?

While the search results don't directly link TMEM106A to neurodegenerative diseases, there is information about TMEM106B, another member of the TMEM106 family:

• TMEM106B as a risk factor: TMEM106B was initially identified as a risk factor for frontotemporal lobar degeneration (FTLD) and has a general role in neurodegenerative diseases .

• Structural insights: The cytoplasmic domain of TMEM106B is intrinsically disordered, which may be relevant for understanding the structural properties of the TMEM106 family .

• Research gap: Given the similarity between TMEM106A and TMEM106B, future research might explore whether TMEM106A also plays a role in neurological conditions.

How do the structural features of TMEM106A contribute to its diverse functions?

The structure-function relationship of TMEM106A is an area of ongoing investigation:

• Domain-specific activities: Mapping studies have shown that the extracellular region of TMEM106A anchored on the plasma membrane is sufficient to inhibit virus infection . This suggests that different domains mediate specific functions.

• Intrinsically disordered regions: While direct structural data for TMEM106A's cytoplasmic domain is limited, studies on the related TMEM106B protein have demonstrated that its cytoplasmic domain is intrinsically disordered . This property may allow for versatile interactions with multiple binding partners.

• Membrane anchoring: Correct membrane anchoring of the extracellular region is required for TMEM106A's antiviral activity, highlighting the importance of proper positioning within the membrane .

• Conformational dynamics: NMR studies of related TMEM106B show that despite having a largely disordered structure, certain regions display weak propensities for secondary structure formation . These transient structural elements might serve as recognition motifs for specific interactions.

What are the potential interactions between TMEM106A and other immune signaling pathways?

Interferon signaling:

• TMEM106A is an interferon-stimulated gene (ISG), indicating its integration within the broader interferon response network .

• Research questions remain about whether TMEM106A provides feedback regulation of the interferon pathway itself.

Toll-like receptor signaling:

• TMEM106A regulates macrophage responses to LPS, a ligand for TLR4 .

• It potentially influences pattern recognition receptor signaling, as Tmem106a deletion enhances responses to LPS stimulation .

MAPK and NF-κB pathways:

• Elevated MAPK and NF-κB signaling are observed in Tmem106a-deficient macrophages during LPS stimulation .

• Future research could address whether TMEM106A directly interacts with components of these signaling pathways or influences their activation indirectly.

How might TMEM106A be leveraged for therapeutic applications?

Antiviral strategies:

• Understanding the mechanism by which TMEM106A blocks SCARB2-virus binding could inform development of novel antivirals against enteroviruses .

• The TMEM106A-SCARB2 interaction represents a potential target for developing therapeutics to prevent EV-A71 infection .

Anti-inflammatory applications:

• Given its role in limiting inflammatory responses, enhancing TMEM106A expression or function might help control excessive inflammation in conditions like sepsis .

• Molecules that mimic TMEM106A's regulatory effects on MAPK and NF-κB signaling could have therapeutic potential.

Cancer treatment:

• As a tumor suppressor, restoration of TMEM106A function in cancers where it is downregulated might represent a treatment strategy .

• Further research is needed to determine whether TMEM106A status could serve as a prognostic marker or therapeutic target in specific cancer types.

What are the functional similarities and differences between TMEM106A and TMEM106B?

How does TMEM106A compare with other interferon-stimulated genes (ISGs) with antiviral functions?

This comparative analysis highlights that while TMEM106A shares mechanistic similarities with established ISGs like BST-2 in restricting enveloped viruses, it has unique additional properties including the ability to block non-enveloped virus receptor interactions and regulate inflammatory responses in macrophages.

What key questions remain unanswered about TMEM106A?

• Structural details: What is the complete three-dimensional structure of TMEM106A, including its transmembrane and extracellular domains? How does this structure relate to its diverse functions?

• Interaction network: What is the full range of TMEM106A's protein-protein interactions in different cell types and under various stimulation conditions?

• Physiological relevance: What is the physiological importance of TMEM106A in immune defense against viral and bacterial infections in vivo?

• Tissue-specific functions: Does TMEM106A have tissue-specific roles beyond what has been observed in macrophages and in the context of viral infections?

• Regulatory mechanisms: What transcription factors and post-translational modifications regulate TMEM106A expression and function?

• Evolutionary aspects: How has TMEM106A evolved across species, and does this evolution correlate with resistance to specific pathogens?

What methodological advances would facilitate TMEM106A research?

• Structural biology approaches: Cryo-electron microscopy or X-ray crystallography studies of TMEM106A alone and in complex with interacting partners would provide critical structural insights.

• Systems biology: Proteomics and interactomics approaches to comprehensively identify TMEM106A-interacting proteins under different cellular conditions.

• Advanced imaging: Super-resolution microscopy to visualize TMEM106A localization and dynamics in live cells during viral infection or inflammatory responses.

• In vivo models: Development of tissue-specific and conditional Tmem106a knockout models to dissect its role in different physiological contexts.

• Single-cell analysis: Application of single-cell RNA-seq and proteomics to understand cell-to-cell variability in TMEM106A expression and function.

• Therapeutic development: High-throughput screening approaches to identify compounds that modulate TMEM106A expression or function for potential therapeutic applications.