Recombinant Bovine Transmembrane protein 173 (TMEM173)

What is the basic structure of bovine TMEM173 protein?

Bovine TMEM173 (UniProt ID: Q2KI99) is a 378 amino acid transmembrane protein. The full-length sequence includes a transmembrane domain and a cytoplasmic domain responsible for signaling functions. The protein contains multiple structural elements including membrane-spanning regions and cytosolic domains essential for ligand recognition and signal transduction. The amino acid sequence of the full-length bovine TMEM173 is well-characterized (MPHSSLHPSIPQPRGLRAQKAALVLLSACLVALWGLGEPPDYTLKWLVLHLASQQMGLLI KGICSLAEELCHVHSRYHGSYWRAVRACLCSSMRCGALLLLSCYFYCSLPNMADLPFTWM LALLGLSQALNILLGLQGLAPAEVSAICEKRNFNVAHGLAWSYYIGYLRLILPGLPARIQ IYNQFHNNTLQGAGSHRLHILFPLDCGVPDDLNVADPNIRFLHELPQQSADRAGIKGRVY TNSIYELLENGQRAGVCVLEYATPLQTLFAMSQDGRAGFSREDRLEQAKLFCRTLEDILA NAPESQNNCRLIVYQEPAEGSSFSLSQEILQHLRQEEREVTMGSTETSVMPGSSVLSQEP ELLISGLEKPLPLRSDVF) .

What primary functions does TMEM173 serve in innate immunity?

TMEM173 functions as a pattern recognition receptor that detects cytoplasmic nucleic acids and transmits cGAS-related signals to activate host innate immune responses. It plays a crucial role in the detection of cytosolic DNA and subsequently triggers signaling cascades leading to type I interferon production and inflammatory responses. TMEM173 is essential for defense against various pathogens, including bacteria and viruses, by initiating antimicrobial immune responses .

How does TMEM173 signaling differ from other innate immune pathways?

TMEM173 signaling is distinct from other innate immune pathways in several ways. Unlike classical pattern recognition receptors that primarily signal through NF-κB or IRF pathways, TMEM173 can operate through both type I interferon-dependent and independent mechanisms. Research has demonstrated that TMEM173's role in coagulation during bacterial infections occurs independently of the canonical TMEM173-induced type I IFN response, instead working through ER stress-initiated activation of gasdermin D (GSDMD) and subsequent release of coagulation factor III (F3) . This dual functionality allows TMEM173 to regulate diverse biological processes beyond typical innate immune responses.

What are the optimal storage conditions for recombinant bovine TMEM173?

Recombinant bovine TMEM173 should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles. For working aliquots, storage at 4°C for up to one week is recommended. The protein is typically supplied as a lyophilized powder in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0. For reconstitution, it's advised to briefly centrifuge the vial before opening and reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding 5-50% glycerol (final concentration) and aliquoting for long-term storage at -20°C/-80°C is recommended, with 50% being the typical default final concentration of glycerol .

What expression systems are most effective for producing functional recombinant TMEM173?

E. coli expression systems have been successfully used to produce recombinant full-length bovine TMEM173 with N-terminal His tags. This system allows for high protein yield while maintaining structural integrity. The bacterial expression system is particularly useful for producing the cytosolic domain of TMEM173, though expression of the full-length protein with transmembrane domains may require additional optimization. For studies requiring post-translational modifications or membrane incorporation, mammalian or insect cell expression systems might be more appropriate, though these approaches were not specifically detailed in the search results .

How can researchers verify the functional activity of recombinant TMEM173?

Functional activity of recombinant TMEM173 can be assessed through several experimental approaches:

• Binding assays with known ligands such as cyclic dinucleotides

• Downstream signaling activation assessment through:

  • Phosphorylation of TBK1 and IRF3

  • Type I interferon production measurement

  • ER stress marker expression analysis (e.g., Ddit3 and Hspa5)

• Calcium flux assays to measure TMEM173-dependent calcium release

• F3 release measurement in monocyte/macrophage models

• Coagulation activity assessment in relevant cellular systems

Researchers should select verification methods based on which functional aspect of TMEM173 is most relevant to their specific research question .

How does TMEM173 contribute to coagulation pathways in sepsis models?

TMEM173 plays a critical role in dysregulated coagulation leading to lethal sepsis through multiple mechanisms:

• TMEM173 binding to ITPR1 controls calcium release from the endoplasmic reticulum in macrophages and monocytes

• TMEM173-dependent increase in cytosolic calcium drives Gasdermin D (GSDMD) cleavage and activation

• Activated GSDMD triggers the release of coagulation factor III (F3), the key initiator of blood coagulation

• This pathway (TMEM173-GSDMD-F3) operates independently of the classical TMEM173-induced type I IFN response

Studies using conditional ablation of TMEM173 in mice have demonstrated that TMEM173 expressed by myeloid cells (Tmem173Mye−/−), but not by T cells (Tmem173T−/−), is responsible for CLP-induced septic death and coagulation activation. Deletion of Tmem173 in myeloid cells prolonged animal survival in sepsis models, with corresponding improvements in coagulation parameters including increased platelet count and fibrinogen concentration, decreased blood PT, APTT, and D-dimer, and reduced fibrin expression in tissues .

What is the role of TMEM173 in cardiac hypertrophy models?

TMEM173 has been identified as a protective factor against pressure overload-induced cardiac hypertrophy. Studies using Tmem173 global knockout (KO) mice subjected to transverse aortic constriction demonstrated that KO mice exhibited more severe hypertrophy, fibrosis, inflammatory infiltration, and cardiac dysfunction compared to wild-type C57BL/6 mice after 6 weeks.

The protective mechanism appears to involve TMEM173's role in promoting autophagic flux. In the absence of TMEM173, inhibited autophagosome degradation was observed in myocardium under electron microscopy. In vitro experiments with neonatal rat cardiomyocytes under phenylephrine treatment confirmed that Tmem173 gene abundance was negatively related to LC3-II abundance and the number of autophagosomes, indicating that TMEM173 promotes autophagosome degradation and helps maintain cardiac function under stress conditions .

How is TMEM173 regulated in cancer pathways, particularly in breast cancer?

The regulation of TMEM173 in breast cancer involves a newly identified signaling axis:

• The FKBP4/NR3C1 axis functions as a negative regulator of TMEM173 in human breast cancer cells

• FKBP4 suppresses TMEM173 at the transcriptional level by inhibiting its promoter activity

• NR3C1 positively regulates TMEM173 expression, as evidenced by:

  • Positive association between NR3C1 and TMEM173 in basal-like breast cancer patients

  • siRNA targeting NR3C1 leads to downregulation of TMEM173 at both protein and mRNA levels

  • Overexpression of NR3C1 results in upregulation of TMEM173

Clinical data analysis has shown that upregulated FKBP4 was significantly related to luminal, HER2-positive, and basal-like subtypes of breast cancer compared to normal tissue, while downregulated TMEM173 was significantly associated with the basal-like subtype. In basal-like breast cancer patients, increased levels of FKBP4 and decreased levels of TMEM173 were strongly correlated with worse survival outcomes .

How can researchers distinguish between TMEM173-dependent type I IFN and non-IFN signaling pathways?

Distinguishing between TMEM173-dependent type I IFN and non-IFN signaling pathways requires strategic experimental design:

These approaches allow researchers to delineate the distinct signaling pathways downstream of TMEM173 activation .

What are the challenges in studying transmembrane dynamics of TMEM173 in different cell types?

Studying the transmembrane dynamics of TMEM173 presents several significant challenges:

• Structural complexity:

  • TMEM173 is a multi-pass transmembrane protein with complex topology

  • Different conformational states may exist during activation and signaling

• Cell type-specific differences:

  • TMEM173 function varies substantially between cell types (e.g., myeloid cells vs. T cells)

  • Conditional knockout studies have shown that myeloid TMEM173 plays a dominant role in certain contexts, such as sepsis models

  • Different cell types may express varying levels of TMEM173 interacting partners

• Subcellular localization:

  • TMEM173 transitions between different cellular compartments during activation

  • Tracking these movements requires sophisticated imaging techniques

• Integration with other signaling pathways:

  • TMEM173 interacts with multiple proteins (e.g., ITPR1, NR3C1) and signaling networks

  • Isolating TMEM173-specific effects from broader cellular responses is challenging

Researchers should consider employing advanced techniques such as super-resolution microscopy, FRET-based interaction studies, and cell-type specific conditional knockout models to address these challenges .

How can TMEM173 function be studied in the context of complex in vivo disease models?

Studying TMEM173 function in complex in vivo disease models requires sophisticated approaches:

• Conditional knockout models:

  • Cell-type specific deletion (e.g., Tmem173Mye−/− for myeloid-specific deletion) allows assessment of TMEM173 function in particular cell populations

  • These models have revealed that myeloid TMEM173, but not T cell TMEM173, mediates septic death and coagulation activation

• Multiple disease models:

  • Using diverse models provides robust validation of TMEM173 functions

  • For sepsis, multiple models have been employed: cecal ligation and puncture (CLP) or bacteremia with E. coli or S. pneumoniae infection

  • For cardiac hypertrophy, transverse aortic constriction models have been effective

• Comprehensive readouts:

  • Monitor multiple parameters simultaneously (e.g., coagulation factors, inflammatory markers, tissue damage indicators)

  • In sepsis models, measurements included platelet count, fibrinogen concentration, blood PT/APTT/D-dimer, tissue fibrin expression, and plasma F3 levels

  • In cardiac models, assessments included hypertrophy, fibrosis, inflammatory infiltration, and cardiac function

• Pharmacological interventions:

  • Complement genetic approaches with TMEM173-targeting drugs

  • Inhibitors of the TMEM173-GSDMD-F3 pathway have shown promise in blocking systemic coagulation and improving animal survival in sepsis models

This multi-faceted approach enables researchers to delineate the complex in vivo functions of TMEM173 .

What are the recommended experimental controls when working with recombinant TMEM173?

When working with recombinant TMEM173, researchers should implement the following controls:

• Protein quality controls:

  • Purity verification by SDS-PAGE (>90% purity is standard)

  • Functionality testing through binding assays with known ligands

  • Western blot confirmation of protein identity and integrity

• Experimental controls:

  • Negative controls: vector-only or irrelevant protein treatments

  • Positive controls: known TMEM173 activators (e.g., cyclic dinucleotides)

  • Dose-response relationships to establish specificity

• Genetic controls:

  • TMEM173 knockout or knockdown cells alongside wild-type cells

  • Complementation with wild-type TMEM173 to confirm specificity

  • Specific pathway component knockdowns (e.g., TBK1, IRF3, GSDMD) to differentiate pathway contributions

• Pathway validation controls:

  • Assessment of known downstream mediators (IFNβ, ER stress markers)

  • Inhibitors of specific pathway components

  • Time course experiments to establish signaling sequence

These controls ensure experimental rigor and facilitate accurate interpretation of TMEM173-related findings .

What techniques are most effective for studying TMEM173-dependent calcium dynamics?

Several techniques have proven effective for studying TMEM173-dependent calcium dynamics:

• Fluorescent calcium indicators:

  • Fura-2, Fluo-4, or genetically encoded calcium indicators (GECIs) like GCaMP

  • Allow real-time monitoring of cytosolic calcium levels in response to TMEM173 activation

  • Can be combined with confocal microscopy for subcellular resolution

• ER calcium measurement:

  • Since TMEM173 binding to ITPR1 controls calcium release from the ER, specialized ER-targeted calcium sensors are valuable

  • ER-GCaMP or D1ER constructs allow specific monitoring of ER calcium stores

• Calcium chelators and modulators:

  • BAPTA-AM to buffer intracellular calcium

  • Thapsigargin to deplete ER calcium stores

  • These tools help establish causality in TMEM173-calcium-GSDMD-F3 signaling

• Protein interaction studies:

  • Co-immunoprecipitation of TMEM173 with ITPR1

  • FRET-based approaches to monitor dynamic interactions

  • Proximity ligation assays to confirm interactions in situ

These methods have helped establish that TMEM173 binding to ITPR1 controls calcium release from the endoplasmic reticulum in macrophages and monocytes, and that this TMEM173-dependent increase in cytosolic calcium drives Gasdermin D (GSDMD) cleavage and activation, triggering the release of F3 .

How can researchers quantitatively assess TMEM173-mediated autophagy in cardiac models?

Quantitative assessment of TMEM173-mediated autophagy in cardiac models involves multiple complementary approaches:

• Electron microscopy analysis:

  • Gold standard for autophagosome and autolysosome identification

  • Allows visualization and quantification of autophagic structures in myocardium

  • Can distinguish between accumulation due to increased formation versus decreased degradation

• Autophagic flux assessment:

  • LC3-II protein levels with and without lysosomal inhibitors (e.g., bafilomycin A1)

  • p62/SQSTM1 accumulation as an indicator of impaired autophagy

  • Tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) to monitor autophagosome maturation

    • Yellow puncta (merged GFP and RFP) indicate autophagosomes

    • Red puncta (RFP only, as GFP is quenched in acidic environment) indicate autolysosomes

• Gene expression analysis:

  • qPCR measurement of autophagy-related genes

  • Assessment of correlation between Tmem173 expression and autophagy markers

• Functional readouts:

  • Cardiomyocyte size measurement to quantify hypertrophy

  • Fibrosis assessment through histological staining

  • Echocardiography for cardiac function parameters

In cardiac models, studies have shown that Tmem173 knockout mice exhibited inhibited autophagosome degradation in myocardium, and in vitro experiments demonstrated that Tmem173 gene abundance was negatively related to LC3-II abundance and the number of autophagosomes, indicating TMEM173's role in promoting autophagic flux .

What potential therapeutic applications exist for targeting TMEM173 in inflammatory diseases?

Research indicates several promising therapeutic applications for targeting TMEM173 in inflammatory diseases:

• Sepsis intervention:

  • Inhibition of the TMEM173-GSDMD-F3 pathway blocks systemic coagulation

  • This approach has improved animal survival in three models of sepsis (CLP or bacteremia with E. coli or S. pneumoniae infection)

  • Targeting this pathway could potentially address the dysregulated coagulation that contributes to septic death

• Cardiac protection:

  • Enhancement of TMEM173 function may protect against pressure overload-induced cardiac hypertrophy

  • TMEM173's role in promoting autophagic flux provides a mechanistic basis for therapeutic development

  • Targeting TMEM173 could help maintain cardiac function under stress conditions

• Cancer immunotherapy:

  • Modulation of the FKBP4/NR3C1/TMEM173 axis may have potential in breast cancer treatment

  • Upregulating TMEM173 expression could potentially improve survival outcomes in basal-like breast cancer patients

  • This approach might enhance tumor immunity through TMEM173's role in immune cell activation

These therapeutic directions are supported by mechanistic studies of TMEM173 function in different disease contexts, though clinical translation will require further research into drug development and delivery strategies .

How do species differences in TMEM173 structure affect experimental interpretation and translation?

Species differences in TMEM173 structure present important considerations for experimental interpretation and translation:

• Sequence and structural variations:

  • Bovine TMEM173 (378 amino acids) may have structural differences from human TMEM173

  • These differences can affect ligand binding properties, protein-protein interactions, and downstream signaling

• Functional implications:

  • Species-specific differences in TMEM173 activation thresholds

  • Potential variations in binding partners and regulatory mechanisms

  • Differences in subcellular localization or trafficking

• Experimental design considerations:

  • Results from bovine or murine models may not directly translate to human systems

  • Species-appropriate positive controls should be included

  • Confirmation of key findings across multiple species strengthens translational relevance

• Translation to clinical applications:

  • Therapeutic strategies targeting TMEM173 must account for species differences

  • Drug screening should include human TMEM173 testing even if initial discovery used bovine or murine models

  • Species-specific polymorphisms may affect drug efficacy or toxicity profiles

Researchers should remain cognizant of these differences when designing experiments and interpreting results, particularly when translating findings from animal models to human applications .