TICAM2 Human

What is TICAM2 and what are its alternative names in the scientific literature?

TICAM2 (TIR domain-containing adapter molecule 2) is a protein involved in Toll-like receptor signaling pathways. In the scientific literature, TICAM2 is also known as TRAM (TRIF-related adapter molecule), TIRP (Toll/interleukin-1 receptor domain-containing protein), TIRAP3 (Toll-like receptor adaptor protein 3), and MyD88-4 . This protein is encoded by the TICAM2 gene, which in humans is located at cytogenetic position 5q22.3 . Understanding these alternative nomenclatures is essential when conducting comprehensive literature searches or designing experiments targeting this protein.

What are the key structural characteristics of TICAM2 protein?

Human TICAM2 protein has a predicted molecular weight of approximately 27-31 kDa as detected by Western blot analysis . The protein contains a TIR (Toll/IL-1R) domain typical of adaptor proteins involved in Toll receptor signaling . Critical to its function are several conserved motifs, particularly an acidic motif containing E87/D88/D89 that provides interaction surfaces between TICAM-2 and TICAM-1, and the highly conserved D91/E92 residues which regulate TICAM-2 self-activation and signaling . The protein undergoes myristoylation, which contributes to its membrane localization properties, an essential aspect of its function in signal transduction .

How does TICAM2 participate in TLR4 signaling pathways?

TICAM2 functions as a sorting adapter in TLR4 signaling, physically bridging TLR4 and TICAM1 (also called TRIF) and functionally transmitting signals to TICAM1 in early endosomes after endocytosis of TLR4 . The MyD88 pathway mediates early phase responses to LPS on the plasma membrane, whereas the TICAM pathway mediates late-phase responses, which induce the production of type I interferon and activation of inflammasomes . This dual-phase response is critical for mounting appropriate immune defenses against gram-negative bacteria. TICAM2's role in endosomal signaling represents a spatiotemporal regulation mechanism that helps determine the specific outcomes of TLR4 activation.

What is the relationship between TICAM2 and neutrophil exhaustion in inflammation?

TICAM2 contributes to the development of exhausted neutrophils through activating Src family kinases (SFK) . Research has demonstrated that TICAM2-deficient neutrophils exhibit decreased expression of inflammatory adhesion molecules (ICAM1, CD11b) and immune-suppressors (PD-L1), along with reduced aggregation following prolonged LPS challenge compared to wild-type neutrophils . Functionally, TICAM2-deficient mice were protected from developing severe systemic inflammation and multi-organ injury following chemical-induced mucosal damage, suggesting that targeting TICAM2 may represent a potential approach for treating severe systemic inflammation and sepsis .

What methods are most effective for detecting and visualizing TICAM2 in experimental systems?

Multiple complementary approaches are available for detecting and visualizing TICAM2 in experimental systems:

• Western Blot Analysis: Effective for detecting endogenous TICAM2 in human, mouse, and rat samples with appropriate antibodies. Typical band detection occurs at approximately 31 kDa .

• Immunocytochemistry/Immunofluorescence (ICC/IF): Enables visualization of TICAM2 subcellular localization. Studies have successfully visualized TICAM2 in various cell lines including Raji human Burkitt's lymphoma and C2C12 mouse myoblast cell lines, typically showing cytoplasmic localization patterns .

• Recombinant Protein Expression: HEK293T cells can be used to express recombinant TICAM2 with tags such as C-Myc/DDK for purification and detection purposes .

When conducting these experiments, it is important to consider cross-species reactivity of antibodies, appropriate controls, and the specific cellular context being investigated.

How can researchers effectively study TICAM2 mutations and their functional implications?

To study TICAM2 mutations and their functional implications, researchers can employ several strategic approaches:

• Site-Directed Mutagenesis: Create specific mutations such as the D91A/E92A mutant to study the role of conserved motifs in TICAM2 function .

• Cellular Localization Studies: Compare wild-type and mutant TICAM2 distribution within cells to understand how mutations affect membrane localization and trafficking. For example, the D91A/E92A mutant protein is distributed largely to the cytosol despite myristoylation .

• Functional Activation Assays: Assess the ability of wild-type versus mutant TICAM2 to activate downstream signaling components like IRF3. The D91A/E92A mutant fails to self-activate TICAM-1 but retains the ability to pass LPS-mediated IRF3 activation when TLR4 is stimulated .

• Knockout and Reconstitution Models: Generate TICAM2 knockout cell lines and reconstitute with either wild-type or mutant TICAM2 to assess functional differences in signaling outcomes .

What are the distinct steps in TICAM2-mediated endosomal LPS signaling?

Research has identified two distinct steps underlying endosomal LPS signaling through TICAM2 for TICAM1 activation:

• TICAM2 Assembly with TLR4: Upon LPS stimulation, TLR4 is endocytosed, and TICAM2 associates with it in early endosomes .

• TICAM2 Self-Activation: Following assembly with TLR4, TICAM2 undergoes self-activation, which is dependent on specific motifs including the D91/E92 residues .

Interestingly, the D91A/E92A mutant of TICAM2 selectively participates in TLR4-dependent TICAM2 assembly but fails to undergo cytosolic TICAM2 self-aggregation required to activate TICAM1 . This mutant provides a valuable tool for dissecting these two steps in the signaling pathway, as it separates TLR4-dependent and self-activation functions of TICAM2.

How does the acidic motif in TICAM2 regulate its function and signaling capability?

The acidic motif in TICAM2, particularly D91/E92 residues, plays a crucial regulatory role in TICAM2's function and signaling capability. These conserved acidic amino acids:

• Facilitate Membrane Localization: The D91/E92 motif guides TICAM2 membrane localization despite myristoylation. Mutation of these residues (D91A/E92A) results in predominantly cytosolic distribution of the protein .

• Enable Self-Activation: Wild-type TICAM2 forms self-aggregation that is essential for activating TICAM1. The D91A/E92A mutant fails to self-activate TICAM1 .

• Maintain TLR4 Response Capability: Despite lacking self-activation capability, the D91A/E92A mutant retains the ability to respond to LPS stimulation through TLR4 and can still induce IRF3 activation in this context .

This dual functionality highlights how specific structural elements of TICAM2 contribute to different aspects of its signaling role, providing insights into the complex regulation of innate immune responses.

What disease models have been used to study TICAM2's role in systemic inflammation?

Several experimental models have been employed to study TICAM2's role in systemic inflammation:

• In Vitro Neutrophil Exhaustion Model: Murine primary neutrophils cultured with prolonged lipopolysaccharide (LPS) stimulation develop an exhaustive phenotype resembling human septic neutrophils with elevated expression of ICAM1, CD11b, PD-L1, and enhanced swarming and aggregation .

• TICAM2 Knockout Models: TICAM2-deficient mice provide valuable insights into how this protein contributes to systemic inflammation. These models have demonstrated protection from developing severe systemic inflammation and multi-organ injury following chemical-induced mucosal damage .

• Pharmacological Inhibition Studies: Application of SFK inhibitor Dasatinib blocks neutrophil exhaustion triggered by prolonged LPS challenge, mimicking aspects of TICAM2 deficiency and providing insights into potential therapeutic approaches .

These models collectively demonstrate that TICAM2 contributes significantly to pathogenic inflammation and immune suppression, key features underlying sepsis pathogenesis.

How might targeting TICAM2 provide therapeutic benefit in inflammatory conditions?

Research suggests that targeting TICAM2 may provide therapeutic benefits in inflammatory conditions, particularly severe systemic inflammation and sepsis:

• Reduced Neutrophil Exhaustion: TICAM2 deficiency decreases the expression of inflammatory adhesion molecules and immune-suppressors on neutrophils, potentially limiting the pathogenic neutrophil phenotype observed in sepsis .

• Prevention of Organ Damage: TICAM2-deficient mice show protection from developing multi-organ injury following chemical-induced mucosal damage, suggesting that TICAM2 inhibition could limit tissue damage during systemic inflammation .

• Potential Pharmacological Approaches: The finding that SFK inhibitor Dasatinib blocks neutrophil exhaustion triggered by prolonged LPS challenge suggests that targeting the TICAM2-SFK-STAT1 axis could be a viable therapeutic strategy .

• Selective Targeting of Late-Phase Responses: Since TICAM2 is involved specifically in endosomal (late-phase) TLR4 signaling, targeting this pathway might allow selective modulation of inflammatory responses without completely suppressing protective immune functions .

Developing therapeutic agents that specifically target TICAM2 or its downstream signaling pathways could potentially offer new approaches for treating severe inflammatory conditions while minimizing adverse effects on beneficial immune responses.

What are the optimal expression systems for producing recombinant TICAM2 for structural and functional studies?

For producing recombinant TICAM2 for structural and functional studies, researchers have successfully employed HEK293T cells transfected with TICAM2 cDNA clone . This mammalian expression system provides several advantages:

• Post-translational Modifications: Mammalian systems ensure proper folding and post-translational modifications that may be critical for TICAM2 function, including potential myristoylation.

• Yield and Purity: Recombinant TICAM2 can be captured through anti-tag (e.g., DDK) affinity columns followed by conventional chromatography steps, typically achieving >80% purity as determined by SDS-PAGE and Coomassie blue staining .

• Stability Considerations: Recombinant TICAM2 protein is typically stored at -80°C with 10% glycerol for stability and should be used with caution regarding freeze-thaw cycles .

When working with recombinant TICAM2 for cell culture applications, filtration before use is recommended, though researchers should be aware that some protein loss may occur during this process .

What are the key considerations when designing antibodies for TICAM2 detection across different species?

When designing or selecting antibodies for TICAM2 detection across different species, several key considerations should be taken into account:

• Cross-Species Reactivity: Some antibodies, such as the polyclonal goat IgG antibody described in the search results, can detect TICAM2 across human, mouse, and rat samples, making them valuable for comparative studies .

• Epitope Selection: For antibody generation, synthetic peptides within the human TICAM2 sequence have been successfully used as immunogens . Targeting conserved regions can enhance cross-species reactivity.

• Validation Across Applications: Antibodies should be validated for specific applications such as Western blot, immunocytochemistry/immunofluorescence (ICC/IF), or other intended uses. For example, some antibodies have been confirmed to work across these different applications .

• Cell Type Considerations: TICAM2 has been successfully detected in various cell types, including Raji human Burkitt's lymphoma cells, C2C12 mouse myoblast cells, and NRK rat normal kidney cells , indicating broad utility across different experimental systems.