TIFA Human
Description
TIFA is fused to a 24 amino acid His-tag at N-terminus & purified by proprietary chromatographic techniques.
Product Specs
Q&A
What is the molecular structure of human TIFA protein?
Human TIFA contains a forkhead-associated (FHA) domain that recognizes phosphothreonine residues, facilitating protein-protein interactions essential for its signaling function. Crystallographic studies of TIFA and its phosphomimetic mutants (Thr9Asp and Thr9Glu) have provided crucial structural insights into its activation mechanism . The protein also contains intrinsically disordered regions (IDR) that contribute to its phase separation properties, which are increasingly recognized as essential for its signaling function .
Methodologically, researchers investigating TIFA structure typically employ X-ray crystallography to determine three-dimensional structures and nuclear magnetic resonance (NMR) spectroscopy to examine dynamic regions. Site-directed mutagenesis, particularly at the critical Thr9 residue, has been instrumental in understanding TIFA's phosphorylation-dependent activation mechanism and oligomerization properties.
How does TIFA participate in innate immune signaling pathways?
TIFA functions as a critical adapter in innate immune signaling, particularly in response to bacterial pathogen-associated molecular patterns (PAMPs). Upon recognition of ADP-β-D-manno-heptose (ADP-Hep) by ALPK1 (alpha-kinase 1), TIFA becomes phosphorylated at Threonine 9, triggering its oligomerization . This oligomerization creates a platform for the recruitment and activation of TRAF6, leading to NF-κB activation and inflammatory gene expression.
To study this pathway, researchers typically use human cell lines (HEK293, THP-1) stimulated with purified ADP-Hep, followed by assessment of TIFA phosphorylation, oligomerization, and downstream signaling events. CRISPR-Cas9-mediated TIFA knockout cells serve as important negative controls for establishing pathway specificity.
What drives TIFA liquid-liquid phase separation in the context of inflammatory signaling?
Recent research has revealed that TIFA undergoes liquid-liquid phase separation (LLPS) rather than forming rigid higher-order structures during ADP-Hep-induced inflammation . This phase separation is primarily driven by three factors:
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Phosphorylation by ALPK1 at the Thr9 position
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Interactions mediated by the pT9-FHA domain
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Contributions from the intrinsically disordered region (IDR) segments
Interestingly, the E178A TIFA mutant maintains LLPS capabilities despite reduced TRAF6 binding, suggesting that interaction with TRAF6 is not essential for phase separation of TIFA . This indicates that TIFA phase separation is primarily an upstream event that facilitates, but does not depend on, downstream signaling interactions.
The methodological approach to studying TIFA phase separation typically involves fluorescently tagged TIFA constructs expressed in cells, combined with high-resolution microscopy to visualize condensate formation and dynamics. Biophysical techniques such as fluorescence recovery after photobleaching (FRAP) can confirm the liquid-like properties of these condensates.
How do TIFA condensates function as signaling hubs in inflammatory pathways?
TIFA condensates formed through LLPS serve as membraneless microreactors that concentrate signaling components, facilitating efficient signal transduction . These phase-separated compartments:
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Recruit TRAF6 into the condensates
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Facilitate the synthesis of K63-linked polyubiquitin chains
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Concentrate downstream effectors including TAK1, TAB2, and NEMO
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Expedite phosphorylation and ubiquitination processes
The concentration of these signaling molecules within a confined space enhances reaction kinetics and specificity, leading to robust activation of the inflammatory signaling cascade . This compartmentalization helps explain how relatively low concentrations of bacterial PAMPs can trigger strong inflammatory responses.
To experimentally investigate these signaling hubs, researchers employ co-immunoprecipitation, immunofluorescence co-localization, and proximity ligation assays to detect protein-protein interactions within the condensates. Super-resolution microscopy can further reveal the spatial organization of different signaling components within these structures.
What are optimal methods for measuring TIFA phosphorylation and activation in human cells?
Several complementary approaches are recommended for comprehensive analysis of TIFA phosphorylation and activation:
| Technique | Application | Advantages | Considerations |
|---|---|---|---|
| Phospho-specific antibodies | Western blotting | Direct detection of pThr9 | Limited by antibody specificity |
| Phos-tag SDS-PAGE | Mobility shift assay | Detects all phosphorylated forms | Requires optimization |
| Mass spectrometry | Phosphosite mapping | Comprehensive, quantitative | Requires specialized equipment |
| Phosphomimetic mutants | Functional studies | Mimics constitutive phosphorylation | May not fully recapitulate phosphorylation |
For functional readouts of TIFA activation, researchers typically assess:
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TIFA oligomerization using non-denaturing PAGE or crosslinking approaches
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Formation of visible puncta/specks using fluorescence microscopy
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Recruitment of TRAF6 using co-immunoprecipitation or proximity ligation assay
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Downstream signaling through NF-κB reporter assays or inflammatory cytokine production
When designing these experiments, it's crucial to include appropriate controls such as TIFA-knockout cells, non-phosphorylatable TIFA mutants (T9A), and specific ALPK1 inhibitors to establish the specificity of observed responses.
How can researchers effectively modulate TIFA phase separation for experimental purposes?
To experimentally manipulate TIFA phase separation, researchers have several options:
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Chemical modulation: Compound 22 has been identified as a molecular probe that enhances TIFA LLPS in the presence of low concentrations of ADP-LD-Hep . This compound can reinstate TIFA liquid condensation even when ADP-LD-Hep concentrations are insufficient to trigger phase separation alone.
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Genetic approaches: Creating specific TIFA mutants can enhance or disrupt phase separation properties. Mutations in the FHA domain or the IDR segments can be particularly effective at altering phase separation propensity without completely abolishing TIFA's ability to signal.
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Physical parameters: Manipulating cellular conditions such as molecular crowding, salt concentration, or temperature can influence phase separation behavior in both cellular and in vitro reconstituted systems.
Statistical analysis of immunofluorescence data has shown that the combination of low-dose ADP-LD-Hep (1 μM) with compound 22 (25 μM) can restore TIFA's liquid-like state to approximately 70% of the intensity produced by high-dose ADP-LD-Hep (10 μM) . This quantitative approach to measuring phase separation provides a reliable readout for experimental manipulation.
What is the relationship between TIFA phase separation and TRAF6 ubiquitination activity?
The formation of TIFA condensates through LLPS creates specialized compartments where TRAF6 is recruited and activated . Within these condensates, several critical processes occur:
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Concentration of ubiquitination machinery components
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Enhanced synthesis of K63-linked polyubiquitin chains by TRAF6
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Facilitated recruitment of ubiquitin-binding proteins such as TAB2 and NEMO
The membraneless nature of these condensates allows for dynamic exchange of components while maintaining high local concentrations of enzymatic machinery. This creates an environment ideal for processivity in building polyubiquitin chains, which are essential for downstream signal propagation.
Methodologically, researchers can investigate this relationship using in vitro reconstitution of the ubiquitination reaction with purified components in the presence or absence of phase-separated TIFA. Fluorescently labeled ubiquitin can track chain formation within condensates, while ubiquitin linkage-specific antibodies can confirm the generation of K63-linked chains specifically.
How do post-translational modifications beyond phosphorylation affect TIFA function?
While phosphorylation at Thr9 is the best-characterized modification of TIFA, understanding the impact of other post-translational modifications represents an important frontier in TIFA research. Potential modifications that may regulate TIFA include:
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Additional phosphorylation sites that may fine-tune oligomerization or protein interactions
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Ubiquitination that could affect TIFA stability or create additional interaction surfaces
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Acetylation or methylation that might modulate phase separation properties
Research approaches to investigate these modifications typically involve mass spectrometry-based proteomics to identify modification sites, followed by site-directed mutagenesis to assess their functional significance. Temporal analysis of modifications can reveal regulatory hierarchies and feedback mechanisms within the signaling pathway.
The interplay between different modifications may be particularly important in determining the dynamics of TIFA condensate formation and dissolution, potentially providing mechanisms for signal termination or amplification.
How might targeting TIFA phase separation be exploited for immunomodulatory drug development?
The discovery that TIFA undergoes phase separation during inflammatory signaling opens new avenues for therapeutic intervention . Several strategic approaches could be developed:
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Small molecule modulators: Building on the discovery of compound 22, which enhances TIFA LLPS in the presence of low ADP-Hep concentrations, researchers could develop more potent and specific compounds targeting this mechanism . Both enhancers (for immunostimulatory applications) and inhibitors (for anti-inflammatory applications) of phase separation could have therapeutic value.
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Peptide-based interventions: Designed peptides that interfere with specific aspects of TIFA oligomerization or phase separation could provide selective manipulation of inflammatory pathways.
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Targeted degradation: Proteolysis-targeting chimeras (PROTACs) directed against TIFA could provide temporal control over its availability in inflammatory cascades.
Chemical biology investigations using cell-based assays such as the HEK blue reporter system have already identified compounds like compound 22 that synergistically enhance NF-κB activation in combination with low concentrations of ADP-LD-Hep . These findings suggest that targeted modulation of TIFA phase separation is indeed feasible and could lead to the development of the first immunomodulators specifically targeting the ALPK1-TIFA-TRAF6 signaling pathway.
What biomarkers could indicate dysregulated TIFA signaling in human inflammatory diseases?
Identifying biomarkers of aberrant TIFA activity could help diagnose conditions with dysregulated innate immune signaling. Potential biomarkers include:
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Phosphorylated TIFA levels in peripheral blood mononuclear cells (PBMCs)
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Altered ratios of TIFA to TRAF6 in affected tissues
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Presence of TIFA condensates in tissue biopsies detected by immunofluorescence
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Downstream gene expression signatures indicative of TIFA-mediated NF-κB activation
Methodologically, multiplexed approaches combining phospho-flow cytometry, single-cell RNA sequencing, and tissue imaging would provide complementary information about TIFA pathway activation. Correlation with clinical parameters could establish the relevance of these biomarkers to disease progression and treatment response.
Such biomarkers could be particularly valuable in inflammatory conditions where bacterial PAMPs might play a role, including inflammatory bowel disease, sepsis, and certain autoimmune disorders.
What are the most promising unresolved questions regarding TIFA biology in human health and disease?
Several critical questions remain to be addressed in TIFA research:
Addressing these questions will require multidisciplinary approaches combining structural biology, advanced imaging, genetic screening, and systems biology. Integration of findings across these domains will be essential for developing a comprehensive understanding of TIFA's role in human biology.
What methodological advances would most benefit TIFA research?
Several technological innovations could substantially advance TIFA research:
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Optogenetic tools for precisely controlling TIFA phase separation in space and time within living cells
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Expanded chemical probe libraries specifically targeting different aspects of TIFA function
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Cryo-electron microscopy studies of TIFA-TRAF6 complexes to understand their three-dimensional organization
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In vivo imaging approaches to visualize TIFA condensates in intact tissues during inflammatory responses
Development of these tools would enable researchers to move beyond correlative studies and establish causal relationships between TIFA phase separation and downstream inflammatory outcomes. Additionally, they would facilitate translation of basic research findings into therapeutic applications by providing platforms for drug screening and validation.
The combination of chemical biology approaches with structural biology, as exemplified by the studies on compound 22 , represents a particularly promising direction for future TIFA research with therapeutic implications.
Product Science Overview
TRAF-Interacting Protein with Forkhead-Associated Domain (TIFA) is a protein that plays a crucial role in the signaling pathways of the immune system. It is known for its interaction with Tumor Necrosis Factor Receptor-Associated Factors (TRAFs), particularly TRAF6, which is involved in the activation of NF-κB, a transcription factor that regulates the expression of various genes involved in immune and inflammatory responses .
TIFA contains a Forkhead-Associated (FHA) domain, which is a phosphopeptide-binding motif. This domain is essential for its function in signal transduction. The FHA domain allows TIFA to bind to phosphorylated serine or threonine residues on other proteins, facilitating the formation of protein complexes that are necessary for downstream signaling events .
TIFA is involved in several biological processes, including:
- NF-κB Activation: TIFA activates TRAF6, leading to the activation of NF-κB. This activation is crucial for the transcription of genes involved in immune responses .
- DNA Damage Response: TIFA transduces DNA damage-induced activation of NF-κB. It accumulates on damaged chromatin following genotoxic stress and enhances the transcription of NF-κB target genes, such as IL-6 and IL-8 .
- Cell Cycle Regulation: TIFA has been shown to inhibit entry into the S phase of the cell cycle, suggesting a role in cell cycle regulation .
Human recombinant TIFA can be produced using recombinant DNA technology. The gene encoding TIFA is cloned into an expression vector, which is then introduced into a suitable host cell, such as E. coli or mammalian cells. The host cells express the TIFA protein, which can be purified using various chromatographic techniques.
TIFA’s activity is regulated through phosphorylation. Phosphorylation at specific threonine residues is crucial for its function in DNA damage response and NF-κB activation. Additionally, TIFA interacts with TRAF2, an E3 ubiquitin ligase, to mediate the ubiquitination of NF-κB essential modulator (NEMO), further regulating NF-κB signaling .
