FABP6 Human

What is FABP6 and what are its primary functions?

FABP6, also known as ileal bile acid binding protein (I-BABP) and gastrotropin, is a 15 kDa cytoplasmic protein belonging to the fatty acid binding protein family. It primarily functions in binding both fatty acids and bile acids, playing crucial roles in fatty acid transport and metabolism . While traditionally considered an intestinal protein, research has revealed its expression and functional significance in multiple tissues, including ovarian cells and colorectal cancer tissues . The protein's ability to interact with bile acids makes it particularly important for maintaining bile acid homeostasis in enterohepatic circulation.

How does FABP6 conservation compare across species?

Human FABP6 shares significant sequence homology with other mammalian species, demonstrating evolutionary conservation that suggests functional importance. The amino acid sequence of human FABP6 is 80% identical to mouse FABP6, 78% identical to canine FABP6, and 75% identical to porcine FABP6 . This high degree of conservation across species provides researchers with relevant animal models for studying FABP6 function, although species-specific differences should be considered when translating findings across experimental models.

What techniques are most effective for studying FABP6 binding properties?

Multiple complementary techniques have proven effective for investigating FABP6 binding properties:

• Surface Plasmon Resonance (SPR): Used successfully to identify fragment hits that bind to FABP6 and to demonstrate displacement by natural ligands such as taurocholate .

• Native Mass Spectrometry: Effective for detecting binding of compounds to FABP6 and for conducting competition studies between different ligands (e.g., between GCDC and synthetic compounds) .

• X-ray Crystallography: Critical for resolving FABP6 structure in both ligand-bound and unbound states, providing atomic-level details of binding interactions .

When designing binding studies, researchers should consider using multiple methodologies to provide complementary data. For instance, SPR can establish initial binding, while crystallography can confirm binding mode and specific interaction sites.

How can FABP6 expression be accurately assessed in tissue samples?

Based on published methodologies, several approaches have proven reliable for FABP6 detection:

• Immunohistochemistry Protocol:

  • Fix tissues in 10% neutral buffered formalin

  • Embed in paraffin and cut into 4 μm sections

  • Perform deparaffinization, dehydration, and antigen retrieval

  • Block with solution containing 3% bovine serum albumin, 0.4% Triton X-100, and 10% normal goat serum

  • Incubate with murine Fabp6 anti-serum (1:500) for 1 hour

  • Wash with PBS and incubate with anti-rabbit secondary antibody (1:200) for 1 hour

  • Visualize using DAB chromogen system

  • Counterstain with hematoxylin to reveal cell nuclei

• Quantitative PCR: For accurate mRNA quantification, designing primers specific for the open reading frame of FABP6 has proven effective. This approach can detect tissue-specific expression patterns and responses to treatments .

• Protein Purification Assessment: Recombinant FABP6 purity can be verified by SDS-PAGE, with quality research-grade protein showing >95% purity .

What is the role of FABP6 in ovarian function and fertility?

Research using Fabp6-deficient mice has revealed an unexpected and important role for this protein in reproductive function. FABP6 is expressed in granulosa and luteal cells of the mouse ovary, with expression patterns changing in response to gonadotropin stimulation. The highest expression levels are observed at eCG-48h and hCG-1h timepoints, suggesting that both gonadotropins stimulate FABP6 gene expression .

Most significantly, Fabp6-deficient mice showed markedly decreased ovulation rates in response to superovulatory treatment compared to wildtype mice, indicating that FABP6 expression in granulosa cells serves a previously unrecognized function in fertility . This finding opens new research directions for investigating FABP6's role in reproductive physiology and potential implications for fertility disorders.

The expression pattern of FABP6 follows that of Cyp19a1 in follicular granulosa cells, suggesting that its expression may be estrogen-dependent . This relationship merits further investigation to understand the hormonal regulation of FABP6 in reproductive tissues.

How does FABP6 impact immune function in colorectal cancer?

FABP6 has emerged as a significant factor in the tumor microenvironment (TME) of colorectal cancer, with important implications for immunotherapy. Research has revealed that FABP6 is negatively correlated with immune infiltration in colorectal cancer tissues . Experimental knockdown of FABP6 produced several immunologically relevant effects:

• Increased major histocompatibility complex (MHC) class 1 expression

• Promoted immune-related chemokine secretion

• Enhanced immunogenicity of tumor cells

• Promoted recruitment of CD8+ T cells

These findings suggest that FABP6 may serve as a potential target for improving immunotherapy responses in colorectal cancer patients. When analyzing immune infiltration patterns in colorectal cancer, researchers should consider FABP6 expression as a potential modulating factor, particularly when evaluating "hot" versus "cold" tumor phenotypes.

What experimental approaches are recommended for FABP6 knockdown studies?

Based on successful implementations in published research, the following considerations are important when designing FABP6 knockdown experiments:

• Model Selection: Both in vitro cell line models and in vivo mouse models have been used successfully. Cell line experiments allow for mechanistic studies of direct effects on cell behavior, while mouse models permit assessment of systemic effects.

• Outcome Assessment: When knocking down FABP6, researchers should assess multiple potential outcomes:

  • Changes in cell proliferation, apoptosis, and migration

  • Alterations in MHC class I expression

  • Modifications to immune-related chemokine secretion

  • Effects on immune cell recruitment, particularly CD8+ T cells

• Control Experiments: It's important to note that FABP6 knockdown did not significantly alter tumor cell proliferation, apoptosis, and migration in colorectal cancer models , suggesting that its primary effects may be immune-related rather than directly affecting cancer cell behavior.

What is known about the druggability of FABP6 for therapeutic applications?

FABP6 has been identified as a potential drug discovery target, particularly for diabetes treatment . Research advances in this area include:

• Crystal Structure Determination: The first crystal structure of human FABP6 has been reported in three forms: unbound, in complex with cholate, and with a novel binding fragment. These structures provide essential information for structure-based drug design .

• Fragment-Based Drug Discovery: FABP6 has been found amenable to fragment-based drug discovery approaches, with several fragment hits identified using surface plasmon resonance (SPR) .

• Natural Ligand Displacement: Fragment hits have been validated through structure-activity relationship (SAR) studies and shown to be displaced by the natural ligand taurocholate, confirming binding to the physiologically relevant site .

When designing inhibitor screening campaigns, researchers should consider both the bile acid binding pocket and potential allosteric sites identified in crystallographic studies.

What are the optimal conditions for handling recombinant human FABP6?

For researchers working with recombinant FABP6, the following handling recommendations should be followed:

• Storage Conditions:

  • Products can be stored at -20°C to -80°C for up to 1 year

  • Aliquot the protein into smaller quantities upon first use

  • Avoid repeated freeze-thaw cycles

• Reconstitution Protocol:

  • Briefly centrifuge vials prior to opening

  • Reconstitute in sterile distilled water or aqueous buffer containing 0.1% BSA

  • Prepare to a concentration of 0.1-1.0 mg/mL

  • Apportion stock solutions into working aliquots

  • Store at ≤ -20°C

  • Make further dilutions in appropriate buffered solutions

• Quality Control Parameters:

  • Verify >95% purity by SDS-PAGE

  • Ensure endotoxin levels <1.0 EU per 1μg of protein by LAL method

How can researchers effectively analyze FABP6's role in gonadotropin-stimulated ovarian function?

When investigating FABP6's role in ovarian function, researchers should consider the following methodological approach:

• Timing of Sample Collection: Collect granulosa and luteal cells at specific time-points during gonadotropin-induced follicular development and ovulation. Critical timepoints include:

  • After eCG treatment (especially at 48h)

  • After hCG treatment (particularly at 1h)

• Validation Markers: Include analysis of Cyp19a1 and Ptgs2 mRNA abundance by qPCR to confirm proper cell populations representing precise stages of granulosa cell differentiation .

• Statistical Analysis:

  • Use one-way analysis of variance followed by Tukey's multiple comparisons post-hoc test for comparing mRNA abundance at different time points

  • Apply unpaired Student's t-test for comparing ovulation rates

  • Consider differences significant when P < 0.05

• Control Considerations: When using Fabp6-deficient models, appropriate wildtype controls (e.g., C57BL/6 mice) should be included for meaningful comparisons .

What are the most promising areas for future FABP6 research?

Based on current findings, several high-priority research directions emerge:

• Further Characterization of FABP6 in Reproductive Biology: Given the unexpected finding that FABP6 plays a role in fertility, further research into its mechanisms in ovarian function could yield valuable insights for reproductive medicine .

• Development of Selective FABP6 Inhibitors: The availability of crystal structures and validated fragment hits provides a foundation for developing selective inhibitors that could be valuable both as research tools and potential therapeutic agents .

• Exploration of FABP6 as an Immunomodulatory Target: The connection between FABP6 and immune infiltration in colorectal cancer suggests potential applications in cancer immunotherapy. Further research into how FABP6 modulates the tumor immune microenvironment could lead to novel therapeutic strategies .

• Investigation of FABP6's Role in Other Diseases: While much research has focused on colorectal cancer, diabetes, and reproduction, FABP6's presence in multiple tissues suggests it may have undiscovered roles in other physiological and pathological processes.

By addressing these research priorities, the scientific community can advance understanding of this multifunctional protein and potentially develop new therapeutic approaches for associated diseases.