FABP6 Human, His

What is FABP6 and what are its primary functions in human biology?

FABP6, also known as ileal fatty acid binding protein (I-BABP), gastrotropin, and several other names (including I-15P, ILBP, ILBP3, I-BALB, I-BAP, ILLBP), is a member of the fatty acid binding protein family . This protein is a small, highly conserved cytoplasmic protein that primarily binds long-chain fatty acids and other hydrophobic ligands. Unlike most FABPs, FABP6 has a unique ability to bind bile acids, sharing this characteristic with only FABP1 (liver fatty acid binding protein) .

The primary functions of FABP6 include:

• Facilitation of fatty acid uptake, transport, and metabolism

• Component of the bile acid recovery system in the small intestine

• Aiding in the digestion and absorption of dietary lipids

Human FABP6 is encoded by the FABP6 gene located on chromosome 5 (NC_000005.10), with NCBI Gene ID 2172 .

How does the structure of His-tagged FABP6 compare to the native protein?

The addition of a histidine tag to FABP6 creates a fusion protein that maintains the core structural features of native FABP6 while providing advantages for purification and detection. Native human FABP6 consists of 128 amino acids (Ala2-Ala128, Accession # P51161) , with a characteristic β-barrel structure typical of the FABP family.

When working with His-tagged FABP6:

• The His-tag typically adds 6-10 histidine residues to either the N- or C-terminus

• The tag generally has minimal impact on the protein's tertiary structure

• Crystal structures have been determined for human FABP6 in unbound form, in complex with cholate, and with binding fragments

• The internal binding pocket structure remains preserved, allowing for normal ligand interactions

What experimental advantages does His-tagged FABP6 offer for research applications?

His-tagged FABP6 provides several methodological advantages in research settings:

What are the optimal conditions for expressing and purifying His-tagged human FABP6?

Based on research protocols, the following methodological approach is recommended:

Expression System:

• E. coli is the preferred expression system for recombinant human FABP6

• BL21(DE3) strain is commonly used for high-level expression

• Expression vector should contain a T7 promoter and His-tag sequence

Expression Conditions:

• Induction with 0.5-1 mM IPTG when culture reaches OD600 of 0.6-0.8

• Post-induction culture at 25-30°C for 4-6 hours (rather than 37°C) to enhance soluble protein production

• Supplementation with 0.2% glucose can reduce basal expression before induction

Purification Protocol:

• Cell lysis using sonication or French press in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors

• Clarification by centrifugation at 20,000 × g for 30 minutes

• IMAC purification using Ni-NTA resin with stepwise imidazole elution (50 mM, 100 mM, 250 mM)

• Size exclusion chromatography for further purification and buffer exchange

• Final storage in 20 mM phosphate buffer pH 7.4, 150 mM NaCl, with optional addition of 10% glycerol for stability

What techniques are most effective for studying FABP6 protein-ligand interactions?

Several complementary techniques have proven valuable for investigating FABP6 interactions with ligands:

Methodological Insights: Fragment-based approaches have successfully identified novel binding fragments in FABP6, as demonstrated by Hendrick et al. who validated hits using SPR and obtained crystal structures of complexes .

How can I investigate FABP6's role in bile acid transport and metabolism?

To study FABP6's role in bile acid transport and metabolism, consider these methodological approaches:

In Vitro Approaches:

• Binding assays with radiolabeled or fluorescently labeled bile acids

• Transport assays using membrane vesicles or cell monolayers expressing FABP6

• Co-immunoprecipitation to identify protein interaction partners in the transport pathway

Cellular Models:

• T84 human colon carcinoma cell line has been validated for FABP6 expression studies

• Intestinal epithelial cell models like Caco-2 cells for transport studies

• FABP6 knockdown/knockout cell lines using siRNA or CRISPR-Cas9

In Vivo Models:

• Fabp6-deficient mice show enhanced excretion of both bile acids and fat on Western-style diet (WSD)

• Sex-specific differences should be considered, as male and female Fabp6-knockout mice show different phenotypes

• Analysis of fecal bile acid content using HPLC-MS methods

Gut Microbiome Analysis:

• 16S rRNA sequencing to examine changes in gut microbiota composition

• Studies have shown sex-specific changes in major bacterial phyla in response to Fabp6 deficiency

What approaches should be considered when investigating FABP6 as a potential therapeutic target?

FABP6 has been identified as a potential drug discovery target that may have therapeutic benefits for diabetes treatment . Researchers should consider the following methodological framework:

Target Validation:

• Confirm disease relevance through gene expression and protein level analysis in relevant tissues

• Develop and characterize Fabp6 knockout models to understand phenotypic consequences

• Investigate tissue-specific effects, noting sex-specific differences observed in mice

• Evaluate impact on relevant metabolic parameters (glucose tolerance, insulin sensitivity)

High-Throughput Screening Strategy:

• Fragment-based drug discovery has proven successful for FABP6

• SPR-based screening can identify fragment hits with millimolar affinity

• Follow-up with SAR studies to confirm specific binding

• Validate hits through displacement assays with natural ligands (e.g., taurocholate)

Structure-Based Drug Design:

• Utilize the crystal structure of human FABP6 for rational design approaches

• Focus on the lipid-binding pocket identified in crystal structures

• Consider binding modes observed with natural ligands like cholate

• Design compounds with improved potency based on fragment hits

Physiological Relevance Assessment:

• Evaluate effects on bile acid homeostasis and lipid metabolism

• Monitor for potential malabsorption of dietary lipids (observed in knockout mice)

• Consider sex-specific effects on adiposity and gut microbiota composition

How can crystallography be used to investigate novel binding fragments in FABP6?

Based on successful crystallographic studies of FABP6 , the following methodological workflow is recommended:

Protein Preparation:

• Express His-tagged FABP6 in E. coli and purify to homogeneity

• Remove the His-tag if necessary using appropriate protease

• Concentrate protein to 10-20 mg/ml in crystallization buffer

Crystallization Strategy:

• Screen multiple conditions using sitting or hanging drop vapor diffusion

• Optimize promising conditions by varying pH, precipitant concentration, and additives

• Consider co-crystallization with ligands or fragment soaking experiments

Fragment Screening Approach:

• Select a diverse fragment library (typically 500-2000 compounds)

• Perform initial SPR-based screening to identify potential binders

• Validate hits through SAR and displacement assays with natural ligands

• Obtain crystals of FABP6 and soak with validated fragments

• Collect diffraction data and solve structures by molecular replacement

Structure Analysis and Optimization:

• Identify binding modes and key interactions of fragments

• Perform structure-based fragment growing or merging

• Design and synthesize compounds with improved binding properties

• Iterate through design-synthesis-testing cycle

Hendrick et al. successfully applied this approach to identify novel binding fragments in FABP6 and obtained crystal structures of complexes .

What methodological approaches can be used to investigate the potential role of FABP6 in cancer progression?

Studies have suggested potential roles for FABP6 in cancer, particularly prostate cancer . To investigate these associations, consider the following methodological framework:

Expression Analysis:

• Immunohistochemistry of tissue microarrays containing cancer and normal tissues

• RT-qPCR for mRNA expression quantification

• Western blotting for protein level analysis

• Mining public gene expression databases for correlations with clinical outcomes

Functional Studies in Cancer Cell Models:

• FABP6 overexpression in cancer cell lines

  • Assess effects on proliferation, migration, invasion, and colony formation

  • Analyze changes in lipid metabolism and signaling pathways

• FABP6 knockdown/knockout approaches

  • siRNA-mediated knockdown for transient suppression

  • CRISPR-Cas9 for stable knockout models

  • Evaluate phenotypic changes in cancer-related behaviors

Mechanism Investigation:

• Identify downstream targets using RNA-seq or proteomics

• Investigate interaction with known oncogenic pathways

• Examine effects on PPAR signaling (suggested in prostate cancer research)

• Study potential involvement in angiogenesis through VEGF regulation

In Vivo Models:

• Xenograft models using cells with modified FABP6 expression

• Patient-derived xenografts to maintain tumor heterogeneity

• Genetically engineered mouse models with tissue-specific FABP6 alterations

• Monitor tumor growth, metastasis, and response to therapies

How should I interpret contradictory data regarding FABP6 expression in different cancer types?

When facing contradictory data about FABP6 expression across cancer types, implement this analytical framework:

Methodological Considerations:

• Evaluate detection methods used (antibody specificity, primer design, detection thresholds)

• Consider heterogeneity within tumor samples (cell types, tumor microenvironment)

• Assess sample size and statistical power of conflicting studies

• Review normalization methods used for quantification

Biological Context Analysis:

• Analyze expression in the context of tissue-specific functions

• Consider FABP6's normal expression pattern (primarily in ileum)

• Evaluate correlation with bile acid metabolism in different tissues

• Investigate potential isoform-specific expression patterns

Resolving Contradictions:

• Perform meta-analysis of available data with clear inclusion criteria

• Design validation studies with well-characterized reagents

• Use multiple detection methods on the same samples

• Correlate expression with functional readouts in cell models

• Consider sex-specific differences (as observed in animal models)

Reporting Guidelines:

• Present contradictory findings transparently

• Describe methodological differences that might explain discrepancies

• Propose testable hypotheses to resolve contradictions

• Avoid overinterpretation of limited data sets

What statistical approaches are recommended for analyzing FABP6 binding assay data?

For robust analysis of FABP6 binding data, consider these statistical methodologies:

Methodological Recommendations:

• Include appropriate positive and negative controls in each experiment

• Perform experiments in at least triplicate (technical and biological replicates)

• Test for normality before applying parametric statistics

• Use Bland-Altman plots to assess agreement between different binding methods

• Apply Bonferroni or false discovery rate corrections for multiple comparisons

How can I validate my findings on FABP6 function using both in vitro and in vivo models?

A comprehensive validation strategy should integrate in vitro and in vivo approaches:

In Vitro Validation:

• Use multiple cell lines or primary cells to ensure robustness of findings

• Apply complementary techniques (e.g., binding assays, cellular localization, transport studies)

• Include rescue experiments in knockout/knockdown models

• Validate key findings with different methodological approaches

In Vivo Validation:

• Select appropriate animal models (Fabp6-knockout mice have been characterized)

• Consider sex-specific effects (documented for Fabp6-deficient mice)

• Use diet interventions to challenge the system (e.g., Western-style diet vs. low-fat diet)

• Measure multiple physiological parameters:

  • Bile acid excretion

  • Fat excretion

  • Energy metabolism (indirect calorimetry)

  • Gut microbiota composition

Integrative Analysis:

• Examine correlation between in vitro and in vivo findings

• Develop mechanistic models that explain observations at both levels

• Address discrepancies through additional targeted experiments

• Consider translational relevance to human physiology

Translational Considerations:

• Investigate FABP6 expression and function in human samples when available

• Use human intestinal organoids for more physiologically relevant in vitro models

• Consider population variations in FABP6 function or expression

What cellular models are most appropriate for studying FABP6 function?

Select cellular models based on research objectives and FABP6's physiological context:

Methodological Recommendations:

• Validate FABP6 expression in your chosen model by Western blot or qPCR

• Consider generating stable cell lines with controlled FABP6 expression

• Use fluorescently-tagged FABP6 for live-cell imaging studies

• Implement polarized cell models where appropriate for transport studies

• Complement cell line studies with primary cells or organoids when possible

How can I establish FABP6 knockout or knockdown models to study its function?

siRNA Knockdown Approach:

• Design 3-4 siRNA sequences targeting different regions of FABP6 mRNA

• Optimize transfection conditions for your cell model

• Validate knockdown efficiency by qPCR and Western blot

• Assess phenotypic changes 48-72 hours post-transfection

• Include scrambled siRNA controls and rescue experiments

CRISPR-Cas9 Knockout Strategy:

• Design 3-4 guide RNAs targeting early exons of FABP6

• Clone into appropriate CRISPR vector system

• Generate stable cell lines through selection

• Validate knockout through sequencing, Western blot, and functional assays

• Create single-cell clones and characterize multiple independent lines

• Consider generating conditional knockout systems for temporal control

Viral Vector Systems:

• Develop shRNA constructs for stable knockdown

• Use inducible promoters (e.g., Tet-On/Off) for controlled expression

• Validate with appropriate controls and rescue experiments

In Vivo Models:

• Fabp6-knockout mice have been characterized and show phenotypes related to bile acid and fat metabolism

• Consider diet interventions to reveal phenotypes (Western-style diet vs. low-fat diet)

• Analyze sex-specific differences in experimental outcomes

What techniques can be used to track FABP6-mediated bile acid transport in cells?

Fluorescent Bile Acid Analogs:

• Use fluorescently labeled bile acids (e.g., NBD-bile acids) for live-cell imaging

• Track uptake and intracellular distribution using confocal microscopy

• Perform quantitative analysis of fluorescence intensity and distribution

• Compare dynamics in FABP6-expressing and knockout/knockdown cells

Radiolabeled Assays:

• Use 3H- or 14C-labeled bile acids for transport studies

• Measure uptake, efflux, and transcellular transport in polarized models

• Analyze binding to recombinant FABP6 through equilibrium dialysis or filter binding

Mass Spectrometry-Based Approaches:

• Develop targeted LC-MS/MS methods for bile acid quantification

• Apply metabolic labeling for flux analysis

• Analyze bile acid profiles in cellular compartments and culture media

• Compare profiles between FABP6-manipulated and control cells

FRET-Based Biosensors:

• Develop FRET sensors using FABP6 conjugated with appropriate fluorophores

• Monitor conformational changes upon bile acid binding

• Track real-time changes in bile acid concentrations in living cells

• Correlate with physiological responses and transport activities