FABP5 Human, His

What is FABP5 and what are its primary functions in human cells?

FABP5 (also known as E-FABP or PA-FABP) is a 15kDa member of the intracellular fatty acid binding protein family that transports fatty acids to various cellular compartments including membranes, enzymes, and nuclear receptors . It plays critical roles in:

• Regulating lipid metabolism and signaling by modulating intracellular lipid availability

• Shuttling ligands to the nuclear receptor PPARβ/δ

• Facilitating the metabolism of anandamide (AEA) via fatty acid amide hydrolase (FAAH)

• Influencing autophagy pathways in neuronal cells

FABP5 is predominantly expressed in epidermal cells but shows significant expression in dopaminergic neurons within the substantia nigra and in SH-SY5Y cells .

How does FABP5's structure relate to its function?

FABP5 Human Recombinant is a single, non-glycosylated polypeptide chain containing 135 amino acids with a molecular mass of approximately 19.66kDa when fused to a His-tag . The protein contains an internal cavity designed to bind fatty acids and related compounds (bile acids or retinoids) . This structural feature enables FABP5 to:

• Solubilize and transport hydrophobic fatty acids through aqueous cellular environments

• Interact with specific fatty acids that trigger downstream signaling pathways

• Deliver bound ligands to nuclear receptors such as PPARβ/δ

What are the established ligands for FABP5 and how were they identified?

Several key ligands have been identified through lipidomic screening and biochemical studies:

These ligands were identified using techniques including co-immunoprecipitation followed by lipidomic analysis and virtual screening using DOCK with footprint similarity scoring .

What are optimal conditions for working with recombinant FABP5-His protein?

Based on established protocols, optimal handling conditions include:

• Storage: 4°C if using within 2-4 weeks; -20°C for longer periods; avoid freeze-thaw cycles

• Buffer: 20mM Tris HCl pH-8 and 50% glycerol

• Purity: >95% as determined by SDS-PAGE

• Expression system: E. coli with induction using 1mM isopropyl-1-thio-β-d-galactopyranoside

For experimental applications, functional FABP5 assays have been successfully conducted in neuroblastoma cells (SH-SY5Y, Neuro-2A) and HEK 293T cells .

How can researchers effectively study FABP5 expression and localization in cells?

Several validated methodological approaches include:

• Western blotting: Using specific antibodies against FABP5 (e.g., Cell Signaling Technology, 39926S) with appropriate secondary antibodies

• Tagged protein expression: Generating FABP5-V5 tagged or His-tagged constructs for localization and interaction studies

• Immunoprecipitation: Using V5 antibody (D3H8Q, Cell Signaling Technology) or similar for pull-down assays

• Subcellular fractionation: To determine compartmentalization of FABP5 within cells

• Knockdown approaches: Using siRNA/shRNA to evaluate functional effects of FABP5 depletion

For neuronal studies, SH-SY5Y cells can be differentiated into dopaminergic neuron-like cells using 10μM retinoic acid for 7 days prior to experimentation .

How does FABP5 contribute to cancer progression and what are the underlying mechanisms?

FABP5 demonstrates context-dependent roles in cancer through multiple mechanisms:

Pro-tumorigenic mechanisms:

• Promotes cell proliferation and invasion in human intrahepatic cholangiocarcinoma

• Forms a fatty-acid-induced FABP5/HIF-1 pathway that reprograms lipid metabolism in liver cancer cells

• Enhances hepatocellular carcinoma progression through degradation of Krüppel-like factor 9 mediated by miR-889-5p

• Activates nuclear fatty acid receptor PPARγ in prostate cancer cells

Anti-tumorigenic mechanisms:

• Restricts tumor growth by promoting IFN-β responses in tumor-associated macrophages

• Suppresses HER2-induced mammary tumorigenesis when genetically ablated

• Controls lung tumor metastasis by regulating natural killer cell maturation

These divergent functions suggest that FABP5 may serve as either a therapeutic target or a prognostic biomarker depending on cancer type and microenvironment.

What evidence links FABP5 to neurodegenerative pathologies?

FABP5 has been implicated in several neurodegenerative mechanisms:

• Parkinson's disease: FABP5 is crucial for mitochondrial dysfunction related to α-synuclein (αSyn) oligomerization/aggregation induced by oxidative stress in neurons . Co-expression of FABP5 with αSyn reduces cell viability, upregulates αSyn oligomerization and aggregation under oxidative stress conditions .

• Autophagy regulation: Knockdown of FABP5 suppresses autophagy in differentiated SH-SY5Y cells, a dopaminergic neuron-like model relevant to Parkinson's disease where autophagy dysfunction is closely linked to pathogenesis .

• Cognitive function: FABP5-null mice display impaired learning and memory, with high brain levels of anandamide and low PPARβ/δ activation . This suggests FABP5 regulates cognitive function through dual mechanisms: by shuttling ligands to PPARβ/δ and by modulating endocannabinoid metabolism .

How is FABP5 involved in pain perception and what therapeutic potential does this present?

FABP5 functions as a key regulator in pain perception through its role in endocannabinoid signaling:

• FABP5 transports anandamide (AEA) within cells and facilitates its metabolism via fatty acid amide hydrolase (FAAH)

• By regulating AEA levels, FABP5 indirectly modulates cannabinoid receptor signaling, particularly CB1 activation

• Inhibition of FABP5 prevents AEA degradation, elevating AEA levels and enhancing CB1 activation

• This mechanism promotes increased pain tolerance and exerts anti-inflammatory effects

The therapeutic potential lies in developing FABP5 inhibitors as novel pain management agents that avoid limitations associated with traditional analgesics:

• Unlike opioids, FABP5 inhibitors would not carry addiction potential or overdose risk

• Unlike NSAIDs, they would avoid gastrointestinal bleeding and other adverse effects

Researchers have used virtual screening to identify small molecules with binding profiles similar to oleic acid (a natural FABP substrate) as potential FABP5 inhibitors .

How do FABP5-binding lipids regulate autophagy in neuronal cells?

A sophisticated lipidomic analysis revealed that specific FABP5-binding lipids differentially regulate autophagy:

RNA-Seq analysis revealed both shared and divergent signaling pathways activated by these lipids in SH-SY5Y cells . Interestingly, FABP5 knockdown suppressed autophagy, suggesting that the protein normally promotes autophagy in neuronal cells - a finding with potential implications for Parkinson's disease where autophagy dysfunction is a key pathological feature .

What is the molecular mechanism underlying FABP5's influence on α-synuclein aggregation?

Research using neuronal models has elucidated the following mechanism:

• Under oxidative stress conditions (induced by rotenone, a mitochondrial complex I inhibitor), FABP5 co-localizes with α-synuclein (αSyn) in mitochondria

• This co-localization significantly reduces mitochondrial membrane potential

• FABP5 co-expression with αSyn reduces cell viability compared to αSyn alone

• FABP5 upregulates αSyn oligomerization and aggregation specifically under oxidative stress conditions

• A high-affinity FABP5 ligand can abolish the co-localization of FABP5 and αSyn in mitochondria, suggesting a potential therapeutic intervention point

This mechanism provides critical insight into how FABP5 might contribute to Parkinson's disease pathogenesis, where αSyn accumulation is a causal factor and mitochondrial dysfunction is a primary driver of dopaminergic neuronal death .

How do FABP5 and the endocannabinoid system interact to influence cognitive function?

FABP5 regulates cognitive function through two distinct but interconnected mechanisms:

• Endocannabinoid regulation: FABP5 enhances the degradation of anandamide (AEA) by facilitating its delivery to fatty acid amide hydrolase (FAAH) . In FABP5-null mice, brain levels of AEA are elevated, which affects signaling through cannabinoid receptors CB1 and CB2 .

• Nuclear receptor signaling: FABP5 shuttles ligands (potentially including arachidonic acid, a metabolite of AEA) to the nuclear receptor PPARβ/δ . In FABP5-null mice, PPARβ/δ activation is reduced .

The dual impairment of these pathways in FABP5-null mice results in learning and memory deficits, suggesting that balanced endocannabinoid signaling and PPARβ/δ activation are necessary for optimal cognitive function .

This mechanistic understanding positions FABP5 as a potential therapeutic target for cognitive dysfunction, particularly in conditions where endocannabinoid signaling or PPARβ/δ activity is disrupted .

What experimental models are most suitable for studying FABP5 function?

Based on published research, the following experimental models have proven effective:

Cell Models:

• SH-SY5Y neuroblastoma cells: Differentiated with 10μM retinoic acid for 7 days to create dopaminergic neuron-like cells; ideal for Parkinson's disease-related studies

• Neuro-2A cells: Useful for studying αSyn aggregation and mitochondrial dysfunction

• HEK 293T cells: Suitable for overexpression studies and protein production

Animal Models:

• FABP5-null mice: Display impaired learning and memory, elevated brain anandamide levels, and reduced PPARβ/δ activation

• Tissue-specific FABP5 transgenic mice: Models expressing high levels of FABP5 in specific tissues show altered insulin sensitivity and metabolic phenotypes

Molecular Tools:

• FABP5-V5 tagged constructs: Enable immunoprecipitation studies to identify interacting partners

• His-tagged FABP5 expression systems: Facilitate protein purification and biochemical studies

• CRISPR/Cas9 screening: Identifies FABP5-related pathways in cancer and other contexts

What approaches are most effective for identifying and validating FABP5 inhibitors?

A systematic approach to FABP5 inhibitor discovery includes:

• Virtual screening: Using DOCK software with footprint similarity scoring to identify compounds with binding profiles similar to natural FABP5 substrates like oleic acid

• Binding assays: Measuring displacement of known FABP5 ligands by potential inhibitors

• Functional validation:

  • Measuring effects on anandamide metabolism

  • Assessing pain perception in animal models

  • Evaluating mitochondrial function in neuronal cells

  • Testing effects on autophagy in SH-SY5Y cells

• Specificity determination: Confirming selectivity for FABP5 over other FABP family members

• Mechanism verification: Demonstrating that inhibitors prevent specific FABP5-mediated processes such as:

  • αSyn aggregation in mitochondria under oxidative stress

  • Delivery of anandamide to FAAH

  • Transport of signaling lipids to PPARβ/δ

This multifaceted approach ensures that identified inhibitors are both potent and specific, with clear mechanisms of action relevant to therapeutic applications.