FABP7 Human, His

What is FABP7 and what are its primary functions in human biology?

FABP7, also known as BLBP, FABPB, B-FABP, or Brain lipid-binding protein, is a highly conserved cytoplasmic protein belonging to the fatty acid binding protein family. It consists of 132 amino acids and has a beta-barrel structure with a hydrophobic binding pocket .

FABP7 is primarily expressed in neural stem cells and astrocytes in the brain, where it performs several key functions:

• Transport and metabolism of long-chain fatty acids, particularly docosahexaenoic acid (DHA)

• Participation in neurogenesis and establishment of the radial glial fiber system

• Regulation of sleep patterns and quality

• Protection against oxidative stress via lipid droplet formation

• Support of brain development through DHA transport

The protein is highly expressed during brain development and plays crucial roles in neuronal migration and cortical layer formation .

What are the structural characteristics of recombinant human FABP7-His protein?

Recombinant human FABP7-His protein has several distinctive structural features:

• A single, non-glycosylated polypeptide chain of 132 amino acids

• Molecular mass of approximately 19.39 kDa (with the His-tag)

• N-terminal His-tag for purification purposes

• Beta-barrel structure with 10 anti-parallel beta-strands

• Two alpha-helices that form a "lid" over the binding pocket

• Hydrophobic binding cavity that accommodates fatty acids, particularly DHA

• Conserved Thr61 residue located near the fatty acid binding site

The protein's three-dimensional structure creates a specific binding environment that enables its high affinity for DHA, distinguishing it from other FABP family members .

What are the recommended storage and handling conditions for FABP7-His protein?

For optimal stability and activity of recombinant FABP7-His protein, researchers should follow these storage and handling recommendations:

Important handling considerations:

• Avoid multiple freeze-thaw cycles as they can degrade the protein

• Aliquot into single-use volumes before freezing

• Thaw samples on ice when ready to use

• Centrifuge briefly before opening the vial to collect contents at the bottom

How does FABP7 contribute to neurogenesis and brain development?

FABP7 plays critical roles in neurogenesis and brain development through several mechanisms:

• Radial glial system: FABP7 is required for establishing the radial glial fiber system, which serves as scaffolding for migrating neurons during cortical layer formation

• DHA transport: FABP7 binds and transports DHA with high affinity, facilitating its availability for neuronal membrane development and synaptogenesis

• Neural stem cell function: FABP7 is expressed in neural stem cells and regulates their proliferation and differentiation, with FABP7 knockout mice showing decreased neurogenesis

• Recovery after injury: After spinal cord injury, FABP7 knockout mice produce fewer neurons compared to wild-type mice, suggesting a role in neuronal regeneration

• Notch signaling: FABP7 expression in radial glia is activated by Notch receptors, integrating it into broader developmental signaling pathways

These functions collectively establish FABP7 as a crucial factor in proper brain development and neuronal organization .

How does the Thr61Met polymorphism affect FABP7 function and sleep patterns?

The Thr61Met polymorphism in FABP7 has significant implications for protein function and sleep regulation:

Methodological approaches to study this polymorphism include:

• Sleep monitoring systems to quantify sleep fragmentation patterns

• Structural analysis to determine how the substitution affects fatty acid binding

• Binding assays comparing wild-type and mutant protein affinity for various fatty acids

• Electrophysiological recordings to assess neural circuit activity in models expressing the variant

The mechanism likely involves altered binding and transport of fatty acids, particularly DHA, which subsequently affects neural circuits involved in sleep regulation .

What methodologies are most effective for studying FABP7's role in metabolic reprogramming in cancer cells?

Research on FABP7's role in cancer metabolic reprogramming, particularly in HER2+ breast cancer brain metastasis, has employed several effective methodologies:

• Genetic manipulation approaches:

  • Stable knockdown using shRNAs in cancer cell lines (BT474-Br, HCC 1569)

  • Complementary overexpression studies to validate phenotypic changes

  • CRISPR-Cas9 gene editing for complete knockout studies

• Metabolic analysis techniques:

  • Extracellular flux analysis measuring oxygen consumption rate (OCR) for OXPHOS activity

  • Measuring extracellular acidification rate (ECAR) for glycolytic activity

  • Metabolic stress tests using oligomycin, FCCP, and other inhibitors to assess specific aspects of energy metabolism

• Multi-omics approaches:

  • Liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify differentially expressed proteins

  • Gene ontology analysis to identify affected metabolic pathways

  • Western blotting to validate changes in expression of metabolic enzymes and invasion-related proteins

• Functional assays:

  • Invasion assays to correlate metabolic changes with invasive capacity

  • Lipid droplet visualization and quantification

  • Assessment under both normoxic and hypoxic conditions to mimic tumor microenvironment

These methodologies have revealed that FABP7 promotes a glycolytic phenotype and lipid droplet storage, enabling cancer cell adaptation and survival in the brain microenvironment .

How can researchers differentiate between FABP7-mediated effects and those of other FABP family members?

Distinguishing FABP7-specific effects from those of other FABP family members requires careful experimental design:

When studying FABP7 in the context of cancer metabolic reprogramming, researchers should particularly focus on its unique roles in promoting glycolysis and lipid droplet formation that may not be shared by other FABP family members .

What is FABP7's role in integrin-Src signaling and cancer metastasis?

FABP7 has emerged as a significant regulator of the integrin-Src signaling pathway, which plays a crucial role in cancer metastasis, particularly in HER2+ breast cancer brain metastasis:

• Pathway involvement:

  • FABP7 promotes integrin-Src signaling, which coordinates multiple pathways involved in tumor progression

  • This pathway is activated downstream of integrin family cell adhesion receptors

  • Hyperactivation of this pathway promotes proliferation, survival, angiogenesis, and metastasis

• Research evidence:

  • Mass spectrometry analysis of FABP7 knockdown cells showed decreased levels of invasion-related proteins

  • FABP7 knockdown resulted in increased expression of tight junction-related proteins, which typically prevent metastasis

  • Overexpression of FABP7 had opposite effects, decreasing tight junction proteins like ZO-2

• Experimental approaches for investigation:

  • Western blotting to assess phosphorylation status of key pathway components

  • Immunoprecipitation to detect protein-protein interactions

  • Invasion assays under normoxic and hypoxic conditions

  • Combined inhibition of FABP7 and pathway components

• Impact on metastasis:

  • FABP7 is required for up-regulation of key metastatic genes and pathways, including Integrins-Src and VEGFA

  • It promotes the growth of HER2+ breast cancer cells in the brain microenvironment in vivo

  • FABP7 expression correlates with poor survival and increased incidence of brain metastases in breast cancer patients

These findings position FABP7 as a potential therapeutic target for preventing or treating brain metastasis in HER2+ breast cancer patients.

How does FABP7 contribute to lipid droplet formation and oxidative stress protection?

FABP7 plays a crucial role in lipid droplet formation and protection against oxidative stress:

• Lipid transport and storage:

  • FABP7 binds and transports fatty acids, particularly long-chain fatty acids like DHA

  • This transport function facilitates the incorporation of fatty acids into lipid droplets

  • Proper lipid trafficking is essential for maintaining cellular lipid homeostasis

• Oxidative stress protection in neural cells:

  • Mammalian cell line studies have shown that FABP7 protects astrocytes from oxidative stress via lipid droplet accumulation

  • Lipid droplets sequester potentially toxic fatty acids, preventing their oxidation and subsequent generation of reactive oxygen species

  • This protective mechanism is particularly important in the brain, where oxidative stress is implicated in neurodegenerative diseases

• Cancer cell adaptation:

  • In cancer cells, FABP7-mediated lipid droplet formation serves as:

    • Energy storage hubs for metabolic adaptation

    • Protection against reactive oxygen species

    • Support for survival during reoxygenation after hypoxia

  • This adaptation mechanism enables cancer cells to survive in challenging microenvironments like the brain

• Metabolic reprogramming:

  • FABP7's role in promoting lipid droplet formation contributes to metabolic flexibility

  • This flexibility is crucial for cancer cells to adapt to varying nutrient availability and oxygen levels

  • FABP7 knockdown studies show altered expression of glycolytic enzymes, suggesting a link between lipid metabolism and glycolytic activity

Understanding these mechanisms provides potential avenues for therapeutic intervention in both neurodegenerative diseases and FABP7-expressing cancers.

What experimental systems are optimal for studying FABP7 function?

Researchers investigating FABP7 function can utilize various experimental systems, each with specific advantages:

For comprehensive investigation, combining multiple systems is recommended. For instance, mechanistic insights gained from recombinant protein and cell culture studies can inform the design and interpretation of animal model experiments, while findings from patient samples can validate the clinical relevance of experimental results .

How can FABP7 reporter systems be optimized for tracking neural differentiation?

FABP7 reporter systems, such as the FABP7 pGreenZeo differentiation reporter, can be optimized for tracking neural differentiation through several strategic approaches:

• Reporter design enhancements:

  • Incorporate bright, fast-maturing fluorescent proteins for superior signal detection

  • Add destabilization domains to ensure signal accurately reflects current expression

  • Include nuclear localization signals to concentrate fluorescence for easier detection

  • Design dual reporters that track FABP7 alongside other neural markers

• Optimized promoter elements:

  • Include not only the core FABP7 promoter but also crucial enhancer regions that respond to neural differentiation signals

  • Consider using synthetic promoters that amplify signal while maintaining specificity

  • Engineer inducible elements for temporal control of reporter activation

• Advanced imaging strategies:

  • Employ automated high-content imaging systems for continuous monitoring

  • Implement incubator-integrated microscopy for long-term tracking

  • Utilize machine learning algorithms for automated cell tracking and lineage analysis

  • Develop 3D imaging capabilities for assessing differentiation in organoids

• Validation techniques:

  • Correlate reporter signal with endogenous FABP7 expression via qPCR and immunostaining

  • Create standard curves relating signal intensity to absolute FABP7 expression levels

  • Use flow cytometry to quantify reporter activity in heterogeneous populations

These optimizations enable researchers to effectively track neural differentiation in real-time, facilitating studies of developmental processes, drug screening for neurogenic compounds, and optimization of differentiation protocols for regenerative medicine applications .

What are the limitations of current FABP7 knockout models for studying neurological disorders?

Current FABP7 knockout models present several limitations for neurological disorder research:

• Compensatory mechanisms:

  • Other FABP family members may compensate for FABP7 loss, masking phenotypes

  • Long-term adaptation to FABP7 absence may not reflect acute loss in adult or disease contexts

  • These compensations vary between genetic backgrounds, as seen in schizophrenia studies

• Species-specific differences:

  • Mouse FABP7 may not perfectly recapitulate all functions of human FABP7

  • Human-specific neurological features may be inadequately modeled

  • Different brain development timelines between species affect result interpretation

• Background strain effects:

  • Different mouse strains show variable phenotypes when FABP7 is knocked out

  • Studies on schizophrenia demonstrated that knockouts on different backgrounds yielded conflicting results regarding association with the disorder

• Developmental vs. adult functions:

  • Germline knockouts affect development from conception

  • Difficult to distinguish developmental effects from adult functions

  • Conditional knockouts would be more appropriate for studying adult-onset disorders

• Human polymorphism modeling:

  • Complete knockout does not accurately model effects of human variants like Thr61Met

  • Knock-in models of specific human variants would better recapitulate human genetic conditions

To address these limitations, researchers should consider:

• Developing conditional and inducible knockout models

• Creating knock-in models of human FABP7 variants

• Implementing cell-type specific manipulations

• Incorporating environmental factors relevant to specific disorders

• Using multiple genetic backgrounds to assess strain-dependent effects