Recombinant Macaca fascicularis Long-chain fatty acid transport protein 4 (SLC27A4)

What is the fundamental function of SLC27A4 in cellular metabolism?

SLC27A4 (solute carrier family 27 member 4) functions primarily as a transmembrane protein that facilitates the translocation of long-chain fatty acids across the plasma membrane. Beyond simple transport, it possesses acyl-CoA synthetase activity, enabling it to catalyze the ATP-dependent esterification of long-chain and very-long-chain fatty acids with coenzyme A. This dual functionality makes SLC27A4 particularly important in cells with high fatty acid metabolism requirements. The protein demonstrates significant activity towards palmitic acid (C16:0) and considerably greater activity towards lignoceric acid (C24:0), while also activating arachidonic acid (C20:4n-6) . Through these mechanisms, SLC27A4 plays crucial roles in lipid biosynthesis, fatty acid oxidation, and cellular energy homeostasis.

What pathways and processes involve SLC27A4 in cellular biology?

SLC27A4 participates in multiple cellular pathways critical to lipid metabolism and energy homeostasis. Key pathways include:

These pathways highlight SLC27A4's integration in broader metabolic networks beyond simple fatty acid transport.

What are the optimal expression systems for producing functional recombinant Macaca fascicularis SLC27A4?

The production of functional recombinant Macaca fascicularis SLC27A4 requires careful consideration of expression systems to maintain proper folding and activity of this transmembrane protein. Based on current research approaches:

• E. coli expression systems: While cost-effective, E. coli-based expression (as in the GST-fusion approach) is suitable primarily for producing soluble domains or when partial functionality is sufficient . The PGEX-4T vector system with GST tags has been successfully used, though proper refolding protocols are essential for obtaining functional protein.

• Mammalian cell expression systems: HEK293 cells provide superior post-translational modifications and proper membrane insertion, making them preferable for full-length, fully functional SLC27A4 studies . This system is particularly recommended for functional assays and structural studies.

• Insect cell systems: Baculovirus-infected insect cells offer a compromise between yield and proper folding, suitable for large-scale production while maintaining most post-translational modifications .

The choice of expression system should be guided by the specific experimental requirements, with mammalian systems generally preferred when complete functionality is essential, particularly for enzymatic activity assays or interaction studies.

What methodologies are most effective for assessing SLC27A4 transport activity in vitro?

Evaluating the transport activity of recombinant SLC27A4 requires specialized assays that can distinguish between simple binding and actual translocation of fatty acids. The most effective methodologies include:

• Fluorescent fatty acid analog uptake: Using fluorescently labeled fatty acids (e.g., BODIPY-labeled fatty acids) to track transport kinetics in real-time through fluorescence microscopy or plate-reader assays. This approach allows for continuous monitoring but may be affected by the modified structure of labeled substrates.

• Radiolabeled fatty acid transport assays: Employing 14C or 3H-labeled fatty acids provides the most direct quantification of transport activity. This method typically involves rapid separation of internalized versus external fatty acids using oil-stop techniques or cell washing protocols followed by scintillation counting.

• Acyl-CoA synthetase activity coupling: Since SLC27A4 possesses intrinsic acyl-CoA synthetase activity, assays measuring AMP production or CoA consumption can indirectly quantify transport coupled to activation. These enzymatic assays can be performed using purified recombinant protein incorporated into liposomes or proteoliposomes.

• Competitive inhibition assays: Using known SLC27A4 inhibitors to establish specificity of the observed transport, which helps distinguish SLC27A4-mediated transport from passive diffusion or other transporters.

For recombinant Macaca fascicularis SLC27A4 specifically, researchers should optimize buffer conditions (pH 7.2-7.4), temperature (37°C), and substrate concentrations based on the known kinetic parameters of the protein.

How can researchers effectively reconstitute lyophilized SLC27A4 protein while maintaining functionality?

Proper reconstitution of lyophilized recombinant SLC27A4 is critical for downstream applications. To maintain optimal functionality:

The reconstituted protein should be stored in small aliquots to avoid repeated freeze-thaw cycles, with storage at -80°C for long-term stability.

How do functional properties of Macaca fascicularis SLC27A4 compare to human and murine orthologs?

The functional properties of SLC27A4 demonstrate both conservation and species-specific differences that researchers should consider when using Macaca fascicularis as a model:

These differences are particularly important when designing inhibitor studies or when investigating tissue-specific functions. The Macaca fascicularis model offers advantages over murine models for skin and neurological studies due to its closer physiological similarity to humans, while maintaining sufficient homology for molecular-level investigations.

What are the key considerations when using antibodies raised against human SLC27A4 for detection of the Macaca fascicularis ortholog?

When utilizing antibodies developed against human SLC27A4 for detecting the Macaca fascicularis ortholog, researchers should consider several critical factors:

These considerations will help ensure specific and reliable detection of Macaca fascicularis SLC27A4 when using human-targeted antibodies.

How does SLC27A4 expression in glioblastoma differ from normal brain tissue, and what methodologies best detect these differences?

Research has revealed significant alterations in SLC27A4 expression in glioblastoma compared to normal brain tissue, with important implications for understanding tumor metabolism:

The expression of SLC27A4 is demonstrably lower in glioblastoma tumor tissue compared to peritumoral areas, suggesting altered fatty acid metabolism in these aggressive brain tumors . This reduced expression may contribute to the metabolic reprogramming observed in glioblastoma, potentially shifting energy production away from fatty acid oxidation toward increased glycolysis.

For optimal detection and quantification of these expression differences, researchers should consider:

• Quantitative Real-Time PCR (qRT-PCR): This technique provides sensitive detection of SLC27A4 transcript levels and has been successfully employed to identify decreased expression in tumor samples . Key methodological considerations include:

  • Using multiple reference genes (GAPDH, ACTB, and TBP) for normalization

  • Employing the 2^-ΔΔCt method for relative quantification

  • Designing primers that span exon-exon junctions to avoid genomic DNA amplification

• Immunohistochemistry/Immunofluorescence: These techniques enable spatial visualization of SLC27A4 protein expression within the tumor and peritumoral tissue architecture, revealing cell type-specific expression patterns. Optimal protocols include:

  • Antigen retrieval using citrate buffer (pH 6.0) with pressure cooker treatment

  • Signal amplification with tyramide signal amplification for low-abundance detection

  • Co-staining with cell type-specific markers to identify expressing cell populations

• Western Blotting with Region-Specific Sampling: This approach allows quantitative comparison between tumor regions and control tissue, though careful microdissection is required to separate tumor core from periphery and peritumoral regions.

Importantly, correlative analyses have revealed that SLC27A4 expression patterns in glioblastoma appear to be influenced by patient factors including BMI and smoking history, with different correlations observed between men and women .

What experimental approaches can determine if SLC27A4 functionally interacts with other fatty acid transport proteins (FATPs) in metabolic pathways?

Investigating functional interactions between SLC27A4 and other fatty acid transport proteins requires multi-faceted experimental approaches:

• Co-immunoprecipitation and Proximity Ligation Assays: These techniques can identify physical interactions between SLC27A4 and other FATPs like SLC27A1, with which it has been shown to cooperate in fatty acid transport across the blood-brain barrier . Key methodological considerations include:

  • Using membrane-compatible detergents (digitonin or n-dodecyl β-D-maltoside) that preserve protein-protein interactions

  • Performing reciprocal pulldowns to confirm specificity of interactions

  • Including appropriate controls for non-specific binding

• FRET/BRET Analysis: Förster/Bioluminescence Resonance Energy Transfer approaches can detect close proximity between tagged proteins in living cells, providing evidence for direct interactions. This requires:

  • Generation of fusion constructs with appropriate fluorescent/luminescent tags

  • Careful selection of tag positions to avoid disrupting protein function

  • Controls for random collision versus specific interaction

• Functional Compensation Studies: RNA interference or CRISPR-based approaches targeting individual or multiple FATP family members can reveal functional redundancy or cooperation:

  • Knockdown of SLC27A4 followed by measurement of fatty acid uptake

  • Simultaneous knockdown of SLC27A4 and other FATPs (particularly SLC27A1)

  • Rescue experiments with wild-type or mutant constructs

• Pathway Flux Analysis: Metabolic labeling with stable isotope-labeled fatty acids can track the contribution of different transporters to downstream metabolic pathways:

  • Incorporation of labeled fatty acids into triglycerides, phospholipids, and ceramides

  • Mass spectrometry-based quantification of labeled metabolites

  • Comparative analysis under conditions of FATP knockdown or overexpression

These approaches collectively provide complementary data on whether SLC27A4 functions independently or as part of a coordinated network with other FATPs in specific tissues or metabolic conditions.

How can researchers effectively analyze the role of SLC27A4 in the PPAR signaling pathway?

The relationship between SLC27A4 and the PPAR signaling pathway represents a complex regulatory network that can be investigated through several complementary approaches:

• Reporter Gene Assays: Utilizing PPAR-responsive element (PPRE) luciferase constructs to measure transcriptional activation:

  • Transfect cells with PPRE-luciferase reporter alongside SLC27A4 expression vectors

  • Treat with PPAR agonists (e.g., fibrates for PPARα, thiazolidinediones for PPARγ)

  • Measure changes in reporter activity with SLC27A4 overexpression or knockdown

• Chromatin Immunoprecipitation (ChIP): To determine if PPARs directly regulate SLC27A4 expression:

  • Perform ChIP using antibodies against different PPAR isoforms

  • Analyze binding to putative PPREs in the SLC27A4 promoter

  • Conduct sequential ChIP to identify co-regulatory complexes

• Metabolomic Profiling: Since SLC27A4 generates fatty acid-CoA that may serve as PPAR ligands:

  • Compare lipid profiles in cells with normal versus altered SLC27A4 expression

  • Identify specific lipid species that may activate PPARs

  • Correlate changes in lipid profiles with PPAR target gene expression

• Integrated Pathway Analysis: Using systems biology approaches:

  • RNA-seq to identify transcriptome-wide effects of SLC27A4 modulation

  • Pathway enrichment analysis focusing on PPAR-regulated genes

  • Network modeling to position SLC27A4 within the broader PPAR signaling network

The PPAR pathway analysis should include examination of key proteins known to interact with SLC27A4 in this context, including RXRA, PPARD, CD36, and ANGPTL4 . This comprehensive approach will help determine whether SLC27A4 functions primarily as a PPAR target gene, as a modulator of PPAR activity through lipid metabolism, or both.

How might alterations in SLC27A4 function contribute to metabolic disorders, and what models best study these mechanisms?

SLC27A4 dysfunction has been implicated in several metabolic disorders, with research suggesting multiple mechanistic pathways:

• Insulin Resistance and Type 2 Diabetes: Clinical studies have identified SLC27A4 as a candidate gene for insulin resistance syndrome . The mechanisms likely involve:

  • Altered fatty acid composition in cellular membranes affecting insulin receptor signaling

  • Dysregulated ceramide production leading to impaired insulin signaling cascade

  • Changes in lipid droplet formation affecting adipocyte function and adipokine secretion

• Skin Disorders: Mutations in SLC27A4 have been associated with ichthyosis prematurity syndrome , demonstrating its importance in:

  • Epidermal barrier formation through proper ceramide synthesis

  • Keratinocyte differentiation processes

  • Maintenance of skin lipid homeostasis

• Neurological Disorders: Given its high expression in the brain and role in fatty acid transport across the blood-brain barrier, SLC27A4 dysfunction may contribute to:

  • Altered brain energy metabolism

  • Disrupted myelination processes

  • Neuroinflammatory responses through modified eicosanoid production

The most effective models for studying these mechanisms include:

When selecting a model, researchers should consider the specific aspect of SLC27A4 function being studied and the translational goals of the research program.

What are the most promising approaches for targeting SLC27A4 in therapeutic development for metabolic diseases?

The unique dual functionality of SLC27A4 as both a transporter and an enzyme offers multiple avenues for therapeutic intervention in metabolic diseases:

• Small Molecule Inhibitors: Development of compounds that selectively inhibit either the transport function or the acyl-CoA synthetase activity:

  • Transport inhibitors: Focus on the transmembrane domains and substrate binding pocket

  • Enzymatic inhibitors: Target the ATP-binding region or CoA-binding site

  • Allosteric modulators: Identify regulatory sites that influence protein conformation

  Key considerations include selectivity over other FATP family members and tissue-specific targeting to avoid unwanted side effects.

• Gene Therapy Approaches: For genetic disorders associated with SLC27A4 mutations:

  • AAV-mediated gene delivery to affected tissues (skin, intestine)

  • CRISPR-based gene correction in stem cells for autologous transplantation

  • Antisense oligonucleotides for modulating splicing in specific mutations

• Indirect Targeting Through PPAR Pathway Modulation:

  • Development of tissue-specific PPAR modulators that regulate SLC27A4 expression

  • Combined approaches targeting both SLC27A4 and other components of fatty acid metabolism

• Repurposing Existing Compounds: Several existing drugs have been shown to modulate FATP function:

  • Thiazolidinediones affect FATP expression through PPAR activation

  • Certain lipid-lowering agents may interact with fatty acid transport systems

  • Anti-inflammatory compounds that modulate lipid mediator production

The therapeutic potential of targeting SLC27A4 is particularly promising for conditions including insulin resistance, inflammatory skin disorders, and certain neurological conditions where altered fatty acid metabolism plays a pathogenic role.

How can researchers effectively evaluate the role of SLC27A4 in cancer metabolism, particularly in brain tumors?

The emerging role of SLC27A4 in cancer metabolism, particularly its reduced expression in glioblastoma , presents important research opportunities. To effectively evaluate its contribution to cancer biology:

• Multi-omics Profiling of Patient Samples:

  • Integrate transcriptomic, proteomic, and lipidomic analyses of tumor versus normal tissue

  • Correlate SLC27A4 expression with lipid composition profiles

  • Stratify tumors based on SLC27A4 expression patterns and correlate with clinical outcomes

• Functional Studies in Patient-Derived Xenografts and Organoids:

  • Manipulate SLC27A4 expression in patient-derived models

  • Assess changes in tumor growth, invasion, and response to therapies

  • Perform metabolic flux analysis using stable isotope-labeled fatty acids

• Mechanistic Investigations in Cell Culture Models:

  • Engineer isogenic cell lines with varying levels of SLC27A4 expression

  • Examine effects on cancer hallmark processes (proliferation, migration, resistance to apoptosis)

  • Determine metabolic dependencies through nutrient restriction studies

• Therapeutic Targeting Approaches:

  • Exploit the differential expression of SLC27A4 between tumor and normal tissue

  • Test metabolic vulnerabilities created by altered fatty acid transport

  • Evaluate combination approaches targeting multiple metabolic pathways

A particularly important consideration is the relationship between SLC27A4 expression and patient characteristics such as BMI and smoking history, which have shown gender-specific correlations in glioblastoma studies . These demographic factors should be incorporated into experimental designs and data analysis to capture the full complexity of SLC27A4's role in cancer metabolism.

What are the latest techniques for structural characterization of membrane proteins like SLC27A4?

Recent technological advances have significantly improved our ability to characterize the structure of challenging membrane proteins like SLC27A4:

• Cryo-Electron Microscopy (Cryo-EM): This technique has revolutionized membrane protein structural biology:

  • Sample preparation using nanodiscs or amphipols to maintain native-like lipid environments

  • Direct electron detectors and improved image processing allowing for near-atomic resolution

  • Time-resolved cryo-EM to capture different conformational states during transport cycles

• Advanced NMR Methods:

  • Solid-state NMR techniques compatible with membrane proteins in lipid bilayers

  • Selective isotopic labeling strategies to study specific domains or residues

  • Methyl-TROSY approaches for studying large membrane protein dynamics

• Computational Structure Prediction:

  • AI-driven platforms like AlphaFold2 and RoseTTAFold showing remarkable accuracy for membrane proteins

  • Molecular dynamics simulations in explicit membrane environments

  • Enhanced sampling techniques to explore conformational landscapes

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Provides information on protein dynamics and solvent accessibility

  • Compatible with detergent-solubilized membrane proteins

  • Reveals ligand-binding sites and conformational changes

How can researchers effectively determine the physiological substrates and substrate specificity profile of SLC27A4?

Determining the true physiological substrates and substrate specificity of SLC27A4 requires sophisticated experimental approaches that go beyond simple transport assays:

• Comprehensive Substrate Screening Platforms:

  • High-throughput competition assays using a reference substrate

  • Mass spectrometry-based untargeted lipidomics to identify enriched lipid species

  • Isothermal titration calorimetry (ITC) for direct binding measurements

• Cellular Metabolic Tracing:

  • Stable isotope-labeled fatty acid incorporation studies

  • Pulse-chase experiments to track substrate metabolism

  • Comparative analysis across tissues and cell types

• Structure-Activity Relationship Studies:

  • Systematic testing of fatty acids varying in chain length, saturation, and functional groups

  • Molecular docking simulations using structural models

  • Site-directed mutagenesis of predicted binding pocket residues

• Physiological Context Consideration:

  • Tissue-specific metabolomic profiling in SLC27A4-expressing tissues

  • Comparison of substrate profiles under different physiological conditions (fed vs. fasted)

  • Evaluation of competitive effects of physiological fatty acid mixtures

What are the emerging research directions and unanswered questions regarding SLC27A4 function?

Despite significant advances in our understanding of SLC27A4, several critical knowledge gaps and emerging research directions merit attention:

• Structural Dynamics During Transport:

  • How does SLC27A4 undergo conformational changes during the transport cycle?

  • What is the molecular mechanism coupling transport to acyl-CoA synthetase activity?

  • How do lipid environments modulate SLC27A4 function?

• Regulatory Networks:

  • What transcriptional and post-translational mechanisms regulate SLC27A4 activity?

  • How is SLC27A4 function integrated with cellular nutrient sensing pathways?

  • What protein-protein interactions modulate its activity in different tissues?

• Pathophysiological Roles:

  • Why is SLC27A4 expression reduced in glioblastoma, and what are the functional consequences?

  • How do polymorphisms in SLC27A4 contribute to metabolic disease susceptibility?

  • What is the role of SLC27A4 in inflammation and immune cell function?

• Therapeutic Targeting:

  • Can selective modulators of SLC27A4 be developed with therapeutic potential?

  • Would targeting SLC27A4 in metabolic disorders produce beneficial effects with acceptable side effect profiles?

  • How might combination approaches targeting multiple aspects of fatty acid metabolism be optimized?