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

Basic Research Questions

• What is the genomic structure and expression profile of SLC27A4?

  SLC27A4 (solute carrier family 27 member 4), also known as FATP4, is encoded by a gene consisting of 13 exons . The protein is broadly distributed across multiple tissues, with particularly high expression in the small intestine, skin, heart, brain, kidney, adipose tissue, and skeletal muscle . It is notably the only FATP family member expressed in the small intestine, where it appears to be the principal fatty acid transporter in enterocytes . SLC27A4 is predominantly localized to the apical side of mature enterocytes and in various subcellular compartments including the endoplasmic reticulum, mitochondria, mitochondria-associated membranes, and peroxisomes in other cell types .

• What are the structural characteristics and functional domains of SLC27A4?

  SLC27A4 is a 72-kDa transmembrane protein (643 amino acids in humans) that contains:

  • An N-terminal transmembrane domain

  • An ER localization signal

  • A highly conserved AMP-binding domain (amino acids 103-536) which includes:

    • The ATP/AMP motif (amino acids 243-345) involved in ATP binding and adenylate formation

    • The FATP/VLACS motif (amino acids 500-551) involved in fatty acid binding

  • A C-terminal domain that is important for function

  The protein's structural arrangement includes at least one transmembrane domain with the N-terminus located on the extracellular/luminal side and the C-terminus on the cytosolic side .

• What are the primary biochemical functions of SLC27A4?

  SLC27A4 has dual functionality in cellular fatty acid metabolism:

  1. Transport function: Facilitates the translocation of long-chain fatty acids (LCFA) across the plasma membrane

  2. Enzymatic function: Acts as an acyl-CoA synthetase that catalyzes the ATP-dependent formation of fatty acyl-CoA, using both long-chain fatty acids (LCFA) and very long-chain fatty acids (VLCFA) as substrates

  This dual functionality helps regulate cellular fatty acid levels by both facilitating uptake and preventing efflux through conversion to acyl-CoA forms .

  Function	Related Proteins
  Fatty acid transporter activity	SLC27A1A, SLC27A2, SLC27A1B, SLC27A5
  Long-chain fatty acid-CoA ligase activity	ACSL5, ACSBG1, SLC27A5, SLC27A2, ACSL4, SLC27A1B, SLC27A3, ACSL1, ACSBG2, ACSL3
  Very long-chain fatty acid-CoA ligase activity	SLC27A1A, SLC27A2, ACSBG1, SLC27A5, ACSL4, SLC27A3, SLC27A1B
  Nucleotide binding	Various proteins including RAD51L1, RHOAC, UBE2D1B, etc.

• What pathways involve SLC27A4 and how does it contribute to lipid metabolism?

  SLC27A4 participates in several metabolic pathways:

  Pathway Name	Related Proteins
  Transport of fatty acids	LCN9, LCN15, LCN12, APOD, LCN1
  PPAR signaling pathway	RXRA, GK2, CYP8B1, HMGCS2, PPARD, CD36, ANGPTL4, ACSL4B, ACSL3, RXRAA
  Fat digestion and absorption	PLA2G5, DGAT1, PLA2G2A, MOGAT3, PNLIPRP2, PLA2G1B, CD36, Cel, DGAT2, FABP2
  Insulin resistance	AKT1, SLC27A1A, PRKCBB, PIK3R3B, SLC2A4, PIK3R1, MAPK9, MAPK10, PPP1R3A, IRS2

  In lipid metabolism, SLC27A4 impacts:

  • Activation of very long-chain fatty acids (VLCFA)

  • Incorporation of fatty acids into neutral lipids (triglycerides, cholesterol esters) and phospholipids

  • Formation of the epidermal barrier through VLCFA metabolism

  • Potentially fat absorption and transport across the blood barrier

Advanced Research Questions

• What phenotypes result from SLC27A4 deficiency in animal models?

  Studies of SLC27A4 knockouts in mice reveal several phenotypes:

  • Embryonic lethality: Complete deletion of both SLC27A4 alleles results in embryonic death as early as day 9.5 of gestation

  • Skin abnormalities: Mice with targeted disruptions show:

    • Tight, thick, shiny, wrinkle-free skin

    • Impaired hair growth

    • Severe breathing difficulties

    • Neonatal death due to dehydration and restricted movement

  • Barrier dysfunction: Altered epidermal barrier with modified lipid composition

  • Biochemical changes: Fibroblasts from FATP4-deficient mice show:

    • 83% decrease in C24:0 activation

    • 58% reduction in peroxisomal degradation of C24:0

    • 54-64% decrease in C24:0 incorporation into major phospholipids

    • 64% decrease in C24:0 incorporation into triglycerides

    • 58% decrease in C24:0 incorporation into cholesterol esters

    • Abnormal neutral lipid droplets

    • Reduced uptake of fluorescent long-chain fatty acids

• What human diseases are associated with SLC27A4 mutations?

  SLC27A4 mutations are associated with:

  1. Ichthyosis Prematurity Syndrome (IPS): An autosomal recessive disorder characterized by:

     • Premature birth

     • Neonatal asphyxia

     • Lifelong non-scaly ichthyosis

     • Atopic manifestations

  2. Metabolic disorders: Polymorphisms in SLC27A4 have been linked to:

     • Insulin resistance

     • Lower body mass index (BMI)

     • Reduced systolic blood pressure

     • Lower plasma triglyceride levels

     • Decreased insulin concentrations

  Specific mutations identified include:

  • p.C168X nonsense mutation in exon 3 (common in Scandinavian populations)

  • c.716-1g>a splice site mutation

  • p.S247P and p.Q300R missense mutations in the AMP binding domain

  • p.R583H missense mutation in the C-terminal domain

  • G209S polymorphism associated with insulin resistance symptoms

• How can researchers effectively study SLC27A4 function in experimental systems?

  Methodological approaches for studying SLC27A4 include:

  Cellular models:

  • Fibroblast cell lines from SLC27A4-deficient mice

  • SLC27A4-transfected cell lines for overexpression studies

  • siRNA knockdown in cultured cells

  • Human neural stem cells derived from induced pluripotent stem cells

  Biochemical assays:

  • LCFA-CoA and VLCFA-CoA synthetase activity measurements

  • Radiolabeled fatty acid incorporation assays into lipid fractions

  • Fluorescently-labeled fatty acid uptake assays (e.g., C1-BODIPY-C12)

  • BODIPY 493/503 staining for neutral lipid accumulation

  Animal models:

  • Targeted gene disruption in mice

  • Tissue-specific knockout using Cre-lox systems

  • Transgenic rescue experiments

  Protein analysis:

  • Subcellular fractionation with Western blotting

  • Immunofluorescence for localization studies

  • Flow cytometry with specific antibodies

• What is the controversy regarding SLC27A4's role in intestinal lipid absorption?

  The role of SLC27A4 in intestinal lipid absorption remains controversial:

  Evidence supporting a critical role:

  • SLC27A4 is the only FATP expressed in small intestine

  • In vitro antisense experiments suggested it's required for fatty acid uptake into intestinal epithelial cells

  • Heterozygous FATP4+/- mice show 48% reduction in FATP4 protein and 40% reduction in fatty acid uptake by isolated enterocytes

  • It's localized to the brush-border membrane of enterocytes, positioning it ideally for fatty acid uptake

  Evidence against a critical role:

  • Heterozygous FATP4+/- mice show no detectable effects on fat absorption despite reduced fatty acid uptake

  • When SLC27A4 was overexpressed in keratinocytes of SLC27A4 knockout mice, there were no differences in growth, weight, food consumption, intestinal triglyceride absorption, fecal fat losses, or cholesterol absorption compared to controls

  • Studies found "no evidence for a physiological role of intestinal FATP4 in dietary lipid absorption in mice"

  These contradictory findings suggest SLC27A4 may have complex, context-dependent functions or possible compensatory mechanisms may exist when it's deleted.

• How does SLC27A4 interact with regulatory pathways and other transport systems?

  SLC27A4 interacts with several regulatory pathways:

  Transcriptional regulation:

  • The SLC27A1 promoter contains a PPAR response element, suggesting similar regulation may occur for SLC27A4

  • TR4, a nuclear receptor family member, may regulate FATP gene expression

  • SLC27A4 is upregulated in acquired obesity

  Metabolic pathway interactions:

  • RabGAPs TBC1D1 and TBC1D4 control uptake of long-chain fatty acids via SLC27A4/FATP4

  • Depletion of SLC27A4/FATP4 but not FAT/CD36 abrogated enhanced fatty acid oxidation in RabGAP-deficient skeletal muscle

  • RabGAP-mediated control involves regulated transport of LCFAs via SLC27A4/FATP4

  • Rab8, Rab10, and Rab14 knockdown decreased LCFA uptake, suggesting interaction with these small GTPases

  Interplay with other transport systems:

  • SLC27A4 may compete with or complement other fatty acid transporters like CD36

  • It indirectly inhibits RPE65 via substrate competition and production of VLCFA derivatives like lignoceroyl-CoA

• What are the latest applications of recombinant SLC27A4 in research?

  Recombinant SLC27A4 is utilized in various research applications:

  Protein interaction studies:

  • Investigating binding partners and regulatory proteins

  • Characterizing interactions with Rab proteins and other small GTPases

  Functional assays:

  • Enzymatic activity measurements to assess acyl-CoA synthetase function

  • Transport assays to quantify fatty acid uptake capacity

  Antibody development and validation:

  • Flow cytometry applications for detecting SLC27A4 in cells

  • Western blotting, ELISA, and other immunodetection methods

  Variant analysis:

  • Testing functional consequences of disease-associated mutations

  • Characterizing polymorphisms like G209S that affect function

  Neural development research:

  • Expression studies in human neural stem cells derived from induced pluripotent stem cells

  • Investigation of potential roles in central nervous system development

• How do mutations in SLC27A4 affect its biochemical function at the molecular level?

  Mutations in SLC27A4 impact its function through several mechanisms:

  Complete loss of protein expression:

  • The p.C168X nonsense mutation results in no detectable SLC27A4 protein in patient cells

  • This leads to complete loss of SLC27A4-mediated very long-chain fatty acid activation

  Altered substrate binding and catalysis:

  • Mutations in the AMP binding domain (p.S247P, p.Q300R) disrupt ATP binding and adenylate formation

  • These mutations repress both fatty acid import and activation functions

  Functional changes in transport capacity:

  • The G209S polymorphism results in significantly higher fatty acid uptake into cells compared to G209 variant

  • This altered transport capacity may contribute to disease pathophysiology

  Pathway-specific disruptions:

  • Patient fibroblasts with SLC27A4 mutations show:

    • 55% reduction in acyl-CoA formation with erucic acid (C22:1)

    • 69% decrease in erucic acid incorporation into cholesterol esters

    • 60% decrease in erucic acid incorporation into triglycerides

    • 37% decrease in erucic acid incorporation into phospholipids