Heather A. Hostetler

Liver type fatty acid binding protein (L-FABP) gene ablation reduces nuclear ligand distribution and peroxisome proliferator-activated receptor-α activity in cultured primary hepatocytes

The effect of liver type fatty acid binding protein (L-FABP) gene ablation on the uptake and distribution of long chain fatty acids (LCFA) to the nucleus by real-time laser scanning confocal imaging and peroxisome proliferator-activated receptor-alpha (PPARalpha) activity was examined in cultured primary hepatocytes from livers wild-type L-FABP+/+ and gene ablated L-FABP-/- mice. Cultured primary hepatocytes from livers of L-FABP-/- mice exhibited: (i) reduced oxidation of palmitic acid, a common dietary long chain fatty acid (LCFA); (ii) reduced expression of fatty acid oxidative enzymes-proteins transcriptionally regulated by PPARalpha; (iii) reduced palmitic acid-induced PPARalpha co-immunoprecipitation with coactivator SRC-1 concomitant with increased PPARalpha co-immunoprecipitation with coinhibitor N-CoR; (iv) reduced palmitic acid-induced PPARalpha. Diminished PPARalpha activation in L-FABP null hepatocytes was associated with lower uptake of common dietary LCFA (palmitic acid as well as its fluorescent derivative BODIPY FL C(16)), reduced level of total unesterified LCFA, and real-time redistribution of BODIPY FL C(16) from the central nucleoplasm to the nuclear envelope. Taken together, these studies support the hypothesis that L-FABP may facilitate ligand (LCFA)-activated PPARalpha transcriptional activity at least in part by increasing total LCFA ligand available to PPARalpha for inducing PPARalpha-mediated transcription of proteins involved in LCFA metabolism.

Glucose regulates fatty acid binding protein interaction with lipids and peroxisome proliferator-activated receptor α

Although the pathophysiology of diabetes is characterized by elevated levels of glucose and long-chain fatty acids (LCFA), nuclear mechanisms linking glucose and LCFA metabolism are poorly understood. As the liver fatty acid binding protein (L-FABP) shuttles LCFA to the nucleus, where L-FABP directly interacts with peroxisome proliferator-activated receptor-α (PPARα), the effect of glucose on these processes was examined. In vitro studies showed that L-FABP strongly bound glucose and glucose-1-phosphate (Kd = 103 ± 19 nM and Kd = 20 ± 3 nM, respectively), resulting in altered L-FABP conformation, increased affinity for lipid ligands, and enhanced interaction with PPARα. In living cells, glucose stimulated cellular uptake and nuclear localization of a nonmetabolizable fluorescent fatty acid analog (BODIPY C-16), particularly in the presence of L-FABP. These data suggest for the first time a direct role of glucose in facilitating L-FABP-mediated uptake and distribution of lipidic ligands to the nucleus for regulation of PPARα transcriptional activity.

Glucose Directly Links to Lipid Metabolism through High Affinity Interaction with Peroxisome Proliferator-activated Receptor α

The pathophysiology of diabetes is characterized not only by elevated glucose but also elevated long chain fatty acid levels. We show for the first time that the peroxisome proliferator-activated receptor-α (PPARα) binds glucose and glucose metabolites with high affinity, resulting in significantly altered PPARα secondary structure. Glucose decreased PPARα interaction with fatty acid metabolites and steroid receptor coactivator-1 while increasing PPARα interaction with DNA. Concomitantly, glucose increased PPARα interaction with steroid receptor coactivator-1, DNA binding, and transactivation of β-oxidation pathways in the presence of activating ligands. Heterodimerization of PPARα to the retinoid X receptor-α resulted in even larger increases in transactivation with the addition of glucose. These data suggest that PPARα is responsible for maintaining energy homeostasis through a concentration-dependent regulation of both lipids and sugars and that hyperglycemic injury mediated by PPARα occurs not only indirectly through elevated long chain fatty acid levels but also through direct action of glucose on PPARα.

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence

Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, and fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2s affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts and caveolae (AF488-CTB); 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488 antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting.

Divergence between human and murine peroxisome proliferator-activated receptor alpha ligand specificities

Peroxisome proliferator-activated receptor α (PPARα) belongs to the family of ligand-dependent nuclear transcription factors that regulate energy metabolism. Although there exists remarkable overlap in the activities of PPARα across species, studies utilizing exogenous PPARα ligands suggest species differences in binding, activation, and physiological effects. While unsaturated long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) function as ligands for recombinant mouse PPARα (mPPARα), no such studies have been conducted with full-length human PPARα (hPPARα). The objective of the current study was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for hPPARα or whether there exist species differences for ligand specificity and affinity. Both hPPARα and mPPARα bound with high affinity to LCFA-CoA; however, differences were noted in LCFA affinities. A fluorescent LCFA analog was bound strongly only by mPPARα, and naturally occurring saturated LCFA was bound more strongly by hPPARα than mPPARα. Similarly, unsaturated LCFA induced transactivation of both hPPARα and mPPARα, whereas saturated LCFA induced transactivation only in hPPARα-expressing cells. These data identified LCFA and LCFA-CoA as endogenous ligands of hPPARα, demonstrated species differences in binding specificity and activity, and may help delineate the role of PPARα as a nutrient sensor in metabolic regulation.

The PDZ1 and PDZ3 Domains of MAGI-1 Regulate the Eight Exon Isoform of the Coxsackievirus and Adenovirus Receptor

ABSTRACT Epithelial integrity is essential for homeostasis and poses a formidable barrier to pathogen entry. Major factors for viral entry into epithelial cells are the localization and abundance of the primary receptor. The coxsackievirus and adenovirus receptor (CAR) is a primary receptor for these two pathogenic groups of viruses. In polarized epithelia, a low-abundance, alternatively spliced eight-exon isoform of CAR, CAREx8, is localized apically where it can support viral infection from the air-exposed surface. Using biochemical, cell biology, genetic, and spectroscopic approaches, we show that the levels of apical CAREx8 are negatively regulated by the PDZ domain-containing protein MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1) and that two MAGI-1 PDZ domains, PDZ1 and PDZ3, regulate CAREx8 levels in opposing ways. Similar to full-length MAGI-1, expression of the isolated PDZ3 domain significantly reduces cell surface CAREx8 abundance and adenovirus infection. In contrast, the PDZ1 domain is able to rescue CAREx8 and adenovirus infection from MAGI-1-mediated suppression. These data suggest a novel cell-based strategy to either suppress viral infection or augment adenovirus-based gene therapy.

Liver-type fatty acid binding protein interacts with hepatocyte nuclear factor 4α.

Hepatocyte nuclear factor 4α (HNF4α) regulates liver type fatty acid binding protein (L‐FABP) gene expression. Conversely as shown herein, L‐FABP structurally and functionally also interacts with HNF4α. Fluorescence resonance energy transfer (FRET) between Cy3‐HNF4α (donor) and Cy5‐L‐FABP (acceptor) as well as FRET microscopy detected L‐FABP in close proximity (∼80 Å) to HNF4α, binding with high affinity Kd ∼250–300 nM. Circular dichroism (CD) determined that the HNF4α/L‐FABP interaction altered protein secondary structure. Finally, L‐FABP potentiated transactivation of HNF4α in COS7 cells. Taken together, these data suggest that L‐FABP provides a signaling path to HNF4α activation in the nucleus.

Phosphatidic Acid (PA) can Displace PPARα/LXRα Binding to The EGFR Promoter Causing its Transrepression in Luminal Cancer Cells

The expression of the epidermal growth factor receptor (EGFR) is highly regulated in normal cells, whereas some cancer cells have high constitutive levels. Understanding naturally-occurring ways of downregulating EGFR in cancer cells was investigated. Phosphatidic acid (PA) or Nuclear Receptors (NR) PPARα/RXRα/LXRα, enhance EGFR expression, mediated by the promoter region -856(A) to -226(T). Unexpectedly, the combination of NRs and PA caused repression. PA induces a conformational change in the nuclear receptor PPARα (increase of alpha-helices at the expense of decreasing beta-sheets), as evidenced by circular dichroism. This represses the naturally-enhancing capability of PPARα on EGFR transcription. PPARα-overexpressing cells in the presence of PA > 300 nM or the enzyme that produces it, phospholipase D (PLD), downregulate EGFR expression. The reasons are two-fold. First, PA displaces PPARα binding to the EGFR promoter at those concentrations. Second, NR heterodimer-dependent promoter activity is weakened in the presence of PA in vivo. Since other genes considered (β-catenin, cyclin D3, PLD2 and ACOX-1) are also downregulated with a PA + PPARα combination, the transrepression appears to be a global phenomenon. Lastly, the reported effect is greater in MCF-7 than in MDA-MB-231 breast cancer cells, which could provide a novel basis for regulating excessive expression of EGFR in luminal cancer cells.

Fatty acid binding profile of the liver X receptor α

Liver X receptor (LXR)α is a nuclear receptor that responds to oxysterols and cholesterol overload by stimulating cholesterol efflux, transport, conversion to bile acids, and excretion. LXRα binds to and is regulated by synthetic (T-0901317, GW3695) and endogenous (oxysterols) ligands. LXRα activity is also modulated by FAs, but the ligand binding specificity of FA and acyl-CoA derivatives for LXRα remains unknown. We investigated whether LXRα binds FA or FA acyl-CoA with affinities that mimic in vivo concentrations, examined the effect of FA chain length and the degree of unsaturation on binding, and investigated whether FAs regulate LXRα activation. Saturated medium-chain FA (MCFA) displayed binding affinities in the low nanomolar concentration range, while long-chain fatty acyl-CoA did not bind or bound weakly to LXRα. Circular dichroic spectra and computational docking experiments confirmed that MCFA bound to the LXRα ligand binding pocket similar to the known synthetic agonist of LXRα (T0901317), but with limited change to the conformation of the receptor. Transactivation assays showed that MCFA activated LXRα, whereas long-chain FA caused no effect. Our results suggest that LXRα functions as a receptor for saturated FA or acyl-CoA of C10 and C12 in length.

Silver Nanoparticles Supported on Carbon Nanotube Carpets: Influence of Surface Functionalization

The effectiveness of nanoparticle-based functional devices depends strongly on the surface morphology and area of the support. An emerging powerful approach of increasing the available surface area without decreasing strength or increasing bulk is to attach arrays of suitable nanotubes on the surface, and to attach the necessary nanoparticles to them. Earlier publications by this team have shown that carpet-like arrays of carbon nanotubes (CNTs) can be successfully grown on a variety of larger carbon substrates such as graphite, foams and fabric, which offer hierarchical multiscale supporting architecture suitable for the attachment of silver nanoparticles (AgNPs). A limiting factor of pure CNT arrays in fluid-based applications is their hydrophobicity, which can reduce the percolation of an aqueous medium through individual nanotubes. Previous studies have demonstrated that the treatment of CNT carpets with dry (oxygen) plasma can induce reversible wettability, and treatment with wet (sol-gel) coating can impart permanent wettability. In this paper, we report the influence of such treatments on the attachment of AgNPs, and their effectiveness in water disinfection treatments. Both types of hydrophilic surface treatment show an increase in silver loading on the CNT carpets. Oxygen-plasma treated surfaces (O-CNT) show fine and densely packed AgNPs, whereas silica-coated nanotubes (silica-CNT) show uneven clusters of AgNPs. However, O-CNT surfaces lose their hydrophilicity during AgNP deposition, whereas silica-CNT surfaces remain hydrophilic. This difference significantly impacts the antibacterial effectiveness of these materials, as tested in simulated water containing Gram negative Escherichia coli (E. coli, JM109). AgNPs on silica-coated CNT substrates showed significantly higher reduction rates of E. coli compared to AgNPs on plasma-treated CNT substrates, despite the finer and better dispersed AgNP distribution in the latter. These results provide important insights into different aspects of surface modification approaches that can control the wettability of CNT carpets, and their applicability in water treatment applications.