Mammalian Fatty Acid-binding Proteins Research Articles

The Multifunctional Family of Mammalian Fatty Acid-Binding Proteins.

Fatty acid-binding proteins (FABPs) are small lipid-binding proteins abundantly expressed in tissues that are highly active in fatty acid (FA) metabolism. Ten mammalian FABPs have been identified, with tissue-specific expression patterns and highly conserved tertiary structures. FABPs were initially studied as intracellular FA transport proteins. Further investigation has demonstrated their participation in lipid metabolism, both directly and via regulation of gene expression, and in signaling within their cells of expression. There is also evidence that they may be secreted and have functional impact via the circulation. It has also been shown that the FABP ligand binding repertoire extends beyond long-chain FAs and that their functional properties also involve participation in systemic metabolism. This article reviews the present understanding of FABP functions and their apparent roles in disease, particularly metabolic and inflammation-related disorders and cancers.

Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a “closed” FakB1 state to an “open” state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.

Fatty acid binding proteins in brain development and disease

Long chain polyunsaturated fatty acids (PUFAs) are critical structural components of the brain and essential for normal brain development. The cellular transportation and physiological actions of PUFAs are mediated by fatty acid binding proteins (FABPs) which are encoded by the intracellular lipid-binding protein gene family. Three of the ten mammalian FABPs identified to date (FABP3, FABP5, FABP7) are expressed in the brain. These three FABPs, along with their fatty acid ligands, have distinct and dynamic spatio-temporal expression profiles that correlate with specific developmental stages and processes in the brain. Functional studies have revealed a variety of roles for FABPs in brain development including the generation of neuronal and/or glial cells, differentiation, neuronal cell migration and axis patterning. A number of transcription factors have been shown to be involved in the developmental regulation of FABP gene expression in the brain. Furthermore, FABPs appear to be major downstream effectors of signaling pathways such as Reelin-Dab1/Notch which mediate neuron-glia crosstalk during brain development. As PUFAs and FABPs play critical roles in brain development, considerable effort has been placed in elucidating their function in the pathogenesis and progression of brain cancers and neuropsychiatric disorders.

Fatty Acid- and Retinoid-binding Proteins Have Distinct Binding Pockets for the Two Types of Cargo

Parasitic nematodes cause serious diseases in humans, animals, and plants. They have limited lipid metabolism and are reliant on lipid-binding proteins to acquire these metabolites from their hosts. Several structurally novel families of lipid-binding proteins in nematodes have been described, including the fatty acid- and retinoid-binding protein family (FAR). In Caenorhabditis elegans, used as a model for studying parasitic nematodes, eight C. elegans FAR proteins have been described. The crystal structure of C. elegans FAR-7 is the first structure of a FAR protein, and it exhibits a novel fold. It differs radically from the mammalian fatty acid-binding proteins and has two ligand binding pockets joined by a surface groove. The first can accommodate the aliphatic chain of fatty acids, whereas the second can accommodate the bulkier retinoids. In addition to demonstrating lipid binding by fluorescence spectroscopy, we present evidence that retinol binding is positively regulated by casein kinase II phosphorylation at a conserved site near the bottom of the second pocket. far-7::GFP (green fluorescent protein) expression shows that it is localized in the head hypodermal syncytia and the excretory cell but that this localization changes under starvation conditions. In conclusion, our study provides the basic structural and functional information for investigation of inhibitors of lipid binding by FAR proteins.

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The Emerging Functions and Mechanisms of Mammalian Fatty Acid–Binding Proteins

Fatty acid-binding proteins (FABPs) are abundant intracellular proteins that bind long-chain fatty acids with high affinity. Nine separate mammalian FABPs have been identified, and their tertiary structures are highly conserved. The FABPs have unique tissue-specific distributions that have long suggested functional differences among them. In the last decade, considerable progress has been made in understanding the specific functions of the FABPs and, in some cases, their mechanisms of action at the molecular level. The FABPs appear to be involved in the extranuclear compartments of the cell by trafficking their ligands within the cytosol via interactions with organelle membranes and specific proteins. Several members of the FABP family have been shown to function directly in the regulation of cognate nuclear transcription factor activity via ligand-dependent translocation to the nucleus. This review will focus on these emerging functions and mechanisms of the FABPs, highlighting the unique functional properties of each as well as the similarities among them.

Two novel Mesocestoides vogae fatty acid binding proteins – functional and evolutionary implications

This work describes two new fatty acid binding proteins (FABPs) identified in the parasite platyhelminth Mesocestoides vogae (syn. corti). The corresponding polypeptide chains share 62% identical residues and overall 90% similarity according to CLUSTALX default conditions. Compared with Cestoda FABPs, these proteins share the highest similarity score with the Taenia solium protein. M. vogae FABPs are also phylogenetically related to the FABP3/FABP4 mammalian FABP subfamilies. The native proteins were purified by chromatographical procedures, and apparent molecular mass and isoelectric point were determined. Immunolocalization studies determined the localization of the expression of these proteins in the larval form of the parasite. The genomic exon-intron organization of both genes is also reported, and supports new insights on intron evolution. Consensus motifs involved in splicing were identified.

Studies of the FABP family: A retrospective

Following my research on the role played by soluble proteins in their function as hydrophobic ligand carriers acting through squalene epoxidase, Dr Odani and I started to work together on low molecular lipid binding proteins. As a result of this collaboration, in 1982 we managed to determine the complete primary structure of Z-protein in rat liver. This was the first report ever to give the complete amino acid sequence of a fatty acid binding protein (FABP). This gave momentum to further such research, and now extensive exploration has been carried out on a whole family of homologous intracellular hydrophobic ligand binding proteins, the product of the expression of an ancient gene family in numerous organisms. Takahashi et al. have determined the primary structures of mammalian FABP family protein in liver, intestine, heart, kidney, and skin through amino acid sequencing as well as through determination of the cDNA sequence. Out of all my research on the FABP family, I believe, my initial study on FABP in liver, my work on kidney FABP, heart type FABP and my discovery of an I-15P (BAPB) and I-FABP application as a diagnostic marker stand out in particular.

Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus

This work describes a new gene coding for a fatty acid binding protein (FABP) in the parasite Echinococcus granulosus, named EgFABP2. The complete gene structure, including the promoter sequence, is reported. The genomic coding domain organisation of the previously reported E. granulosus FABP gene ( EgFABP1) has been also determined. The corresponding polypeptide chains share 76% of identical residues and an overall 96% of similarity. The two EgFABPs present the highest amino acid homologies with the mammalian FABP subfamily containing heart-FABPs (H-FABPs). The coding sequences of both genes are interrupted by a single intron located in the position of the third intron reported for vertebrate FABP genes. Both genes are expressed in the protoscolex stage of the parasite. The promoter region of EgFABP2 presents several consensus putative cis-acting elements found in other members of the family, suggesting interesting possible mechanisms involved in the host–parasite adaptation.

New insights into the structure and function of fatty acid-binding proteins.

Fatty acid-binding proteins (FABPs) are members of a superfamily of lipid-binding proteins, and occur intracellularly in vertebrates and invertebrates. This review presents recent findings on the diversity of these FABPs and their proposed roles in fatty acid (FA) metabolism and other cellular processes. Special attention is paid to the structural features of the different mammalian FABP types and the physiological role of these proteins in FA transport, cell growth and differentiation, cellular signalling, gene transcription and cytoprotection. Additionally, data on FABP knockout mice and the implication of FABP in medicine are discussed.

An embryo-associated fatty acid-binding protein in the filarial nematode Brugia malayi.

Brugia malayi is a filarial nematode parasite that causes lymphatic filariasis, a disease that affects millions of people in the tropics. Sexual reproduction of filarial worms occurs within the lymphatic vessels of the human host and is crucial for transmission of the parasite to the mosquito vector. We have previously identified several B. malayi genes that exhibit apparent gender-specific expression. One of these had significant sequence similarity to the Ascaris suum embryo-associated fatty acid binding protein, As-p18. The full length cDNA for the B. malayi female-associated fatty acid binding protein (Bm-FAB-1) encodes a 17.8 kDa protein (excluding a signal peptide) with 70% sequence identity with mature As-p18 and significant similarity to Caenorhabditis elegans and mammalian fatty acid-binding proteins (FABPs). Antibodies raised to Bm-FAB-1 bound to developing embryos within female worms, especially around early embryo cells and the surfaces of immature worms within eggs. Functional studies showed that recombinant Bm-FAB-1 binds to several long chain fatty acids including oleate, but not retinol. Taken together, these results demonstrate that Bm-FAB-1 is a member of an unusual nematode-specific, secreted lipid binding protein family. The existence of a novel class of lipid binding proteins in nematode embryos raises the possibility that drugs targeting these proteins could be developed with broad activity against nematode parasites of medical and veterinary importance.

Collision-mediated transfer of long-chain fatty acids by neural tissue fatty acid-binding proteins (FABP): studies with fluorescent analogs.

Mammalian fatty acid-binding proteins (FABP) are a family of intracellular proteins (approx 15 kDa) that bind long-chain fatty acids (FA) with high affinity. They are believed to serve as cytoplasmic transporters of FA and to target FA to specific cellular sites of utilization. Several different FABPs are expressed in neural tissue, including brain FABP (B-FABP), myelin FABP (M-FABP), and heart FABP (H-FABP). We have previously shown that H-FABP transfers FAvia direct collisional interactions with acceptor model membranes. In the present studies, we use a fluorescence resonance energy transfer (FRET) assay to examine the rate and mechanism of transfer of a fluorescent long-chain fatty acid from B-FABP to phospholipid vesicles. The rate of transfer is shown to be independent of buffer ionic strength and dramatically enhanced by the presence of specific anionic phospholipids. These results are consistent with a mechanism by which FA are transferred from B-FABP to phospholipid membranes by a transient collision-based mechanism.

Role of fatty acid-binding protein in lipid metabolism of insect flight muscle.

Since insect flight muscles are among the most active muscles in nature, their extremely high rates of fuel supply and oxidation pose interesting physiological problems. Long-distance flights of species like locusts and hawkmoths are fueled through fatty acid oxidation. The lipid substrate is transported as diacylglycerol in the blood, employing a unique and efficient lipoprotein shuttle system. Following diacyglycerol hydrolysis by a flight muscle lipoprotein lipase, the liberated fatty acids are ultimately oxidized in the mitochondria. Locusta flight muscle cytoplasm contains an abundant fatty acid-binding protein (FABP). The flight muscle FABP of Locusta migratoria is a 15 kDa protein with an isoelectric point of 5.8, binding fatty acids in a 1:1 molar stoichiometric ratio. Binding affinity of the FABP for long-chain fatty acids (apparent dissociation constant Kd = 5.21 +/- 0.16 microM) is however markedly lower than that of mammalian FABPs. The NH2-terminal amino acid sequence shares structural homologies with two insect FABPs recently purified from hawkmoth midgut, as well as with mammalian FABPs. In contrast to all other isolated FABPs, the NH2 terminus of locust flight muscle FABP appeared not to be acetylated. During development of the insect, a marked increase in fatty acid binding capacity of flight muscle homogenate was measured, along with similar increases in both fatty acid oxidation capacity and citrate synthase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Diversity of fatty acid-binding protein structure and function: studies with fluorescent ligands.

The mammalian fatty acid-binding proteins (FABP) are localized in many distinct cell types. They bind long chain fatty acids in vitro, however, their functions and mechanisms of action in vivo remain unknown. The present studies have sought to understand the relationships among these proteins, and to address the possible role of FABP in cellular fatty acid traffic. A series of anthroyloxy-labeled fluorescent fatty acids have been used to examine the physicochemical properties of the fatty acid-binding sites of different members of the FABP family. The fatty acid probes have also been used to study the rate and mechanism of fatty acid transfer from different FABP types to phospholipid membranes. The results of these studies show a number of interesting and potentially important differences between FABP family members. An examination of adipocyte and heart FABP (A- and H-FABP) shows that their fatty acid-binding sites are less hydrophobic than the liver FABP (L-FABP) site, and that the bound ligand experiences less motional constraint within the A- and H-FABP binding sites than within the L-FABP binding site. In keeping with these differences in structural properties, it was found that anthroyloxy-fatty acid transfer from A- and H-FABP to membranes is markedly faster than from L-FABP. Moreover, the mechanism of fatty acid transfer was found to be similar for the highly homologous A- and H-FABP, whereby transfer to phospholipid membranes appears to occur via transient collisional interactions between the FABP and membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure of locust flight muscle fatty acid binding protein

The amino acid sequence of the fatty acid binding protein (FABP) from flight muscle of the locust, Schistocerca gregaria, has been determined. The sequence of the N-terminal 39 amino acid residues, determined by automated Edman degradation, was used to prepare a degenerate oligonucleotide that corresponded to amino acid residues 16–23. cDNA coding for FABP was constructed from flight muscle mRNA and amplified by the polymerase chain reaction using the degenerate oligonucleotide and an oligo dT- Not I primer adapter as primers. The amplification product was cloned and sequenced. Additionally, a cDNA library of flight muscle mRNA was prepared and screened with a 414-bp probe prepared from the clone. The primary structure of locust FABP was compared with the proteins in the Swiss protein databank and found to have significant homology with mammalian FABPs over the entire 133-residue sequence. The best match was versus human heart FABP (41% identity), attesting to the highly conserved nature of this protein. The results suggest that locust muscle FABP is a member of the lipid binding protein superfamily and may provide valuable insight into the evolution of this abundant protein class.

Isolation, characterization, and cDNA sequence of two fatty acid-binding proteins from the midgut of Manduca sexta larvae.

Two abundant fatty acid-binding proteins (MFB1 and MFB2) were isolated from the midgut cytosol of larval Manduca sexta. As isolated, MFB1 and MFB2 were found to contain bound fatty acids in a 1:1 molar stoichiometric ratio. Immunological screening demonstrated that MFB1 and MFB2 were restricted to the midgut in a gradient distribution, with MFB1 more concentrated in the anterior two-thirds of the midgut and MFB2 more concentrated in the posterior two-thirds of the midgut. MFB1 exchanged fatty acid more readily than did MFB2. MFB1 was about 2% and MFB2 about 12% of the cytosolic protein in the midgut. cDNA clones for MFB1 and MFB2 both encode proteins of 131 amino acids that are rich in lysine and acidic residues. Analysis of the amino acid sequence alignment of the MFBs with six mammalian fatty acid-binding proteins revealed a number of shared features: 9 conserved glycines, presumably important in turns of the beta-strands; a basic amino acid in a position corresponding to the residue reported to participate in binding the carboxyl group of the fatty acid (Arg in MFB1 and Lys in MFB2); and conservation of many of the residues important in binding the aliphatic portion of the fatty acid.

Expression of a mammalian fatty acid-binding protein in Escherichia coli.

Rat liver fatty acid-binding protein (FABP) is a 14,200-Da polypeptide that is abundantly represented in the cytosol of liver and small intestinal epithelial cells. It may play an important role in the intracellular transport and metabolism of fatty acids. We have recently determined its sequence from an analysis of cloned cDNAs (Gordon, J.I., Alpers, D.H., Ockner, R. K., and Strauss, A.W. (1983) J. Biol. Chem. 258, 3356-3363). With the availability of full-length cloned FABP cDNAs, genetic manipulation of the coding region introduced into an appropriate expression vector could provide a powerful tool for analyzing the molecular details of fatty acyl-protein interaction. We, therefore, have inserted the coding sequence of rat FABP cDNA into a prokaryotic expression vector. Synthetic oligodeoxynucleotides were used to "properly" space the initiator methionine residue downstream from a Shine-Dalgarno ribosome-binding site. FABP transcription in the chimeric plasmid was under the direction of the leftward promotor of phage lambda. Promotor activity in turn was regulated by a thermolabile repressor specified by a defective lambda prophage contained in the host chromosome. When grown at permissive temperatures, rat liver FABP represented approximately 0.8% of radiolabeled soluble bacterial proteins. Edman degradation of FABP purified from Escherichia coli lysates indicated that it was intact. However, its NH2 terminus was not acetylated as it is in mammalian tissues. A frameshift mutation was introduced in vitro into the coding region of FABP cDNA. The effects of this on fatty acyl-protein interaction were examined using a solid phase (oleic acid-Sepharose) binding assay. Amino acid sequence rearrangements in the COOH-terminal region of rat liver FABP had a significant effect on its ability to bind fatty acids as well as on its stability in bacteria.