Possible involvement of fatty acid binding protein in peroxisomal β-oxidation of fatty acids

The localization of β-oxidation of fatty acids in isolated peroxisomes from rat liver was investigated. The enzyme system is soluble in the luminal compartment and carnitine does not appear to be involved in the transfer of the CoA derivatives through the peroxisomal membrane. Experiments involving proteolysis, inhibitors and competitive inhibition suggest that a fatty acid binding protein is responsible for the carrier process. This carrier protein seems to be present in increased amounts both in the supernatant and in the peroxisomes after clofibrate induction.     

Key amino acid residues of ankyrin-sensitive phosphatidylethanolamine/phosphatidylcholine-lipid binding site of βI-spectrin

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that "opening" of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton.     

Two modes of fatty acid binding to bovine β-lactoglobulin--crystallographic and spectroscopic studies

Lactoglobulin is a natural protein present in bovine milk and common component of human diet, known for binding with high affinity wide range of hydrophobic compounds, among them fatty acids 12–20 carbon atoms long. Shorter fatty acids were reported as not binding to β‐lactoglobulin. We used X‐ray crystallography and fluorescence spectroscopy to show that lactoglobulin binds also 8‐ and 10‐carbon caprylic and capric acids, however with lower affinity. The determined apparent association constant for lactoglobulin complex with caprylic acid is 10.8 ± 1.7 × 10³ M^?1, while for capric acid is 6.0 ± 0.5 × 10³ M^?1. In crystal structures determined with resolution 1.9 Å the caprylic acid is bound in upper part of central calyx near polar residues located at CD loop, while the capric acid is buried deeper in the calyx bottom and does not interact with polar residues at CD loop. In both structures, water molecule hydrogen‐bonded to carboxyl group of fatty acid is observed. Different location of ligands in the binding site indicates that competition between polar and hydrophobic interactions is an important factor determining position of the ligand in β‐barrel. Copyright © 2010 John Wiley & Sons, Ltd.     

Probing the steric requirements of the γ-aminobutyric acid aminotransferase active site with fluorinated analogues of vigabatrin

We have synthesized three analogues of 4-amino-5-fluorohexanoic acids as potential inactivators of γ-aminobutyric acid aminotransferase (GABA-AT), which were designed to combine the potency of their shorter chain analogue, 4-amino-5-fluoropentanoic acid (AFPA), with the greater enzyme selectivity of the antiepileptic vigabatrin (Sabril®). Unexpectedly, these compounds failed to inactivate or inhibit the enzyme, even at high concentrations. On the basis of molecular modeling studies, we propose that the GABA-AT active site has an accessory binding pocket that accommodates the vinyl group of vigabatrin and the fluoromethyl group of AFPA, but is too narrow to support the extra width of the distal methyl group in the synthesized analogues.     

Is the fatty acid composition of Daphnia galeata determined by the fatty acid composition of the ingested diet?

1. The fatty acid (FA) composition of Daphnia galeata and their algal food was analysed and showed many similarities, however, some significant differences were also found in the relative abundance of the FA C16 : 4ω3 and docosahexaenoic acid (DHA). Their relative abundances were much lower in daphnids than in their algal diet.
2. When daphnids were fed three distinct emulsion particles with DHA : eicosapentaenoic acid (EPA) ratios of c. 0.7, 2 and 4, the final DHA : EPA ratio in the daphnids always favoured EPA. The increase of the food DHA : EPA ratio resulted in a minor increase of DHA (to c. 2%). Feeding the animals on emulsion particles with increasing ratios of DHA : EPA, caused a minor ( c. 2%) increase of DHA level but EPA levels remained high ( c. 10%).
3. When labelled with [¹⁴C]linoleic acid and [¹⁴C]linolenic acid daphnids showed low conversion of both essential FA into C20 polyunsaturated fatty acids (PUFAs). This low conversion activity might explain the importance of C20 PUFAs as dietary compounds in the food of Daphnia.
4. The results indicate the insignificance of DHA and C16 : 4ω3 for daphnids. As EPA can be derived from C18 : 3ω3 it is not strictly essential, although it might be a significant factor in food quality for Daphnia.     

Probing the binding sites of resveratrol,genistein, and curcumin with milk β-lactoglobulin

We determined the binding sites of curcumin (cur), resveratrol (res), and genistein (gen) with milk β-lactoglobulin (β-LG) at physiological conditions. Fourier transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of polyphenol–protein complexes. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K_{curcumin–β-LG}?=?4.4 (±?.4)?×?10⁴ M^?1, K_{resveratrol–β-LG}?=?4.2 (±?.2)?×?10⁴ M^?1, and K_{genistein–β-LG}?=?1.2 (±?.2)?×?10⁴?M^?1. The number of polyphenol molecules bound per protein (n) was 1 (cur), 1.1 (res), and 1 (gen). Molecular modeling showed the participation of several amino acid residues in polyphenol–protein complexation with the free binding energy of ?12.67 (curcumin–β-LG), ?12.60 (resveratrol–β-LG), and ?10.68?kcal/mol (genistein–β-LG). The order of binding was cur?>?res?>?gen. Alteration of the protein conformation was observed in the presence of polyphenol with a major reduction of β-sheet and an increase in turn structure, causing a partial protein structural destabilization. β-LG might act as a carrier to transport polyphenol in vitro.     

Activities of fatty acid desaturases and fatty acid composition of liver microsomes in rats fed β-carotene and 13-cis-retinoic acid

The fatty acid composition of microsomal lipids and the activities of Δ9- and Δ6-desaturases in liver microsomes of rats fed diets supplemented with β-carotene and two levels of 13-cis-retinoic acid were studied. Four groups of male, weanling rats were fed semipurified diets containing 0 or 100 mg β-carotene per kg diet, and 20 or 100 mg 13-cis-retinoic acid per kg diet. After 11 weeks of feeding, the rats were killed, liver microsomes were prepared and assayed for Δ9-desaturase and Δ6-desaturase activities. The activity of Δ9-desaturase was lower in liver microsomes of rats fed β-carotene-supplemented diet or the diet supplemented with the higher level of 13-cis-retinoic acid. Microsomal Δ6-desaturase activity was, however, higher in liver of rats fed 13-cis-retinoic acid; there was no effect of β-carotene on Δ6-desaturase activity. The fatty acid compositional data on total lipids of liver microsomes were consistent with the diet-induced changes in fatty acid desaturases. Phospholipid composition of liver microsomes was also altered as a result of feeding β-carotene or 13-cis-retinoic acid-containing diets. The proportions of phosphatidylethanolamine were generally higher, whereas those of phosphatidylcholine were lower in the experimental groups as compared with the control.     

Locating the binding sites of retinol and retinoic acid with milk β-lactoglobulin

β-lactoglobulin (β-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules such as retinoids. We located the binding sites of retinol and retinoic acid on β-LG in aqueous solution at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods, and molecular modeling. The retinoid-binding sites and the binding constants as well as the effect of retinol and retinoic acid complexation on protein stability and secondary structure were determined. Structural analysis showed that retinoids bind strongly to β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K _{retinol- β -LG?}=?6.4 (±?.6)?×?10⁶?M^?1 and K _{retinoic acid- β -LG?}=?3.3 (±?.5)?×?10⁶?M^?1. The number of retinoid molecules bound per protein (n) is 1.1 (±?.2) for retinol and 1.5 (±?.3) for retinoic acid. Molecular modeling showed the participation of several amino acids in the retinoid–protein complexes with the free binding energy of ?8.11?kcal/mol for retinol and ?7.62?kcal/mol for retinoic acid. Protein conformation was altered with reduction of β-sheet from 59 (free protein) to 52–51% and a major increase in turn structure from 13 (free protein) to 24–22%, in the retinoid–β-LG complexes, indicating a partial protein destabilization.     

Locating the binding sites of retinol and retinoic acid with milk β-lactoglobulin

β-lactoglobulin (β-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules such as retinoids. We located the binding sites of retinol and retinoic acid on β-LG in aqueous solution at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods, and molecular modeling. The retinoid-binding sites and the binding constants as well as the effect of retinol and retinoic acid complexation on protein stability and secondary structure were determined. Structural analysis showed that retinoids bind strongly to β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K (retinol-) (β) (-LG?)=?6.4 (±?.6)?×?10(6)?M(-1) and K (retinoic acid-) (β) (-LG?)=?3.3 (±?.5)?×?10(6)?M(-1). The number of retinoid molecules bound per protein (n) is 1.1 (±?.2) for retinol and 1.5 (±?.3) for retinoic acid. Molecular modeling showed the participation of several amino acids in the retinoid-protein complexes with the free binding energy of -8.11?kcal/mol for retinol and -7.62?kcal/mol for retinoic acid. Protein conformation was altered with reduction of β-sheet from 59 (free protein) to 52-51% and a major increase in turn structure from 13 (free protein) to 24-22%, in the retinoid-β-LG complexes, indicating a partial protein destabilization.     

Kinetic advantage of the interaction between the fatty acid β-oxidation enzymes and the complexes of the respiratory chain

Respiration-linked oxidation of 3-hydroxybutyryl-CoA, crotonyl-CoA and saturated fatty acyl (C₄, C₈ and C₁₄)-CoA esters was studied in different mitochondrial preparations. Oxidation of acyl-CoA esters was poor in intact mitochondria; however, it was significant, as well as, NAD⁺ and CoA-dependent in gently and in vigorously sonicated mitochondria. The respiration-linked oxidation of crotonyl-CoA and 3-hydroxybutyryl-CoA proceeded at much higher rates (over 700%) in gently disrupted mitochondria than in completely disrupted mitochondria. The redox dye-linked oxidation of crotonyl-CoA (with inhibited respiratory chain) was also higher in gently disrupted mitochondria (149%) than in disrupted ones. During the respiration-linked oxidation of 3-hydroxybutyryl-CoA the steady-state NADH concentrations in the reaction chamber were determined, and found to be 8 μM in gently sonicated and 15 μM in completely sonicated mitochondria in spite of the observation that the gently sonicated mitochondria oxidized the 3-hydroxybutyryl-CoA much faster than the completely sonicated mitochondria. The NAD⁺-dependence of 3-hydroxybutyryl-CoA oxidation showed that a much smaller NAD⁺ concentration was enough to half-saturate the reaction in gently disrupted mitochondria than in completely disrupted ones. Thus, these observations indicate the positive kinetic consequence of organization of β-oxidation enzyme in situ. Respiration-linked oxidation of bytyryl-, oxtanoyl- and palmitoyl-CoA was also studied and these CoA intermediates were oxidized at approx. 50% of the rate of crotonyl- and 3-hydroxybutyryl-CoA in the gently disrupted mitochondria. In vigorously disrupted mitochondria the oxidation rate of these saturated acyl-CoA intermediates was hardly detectable indicating that the connection between the acyl-CoA dehydrogenase and the respiratory chain had been disrupted.     

Regulating effect of β-ketoacyl synthase domain of fatty acid synthase on fatty acyl chain length in de novo fatty acid synthesis

Fatty acid synthase (FAS) is a multifunctional homodimeric protein, and is the key enzyme required for the anabolic conversion of dietary carbohydrates to fatty acids. FAS synthesizes long-chain fatty acids from three substrates: acetyl-CoA as a primer, malonyl-CoA as a 2 carbon donor, and NADPH for reduction. The entire reaction is composed of numerous sequential steps, each catalyzed by a specific functional domain of the enzyme. FAS comprises seven different functional domains, among which the β-ketoacyl synthase (KS) domain carries out the key condensation reaction to elongate the length of fatty acid chain. Acyl tail length controlled fatty acid synthesis in eukaryotes is a classic example of how a chain building multienzyme works. Different hypotheses have been put forward to explain how those sub-units of FAS are orchestrated to produce fatty acids with proper molecular weight. In the present study, molecular dynamic simulation based binding free energy calculation and access tunnels analysis showed that the C16 acyl tail fatty acid, the major product of FAS, fits to the active site on KS domain better than any other substrates. These simulations supported a new hypothesis about the mechanism of fatty acid production ratio: the geometric shape of active site on KS domain might play a determinate role.     

Is the C-terminal region of bradykinin the binding site of polyphenols?

Bradykinin is a bioactive hormone involved in a variety of physiological processes. In various solvents, this peptide adopts beta-turn structures. The C-terminal turn is a structural feature for the receptor affinity of agonists and antagonists while the N-terminal turn might be important for antagonistic activities. Polyphenols like dimeric proanthocyanidin B3 interact with the peptide. Thus to investigate the effects of polyphenols on bradykinin activity and structure, we studied the interaction in the structuring solvent DMSO which can be a close mimic of aqueous physiological environments like receptor-binding sites. Bradykinin alone presented a folded structure with two turns. B3 interacted with the peptide C-terminus and involved the loss of the bend structure of this region, while the N-terminus turn was maintained. Numerous studies have shown that polyphenolic molecules can act upon various biological targets, and the formation of this type of complex might be one of the possible modes of action.     

Role of fatty acid uptake and fatty acid β-oxidation in mediating insulin resistance in heart and skeletal muscle

Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and β-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial β-oxidation. However, imbalance between fatty acid uptake and β-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid β-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid β-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid β-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.     

Immunohistochemical localization of mitochondrial fatty acid β-oxidation enzymes in Müller cells of the retina

The presence of a mitochondrial fatty acid β-oxidation system in the retina was shown by immunohistochemistry. Fatty acids are considered to serve as a major energy source metabolized by fatty acid β-oxidation together with glucose metabolized by glycolysis in the organs of the entire body, but almost nothing is known about this metabolic system in the retina. Adult rat retinae were subjected to immunofluorescence and immuno-electron microscopy for the localization of fatty acid β-oxidation enzymes, together with western blot analysis for quantitation of the amount of enzyme proteins and DNA microarray analysis for gene expression. All the enzymes examined were shown to be present in the retina, but in small amounts, with the amount of protein and gene expression in the retina being about 1/10 of those in the liver. Immunohistochemistry at light and electron microscopic levels revealed the enzymes to be more preferentially localized to the mitochondria of Müller cells than the retinal neurons. The Müller cells were isolated from the retina and confirmed for the presence of mitochondrial fatty acid β-oxidation enzymes. A mitochondrial fatty acid β-oxidation system was thus shown to be present in the retina heterogeneously.     

The effect of charge reversal mutations in the α-helical region of liver fatty acid binding protein on the binding of fatty-acyl CoAs,lysophospholipids and bile acids

Liver fatty acid binding protein (LFABP) is unique among the various types of FABPs in that it can bind a variety of ligands in addition to fatty acids. LFABP is able to bind long chain fatty acids with a 2:1 stoichiometry and the crystal structure has identified two fatty acid binding sites in the binding cavity. The presumed primary site (site 1) involves the fatty acid binding with the carboxylate group buried in the cavity whereas the fatty acid at site 2 has the carboxylate group solvent-exposed within the ligand portal region and in the vicinity of -helix II. The -helical region contains three cationic residues, K20, K31, K33 and modelling studies suggest that K31 on -helix II could make an electrostatic contribution to anionic ligands binding to site 2. The preparation of three charge reversal mutants of LFABP, K20E, K31E and K33E has allowed an investigation of the role of site 2 in ligand binding, particularly those ligands with a bulky anionic head group. The binding of oleoyl CoA, lysophosphatidic acid, lysophosphatidylcholine, lithocholic acid and taurolithocholate 3-sulphate to LFABP has been studied using the -helical mutants. The results support the concept that such ligands bind at site 2 of LFABP where solvent exposure allows the accommodation of their bulky anionic group.     

What makes a binding site a binding site?

Organic probe molecules have recently been used to define hydrophobic binding sites on the surface of proteins. It appears that the presence of water on the surface of a protein plays a crucial role in the interaction between that protein and its binding site.     

Hemoglobin binding with haptoglobin: Delineation of the haptoglobin binding site on the α-chain of human hemoglobin

Previous studies from this laboratory employing a comprehensive synthetic overlapping peptide strategy showed that the -chain of human hemoglobin (Hb) contains a single haptoglobin (HP) binding region residing within residues 121–135. The present study describes a precise delineation of this Hp-binding site on the -chain. Two overlapping peptides (111–125 and 121–135) spanning this region and a panel of five peptides decreasing at the C-terminal from residue 135 by decrements of two residues (119–135, 119–133, 119–131, 119–129, and 119–127) were synthesized, purified, and characterized. Quantitative radiometric titration of¹²⁵I-labeled human HP (type 2-1) with adsorbents of each of these synthetic peptides showed that the peptide 119–127 retained a Hp-binding activity equivalent to that of peptide 121–135. This finding indicated that Lys-127 marked the C-terminal boundary of the binding site. Another panel of eight peptides was then synthesized, which had their C-terminus fixed at Lys-127 and increased at the N-terminus by one-residue increments from residue 122 up to residue 115 (122–127, 121–127, 120–127, 119–127, 118–127, 117–127, 116–127, and 115–127). The binding of¹²⁵I-Hp to adsorbents of these peptides demonstrated that the N-terminal boundary of the site did not extend beyond Valine 121. It is, therefore, concluded that the Hp-binding site on the -chain of human Hb comprises residues 121–127.     

Heat stress deteriorates mitochondrial β-oxidation of long-chain fatty acids in cultured fibroblasts with fatty acid β-oxidation disorders

Mitochondrial fatty acids β-oxidation disorder (FAOD) has become popular with development of tandem mass spectrometry (MS/MS) and enzymatic evaluation techniques. FAOD occasionally causes acute encephalopathy or even sudden death in children. On the other hand, hyperpyrexia may also trigger severe seizures or encephalopathy, which might be caused by the defects of fatty acid β-oxidation (FAO). We investigated the effect of heat stress on FAO to determine the relationship between serious febrile episodes and defect in β-oxidation of fatty acid in children. Fibroblasts from healthy control and children with various FAODs, were cultured in the medium loaded with unlabelled palmitic acid for 96 h at 37 °C or 41 °C. Acylcarnitine (AC) profiles in the medium were determined by MS/MS, and specific ratios of ACs were calculated. Under heat stress (at 41 °C), long-chain ACs (C12, C14, or C16) were increased, while medium-chain ACs (C6, C8, or C10) were decreased in cells with carnitine palmitoyl transferase II deficiency, very-long-chain acyl-CoA dehydrogenase deficiency and mitochondrial trifunctional protein deficiency, whereas AC species from short-chain (C4) to long-chain (C16) were barely affected in medium-chain acyl-CoA dehydrogenase and control. While long-chain ACs (C12–C16) were significantly elevated, short to medium-chain ACs (C4–C10) were reduced in multiple acyl-CoA dehydrogenase deficiency. These data suggest that patients with long-chain FAODs may be more susceptible to heat stress compared to medium-chain FAOD or healthy control and that serious febrile episodes may deteriorate long-chain FAO in patients with long-chain FAODs.