Fatty acid binding proteins in multiple myeloma

Fatty acid binding proteins (FABPs) transport fatty acids (FA) into cells as an energy source, and their inhibition suppressed tumor proliferation in solid tumors. Multiple myeloma (MM) is a hematologic malignancy, known for disrupted protein metabolism including high proteasome activity, where proteasome inhibitors made a dramatic improvement in its treatment. Recent discovery found FABPs as a novel metabolic pathway in MM, which will have an impact on understanding the biology and on therapeutic application in MM.     

Fatty acid binding sites of rodent adipocyte and heart fatty acid binding proteins: characterization using fluorescent fatty acids

Murine adipocyte and rat heart fatty acid binding proteins (FABP) are closely related members of a family of cytosolic proteins which bind long-chain free fatty acids (ffa). The physical and chemical characteristics of the fatty acid binding sites of these proteins were studied using a series of fluorescent analogues of stearic acid (18:0) with an anthracene moiety covalently attached at seven different positions along the length of the hydrocarbon chain (AOffa). Previously, we used these probes to investigate the binding site of rat liver FABP (L-FABP) [Storch et al. (1989) J. Biol. Chem. 264, 8708-8713]. Here we extend those studies to adipocyte and heart FABP, two members of the FABP family which share a high degree of sequence homology with each other (62% identity) but which are less homologous with L-FABP (approximately 30%). The results show that the fluorescence emission spectra of AOffa bound to adipocyte FABP (A-FABP) are blue-shifted relative to heart FABP (H-FABP), indicating that AOffa bound to A-FABP are held in a more constrained configuration. For both proteins, constraint on the bound ffa probe is highest at the midportion of the acyl chain. Ffa are bound in a hydrophobic environment in both proteins. Excited-state lifetimes and fluorescence quantum yields suggest that the binding site of H-FABP is more hydrophobic than that of A-FABP. Nevertheless, acrylamide quenching experiments indicate that ffa bound to H-FABP are more accessible to the aqueous environment than are A-FABP-bound ffa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fatty acid binding proteins from developing human fetal brain: Characterization and binding properties

Two fatty acid binding proteins (FABPs) of identicalM _r, 13 kDa, have been isolated from developing human fetal brain. A delipidated 105,000 g supernatant was incubated with [1 -¹⁴C]oleate and subjected to a Sephacryl S-200 column followed by gel filtration chromatography on a Sephadex G-75 column and ion-exchange chromatography using a DEAE-Sephacel column. Purity was checked by UV spectroscopy, SDS-PAGE, isoelectric focusing and immunological cross-reactivity. The two FABPs designated as DE-I (pI 5.4) and DE-II (pI 6.9) showed cross-reactivity with each other and no alteration at the antigenic site during intrauterine development. Anti-human fetal brain FABP does not cross-react with purified human fetal heart, gut, lung or liver FABPs. The molecular mass of DE-I and DE-II is lower than those of fetal lung and liver FABPs. Like liver FABP, these proteins bind organic anions, fatty acids and acyl CoAs but differ in their binding affinities. Both DE-I and DE-II have been found to exhibit higher affinity for oleate (K _d = 0.23 μM) than palmitate (K _d = 0.9μM) or palmitoyl-CoA (K _d = 0.96 μM), with DE-I binding less fatty acids than DE-II. DE-II is more efficient in transferring fatty acid from phospholipid vesjcles than DE-I indicating that human fetal brain FABPs may play a significant role in fatty acid transport in developing fetal brain.     

Fatty acid binding proteins from different tissues show distinct patterns of fatty acid interactions

Fatty acid binding proteins (FABP) form a family of proteins displaying tissue-specific expression. These proteins are involved in fatty acid (FA) transport and metabolism by mechanisms that also appear to be tissue-specific. Cellular retinoid binding proteins are related proteins with unknown roles in FA transport and metabolism. To better understand the origin of these tissue-specific differences we report new measurements, using the acrylodated intestinal fatty acid binding protein (ADIFAB) method, of the binding of fatty acids (FA) to human fatty acid binding proteins (FABP) from brain, heart, intestine, liver, and myelin. We also measured binding of FA to a retinoic acid (CRABP-I) and a retinol (CRBP-II) binding protein and we have extended to 19 different FA our characterization of the FA-ADIFAB and FA-rat intestinal FABP interactions. These studies extend our previous analyses of human FABP from adipocyte and rat FABPs from heart, intestine, and liver. Binding affinities varied according to the order brain approximately myelin approximately heart > liver > intestine > CRABP > CRBP. In contrast to previous studies, no protein revealed a high degree of selectivity for particular FA. The results indicate that FA solubility (hydrophobicity) plays a major role in governing binding affinities; affinities tend to increase with increasing hydrophobicity (decreasing solubility) of the FA. However, our results also reveal that, with the exception of the intestinal protein, FABPs exhibit an additional attractive interaction for unsaturated FA that partially compensates for their trend toward lower affinities due to their higher aqueous solubilities. Thermodynamic potentials were determined for oleate and arachidonate binding to a subset of the FABP and retinoid binding proteins. FA binding to all FABPs was enthalpically driven. The DeltaH degrees values for paralogous FABPs, proteins from the same species but different tissues, reveal an exceptionally wide range of values, from -22 kcal/mol (myelin) to -7 kcal/mol (adipocyte). For orthologous FABPs from the same tissue but different species, DeltaH degrees values were similar. In contrast to the enthalpic dominance of FA binding to FABP, binding of FA to CRABP-I was entropically driven. This is consistent with the notion that FA specificity for FABP is determined by the enthalpy of binding. Proteins from different tissues also revealed considerable heterogeneity in heat capacity changes upon FA binding, DeltaC(p) values ranged between 0 and -1.3 kcal mol(-1) K(-1). The results demonstrate that thermodynamic parameters are quite different for paralogous but are quite similar for orthologous FABP, suggesting tissue-specific differences in FABP function that may be conserved across species.     

Fatty acid interactions with native and mutant fatty acid binding proteins

The interactions of long chain fatty acids (FA) with wild type (WT) fatty acid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constants for binding and dissociation. These measurements have been done using the fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of the free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that for WT proteins from adipocyte, heart, intestine, and liver, Kd values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Binding affinities for heart and liver are generally greater than those for adipocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37øC is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FFA, suggests a buffering action of FABPs that helps to maintain the intracellular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature dependence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of the intestinal FABP. These measurements showed that binding affinities did not report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an alanine substitution for arginine 106 yields a 30 fold increase in binding affinity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg106, is more than compensated for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.     

Fatty acid transport proteins

PURPOSE OF REVIEW: Fatty acid transport proteins are a family of proteins involved in fatty acid uptake and activation. This review summarizes recent progress in elucidating the function of fatty acid transport proteins. RECENT FINDINGS: Recent experiments clearly establish FATP1 as a regulated fatty acid transporter in both adipose tissue and muscle with important roles in energy homeostasis, thermogenesis and insulin resistance. Knockout of FATP5 in mice show it to be a bifunctional protein required for both hepatic fatty acid uptake and bile acid reconjugation. The most striking phenotype of FATP4 deletion is a defect in skin homeostasis, which may be due to its very long chain acyl-coenzyme A synthetase activity. Fatty acid transport proteins are increasingly being recognized as multifunctional proteins that can mediate the uptake of fatty acids as well as catalyze the formation of coenzyme A derivatives using long-chain and very-long chain fatty acids, bile acids and bile acid precursors as substrates. SUMMARY: Modulation of fatty acid transport protein function can result in altered energy homeostasis and insulin sensitivity, defective skin homeostasis, and altered bile acid metabolism. Both fatty acid uptake and enzymatic activity of fatty acid transport proteins likely contribute to these phenotypes. Future studies are needed to better understand the molecular mechanism of fatty acid transport protein function and the physiological role of FATP2, FATP3, and FATP6.     

Fatty acid binding proteins in the three types of rat skeletal muscle

By means of Sephadex G-50 column chromatography, a Mr 12,000 fatty acid binding protein (FABP) was found to be present in all three types of skeletal muscle. FABP concentrations in muscle cytosols (105,000g supernatant) were fiber type specific with binding levels (expressed as pmole [14C]oleate bound/mg protein) of 70 +/- 7 in fast-twitch white (FTW) (heart FABP = 469 +/- 33). Cytosols from all three fiber types cross-reacted with antibody to pure heart FABP on Ouchterlony immunodiffusion analysis. FABP content, determined by radial immunodiffusion, followed the same pattern in the muscle types as that in the binding assay. The values (in micrograms/mg protein) were 3.3 +/- 0.1 in FTW, 17.0 +/- 0.4 in FTR, and 31.7 +/- 1.4 in STR fibers (heart = 55). Disc gel electrophoresis revealed a protein band in each fiber type that had migration identical to that of pure heart FABP and was proportional to the amounts determined by Sephadex G-50 chromatography and immunoassay. In addition, Western blots of tissue cytosols, using antibody to heart FABP, detected single protein bands identical in size to pure heart FABP in all three types of skeletal muscle. These results show the presence of a FABP in all skeletal muscle types with an immunologic and electrophoretic characterization identical to that of heart FABP.     

Fatty acid-binding proteins in the heart

Long-chain fatty acids are important fuel molecules for the heart, their oxidation in mitochondria providing the bulk of energy required for cardiac functioning. The low solubility of fatty acids in aqueous solutions impairs their cellular transport. However, cardiac tissue contains several proteins capable of binding fatty acids non-covalently. These fatty acid-binding proteins (FABPs) are thought to facilitate both cellular uptake and intracellular transport of fatty acids. The majority of fatty acids taken up by the heart seems to pass the sarcolemma through a carrier-mediated translocation mechanism consisting of one or more membrane-associated FABPs. Intracellular transport of fatty acids towards sites of metabolic conversion is most likely accomplished by cytoplasmic FABPs. In this review, the roles of membrane-associated and cytoplasmic FABPs in cardiac fatty acid metabolism under (patho)physiological circumstances are discussed.     

Fatty acid binding to plasma albumin.

A review of the available information about fatty acid binding to plasma albumin is presented. Albumin is composed of a single polypeptide chain, folded so as to form three or four spherical units. The strong fatty acid binding sites probably are located in crevices between these spherical regions. The anionic form of the fatty acid binds to albumin. Most of the binding energy comes from nonpolar interactions between the fatty acid hydrocarbon chain and uncharged amino acid side chains that line the binding sites. The binding sites are somewhat pliable, and their configuration can adapt to fit the incoming fatty acid. Stepwise association constants for binding to human albumin of fatty acids containing 6-18 carbon atoms are presented. These data indicate that each mole of fatty acid binds with a different affinity and that the association constants for multiple binding diminish sequentially, i.e., kappa 1 greater than kappa 2 greater than kappa 3 greater ... greater kappan. Because of uncertainties concerning fatty acid association in aqueous solutions, the constants for the 14-18 carbon acids probably are not definitive. In the usual physiological concentration range, free fatty acids do not displace appreciable amounts of a second organic compound from albumin. Sensitive spectrophotometric analyses revealed, however, that even small increases in free fatty acid concentration alter the molecular interaction between human albumin and another organic compound.     

Fatty acid binding proteins stabilize leukotriene A4: competition with arachidonic acid but not other lipoxygenase products

Leukotriene A(4) (LTA(4)) is a chemically reactive conjugated triene epoxide product derived from 5-lipoxygenase oxygenation of arachidonic acid. At physiological pH, this reactive compound has a half-life of less than 3 s at 37 degrees C and approximately 40 s at 4 degrees C. Regardless of this aqueous instability, LTA(4) is an intermediate in the formation of biologically active leukotrienes, which can be formed through either intracellular or transcellular biosynthesis. Previously, epithelial fatty acid binding protein (E-FABP) present in RBL-1 cells was shown to increase the half-life of LTA(4) to approximately 20 min at 4 degrees C. Five FABPs (adipocyte FABP, intestinal FABP, E-FABP, heart/muscle FABP, and liver FABP) have now been examined and also found to increase the half-life of LTA(4) at 4 degrees C to approximately 20 min with protein present. Stabilization of LTA(4) was examined when arachidonic acid was present to compete with LTA(4) for the binding site on E-FABP. Arachidonate has an apparent higher affinity for E-FABP than LTA(4) and was able to completely block stabilization of the latter. When E-FABP is not saturated with arachidonate, FABP can still stabilize LTA(4). Several lipoxygenase products, including 5-hydroxyeicosatetraenoic acid, 5,6-dihydroxyeicosatetraenoic acid, and leukotriene B(4), were found to have no effect on the stability of LTA(4) induced by E-FABP even when present at concentrations 3-fold higher than LTA(4).     

Fatty acid transport proteins and insulin resistance

PURPOSE OF REVIEW: Disturbed fatty acid metabolism and homeostasis is associated with insulin resistance. The aim of this review, therefore, is to summarize recent developments relating to the relevance and importance of the fatty acid transport proteins (FATPs) in the aetiology of insulin resistance. In particular, the potential differences between the six members of the FATP family will be considered. RECENT FINDINGS: FATP1 knockout mice failed to develop insulin resistance associated with lipid infusion or a high-fat diet, as wild-type mice did. FATP1-mediated fatty acid uptake may cause intramuscular lipid accumulation leading to insulin resistance in muscle if the fatty acids are not oxidized. While mouse models demonstrated an absolute requirement for FATP4 for survival, they provided no direct evidence for a role of FATP4 in insulin resistance. However, expression of FATP4 in human adipose tissue was increased in obesity (independent of genetic factors). While other members of the FATP family have important roles in fatty acid metabolism, they have not been clearly linked to insulin resistance. FATP-mediated fatty acid uptake may be driven by intrinsic acyl-CoA synthase activity. SUMMARY: Any role in the development of insulin resistance is likely to be different for each member of the FATP family. So far, both FATP1 and FATP4 have been associated with parameters related to insulin resistance. Whether increased FATP-mediated fatty acid uptake is beneficial or detrimental may be dependent on the tissue in question and on the subsequent fate of the fatty acids. These issues remain to be resolved.     

Fatty acid and lactate metabolism by heart homogenates from alloxan-diabetic dogs

Myocardial homogenates from control of alloxan-diabetic dogs were incubated in the presence of lactate or palmitate as the sole substrate. The rate of lactate oxidation of the homogenates from diabetic dogs was less than half of the oxidation rate of homogenates from control animals. The incorporation of labelled palmitate into triglyceride was greatly enhanced in the heart taken from diabetic animals. The tissue concentration of l-carnitine was twice and that of triglyceride was three times as high in the diabetic animals as in their control counterparts. Thus the myocardium of diabetic animals exhibited both altered substrate utilization and increased rate of triglyceride synthesis.     

Bovine fatty acid binding proteins. Isolation and characterisation of two cardiac fatty acid binding proteins that are distinct from corresponding hepatic proteins

When a 100,000 X g supernatant from bovine heart was incubated with [1-14C]oleic acid and subjected to isoelectric focusing, two fatty acid binding proteins (FABPs) with isoelectric points at 4.9 and 5.1 were detected. The proteins were purified on a large scale first by heat and acid precipitation of a postmitochondrial supernatant, as well as fractionation with ammonium sulfate, then by alternate application of ion-exchange and gel chromatography. The procedure afforded around 60 mg pure proteins from 1.5 kg fresh heart muscle. Relative molecular masses of 15 300 +/- 1600 for both proteins were derived from sodium dodecyl sulfate/polyacrylamide gel electrophoresis, gel chromatography, sedimentation velocity as well as from amino acid analysis. Up to 50% of the proteins' secondary structures consisted of beta-sheet. N-termini of the peptide chains were blocked; the amino acid compositions of the two proteins were similar, but differed considerably from those of the two FABPs isolated from bovine liver [Haunerland et al. (1984) Hoppe Seyler's Z. Physiol. Chem. 365, 365-376]. Whereas hepatic FABPs changed their pI upon binding fatty acids, cardiac FABPs did not. Cardiac FABPs were immunologically identical, but did not cross-react with hepatic proteins. A reversible, concentration-dependent self-association reported for FABP from pig heart [Fournier et al. (1983) Biochemistry 22, 1863-1872] was not observed for FABP from bovine heart. Changes of concentration did not alter secondary structure, intrinsic fluorescence or the sedimentation coefficient of the protein.     

Fatty acid-binding protein-hormone-sensitive lipase interaction. Fatty acid dependence on binding

Adipose lipolysis is mediated, in part, via interaction of fatty acid-binding protein (FABP) with hormone-sensitive lipase (HSL). Mice with reduced FABP content in fat (adipocyte FABP null) exhibit diminished fat cell lipolysis, whereas transgenic mice with increased FABP content in fat (epithelial FABP transgenic) exhibit enhanced lipolysis. To examine the relationship between the binding of FABP to HSL and activation of catalytic activity, isothermal titration microcalorimetry as well as kinetic analysis using a variety of FABP isoforms have been employed. In the absence of fatty acids, no FABP-HSL association could be demonstrated for any FABP form. However, in the presence of 10 microm oleate, A-FABP and E-FABP each bound to HSL with high affinity (Kd of 0.5 and 3 nM, respectively) in a approximately 1:1 molar stoichiometry, whereas liver FABP and intestinal FABP did not exhibit any association. To compare binding to catalysis, each FABP isoform was incubated with HSL in vitro, and enzymatic activity was assessed. Importantly, each FABP form stimulated HSL activity approximately 2-fold using cholesteryl oleate as substrate but exhibited no activation using p-nitrophenyl butyrate. The activation by A-FABP was dependent upon its fatty acid binding properties because a non-fatty acid binding mutant, R126Q, failed to activate HSL. These results suggest that binding and activation of HSL by FABPs are separate and distinct functions and that HSL contains a site for fatty acid binding that allows for FABP association.     

Predicting nucleic acid binding interfaces from structural models of proteins

The function of DNA‐ and RNA‐binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure‐based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high‐resolution three‐dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I‐TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high‐resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I‐TASSER produces high‐quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low‐resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Fatty acid acylation of vaccinia virus proteins.

Labeling of vaccinia virus-infected cells with [3H]myristic acid resulted in the incorporation of label into two viral proteins with apparent molecular weights of 35,000 and 25,000 (designated M35 and M25, respectively). M35 and M25 were expressed in infected cells after the onset of viral DNA replication, and both proteins were present in purified intracellular virus particles. Virion localization experiments determined M25 to be a constituent of the virion envelope, while M35 appeared to be peripherally associated with the virion core. M35 and M25 labeled by [3H]myristic acid were stable to treatment with neutral hydroxylamine, suggesting an amide-linked acylation of the proteins. Chromatographic identification of the protein-bound fatty acid moieties liberated after acid methanolysis of M25, isolated from infected cells labeled during a 4-h pulse, resulted in the recovery of 25% of the protein-bound fatty acid as myristate-associated label and 75% as palmitate, indicating that interconversion of myristate to palmitate had occurred during the labeling period. Similar analyses of M25 and M35, isolated from infected cells labeled during a 0.5-h pulse, determined that 46 and 43%, respectively, of the protein-bound label had been elongated to palmitate even during this brief labeling period. In contrast, M25 and M35 isolated from purified intracellular virions labeled continuously during 24 h of growth contained 75 and 70%, respectively, myristate-associated label, suggesting greater stability of these proteins or a favored interaction of the proteins containing myristate with the maturing or intracellular virion.     

Fatty acid acylation of proteins in Physarum polycephalum

We have investigated the occurrence of protein-fatty acid acylation by metabolic incorporation of [3H]myristic and [3H]palmitic acids in Physarum polycephalum. We show that this organism contains fatty acylated proteins with mainly myristic acid covalently attached in alkali-stable linkages, probably amides. We find no evidence for ester-linked fatty acids, in contrast to the situation in vertebrate cells.     

Fatty acid transfer from intestinal fatty acid binding protein to membranes: electrostatic and hydrophobic interactions

Intestinal fatty acid binding protein (IFABP) is thought to participate in the intracellular transport of fatty acids (FAs). Fatty acid transfer from IFABP to phospholipid membranes is proposed to occur during protein-membrane collisional interactions. In this study, we analyzed the participation of electrostatic and hydrophobic interactions in the collisional mechanism of FA transfer from IFABP to membranes. Using a fluorescence resonance energy transfer assay, we examined the rate and mechanism of transfer of anthroyloxy-fatty acid analogs a) from IFABP to phospholipid membranes of different composition; b) from chemically modified IFABPs, in which the acetylation of surface lysine residues eliminated positive surface charges; and c) as a function of ionic strength. The results show clearly that negative charges on the membrane surface and positive charges on the protein surface are important for establishing the "collisional complex", during which fatty acid transfer occurs. In addition, changes in the hydrophobicity of the protein surface, as well as the hydrophobic volume of the acceptor vesicles, also influenced the rate of fatty acid transfer. Thus, ionic interactions between IFABP and membranes appear to play a primary role in the process of fatty acid transfer to membranes, and hydrophobic interactions can also modulate the rates of ligand transfer.