Evidence that free polyunsaturated fatty acids modify Na+ channels by directly binding to the channel proteins.

The effects of free polyunsaturated fatty acids (PUFA) on the binding of ligands to receptors on voltage-sensitive Na+ channels of neonatal rat cardiac myocytes were assessed. The radioligand was [benzoyl-2,5-(3)H] batrachotoxinin A 20alpha-benzoate ([(3)H]BTXB), a toxin that binds to the Na+ channel. The PUFA that have been shown to be antiarrhythmic, including eicosapentaenoic acid (EPA; C20:5n-3), docosahexaenoic acid (DHA; C22:6n-3), eicosatetraynoic acid (ETYA), linolenic acid (C18:3n-3), and linoleic acid (C18:2n-6), inhibited [(3)H]BTXB binding in a dose-dependent fashion with IC50 values of 28-35 microM, whereas those fatty acids that have no antiarrhythmic effects including saturated fatty acid (stearic acid, C18:0), monounsaturated fatty acid (oleic acid; C18:1n-9), and EPA methyl ester did not have a significant effect on [(3)H]BTXB binding. Enrichment of the myocyte membrane with cholesterol neither affected [(3)H]BTXB binding when compared with control cells nor altered the inhibitory effects of PUFA on [(3)H]BTXB binding. Scatchard analysis of [(3)H]BTXB binding showed that EPA reduced the maximal binding without altering the Kd for [(3)H]BTXB binding, indicating allosteric inhibition. The inhibition by EPA of [(3)H]BTXB binding was reversible (within 30 min) when delipidated bovine serum albumin was added. The binding of the PUFA to this site on the Na+ channel is reversible and structure-specific and occurs at concentrations close to those required for apparent antiarrhythmic effects and a blocking effect on the Na+ current, suggesting that binding of the PUFA at this site relates to their antiarrhythmic action.

Expression of the CD36 homolog (FAT) in fibroblast cells: effects on fatty acid transport.

An adipocyte membrane glycoprotein, (FAT), homologous to human CD36, has been previously implicated in the binding/transport of long-chain fatty acids. It bound reactive derivatives of long-chain fatty acids and binding was specific and associated with significant inhibition of fatty acid uptake. Tissue distribution of the protein and regulation of its expression were also consistent with its postulated role. In this report, we have examined the effects of FAT expression on rates and properties of fatty acid uptake by Ob17PY fibroblasts lacking the protein. Three clones (P21, P22, and P25) were selected based on FAT mRNA and protein levels. Cell surface labeling could be demonstrated with the anti-CD36 antibody FITC-OKM5. In line with this, the major fraction of immunoreactive FAT was associated with the plasma membrane fraction. Assays of oleate and/or palmitate uptake demonstrated higher rates in the three FAT-expressing clones, compared to cells transfected with the empty vector. Clone P21, which had the highest protein levels on Western blots, exhibited the largest increase in transport rates. Fatty acid uptake in FAT-expressing P21 cells reflected two components, a phloretin-sensitive high-affinity saturable component with a Km of 0.004 microM and a basal phloretin-insensitive component that was a linear function of unbound fatty acid. P21 cells incorporated more exogenous fatty acid into phospholipids, indicating that binding of fatty acids was followed by their transfer into the cell and that both processes were increased by FAT expression. The data support the interpretation that FAT/CD36 functions as a high-affinity membrane receptor/transporter for long-chain fatty acids.

Free, long-chain, polyunsaturated fatty acids reduce membrane electrical excitability in neonatal rat cardiac myocytes.

Because previous studies showed that polyunsaturated fatty acids can reduce the contraction rate of spontaneously beating heart cells and have antiarrhythmic effects, we examined the effects of the fatty acids on the electrophysiology of the cardiac cycle in isolated neonatal rat cardiac myocytes. Exposure of cardiomyocytes to 10 microM eicosapentaenoic acid for 2-5 min markedly increased the strength of the depolarizing current required to elicit an action potential (from 18.0 +/- 2.4 pA to 26.8 +/- 2.7 pA, P < 0.01) and the cycle length of excitability (from 525 ms to 1225 ms, delta = 700 +/- 212, P < 0.05). These changes were due to an increase in the threshold for action potential (from -52 mV to -43 mV, delta = 9 +/- 3, P < 0.05) and a more negative resting membrane potential (from -52 mV to -57 mV, delta = 5 +/- 1, P < 0.05). There was a progressive prolongation of intervals between spontaneous action potentials and a slowed rate of phase 4 depolarization. Other polyunsaturated fatty acids--including docosahexaenoic acid, linolenic acid, linoleic acid, arachidonic acid, and its nonmetabolizable analog eicosatetraynoic acid, but neither the monounsaturated oleic acid nor the saturated stearic acid--had similar effects. The effects of the fatty acids could be reversed by washing with fatty acid-free bovine serum albumin. These results show that free polyunsaturated fatty acids can reduce membrane electrical excitability of heart cells and provide an electrophysiological basis for the antiarrhythmic effects of these fatty acids.

13C NMR study of the effects of leptin treatment on kinetics of hepatic intermediary metabolism

The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin’s biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin’s effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive 13C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring flux of 13C-labeled substrate through several multistep pathways. In perfused liver from either ob/ob or lean mice, we found that acute treatment with leptin in vitro modulates pathways controlling carbohydrate flux into 13C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of ob/ob liver also caused a rapid stimulation of long-chain fatty acid synthesis from 13C-labeled acetyl-CoA by the de novo synthesis route. Chronic leptin treatment in vivo induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-13C]pyruvate in ob/ob mouse liver perfused in the absence of the hormone. Consistent with the 13C NMR results, leptin treatment of the ob/ob mouse in vivo resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin in vivo exerted the opposite effect of acute treatment in vitro and markedly decreased hepatic de novo synthesis of fatty acids in ob/ob mouse liver. In agreement with the 13C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the ob/ob mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.

Structure-based conformational preferences of amino acids

Proteins can be very tolerant to amino acid substitution, even within their core. Understanding the factors responsible for this behavior is of critical importance for protein engineering and design. Mutations in proteins have been quantified in terms of the changes in stability they induce. For example, guest residues in specific secondary structures have been used as probes of conformational preferences of amino acids, yielding propensity scales. Predicting these amino acid propensities would be a good test of any new potential energy functions used to mimic protein stability. We have recently developed a protein design procedure that optimizes whole sequences for a given target conformation based on the knowledge of the template backbone and on a semiempirical potential energy function. This energy function is purely physical, including steric interactions based on a Lennard-Jones potential, electrostatics based on a Coulomb potential, and hydrophobicity in the form of an environment free energy based on accessible surface area and interatomic contact areas. Sequences designed by this procedure for 10 different proteins were analyzed to extract conformational preferences for amino acids. The resulting structure-based propensity scales show significant agreements with experimental propensity scale values, both for α-helices and β-sheets. These results indicate that amino acid conformational preferences are a natural consequence of the potential energy we use. This confirms the accuracy of our potential and indicates that such preferences should not be added as a design criterion.

Acylation stabilizes a protease-resistant conformation of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides

Protein acylation is an important way in which a number of proteins with a variety of functions are modified. The physiological role of the acylation of cellular proteins is still poorly understood. Covalent binding of fatty acids to nonintegral membrane proteins is thought to produce transient or permanent enhancement of the association of the polypeptide chains with biological membranes. In this paper, we investigate the functional role for the palmitoylation of an atypical membrane-bound protein, yeast protoporphyrinogen oxidase, which is the molecular target of diphenyl ether-type herbicides. Palmitoylation stabilizes an active heat- and protease-resistant conformation of the protein. Palmitoylation of protoporphyrinogen oxidase has been demonstrated to occur in vivo both in yeast cells and in a heterologous bacterial expression system, where it may be inhibited by cerulenin leading to the accumulation of degradation products of the protein. The thiol ester linking palmitoleic acid to the polypeptide chain was shown to be sensitive to hydrolysis by hydroxylamine and also by the widely used serine-protease inhibitor phenylmethylsulfonyl fluoride.

Dynamics of Gene Expression Revealed by Comparison of Serial Analysis of Gene Expression Transcript Profiles from Yeast Grown on Two Different Carbon SourcesD⃞

We describe a genome-wide characterization of mRNA transcript levels in yeast grown on the fatty acid oleate, determined using Serial Analysis of Gene Expression (SAGE). Comparison of this SAGE library with that reported for glucose grown cells revealed the dramatic adaptive response of yeast to a change in carbon source. A major fraction (>20%) of the 15,000 mRNA molecules in a yeast cell comprised differentially expressed transcripts, which were derived from only 2% of the total number of ∼6300 yeast genes. Most of the mRNAs that were differentially expressed code for enzymes or for other proteins participating in metabolism (e.g., metabolite transporters). In oleate-grown cells, this was exemplified by the huge increase of mRNAs encoding the peroxisomal β-oxidation enzymes required for degradation of fatty acids. The data provide evidence for the existence of redox shuttles across organellar membranes that involve peroxisomal, cytoplasmic, and mitochondrial enzymes. We also analyzed the mRNA profile of a mutant strain with deletions of the PIP2 and OAF1 genes, encoding transcription factors required for induction of genes encoding peroxisomal proteins. Induction of genes under the immediate control of these factors was abolished; other genes were up-regulated, indicating an adaptive response to the changed metabolism imposed by the genetic impairment. We describe a statistical method for analysis of data obtained by SAGE.

Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide

The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features—an optimal C12–C8 chain length, π-unsaturation introduction at the corresponding arachidonoyl Δ8,9/Δ11,12 and oleoyl Δ9,10 location, and an α-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with Kis that drop below 200 pM and are 102–103 times more potent than the corresponding trifluoromethyl ketones.

Regulation of fatty acid homeostasis in cells: Novel role of leptin

It is proposed that an important function of leptin is to confine the storage of triglycerides (TG) to the adipocytes, while limiting TG storage in nonadipocytes, thus protecting them from lipotoxicity. The fact that TG content in nonadipocytes normally remains within a narrow range, while that of adipocytes varies enormously with food intake, is consistent with a system of TG homeostasis in normal nonadipocytes. The facts that when leptin receptors are dysfunctional, TG content in nonadipocytes such as islets can increase 100-fold, and that constitutively expressed ectopic hyperleptinemia depletes TG, suggest that leptin controls the homeostatic system for intracellular TG. The fact that the function and viability of nonadipocytes is compromised when their TG content rises above or falls below the normal range suggests that normal homeostasis of their intracellular TG is critical for optimal function and to prevent lipoapoptosis. Thus far, lipotoxic diabetes of fa/fa Zucker diabetic fatty rats is the only proven lipodegenerative disease, but the possibility of lipotoxic disease of skeletal and/or cardiac muscle may require investigation, as does the possible influence of the intracellular TG content on autoimmune and neoplastic processes.

A quantitative method for evaluating the stabilities of nucleic acids

We report a general method for screening, in solution, the impact of deviations from canonical Watson-Crick composition on the thermodynamic stability of nucleic acid duplexes. We demonstrate how fluorescence resonance energy transfer (FRET) can be used to detect directly free energy differences between an initially formed “reference” duplex (usually a Watson-Crick duplex) and a related “test” duplex containing a lesion/alteration of interest (e.g., a mismatch, a modified, a deleted, or a bulged base, etc.). In one application, one titrates into a solution containing a fluorescently labeled, FRET-active, reference duplex, an unlabeled, single-stranded nucleic acid (test strand), which may or may not compete successfully to form a new duplex. When a new duplex forms by strand displacement, it will not exhibit FRET. The resultant titration curve (normalized fluorescence intensity vs. logarithm of test strand concentration) yields a value for the difference in stability (free energy) between the newly formed, test strand-containing duplex and the initial reference duplex. The use of competitive equilibria in this assay allows the measurement of equilibrium association constants that far exceed the magnitudes accessible by conventional titrimetric techniques. Additionally, because of the sensitivity of fluorescence, the method requires several orders of magnitude less material than most other solution methods. We discuss the advantages of this method for detecting and characterizing any modification that alters duplex stability, including, but not limited to, mutagenic lesions. We underscore the wide range of accessible free energy values that can be defined by this method, the applicability of the method in probing for a myriad of nucleic acid variations, such as single nucleotide polymorphisms, and the potential of the method for high throughput screening.

Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex

Hepatitis C virus (HCV) helicase, non-structural protein 3 (NS3), is proposed to aid in HCV genome replication and is considered a target for inhibition of HCV. In order to investigate the substrate requirements for nucleic acid unwinding by NS3, substrates were prepared by annealing a 30mer oligonucleotide to a 15mer. The resulting 15 bp duplex contained a single-stranded DNA overhang of 15 nt referred to as the bound strand. Other substrates were prepared in which the 15mer DNA was replaced by a strand of peptide nucleic acid (PNA). The PNA–DNA substrate was unwound by NS3, but the observed rate of strand separation was at least 25-fold slower than for the equivalent DNA–DNA substrate. Binding of NS3 to the PNA–DNA substrate was similar to the DNA–DNA substrate, due to the fact that NS3 initially binds to the single-stranded overhang, which was identical in each substrate. A PNA–RNA substrate was not unwound by NS3 under similar conditions. In contrast, morpholino–DNA and phosphorothioate–DNA substrates were utilized as efficiently by NS3 as DNA–DNA substrates. These results indicate that the PNA–DNA and PNA–RNA heteroduplexes adopt structures that are unfavorable for unwinding by NS3, suggesting that the unwinding activity of NS3 is sensitive to the structure of the duplex.

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2′-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with Taq polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XbaI and by mass spectrometry of the PCR products.

Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR

The incorporation of potentially catalytic groups into DNA is of interest for the in vitro selection of novel deoxyribozymes. We have devised synthetic routes to a series of three C7 modified 7-deaza-dATP derivatives with pendant aminopropyl, Z-aminopropenyl and aminopropynyl side chains. These modified triphosphates have been tested as substrates for Taq polymerase during PCR. All the modifications are tolerated by this enzyme, with the aminopropynyl side chain giving the best result. Most protein enzymes have more than one type of catalytic group located in their active site. By using C5-imidazolyl-modified dUTPs together with 3-(aminopropynyl)-7-deaza-dATP in place of the natural nucleotides dTTP and dATP, we have demonstrated the simultaneous incorporation of both amino and imidazolyl moieties into a DNA molecule during PCR. The PCR product containing the four natural bases was fully digested by XbaI, while PCR products containing the modified 7-deaza-dATP analogues were not cleaved. Direct evidence for the simultaneous incorporation during PCR of an imidazole-modified dUTP and an amino-modified 7-deaza-dATP has been obtained using mass spectrometry.

A Saccharomyces cerevisiae homolog of the human adrenoleukodystrophy transporter is a heterodimer of two half ATP-binding cassette transporters.

The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein (PMP70) are half ATP-binding cassette (ABC) transporters in the human peroxisome membrane. ALDP and PMP70 share sequence homology and both are implicated in genetic diseases. PXA1 and YKL741 are Saccharomyces cerevisiae genes that encode homologs of ALDP and PMP70. Pxa1p, a putative ortholog of ALDP, is involved in peroxisomal beta-oxidation of fatty acids while YKL741 is an open reading frame found by the yeast genome sequencing project. Here we designate YKL741 as PXA2 and show that its protein product, Pxa2p, like Pxa1p, is associated with peroxisomes but not required for their assembly. Yeast strains carrying gene disruption of PXA1, PXA2, or both have similar and, in the case of the latter, nonadditive phenotypes. We also find that the stability of Pxa1p, but not Pxa2p, is markedly reduced in the absence of the other. Finally, we find that Pxa1p and Pxa2p coimmuno-precipitate. These genetic and physical data suggest that Pxa1p and Pxa2p heterodimerize to form a complete peroxisomal ABC transporter involved in fatty acid beta-oxidation. This result predicts the presence of similar heterodimeric ABC transporters in the mammalian peroxisome membrane.

Brain lipids that induce sleep are novel modulators of 5-hydroxytrypamine receptors.

Amide derivatives of fatty acids were recently isolated from cerebrospinal fluid of sleep-deprived animals and found to induce sleep in rats. To determine which brain receptors might be sensitive to these novel neuromodulators, we tested them on a range of receptors expressed in Xenopus oocytes. cis-9,10-Octadecenamide (ODA) markedly potentiated the action of 5-hydroxytryptamine (5-HT) on 5-HT2A and 5-HT2C receptors, but this action was not shared by related compounds such as oleic acid and trans-9,10-octacenamide. ODA was active at concentrations as low as 1 nM. The saturated analog, octadecanamide, inhibited rather than potentiated 5-HT2C responses. ODA had either no effect or only weak effects on other receptors, including muscarinic cholinergic, metabotropic glutamate, GABA(A), N-methyl-D-asparate, or alpha-amino-3-hydroxy-5-methyl-4-isoxozolepropionic acid receptors. Modulation of 5-HT2 receptors by ODA and related lipids may represent a novel mechanism for regulation of receptors that activate G proteins and thereby play a role in alertness, sleep, and mood as well as disturbances of these states.