Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s)

The de novo biosynthesis of sphinganine and sphingosine was studied using LM cells incubated with [14C] serine in serum-free media. Most of the radiolabeled long-chain bases were initially found in dihydroceramides (as sphinganine) and the proportion appearing in complex sphingolipids (as sphingosine) increased over time. Since free long-chain bases were not detected (although formation of 3-ketosphinganine, the first condensation product of serine and palmitoyl-CoA, could be demonstrated in vitro), it appears that the first step is rate-limiting for dihydroceramide biosynthesis. The kinetics suggested that after N-acyl-sphinganines were formed they were dehydrogenated to N-acylsphingosines. No evidence was found for the formation in vivo or in vitro of the putative intermediates of the direct biosynthesis of sphingosine from sphinganine (i.e. 3-ketosphingosine and free sphingosine). The conversion of N-acylsphinganines to N-acyl-sphingosines was confirmed by incubating cells with [14C] serine followed by unlabeled serine, which resulted in a rapid increase in the sphingosine-to-sphinganine ratio in amide-linked sphingolipids during the chase. These findings are most consistent with a pathway for long-chain base biosynthesis in which N-acyl-sphinganines are first synthesized by LM cells and the 4-trans-double bond is added to this or subsequent products, as opposed to the most cited pathway wherein sphingosine is made directly from sphinganine.     

Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by beta-chloroalanine

The effects of beta-chloroalanine (beta-Cl-alanine) on serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid (5 mM beta-Cl-alanine caused complete inactivation in 10 min), irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with beta-Cl-L-alanine (not beta-Cl-D-alanine) and was blocked by L-serine. These are characteristics of mechanism-based ("suicide") inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with beta-Cl-alanine (complete inhibition occurred in 15 min with 5 mM), and this treatment inhibited [14C]serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of beta-Cl-L-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, [14C]serine uptake was not blocked, total lipid biosynthesis from [14C]acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and [3H]thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of [3H]leucine, which was only decreased by 14%. Although beta-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that beta-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis.     

Structural requirements for long-chain (sphingoid) base inhibition of protein kinase C in vitro and for the cellular effects of these compounds

Sphingosine, sphinganine, and other long-chain (sphingoid) bases inhibit protein kinase C in vitro and block cellular responses to agonists that are thought to act via this enzyme. To gain further insight into the mechanism of this inhibition, a series of long-chain analogues differing in alkyl chain length (11-20 carbon atoms), stereochemistry, and headgroup were examined for (a) inhibition of protein kinase C activity in vitro, (b) the neutrophil respiratory burst in response to phorbol myristate acetate (PMA), (c) the PMA-induced differentiation of HL-60 cells, and (d) the growth of Chinese hamster ovary cells. In every instance, the effects were maximal with the 18-carbon homologues, which are the same length as the predominant naturally occurring long-chain base (sphingosine). The lower potency of the shorter chain homologues was partially due to decreased uptake by cells. Small differences were obtained with the four stereoisomers of sphingosine (i.e., D and L forms of erythro- and threo-sphingosine), with N-methyl derivatives of the different sphingosine homologues, and with simpler alkylamines (e.g., stearylamine). The potency of the different headgroup analogues may be affected by the degree of protonation at the assay pH. The pKa of sphingosine was measured to be 6.7; the pKa varied among the analogues. These findings establish that the major structural features required for inhibition of protein kinase C and cellular processes dependent on this enzyme are the presence of a free amino group and an aliphatic side chain and that other groups have more subtle effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel biosynthetic pathway providing precursors for fatty acid biosynthesis and secondary metabolite formation in myxobacteria

Short chain carboxylic acids are well known as the precursors of fatty acid and polyketide biosynthesis. Iso-fatty acids, which are important for the control of membrane fluidity, are formed from branched chain starter units (isovaleryl-CoA and isobutyryl-CoA), which in turn are derived from the degradation of leucine and valine, respectively. Branched chain carboxylic acids are also employed as starter molecules for the biosynthesis of secondary metabolites, e.g. the therapeutically important anthelmintic agent avermectin or the electron transport inhibitor myxothiazol. During our studies on myxothiazol biosynthesis in the myxobacterium, Stigmatella aurantiaca, we detected a novel biosynthetic route to isovaleric acid. After cloning and inactivation of the branched chain keto acid dehydrogenase complex, which is responsible for the degradation of branched chain amino acids, the strain is still able to produce iso-fatty acids and myxothiazol. Incorporation studies employing deuterated leucine show that it can only serve as precursor in the wild type strain but not in the esg mutant. Feeding experiments using (13)C-labeled precursors show that isovalerate is efficiently made from acetate, giving rise to a labeling pattern in myxothiazol that provides evidence for a novel branch of the mevalonate pathway involving the intermediate 3,3-dimethylacryloyl-CoA. 3,3-Dimethylacrylic acid was synthesized in deuterated form and fed to the esg mutant, resulting in strong incorporation into myxothiazol and iso-fatty acids. Similar experiments employing Myxococcus xanthus revealed that the discovered biosynthetic route described is present in other myxobacteria as well.     

Inhibitory effect of undecanoic acid on the biosynthesis of long-chain fatty acids in Mortierella isabellina

Summary Following growth on n-alkanes, undecanoic acid in high concentrations completely inhibits the acylation of fatty acids formed during the terminal oxidation so that the intracellular fatty acid pattern is composed exclusively of components from the de novo synthesis. An inhibitory effect of undecanoic acid stems presumably from the effect it has on the long-chain acyl-coenzyme A synthetase I, whereas the corresponding long-chain acyl-coenzyme A synthetase II, which is bound to specific cell organelles remains untouched by this inhibition. The strongly reduced growth, even following glucose oxidation, probably comes from the effect of C₁₁-acid on specific intramitochondrial situated enzymes.Prof. Dr. H. G. Schlegel dedicated to his 60th birthday     

Membrane lipid biosynthesis in Acholeplasma laidlawii b: elongation of medium- and long-chain exogenous fatty acids in growing cells

The chain elongation of a wide variety of exogenous fatty acids and the subsequent incorporation of the chain elongation products into the total membrane lipids of Acholeplasma laidlawii B were systematically studied. Within each chemical class of fatty acids examined, the extent of chain elongation increased with increases in chain length, reached a maximum value, and then declined with further increases in chain length. Depending on chemical structure, exogenous fatty acids containing less than 6 to 9 carbon atoms or more than 15 to 18 carbon atoms were not substrates for the chain elongation system. The substrate specificity of this fatty acid elongation system was strikingly broad, and straight-chain, methyl isobranched, and methyl anteisobranched saturated fatty acids, as well as cis- and trans-monounsaturated, cis-cyclopropane, and cis-polyunsaturated fatty acids, underwent chain elongation in vivo. The extent of chain elongation and the average chain length of the primary elongation products correlated well with the physical properties (melting temperatures) of the exogenous fatty acid substrates. The specificity of fatty acid chain elongation in A. laidlawii B maintained the fluidity and physical state of the membrane lipids within a rather wide but definitely limited range. The fatty acid chain elongation system of this organism could be markedly influenced by the presence of a second exogenous fatty acid that was not itself a substrate for the chain elongation system but was incorporated directly into the membrane lipids. The presence of a relatively low-melting exogenous fatty acid increased both the extent of chain elongation and the average chain length of the elongation products generated, whereas the presence of a relatively high-melting fatty acid had the opposite effect. The extent of chain elongation and nature of the elongation products formed were not, however, dependent on the fluidity and physical state of the membrane lipids per se. The second exogenous fatty acid appeared instead to exert its characteristic effect by competing with the chain elongation substrate and elongation products for the stereospecific acylation of positions 1 and 2 of sn-glycerol-3-phosphate. The similar effects of alterations in environmental temperature, cholesterol content, and exposure to the antibiotic cerulenin on the fatty acid chain elongation and de novo biosynthetic activities suggested that the chain elongation system of this organism may be a component of the de novo biosynthetic system.     

Uptake of long-chain fatty acids in HepG2 cells involves caveolae: analysis of a novel pathway

We investigated the role of caveolae in uptake and intracellular trafficking of long chain fatty acids (LCFA) in HepG2 human hepatoma cells. The uptake of [(3)H]oleic acid and [(3)H]stearic acid into HepG2 cells was measured by radioactive assays and internalization of the non-metabolizable fluorescent fatty acid 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] (12-NBD) stearate into single HepG2 cells was semi-quantitatively assessed by laser scanning microscopy. The initial rate of [(3)H]oleic acid uptake (V(0)) in HepG2 cells exhibited saturable transport kinetics with increasing concentrations of free oleic acid (V(max) 854 +/- 46 pmol mg protein(-1) min(-1), K(m) 100 +/- 14 nmol/l). While inhibition of clathrin coated pits did not influence LCFA uptake in HepG2, inhibition of caveolae formation by filipin III, cyclodextrin, and caveolin-1 antisense oligonucleotides resulted in reduction of [(3)H]oleic acid uptake by 54%, 45%, and 23%, respectively. Furthermore, filipin III inhibited the uptake of [(3)H]stearic acid and its fluorescent derivative 12-NBD stearate by 44% and 50%, respectively. Transfection studies with alpha-caveolin-1/cyanofluorescent protein chimeras showed significant colocalization of caveolae and internalized 12-NBD stearate. In conclusion, these data suggest a significant role for caveolae mediated uptake and intracellular trafficking of LCFA in HepG2 cells.     

Kinetics of the inhibition of hog kidney D-amino acid oxidase by short-, medium- and long-chain fatty acids

Various fatty acids were studied in vitro as inhibitors of pure hog kidney D-amino acid oxidase by means of a spectrophotometric peroxidase-coupling method using D-methionine as a substrate. All the fatty acids tested behaved as substrate-competitive inhibitors of the enzyme. The affinity of the saturated aliphatic acids for D-amino acid oxidase decreased from pentanoate (5:0; Ki = 220 microM) to laurate (12:0; Ki = 675 microM), then rose to a maximum with stearate (18:0; Ki = 36 microM), suggesting the presence of a site in the active center of the enzyme that accepts long-chain fatty acid alkyl groups. Unsaturation did not further increase the affinity of the fatty acid for this binding site.     

Pathways and subcellular compartmentation of NAD biosynthesis in human cells: from entry of extracellular precursors to mitochondrial NAD generation

NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule.     

Characteristics of the growth inhibition and cytotoxicity of long-chain (sphingoid) bases for Chinese hamster ovary cells: evidence for an involvement of protein kinase C

Long-chain bases are potent inhibitors of protein kinase C and cellular processes mediated by this enzyme. However, when added to cells they usually cause some degree of growth inhibition and cytotoxicity and it is unclear whether this reflects inhibition of protein kinase C or nonspecific detergent effects of these amphipathic compounds. This study examined the effects of sphinganine on Chinese hamster ovary (CHO) cells to gain more insight into these possibilities. Sphinganine concentrations between 0.75 and 4 microM resulted in a combination of growth inhibition and cytotoxicity that correlated with protein kinase C inhibition by five criteria: (1) the effective concentrations were comparable to those for protein kinase C inhibition in vitro and in other intact cells; (2) the structural specificity for the long-chain base moiety paralleled the potency of protein kinase C inhibition; (3) sphinganine blocked changes in protein phosphorylation patterns that occurred in response to phorbol 12-myristate 13-acetate (and vice versa); whereas (4) a mutant cell line that exhibited increased resistance to sphinganine cytotoxicity lacked both phorbol ester- and sphinganine-induced phosphorylation changes and differed somewhat in the behavior of protein kinase C assayed in vitro; and (5) sphinganine did not appear to be acting as a detergent (except at higher concentrations) nor as a lysosomotrophic agent. While the complexity of this cellular behavior mandates caution in interpreting these results, they suggest that the cytotoxicity and growth inhibition may be a consequence of protein kinase C inhibition.     

Hypocholesterolemic effects of fatty acid bile acid conjugates (FABACs) in mice

Fatty acid bile acid conjugates (FABACs) prevent and dissolve cholesterol gallstones and prevent diet induced fatty liver, in mice. The present studies aimed to test their hypocholesterolemic effects in mice. Gallstone susceptible (C57L/J) mice, on high fat (HFD) or regular diet (RD), were treated with the conjugate of cholic acid with arachidic acid (FABAC; Aramchol). FABAC reduced the elevated plasma cholesterol levels induced by the HFD. In C57L/J mice, FABAC reduced plasma cholesterol by 50% (p < 0.001). In mice fed HFD, hepatic cholesterol synthesis was reduced, whereas CYP7A1 activity and expression were increased by FABAC. The ratio of fecal bile acids/neutral sterols was increased, as was the total fecal sterol excretion. In conclusion, FABACs markedly reduce elevated plasma cholesterol in mice by reducing the hepatic synthesis of cholesterol, in conjunction with an increase of its catabolism and excretion from the body.     

Degradation of sphingoid long-chain base 1-phosphates (LCB-1Ps): functional characterization and expression of AtDPL1 encoding LCB-1P lyase involved in the dehydration stress response in Arabidopsis

Sphingoid long-chain base (LCB) 1-phosphates are degradated by LCB 1-phosphate lyase to C(16) fatty aldehydes and phosphoethanolamine. Here, we confirmed that the At1g27980 gene product, AtDPL1, is a functional LCB-1-phosphate lyase. Expression of green fluorescent protein fusion products in suspension-cultured Arabidopsis cells showed that AtDPL1 is located to the endoplasmic reticulum. The rates of fresh weight decreases of dpl1-1 and dpl1-2 mutants were significantly slower than those of the wild-type plants. This ability to limit their transpiration reflected the leaf temperature of the mutant plants more than that of wild-type plants, suggesting that AtDPL1 plays a role in dehydration stress.     

Improved production of long-chain fatty acid in Escherichia coli by an engineering elongation cycle during fatty acid synthesis (FAS) through genetic manipulation

The microbial biosynthesis of fatty acid of lipid metabolism, which can be used as precursors for the production of fuels of chemicals from renewable carbon sources, has attracted significant attention in recent years. The regulation of fatty acid biosynthesis pathways has been mainly studied in a model prokaryote, Escherichia coli. During the recent period, global regulation of fatty acid metabolic pathways has been demonstrated in another model prokaryote, Bacillus subtilis, as well as in Streptococcus pneumonia. The goal of this study was to increase the production of long-chain fatty acids by developing recombinant E. coli strains that were improved by an elongation cycle of fatty acid synthesis (FAS). The fabB, fabG, fabZ, and fabI genes, all homologous of E. coli, were induced to improve the enzymatic activities for the purpose of overexpressing components of the elongation cycle in the FAS pathway through metabolic engineering. The beta-oxoacyl-ACP synthase enzyme catalyzed the addition of acyl-ACP to malonyl-ACP to generate beta- oxoacyl-ACP. The enzyme encoded by the fabG gene converted beta-oxoacyl-ACP to beta-hydroxyacyl-ACP, the fabZ catalyzed the dehydration of beta-3-hydroxyacyl-ACP to trans-2-acyl-ACP, and the fabI gene converted trans-2- acyl-ACP to acyl-ACP for long-chain fatty acids. In vivo productivity of total lipids and fatty acids was analyzed to confirm the changes and effects of the inserted genes in E. coli. As a result, lipid was increased 2.16-fold higher and hexadecanoic acid was produced 2.77-fold higher in E. coli JES1030, one of the developed recombinants through this study, than those from the wild-type E. coli.     

A new inhibitor of the long-chain fatty acid transfer across the mitochondrial membrane: 2-(3-methylcinnamylhydrazono)-propionate (BM 42.304)

Using two different assay systems to distinguish between overt and inner forms of carnitine palmitoyl transferase (CPT, EC 2.3.1.21) of intact guinea-pig liver mitochondria, we have shown that the hypoglycemic agent 2-(3-methylcinnamylhydrazono)-propionate (BM 42.304) inhibits the activity of carnitine-acylcarnitine translocase of liver mitochondria. The results offer an explanation for the inhibitory effect of the compound on ketogenesis with oleate but not with octanoate in the perfused guinea-pig liver, previously reported by us (Biochem. Pharmacol. 32, 3405-3412, 1983).     

The assimilation of nucleic acid precursors by intact cells and protoplasts of the blue-green alga Anacystis nidulans

Summary The entry of both nucleotides and base precursors into whole cells and the nucleic acid fraction of the blue-green alga Anacystis nidulans has been examined. Very slow rates of uptake were observed with intact organisms but protoplast preparations assimilated the nucleic acid precursors much more readily.