Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA Δ11-desaturase of the cabbage looper moth, Trichoplusia ni

Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera. The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Δ9-desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acyl-CoA Δ9-desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group. In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni, a species in which all unsaturated pheromone products are produced via a Δ11Z-desaturation mechanism. The largest ORF of the ≈1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Δ11Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast Δ9-desaturases, suggesting conserved transmembrane topology. A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Δ9Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an ≈1,250-nt PDesat-Tn Δ11Z mRNA that is consistent with the spatial and temporal distribution of Δ11-desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Δ11Z resulted in complementation of the strain’s fatty acid auxotrophy and the production of Δ11Z-unsaturated fatty acids.

Lamellar lipoproteins uniquely contribute to hyperlipidemia in mice doubly deficient in apolipoprotein E and hepatic lipase

Remnants of triglyceride-rich lipoproteins containing apolipoprotein (apo) B-48 accumulate in apo E-deficient mice, causing pronounced hypercholesterolemia. Mice doubly deficient in apo E and hepatic lipase have more pronounced hypercholesterolemia, even though remnants do not accumulate appreciably in mice deficient in hepatic lipase alone. Here we show that the doubly deficient mice manifest a unique lamellar hyperlipoproteinemia, characterized by vesicular particles 600 Å–1,300 Å in diameter. As seen by negative-staining electron microscopy, these lipoproteins also contain an electron-lucent region adjacent to the vesicle wall, similar to the core of typical lipoproteins. Correlative chemical analysis indicates that the vesicle wall is composed of a 1:1 molar mixture of cholesterol and phospholipids, whereas the electron-lucent region appears to be composed of cholesteryl esters (about 12% of the particle mass). Like the spherical lipoproteins of doubly deficient mice, the vesicular particles contain apo B-48, but they are particularly rich in apo A-IV. We propose that cholesteryl esters are removed from spherical lipoproteins of these mice by scavenger receptor B1, leaving behind polar lipid-rich particles that fuse to form vesicular lipoproteins. Hepatic lipase may prevent such vesicular lipoproteins from accumulating in apo E-deficient mice by hydrolyzing phosphatidyl choline as scavenger receptor B1 removes the cholesteryl esters and by gradual endocytosis of lipoproteins bound to hepatic lipase on the surface of hepatocytes.

Inhibition of the visual cycle in vivo by 13-cis retinoic acid protects from light damage and provides a mechanism for night blindness in isotretinoin therapy

Isotretinoin (13-cis retinoic acid) is frequently prescribed for severe acne [Peck, G. L., Olsen, T. G., Yoder, F. W., Strauss, J. S., Downing, D. T., Pandya, M., Butkus, D. & Arnaud-Battandier, J. (1979) N. Engl. J. Med. 300, 329–333] but can impair night vision [Fraunfelder, F. T., LaBraico, J. M. & Meyer, S. M. (1985) Am. J. Ophthalmol. 100, 534–537] shortly after the beginning of therapy [Shulman, S. R. (1989) Am. J. Public Health 79, 1565–1568]. As rod photoreceptors are responsible for night vision, we administered isotretinoin to rats to learn whether night blindness resulted from rod cell death or from rod functional impairment. High-dose isotretinoin was given daily for 2 months and produced systemic toxicity, but this caused no histological loss of rod photoreceptors, and rod-driven electroretinogram amplitudes were normal after prolonged dark adaptation. Additional studies showed, however, that even a single dose of isotretinoin slowed the recovery of rod signaling after exposure to an intense bleaching light, and that rhodopsin regeneration was markedly slowed. When only a single dose was given, rod function recovered to normal within several days. Rods and cones both showed slow recovery from bleach after isotretinoin in rats and in mice. HPLC analysis of ocular retinoids after isotretinoin and an intense bleach showed decreased levels of rhodopsin chromophore, 11-cis retinal, and the accumulation of the biosynthetic intermediates, 11-cis and all-trans retinyl esters. Isotretinoin was also found to protect rat photoreceptors from light-induced damage, suggesting that strategies of altering retinoid cycling may have therapeutic implications for some forms of retinal and macular degeneration.

Regulation of Ferulate-5-Hydroxylase Expression in Arabidopsis in the Context of Sinapate Ester Biosynthesis1

Sinapic acid is an intermediate in syringyl lignin biosynthesis in angiosperms, and in some taxa serves as a precursor for soluble secondary metabolites. The biosynthesis and accumulation of the sinapate esters sinapoylglucose, sinapoylmalate, and sinapoylcholine are developmentally regulated in Arabidopsis and other members of the Brassicaceae. The FAH1 locus of Arabidopsis encodes the enzyme ferulate-5-hydroxylase (F5H), which catalyzes the rate-limiting step in syringyl lignin biosynthesis and is required for the production of sinapate esters. Here we show that F5H expression parallels sinapate ester accumulation in developing siliques and seedlings, but is not rate limiting for their biosynthesis. RNA gel-blot analysis indicated that the tissue-specific and developmentally regulated expression of F5H mRNA is distinct from that of other phenylpropanoid genes. Efforts to identify constructs capable of complementing the sinapate ester-deficient phenotype of fah1 mutants demonstrated that F5H expression in leaves is dependent on sequences 3′ of the F5H coding region. In contrast, the positive regulatory function of the downstream region is not required for F5H transcript or sinapoylcholine accumulation in embryos.

Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the α-secretase ADAM 10

Biochemical, epidemiological, and genetic findings demonstrate a link between cholesterol levels, processing of the amyloid precursor protein (APP), and Alzheimer's disease. In the present report, we identify the α-secretase ADAM 10 (a disintegrin and metalloprotease) as a major target of the cholesterol effects on APP metabolism. Treatment of various peripheral and neural cell lines with either the cholesterol-extracting agent methyl-β-cyclodextrin or the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin resulted in a drastic increase of secreted α-secretase cleaved soluble APP. This strong stimulatory effect was in the range obtained with phorbol esters and was further increased in cells overexpressing ADAM 10. In cells overexpressing APP, the increase of α-secretase activity resulted in a decreased secretion of Aβ peptides. Several mechanisms were elucidated as being the basis of enhanced α-secretase activity: increased membrane fluidity and impaired internalization of APP were responsible for the effect observed with methyl-β-cyclodextrin; treatment with lovastatin resulted in higher expression of the α-secretase ADAM 10. Our results demonstrate that cholesterol reduction promotes the nonamyloidogenic α-secretase pathway and the formation of neuroprotective α-secretase cleaved soluble APP by several mechanisms and suggest approaches to prevention of or therapy for Alzheimer's disease.

Developmental Expression and Substrate Specificities of Alfalfa Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase in Relation to Lignification1

The biosynthesis of monolignols can potentially occur via two parallel pathways involving free acids or their coenzyme A (CoA) esters. Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze functionally identical reactions in these two pathways, resulting in the formation of mono- or dimethoxylated lignin precursors. The activities of the two enzymes increase from the first to the sixth internode in stems of alfalfa (Medicago sativa L.), preceding the deposition of lignin. Alfalfa CCOMT is highly similar at the amino acid sequence level to the CCOMT from parsley, although it contains a six-amino acid insertion near the N terminus. Transcripts encoding both COMT and CCOMT are primarily localized to vascular tissue in alfalfa stems. Alfalfa CCOMT expressed in Escherichia coli catalyzes O-methylation of caffeoyl and 5-hydroxyferuloyl CoA, with preference for caffeoyl CoA. It has low activity against the free acids. COMT expressed in E. coli is active against both caffeic and 5-hydroxyferulic acids, with preference for the latter compound. Surprisingly, very little extractable O-methyltransferase activity versus 5-hydroxyferuloyl CoA is present in alfalfa stem internodes, in which relative O-methyltransferase activity against 5-hy-droxyferulic acid increases with increasing maturity, correlating with increased lignin methoxyl content.

The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phospholipid binding site.

The Raf-1 protein kinase is the best-characterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals. The details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the involvement of both Ras-binding sites in effective Raf-1-mediated transformation provides insight into the molecular aspects and consequences of Ras-Raf interactions. The Raf-1 CRD is a member of an emerging family of domains, many of which are found within signal transducing proteins. Several contain binding sites for diacylglycerol (or phorbol esters) and phosphatidylserine and are believed to play a role in membrane translocation and enzyme activation. The CRD from Raf-1 does not bind diacylglycerol but interacts with Ras and phosphatidylserine. To investigate the ligand-binding specificities associated with CRDs, we have determined the solution structure of the Raf-1 CRD using heteronuclear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PKC). The differences are confined to regions of the CRDs involved in binding phorbol ester in the PKC domains. Since phosphatidylserine is a common ligand, we expect its binding site to be located in regions where the structures of the Raf-1 and PKC domains are similar. The structure of the Raf-1 CRD represents an example of this family of domains that does not bind diacylglycerol and provides a framework for investigating its interactions with other molecules.

Protein kinase cross-talk: membrane targeting of the beta-adrenergic receptor kinase by protein kinase C.

The beta-adrenergic receptor kinase (betaARK) is the prototypical member of the family of cytosolic kinases that phosphorylate guanine nucleotide binding-protein-coupled receptors and thereby trigger uncoupling between receptors and guanine nucleotide binding proteins. Herein we show that this kinase is subject to phosphorylation and regulation by protein kinase C (PKC). In cell lines stably expressing alpha1B- adrenergic receptors, activation of these receptors by epinephrine resulted in an activation of cytosolic betaARK. Similar data were obtained in 293 cells transiently coexpressing alpha1B- adrenergic receptors and betaARK-1. Direct activation of PKC with phorbol esters in these cells caused not only an activation of cytosolic betaARK-1 but also a translocation of betaARK immunoreactivity from the cytosol to the membrane fraction. A PKC preparation purified from rat brain phospborylated purified recombinant betaARK-1 to a stoichiometry of 0.86 phosphate per betaARK-1. This phosphorylation resulted in an increased activity of betaARK-1 when membrane-bound rhodopsin served as its substrate but in no increase of its activity toward a soluble peptide substrate. The site of phosphorylation was mapped to the C terminus of betaARK-1. We conclude that PKC activates betaARK by enhancing its translocation to the plasma membrane.

Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants.

It is becoming clear that an adequate level of long-chain highly unsaturated fatty acids in the nervous system is required for optimal function and development; however, the ability of infants to biosynthesize long-chain fatty acids is unknown. This study explores the capacity of human infants to convert 18-carbon essential fatty acids to their elongated and desaturated forms, in vivo. A newly developed gas chromatography/negative chemical ionization/mass spectrometry method employing 2H-labeled essential fatty acids allowed assessment of this in vivo conversion with very high sensitivity and selectivity. Our results demonstrate that human infants have the capacity to convert dietary essential fatty acids administered enterally as 2H-labeled ethyl esters to their longer-chain derivatives, transport them to plasma, and incorporate them into membrane lipids. The in vivo conversion of linoleic acid (18:2n6) to arachidonic acid (20:4n6) is demonstrated in human beings. All elongases/desaturases necessary for the conversion of linolenic acid (18:3n3) to docosahexaenoic acid (22:6n3) are also active in the first week after birth. Although the absolute amounts of n-3 fatty acid metabolites accumulated in plasma are greater than those of the n-6 family, estimates of the endogenous pools of 18:2n6 and 18:3n3 indicate that n-6 fatty acid conversion rates are greater than those of the n-3 family. While these data clearly demonstrate the capability of infants to biosynthesize 22:6n3, a lipid that is required for optimal neural development, the amounts produced in vivo from 18:3n3 may be inadequate to support the 22:6n3 level observed in breast-fed infants.

Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase.

Protein kinase C (PKC), a major cellular receptor for tumor-promoting phorbol esters and diacylglycerols (DGs), appears to be involved in a variety of cellular functions, although its activation mechanism in vivo is not yet fully understood. To evaluate the signaling pathways involved in the activation of PKC epsilon upon stimulation by platelet-derived growth factor (PDGF) receptor (PDGFR), we used a series of PDGFR "add-back" mutants. Activation of a PDGFR mutant (Y40/51) that binds and activates phosphatidylinositol 3-kinase (PI 3-kinase) caused translocation of PKC epsilon from the cytosol to the membrane in response to PDGF. A PDGFR mutant (Y1021) that binds and activates phospholipase C gamma (PLC gamma), but not PI 3-kinase, also caused the PDGF-dependent translocation of PKC epsilon. The translocation of PKC epsilon upon stimulation of PDGFR (Y40/51) was inhibited by wortmannin, an inhibitor of PI 3-kinase. Activation of PKC epsilon was further confirmed in terms of PKC epsilon-dependent expression of a phorbol 12-tetradecanoate 13-acetate response element (TRE)-luciferase reporter. Further, purified PKC epsilon was activated in vitro by either DG or synthetic phosphatidylinositol 3,4,5-trisphosphate. These results clearly demonstrate that PKC epsilon is activated through redundant and independent signaling pathways which most likely involve PLC gamma or PI 3-kinase in vivo and that PKC epsilon is one of the downstream mediators of PI 3-kinase whose downstream targets remain to be identified.

Synthesis and coupling reactions of alpha,alpha-dialkylated amino acids with nucleobase side chains.

Several di- and tripeptides containing protected purine (adenine) and pyrimidine (thymine) residues on their side chains were synthesized. The parent amino acids alpha, alpha-dialkylated in a symmetrical manner. An effective coupling procedure was developed for these sterically hindered amino acids: the fluoren-9-ylmethyloxycarbonyl-protected amino acid was dehydrated to its oxazolinone form, which was coupled in good yields with amino esters in hot tetrachloroethane.

Crystal structure of the complex of a catalytic antibody Fab fragment with a transition state analog: structural similarities in esterase-like catalytic antibodies.

The x-ray structure of the complex of a catalytic antibody Fab fragment with a phosphonate transition-state analog has been determined. The antibody (CNJ206) catalyzes the hydrolysis of p-nitrophenyl esters with significant rate enhancement and substrate specificity. Comparison of this structure with that of the uncomplexed Fab fragment suggests hapten-induced conformational changes: the shape of the combining site changes from a shallow groove in the uncomplexed Fab to a deep pocket where the hapten is buried. Three hydrogen-bond donors appear to stabilize the charged phosphonate group of the hapten: two NH groups of the heavy (H) chain complementarity-determining region 3 (H3 CDR) polypeptide chain and the side-chain of histidine-H35 in the H chain (His-H35) in the H1 CDR. The combining site shows striking structural similarities to that of antibody 17E8, which also has esterase activity. Both catalytic antibody ("abzyme") structures suggest that oxyanion stabilization plays a significant role in their rate acceleration. Additional catalytic groups that improve efficiency are not necessarily induced by the eliciting hapten; these groups may occur because of the variability in the combining sites of different monoclonal antibodies that bind to the same hapten.

Initial hepatic removal of chylomicron remnants is unaffected but endocytosis is delayed in mice lacking the low density lipoprotein receptor.

Two endocytic receptors, the low density lipoprotein (LDL) receptor (LDLR) and the LDLR-related protein (LRP), are thought to act in concert in the hepatic uptake of partially metabolized dietary lipoproteins, the chylomicron remnants. We have evaluated the role of these two receptors in the hepatic metabolism of chylomicron remnants in normal mice and in LDLR-deficient [LDLR (-/-)] mice. The rate of chylomicron remnant removal by the liver was normal up to 30 min after intravenous injection of chylomicrons into LDLR (-/-) mice and was unaffected by receptor-associated protein (RAP), a potent inhibitor of ligand binding to LRP. In contrast, endocytosis of the remnants by the hepatocytes, measured by their accumulation in the endosomal fraction and by the rate of hydrolysis of component cholesteryl esters, was dramatically reduced in the absence of the LDLR. Coadministration of RAP prevented the continuing hepatic removal of chylomicron remnants in LDL (-/-) mice after 30 min, consistent with blockade of the slow endocytosis by a RAP-sensitive process. Taken together with previous studies, our results are consistent with a model in which the initial hepatic removal of chylomicron remnants is primarily mediated by mechanisms that do not include LDLR or LRP, possibly involving glycosaminoglycan-bound hepatic lipase and apolipoprotein E. After the remnants bind to these alternative sites on the hepatocyte surface, endocytosis is predominantly mediated by the LDLR and also by a slower and less efficient backup process that is RAP sensitive and therefore most likely involves LRP.

Use of binding energy by an RNA enzyme for catalysis by positioning and substrate destabilization.

A fundamental catalytic principle for protein enzymes in the use of binding interactions away from the site of chemical transformation for catalysis. We have compared the binding and reactivity of a series of oligonucleotide substrates and products of the Tetrahymena ribozyme, which catalyzes a site-specific phosphodiester cleavage reaction: CCCUCUpA+G<-->CCCUCU-OH+GpA. The results suggest that this RNA enzyme, like protein enzymes, can utilize binding interactions to achieve substantial catalysis via entropic fixation and substrate destabilization. The stronger binding of the all-ribose oligonucleotide product compared to an analog with a terminal 3' deoxyribose residue gives an effective concentration of 2200 M for the 3' hydroxyl group, a value approaching those obtained with protein enzymes and suggesting the presence of a structurally well defined active site capable of precise positioning. The stabilization from tertiary binding interactions is 40-fold less for the oligonucleotide substrate than the oligonucleotide product, despite the presence of the reactive phosphoryl group in the substrate. This destabilization is accounted for by a model in which tertiary interactions away from the site of bond cleavage position the electron-deficient 3' bridging phosphoryl oxygen of the oligonucleotide substrate next to an electropositive Mg ion. As the phosphodiester bond breaks and this 3' oxygen atom develops a negative charge in the transition state, the weak interaction of the substrate with Mg2+ becomes strong. These strategies of "substrate destabilization" and "transition state stabilization" provide estimated rate enhancements of approximately 280- and approximately 60-fold, respectively. Analogous substrate destabilization by a metal ion or hydrogen bond donor may be used more generally by RNA and protein enzymes catalyzing reactions of phosphate esters.

Neurosteroids, via sigma receptors, modulate the [3H]norepinephrine release evoked by N-methyl-D-aspartate in the rat hippocampus.

N-Methyl-D-aspartate (NMDA, 200 microM) evokes the release of [3H]norepinephrine ([3H]NE) from preloaded hippocampal slices. This effect is potentiated by dehydroepiandrosterone sulfate (DHEA S), whereas it is inhibited by pregnenolone sulfate (PREG S) and the high-affinity sigma inverse agonist 1,3-di(2-tolyl)guanidine, at concentrations of > or = 100 nM. Neither 3 alpha-hydroxy-5 alpha-pregnan-20-one nor its sulfate ester modified NMDA-evoked [3H]NE overflow. The sigma antagonists haloperidol and 1-[2-(3,4-dichlorophenyl)-ethyl]-4-methylpiperazine, although inactive by themselves, completely prevented the effects of DHEA S, PREG S, and 1,3-di(2-tolyl)guanidine on NMDA-evoked [3H]NE release. Progesterone (100 nM) mimicked the antagonistic effect of haloperidol and 1-[2-(3,4-dichlorophenyl)ethyl]-4-methyl-piperazine. These results indicate that the tested steroid sulfate esters differentially affected the NMDA response in vitro and suggest that DHEA S acts as a sigma agonist, that PREG S acts as a sigma inverse agonist, and that progesterone may act as a sigma antagonist. Pertussis toxin, which inactivates the Gi/o types of guanine nucleotide-binding protein (Gi/o protein) function, suppresses both effects of DHEA S and PREG S. Since sigma 1 but not sigma 2 receptors are coupled to Gi/o proteins, the present results suggest that DHEA S and PREG S control the NMDA response via sigma 1 receptors.