Study of the 3-Hydroxy Eicosanoyl-Coenzyme A Dehydratase and (E)-2,3 Enoyl-Coenzyme A Reductase Involved in Acyl-Coenzyme A Elongation in Etiolated Leek Seedlings1

(R,S)-[1-14C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-14C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-14C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-14C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-14C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-14C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-14C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg−1 h−1, which is the highest ever reported for a plant system.

Genetic complexity of pathogen perception by plants: The example of Rcr3, a tomato gene required specifically by Cf-2

Genetic analysis of plant–pathogen interactions has demonstrated that resistance to infection is often determined by the interaction of dominant plant resistance (R) genes and dominant pathogen-encoded avirulence (Avr) genes. It was postulated that R genes encode receptors for Avr determinants. A large number of R genes and their cognate Avr genes have now been analyzed at the molecular level. R gene loci are extremely polymorphic, particularly in sequences encoding amino acids of the leucine-rich repeat motif. A major challenge is to determine how Avr perception by R proteins triggers the plant defense response. Mutational analysis has identified several genes required for the function of specific R proteins. Here we report the identification of Rcr3, a tomato gene required specifically for Cf-2-mediated resistance. We propose that Avr products interact with host proteins to promote disease, and that R proteins “guard” these host components and initiate Avr-dependent plant defense responses.

Cytochrome P450 Monooxygenases for Fatty Acids and Xenobiotics in Marine Macroalgae1

The metabolism of xenobiotics has mainly been investigated in higher plant species. We studied them in various marine macroalgae of the phyla Chlorophyta, Chromophyta, and Rhodophyta. Microsomes contained high oxidative activities for known cytochrome (Cyt) P450 substrates (fatty acids, cinnamic acid, 3- and 4-chlorobiphenyl, 2,3-dichlorobiphenyl, and isoproturon; up to 54 pkat/mg protein). The presence of Cyt P450 (approximately 50 pmol/mg protein) in microsomes of the three algal families was demonstrated by CO-difference absorption spectra. Intact algal tissue converted 3-chlorobiphenyl to the same monohydroxy-metabolite formed in vitro. This conversion was 5-fold stimulated upon addition of phenobarbital, and was abolished by the known P450 inhibitor, 1-aminobenzotriazole. It is concluded that marine macroalgae contain active species of Cyt P450 and could act as a metabolic sink for marine pollutants.

Co-Permeability of 3H-Labeled Water and 14C-Labeled Organic Acids across Isolated Plant Cuticles1 : Investigating Cuticular Paths of Diffusion and Predicting Cuticular Transpiration

Penetration of 3H-labeled water (3H2O) and the 14C-labeled organic acids benzoic acid ([14C]BA), salicylic acid ([14C]SA), and 2,4-dichlorophenoxyacetic acid ([14C]2,4-D) were measured simultaneously in isolated cuticular membranes of Prunus laurocerasus L., Ginkgo biloba L., and Juglans regia L. For each of the three pairs of compounds (3H2O/[14C]BA, 3H2O/[14C]SA, and 3H2O/[14C]2,4-D) rates of cuticular water penetration were highly correlated with the rates of penetration of the organic acids. Therefore, water and organic acids penetrated the cuticles by the same routes. With the combination 3H2O/[14C]BA, co-permeability was measured with isolated cuticles of nine other plant species. Permeances of 3H2O of all 12 investigated species were highly correlated with the permeances of [14C]BA (r2 = 0.95). Thus, cuticular transpiration can be predicted from BA permeance. The application of this experimental method, together with the established prediction equation, offers the opportunity to answer several important questions about cuticular transport physiology in future investigations.

Polyunsaturated fatty acids modulate sodium and calcium currents in CA1 neurons.

Recent evidence indicates that long-chain polyunsaturated fatty acids (PUFAs) can prevent cardiac arrhythmias by a reduction of cardiomyocyte excitability. This was shown to be due to a modulation of the voltage-dependent inactivation of both sodium (INa) and calcium (ICa) currents. To establish whether PUFAs also regulate neuronal excitability, the effects of PUFAs on INa and ICa were assessed in CA1 neurons freshly isolated from the rat hippocampus. Extracellular application of PUFAs produced a concentration-dependent shift of the voltage dependence of inactivation of both INa and ICa to more hyperpolarized potentials. Consequently, they accelerated the inactivation and retarded the recovery from inactivation. The EC50 for the shift of the INa steady-state inactivation curve was 2.1 +/- 0.4 microM for docosahexaenoic acid (DHA) and 4 +/- 0.4 microM for eicosapentaenoic acid (EPA). The EC50 for the shift on the ICa inactivation curve was 2.1 +/- 0.4 for DHA and > 15 microM for EPA. Additionally, DHA and EPA suppressed both INa and ICa amplitude at concentrations > 10 microM. PUFAs did not affect the voltage dependence of activation. The monounsaturated oleic acid and the saturated palmitic acid were virtually ineffective. The combined effects of the PUFAs on INa and ICa may reduce neuronal excitability and may exert anticonvulsive effects in vivo.

Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.

Suppression of tumorigenicity by the wild-type tuberous sclerosis 2 (Tsc2) gene and its C-terminal region.

The Tsc2 gene, which is mutationally inactivated in the germ line of some families with tuberous sclerosis, encodes a large, membrane-associated GTPase activating protein (GAP) designated tuberin. Studies of the Eker rat model of hereditary cancer strongly support the role of Tsc2 as a tumor suppressor gene. In this study, the biological activity of tuberin was assessed by expressing the wild-type Tsc2 gene in tumor cell lines lacking functional tuberin and also in rat fibroblasts with normal levels of endogenous tuberin. The colony forming efficiency of Eker rat-derived renal carcinoma cells was significantly reduced following reintroduction of wild-type Tsc2. Tumor cells expressing the transfected Tsc2 gene became more anchorage-dependent and lost their ability to form tumors in severe combined immunodeficient mice. At the cellular level, restoration of tuberin expression caused morphological changes characterized by enlargement of the cells and increased contact inhibition. As with the full-length Tsc2 gene, a clone encoding only the C terminus of tuberin (amino acids 1049-1809, including the GAP domain) was capable of reducing both colony formation and in vivo tumorigenicity when transfected into the Eker rat tumor cells. In normal Rat1 fibroblasts, conditional overexpression of tuberin also suppressed colony formation and cell growth in vitro. These results provide direct experimental evidence for the tumor suppressor function of Tsc2 and suggest that the tuberin C terminus plays an important role in this activity.

Constitutive receptor activation by Crouzon syndrome mutations in fibroblast growth factor receptor (FGFR)2 and FGFR2/Neu chimeras.

Crouzon syndrome is an autosomal dominant condition primarily characterized by craniosynostosis. This syndrome has been associated with a variety of amino acid point mutations in the extracellular domain of fibroblast growth factor receptor 2 (FGFR2). FGFR2/Neu chimeras were generated by substituting the extracellular domain of Neu with that of FGFR2 containing the following Crouzon mutations: Tyr-340-->His; Cys-342-->Tyr; Cys-342-->Arg; Cys-342-->Ser; Ser-354-->Cys: and delta17 (deletion of amino acids 345-361). Each of the mutant chimeric FGFR2/Neu constructs stimulated focus formation in NIH 3T3 cells, indicating that Crouzon mutations can stimulate signal transduction through a heterologous receptor tyrosine kinase. In vitro kinase assay results indicate that FGFR2 receptors containing Crouzon mutations have increased tyrosine kinase activity and, when analyzed under nonreducing conditions, exhibited disulfide-bonded dimers. Thus the human developmental abnormality Crouzon syndrome arises from constitutive activation of FGFR2 due to aberrant intermolecular disulfide-bonding. These results together with our earlier observation that achondroplasia results from constitutive activation of the related receptor FGFR3, leads to the prediction that other malformation syndromes attributed to FGFRs, such as Pfeiffer syndrome and Thanatophoric dysplasia, also arise from constitutive receptor activation.

Binding site size limit of the 2:1 pyrrole-imidazole polyamide-DNA motif.

Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids can be combined in antiparallel side-by-side dimeric complexes for sequence-specific recognition in the minor groove of DNA. Six polyamides containing three to eight rings bind DNA sites 5-10 bp in length, respectively. Quantitative DNase I footprint titration experiments demonstrate that affinity maximizes and is similar at ring sizes of five, six, and seven. Sequence specificity decreases as the length of the polyamides increases beyond five rings. These results provide useful guidelines for the design of new polyamides that bind longer DNA sites with enhanced affinity and specificity.

Matrix metalloproteinase 2 releases active soluble ectodomain of fibroblast growth factor receptor 1.

Recent studies have demonstrated the existence of a soluble fibroblast growth factor (FGF) receptor type 1 (FGFR1) extracellular domain in the circulation and in vascular basement membranes. However, the process of FGFR1 ectodomain release from the plasma membrane is not known. Here we report that the 72-kDa gelatinase A (matrix metalloproteinase type 2, MMP2) can hydrolyze the Val368-Met369 peptide bond of the FGFR1 ectodomain, eight amino acids upstream of the transmembrane domain, thus releasing the entire extracellular domain. Similar results were obtained regardless of whether FGF was first bound to the receptor or not. The action of MMP2 abolished binding of FGF to an immobilized recombinant FGFR1 ectodomain fusion protein and to Chinese hamster ovary cells overexpressing FGFR1 The released recombinant FGFR1 ectodomain was able to bind FGF after MMP2 cleavage, suggesting that the cleaved soluble receptor maintained its FGF binding capacity. The activity of MMP2 could not be reproduced by the 92-kDa gelatinase B (MMP9) and was inhibited by tissue inhibitor of metalloproteinase type 2. These studies demonstrate that FGFR1 may be a specific target for MMP2 on the cell surface, yielding a soluble FGF receptor that may modulate the mitogenic and angiogenic activities of FGF.

Protonatable residues at the cytoplasmic end of transmembrane helix-2 in the signal transducer HtrI control photochemistry and function of sensory rhodopsin I.

Neutral residue replacements were made of 21 acidic and basic residues within the N-terminal half of the Halobacterium salinarium signal transducer HtrI [the halobacterial transducer for sensory rhodopsin I (SRI)] by site-specific mutagenesis. The replacements are all within the region of HtrI that we previously concluded from deletion analysis to contain sites of interaction with the phototaxis receptor SRI. Immunoblotting shows plasmid expression of the htrI-sopI operon containing the mutations produces SRI and mutant HtrI in cells at near wild-type levels. Six of the HtrI mutations perturb photochemical kinetics of SRI and one reverses the phototaxis response. Substitution with neutral amino acids of Asp-86, Glu-87, and Glu-108 accelerate, and of Arg-70, Arg-84, and Arg-99 retard, the SRI photocycle. Opposite effects on photocycle rate cancel in double mutants containing one replaced acidic and one replaced basic residue. Laser flash spectroscopy shows the kinetic perturbations are due to alteration of the rate of reprotonation of the retinylidene Schiff base. All of these mutations permit normal attractant and repellent signaling. On the other hand, the substitution of Glu-56 with the isosteric glutamine converts the normally attractant effect of orange light to a repellent signal in vivo at neutral pH (inverted signaling). Low pH corrects the inversion due to Glu-56 -> Gln and the apparent pK of the inversion is increased when arginine is substituted at position 56. The results indicate that the cytoplasmic end of transmembrane helix-2 and the initial part of the cytoplasmic domain contain interaction sites with SRI. To explain these and previous results, we propose a model in which (i) the HtrI region identified here forms part of an electrostatic bonding network that extends through the SRI protein and includes its photoactive site; (ii) alteration of this network by photoisomerization-induced Schiff base deprotonation and reprotonation shifts HtrI between attractant and repellent conformations; and (iii) HtrI mutations and extracellular pH alter the equilibrium ratios of these conformations.

Cloning and expression in Escherichia coli of the OGG1 gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine.

A spontaneous mutator strain of Escherichia coli (fpg mutY) was used to clone the OGG1 gene of Saccharomyces cerevisiae, which encodes a DNA glycosylase activity that excises 7,8-dihydro-8-oxoguanine (8-OxoG). E. coli (fpg mutY) was transformed by a yeast DNA library, and clones that showed a reduced spontaneous mutagenesis were selected. The antimutator activity was associated with pYSB10, an 11-kbp recombinant plasmid. Cell-free extracts of E. coli (fpg mutY) harboring pYSB10 possess an enzymatic activity that cleaves a 34-mer oligonucleotide containing a single 8-oxoG opposite a cytosine (8-OxoG/C). The yeast DNA fragment of 1.7 kbp that suppresses spontaneous mutagenesis and overproduces the 8-OxoG/C cleavage activity was sequenced and mapped to chromosome XIII. DNA sequencing identified an open reading frame, designated OGG1, which encodes a protein of 376 amino acids with a molecular mass of 43 kDa. The OGG1 gene was inserted in plasmid pUC19, yielding pYSB110. E. coli (fpg) harboring pYSB110 was used to purify the Ogg1 protein of S. cerevisiae to apparent homogeneity. The Ogg1 protein possesses a DNA glycosylase activity that releases 8-OxoG and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. The Ogg1 protein preferentially incises DNA that contains 8-OxoG opposite cytosine (8-OxoG/C) or thymine (8-OxoG/T). In contrast, Ogg1 protein does not incise the duplex where an adenine is placed opposite 8-OxoG (8-OxoG/A). The mechanism of strand cleavage by Ogg1 protein is probably due to the excision of 8-OxoG followed by a beta-elimination at the resulting apurinic/apyrimidinic site.

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.

Repression of a matrix metalloprotease gene by E1A correlates with its ability to bind to cell type-specific transcription factor AP-2.

Adenovirus E1A 243-amino acid protein can repress a variety of enhancer -linked viral and cellular promoters. This repression is presumed to be mediated by its interaction with and sequestration of p3OO, a transcriptional coactivator. Type IV 72-kDa collagenase is one of the matrix metalloproteases that has been implicated in differentiation, development, angiogenesis, and tumor metastasis. We show here that the cell type-specific transcription factor AP-2 is an important transcription factor for the activation of the type IV 72-kDa collagenase promoter and that adenovirus E1A 243-amino acid protein represses this promoter by targeting AP-2. Glutathione S-transferase-affinity chromatography studies show that the E1A protein interacts with the DNA binding/dimerization region of AP-2 and that the N-terminal amino acids of E1A protein are required for this interaction. Further, E1A deletion mutants which do not bind to p3OO can repress this collagenase promoter as efficiently as the wildtype E1A protein. Because the AP-2 element is present in a variety of viral and cellular enhancers which are repressed by E1A, these studies suggest that E1A protein can repress cellular and viral promoter/enhancers by forming a complex with cellular transcription factors and that this repression mechanism may be independent of its interaction with p3OO.

Betidamino acids: versatile and constrained scaffolds for drug discovery.

Betidamino acids (a contraction of "beta" position and "amide") are N'-monoacylated (optionally, N'-monoacylated and N-mono- or N,N'-dialkylated) aminoglycine derivatives in which each N'acyl/alkyl group may mimic naturally occurring amino acid side chains or introduce novel functionalities. Betidamino acids are most conveniently generated on solid supports used for the synthesis of peptides by selective acylation of one of the two amino functions of orthogonally protected aminoglycine(s) to generate the side chain either prior to or after the elongation of the main chain. We have used unresolved Nalpha-tert-butyloxycarbonyl-N'alpha-fluorenylmethoxycarbonyl++ + aminoglycine, and Nalpha-(Nalpha-methyl)-tert-butyloxycarbonyl-N'alpha-fluo renylmethoxycarbonyl aminoglycine as the templates for the introduction of betidamino acids in Acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(A c)-Leu-Ilys-Pro-DAla-NH2, where 2Nal is 2-naphthylalanine, 4Cpa is 4-chlorophenylalanine, 3Pal is 3-pyridylalanine, Aph is 4-aminophenylalanine, and Ilys is Nepsilon-isopropyllysine], a potent gonadotropin-releasing hormone antagonist, in order to test biocompatibility of these derivatives. Diasteremneric peptides could be separated in most cases by reverse-phase HPLC. Biological results indicated small differences in relative potencies (<5-fold) between the D and L nonalkylated betidamino acid-containing Acyline derivatives. Importantly, most betide diastereomers were equipotent with Acyline. In an attempt to correlate structure and observed potency, Ramachandran-type plots were calculated for a series of betidamino acids and their methylated homologs. According to these calculations, betidamino acids have access to a more limited and distinct number of conformational states (including those associated with alpha-helices, beta-sheets, or turn structures), with deeper minima than those observed for natural amino acids.