Intermolecular crosslinking of fatty acyl chains in phospholipids: Use of photoactivable carbene precursors

Phospholipids containing photolysable carhene precursors (beta-trifluoro-a-diazopropionoxy and m-diazirinophenoxy groups) in w-positions of sn-2 fatty acyl chains were prepared. Photolysis of their vesicles produced crosslinked products in 40-60 % yields. Crosslinking was mostly intermolecular and occurred bv carbene insertion into the C-H bonds of a second fatty acyl chain. Crosslinking products were characterized by (i) their gel permeation behavior, (ii) analysis of produets formed by base-catalyzed transesterification. (iii) degradation with phosphoiipases A2 and C, (iv) gas chromatography/mass spectrometry, and-(v) use of mixtures of phospholipids carrying thf' carhene precursors and a phospholipid containing radioactively labeled fatty acyl groups. Nitrenes generated from the aliphatic or aromatic azido groups in phospholipids were unsatisfactory for forming crosslinks by insertion in C-H bond

Sites of intermolecular crosslinking of fatty acyl chains in phospholipids carrying a photoactivable carbene precursor

Sonicated vesicles of l-fatty acyl-2-w-(2-diazo-3.3,3-trifluoropropionoxy) fatty acyl sn-glycero-3-phosphorylcholines were shown recently to form intermolecular crosslinks by insertion of the photogenerated carbene into a C-H bond of a neighboring hydrocarbon chain. We now report that photolysis of multilamellar dispersions gives a second series of products in which carbene insertion is accompanied by elimination of a molecule of hydrogen fluoride. The sites of crosslinking in the latter compounds have been studied by mass spectrometry using phospholipids with varying chain lengths of the fatty acyl groups carrying the carbene precursor. The patterns observed show that the point of maximum crosslinking is consistent with the recent conclusion that in phospholipids the sn-2 fatty acyl chain trails the sn-1 chain by 2-4 atoms.

Western diet changes cardiac acyl-CoA composition in obese rats: a potential role for hepatic lipogenesis.

The "lipotoxic footprint" of cardiac maladaptation in diet-induced obesity is poorly defined. We investigated how manipulation of dietary lipid and carbohydrate influenced potential lipotoxic species in the failing heart. In Wistar rats, contractile dysfunction develops at 48 weeks on a high-fat/high-carbohydrate "Western" diet, but not on low-fat/high-carbohydrate or high-fat diets. Cardiac content of the lipotoxic candidates--diacylglycerol, ceramide, lipid peroxide, and long-chain acyl-CoA species--was measured at different time points by high-performance liquid chromatography and biochemical assays, as was lipogenic capacity in the heart and liver by qRT-PCR and radiometric assays. Changes in membranes fluidity were also monitored using fluorescence polarization. We report that Western feeding induced a 40% decrease in myocardial palmitoleoyl-CoA content and a similar decrease in the unsaturated-to-saturated fatty acid ratio. These changes were associated with impaired cardiac mitochondrial membrane fluidity. At the same time, hepatic lipogenic capacity was increased in animals fed Western diet (+270% fatty acid elongase activity compared with high-fat diet), while fatty acid desaturase activity decreased over time. Our findings suggest that dysregulation of lipogenesis is a significant component of heart failure in diet-induced obesity.

Highly efficient ketone body treatment in multiple acyl-CoA dehydrogenase deficiency-related leukodystrophy.

BACKGROUND Multiple acyl-CoA dehydrogenase deficiency- (MADD-), also called glutaric aciduria type 2, associated leukodystrophy may be severe and progressive despite conventional treatment with protein- and fat-restricted diet, carnitine, riboflavin, and coenzyme Q10. Administration of ketone bodies was described as a promising adjunct, but has only been documented once. METHODS We describe a Portuguese boy of consanguineous parents who developed progressive muscle weakness at 2.5 y of age, followed by severe metabolic decompensation with hypoglycaemia and coma triggered by a viral infection. Magnetic resonance (MR) imaging showed diffuse leukodystrophy. MADD was diagnosed by biochemical and molecular analyses. Clinical deterioration continued despite conventional treatment. Enteral sodium D,L-3-hydroxybutyrate (NaHB) was progressively introduced and maintained at 600 mg/kg BW/d (≈3% caloric need). Follow up was 3 y and included regular clinical examinations, biochemical studies, and imaging. RESULTS During follow up, the initial GMFC-MLD (motor function classification system, 0 = normal, 6 = maximum impairment) level of 5-6 gradually improved to 1 after 5 mo. Social functioning and quality of life recovered remarkably. We found considerable improvement of MR imaging and spectroscopy during follow up, with a certain lag behind clinical recovery. There was some persistent residual developmental delay. CONCLUSION NaHB is a highly effective and safe treatment that needs further controlled studies.

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

The x-ray crystal structures of trans-cinnamoyl–subtilisin, an acyl-enzyme covalent intermediate of the serine protease subtilisin Carlsberg, have been determined to 2.2-Å resolution in anhydrous acetonitrile and in water. The cinnamoyl–subtilisin structures are virtually identical in the two solvents. In addition, their enzyme portions are nearly indistinguishable from previously determined structures of the free enzyme in acetonitrile and in water; thus, acylation in either aqueous or nonaqueous solvent causes no appreciable conformational changes. However, the locations of bound solvent molecules in the active site of the acyl- and free enzyme forms in acetonitrile and in water are distinct. Such differences in the active site solvation may contribute to the observed variations in enzymatic activities. On prolonged exposure to organic solvent or removal of interstitial solvent from the crystal lattice, the channels within enzyme crystals are shown to collapse, leading to a drop in the number of active sites accessible to the substrate. The mechanistic and preparative implications of our findings for enzymatic catalysis in organic solvents are discussed.

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.

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.

Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD +/− mice yielded an abnormally low number of LCAD +/− and −/− offspring, indicating frequent gestational loss. LCAD −/− mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD −/− males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD −/− mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD in vivo.

Identification, Separation, and Characterization of Acyl-Coenzyme A Dehydrogenases Involved in Mitochondrial β-Oxidation in Higher Plants1

The existence in higher plants of an additional β-oxidation system in mitochondria, besides the well-characterized peroxisomal system, is often considered controversial. Unequivocal demonstration of β-oxidation activity in mitochondria should rely on identification of the enzymes specific to mitochondrial β-oxidation. Acyl-coenzyme A dehydrogenase (ACAD) (EC 1.3.99.2,3) activity was detected in purified mitochondria from maize (Zea mays L.) root tips and from embryonic axes of early-germinating sunflower (Helianthus annuus L.) seeds, using as the enzyme assay the reduction of 2,6-dichlorophenolindophenol, with phenazine methosulfate as the intermediate electron carrier. Subcellular fractionation showed that this ACAD activity was associated with mitochondrial fractions. Comparison of ACAD activity in mitochondria and acyl-coenzyme A oxidase activity in peroxisomes showed differences of substrate specificities. Embryonic axes of sunflower seeds were used as starting material for the purification of ACADs. Two distinct ACADs, with medium-chain and long-chain substrate specificities, respectively, were separated by their chromatographic behavior, which was similar to that of mammalian ACADs. The characterization of these ACADs is discussed in relation to the identification of expressed sequenced tags corresponding to ACADs in cDNA sequence analysis projects and with the potential roles of mitochondrial β-oxidation in higher plants.

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.

Depletion of Acyl-Coenzyme A-Binding Protein Affects Sphingolipid Synthesis and Causes Vesicle Accumulation and Membrane Defects in Saccharomyces cerevisiae

Deletion of the yeast gene ACB1 encoding Acb1p, the yeast homologue of the acyl-CoA-binding protein (ACBP), resulted in a slower growing phenotype that adapted into a faster growing phenotype with a frequency >1:105. A conditional knockout strain (Y700pGAL1-ACB1) with the ACB1 gene under control of the GAL1 promoter exhibited an altered acyl-CoA profile with a threefold increase in the relative content of C18:0-CoA, without affecting total acyl-CoA level as previously reported for an adapted acb1Δ strain. Depletion of Acb1p did not affect the general phospholipid pattern, the rate of phospholipid synthesis, or the turnover of individual phospholipid classes, indicating that Acb1p is not required for general glycerolipid synthesis. In contrast, cells depleted for Acb1p showed a dramatically reduced content of C26:0 in total fatty acids and the sphingolipid synthesis was reduced by 50–70%. The reduced incorporation of [3H]myo-inositol into sphingolipids was due to a reduced incorporation into inositol-phosphoceramide and mannose-inositol-phosphoceramide only, a pattern that is characteristic for cells with aberrant endoplasmic reticulum to Golgi transport. The plasma membrane of the Acb1p-depleted strain contained increased levels of inositol-phosphoceramide and mannose-inositol-phosphoceramide and lysophospholipids. Acb1p-depleted cells accumulated 50- to 60-nm vesicles and autophagocytotic like bodies and showed strongly perturbed plasma membrane structures. The present results strongly suggest that Acb1p plays an important role in fatty acid elongation and membrane assembly and organization.

Acyl-homoserine lactone quorum sensing in Gram-negative bacteria: A signaling mechanism involved in associations with higher organisms

Recent advances in studies of bacterial gene expression have brought the realization that cell-to-cell communication and community behavior are critical for successful interactions with higher organisms. Species-specific cell-to-cell communication is involved in successful pathogenic or symbiotic interactions of a variety of bacteria with plant and animal hosts. One type of cell–cell signaling is acyl-homoserine lactone quorum sensing in Gram-negative bacteria. This type of quorum sensing represents a dedicated communication system that enables a given species to sense when it has reached a critical population density in a host, and to respond by activating expression of genes necessary for continued success in the host. Acyl-homoserine lactone signaling in the opportunistic animal and plant pathogen Pseudomonas aeruginosa is a model for the relationships among quorum sensing, pathogenesis, and community behavior. In the P. aeruginosa model, quorum sensing is required for normal biofilm maturation and for virulence. There are multiple quorum-sensing circuits that control the expression of dozens of specific genes that represent potential virulence loci.

Cloning and characterization of a hormonally regulated rat long chain acyl-CoA synthetase

A previously unidentified gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) was cloned and characterized as a 79-kDa cytoplasmic protein expressed in Leydig cells of the rat testis. GR-LACS shares sequence identity with two conserved regions of the LACS and luciferase families, including the ATP/AMP binding domain and the 25-aa fatty acyl-CoA synthetase signature motif, but displays low overall amino acid similarities (23–28%). GR-LACS mRNA is expressed abundantly in Leydig cells of the adult testis and to a lesser degree in the seminiferous tubules in spermatogonia and Sertoli cells. It is also observed in ovary and brain. Immunoreactive protein expression was observed mainly in Leydig cells and minimally in the tubules but was not detected in other tissues. In vivo, treatment with a desensitizing dose of human chorionic gonadotropin caused transcriptional down-regulation of GR-LACS expression in Leydig cells. The expressed protein present in the cytoplasm of transfected cells displayed acyl-CoA synthetase activity for long chain fatty acid substrates. GR-LACS may contribute to the provision of energy requirements and to the biosynthesis of steroid precursors and could participate through acyl-CoA's multiple functions in the regulation of the male gonad.

A Determinant of Substrate Specificity Predicted from the Acyl-Acyl Carrier Protein Desaturase of Developing Cat's Claw Seed1

Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmitoleic acid (16:1Δ9) plus cis-vaccenic acid (18:1Δ11) in its seed oil. To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturases were isolated from developing cat's claw seeds. The predominant acyl-ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Δ9–18:0)-ACP desaturase from castor (Ricinis communis L.) and other species. Upon expression in Escherichia coli, the cat's claw polypeptide functioned as a Δ9 acyl-ACP desaturase but displayed a distinct substrate specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP. Comparison of the predicted amino acid sequence of the cat's claw enzyme with that of the castor Δ9–18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part for the differences in substrate specificity between the two desaturases. Consistent with this prediction, conversion of leucine-118 to tryptophan in the mature castor Δ9–18:0-ACP desaturase resulted in an 80-fold increase in the relative specificity of this enzyme for 16:0-ACP. The alteration in substrate specificity observed in the L118W mutant is in agreement with a crystallographic model of the proposed substrate-binding pocket of the castor Δ9–18:0-ACP desaturase.