Metabolomic and Transcriptomic Analysis of Effects of Three MUFA-Rich Oils on Hepatic Glucose and Lipid Metabolism in Mice.

SCOPE: Olive oil, rapeseed oil, and lard are dietary fats rich in monounsaturated fatty acids, but the effects of dietary oils enriched in monounsaturated fatty acids on hepatic lipid deposition have seldom been compared. METHODS AND RESULTS: Ninety 8-week-old C57BL/6J male mice are randomly divided into six groups and fed diets containing lard, rapeseed oil, or olive oil with a 10% or 45% fat energy supply for 16 weeks. Under high-fat conditions, serum total cholesterol levels in the lard and olive oil groups are significantly higher than those in the rapeseed oil group. Hepatic lipid content in the olive oil group is higher than that in the other two groups. Compared with rapeseed oil, lard increases the liver levels of arachidonic, palmitic, and myristic acids and decreases the levels of eicosapentaenoic linolenic acid and linoleic acid. Olive oil increases the liver levels of docosatrienoic, arachidonic, oleic, and myristic acids; maltose; and fructose and decreases the levels of eicosapentaenoic, linolenic, and linoleic acids. CONCLUSION: Olive oil probably causes hepatic lipid deposition in mice, which may enhance hepatic lipid synthesis by activating the starch and sucrose metabolic pathways. By contrast, rapeseed oil shows a significant anti-lipid deposition effect on the liver.

Temporal evolution of fatty acid content in human milk of lactating mothers from the Philippines.

Fatty acids (FA) play a key role in infant growth and development. The aim of this study was to study the temporal evolution of FA from 3 or 4 weeks to 4 months postpartum in human milk (HM) from Filipino mothers. Mid-morning HM samples (n = 41) were collected after full expression from a single breast and FA were assessed using gas-liquid chromatography coupled to flame ionization detector. The total FA content remained relatively constant over the study period. The most abundant FA in HM were oleic acid (OA), palmitic acid (PA) and linoleic acid (LA), a trend similarly reported in HM from European and Chinese mothers. The former two were unchanged over the course of lactation while there was a slight increase in LA content over time. Similarly, the saturated fatty acid (SFA) and monounsaturated FA (MUFA) contents did not vary over the first four months of lactation. The SFA content was much higher than that reported in HM from Europe and China, mainly driven by PA, lauric and myristic acids. The MUFA content on the other hand, while comparable to that reported in HM from Chinese populations was lower than that reported in Europe. There was a small increase in the polyunsaturated FA (PUFA) content over the study duration. The levels of essential FA, linoleic acid (LA) and α-linolenic acid (ALA) were found to be much lower than that reported in other populations. The concentrations of arachidonic acid (AA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) remained stable over the study duration. AA and DHA in HM from Filipino mothers were comparable to global averages, however in case of the latter the concentration was found to be lower than in previous reports. DHA is of great clinical significance as it plays a key role in infant growth and development. In our study, we observed a wide inter- and intra-individual variability in the levels of DHA in HM, presumably reflecting diverse intakes of DHA rich foods and bioconversion in vivo. Personalized recommendations may help achieve recommended levels of DHA amongst population with levels below global averages. This may help achieve HM sufficiency and therefore be linked to clinical benefits for the mother and the baby. SUMMARY: This study details the temporal evolution of human milk (HM) fatty acids (FA) in Filipino mothers up to four months postpartum. The total FA content remained relatively constant over the study period. The most abundant FA were oleic, palmitic and linoleic acids. HM from Filipino mothers had relatively higher saturated FA content driven by palmitic, lauric and myristic acids, while the levels of essential FA, linoleic and α-linoleic acids were lower compared to other populations. Similarly, the concentration of monounsaturated FA were also lower than that reported in HM from European mothers. Arachidonic acid and docosahexaenoic acid (DHA) concentrations were comparable to global averages however the HM DHA levels were seen to have decreased when compared to previous reports from the Philippines. Additionally, a wide variability was seen in HM DHA levels suggesting a need for strategies such as personalized recommendations in order to ensure HM DHA sufficiency.

Producing high value unsaturated fatty acid by whole-cell catalysis using microalga: A case study with Tribonema minus.

High value unsaturated fatty acids can be produced by de novo synthesis in microalgal cells, especially via heterotrophic cultivation. Unfortunately, the lipid accumulation of heterotrophic microalgae cannot be improved efficiently in conventional ways. Here we reported heterotrophic Tribonema minus, a promising resource for the production of palmitoleic acid which has increasing demands in health service for patients with metabolic syndrome, as whole-cell biocatalyst to develop a novel way of shifting low value exogenous saturated fatty acids to high value ones. Results showed that myristic acid is the best precursor for whole-cell catalysis; it elevated the lipid content of T. minus to 42.2%, the highest among the tried precursors. The influences of cultivation condition on the utilization of extrinsic myristic acid and lipid accumulation were also determined. Under the optimized condition, the lipid content reached as high as 48.9%. In addition, our findings showed that ~13.0% of C16:1 in T. minus is derived from extrinsic myristic acid, and 30.1% of metabolized precursor is converted into heterologous fatty acids. Thus, a feasible approach for both increasing the value of low value saturated fatty acid by bioconversion and enhancing the lipid accumulation in microalgae is proposed by supplementing extrinsic myristic acid.

Two AMP-Binding Domain Proteins from Rhizophagus irregularis Involved in Import of Exogenous Fatty Acids.

Arbuscular mycorrhizal fungi (AMF) colonize roots, where they provide nutrients in exchange for sugars and lipids. Because AMF lack genes for cytosolic fatty acid de novo synthase (FAS), they depend on host-derived fatty acids. AMF colonization is accompanied by expression of specific lipid genes and synthesis of sn-2 monoacylglycerols (MAGs). It is unknown how host-derived fatty acids are taken up by AMF. We describe the characterization of two AMP-binding domain protein genes from Rhizophagus irregularis, RiFAT1 and RiFAT2, with sequence similarity to Saccharomyces cerevisiae fatty acid transporter 1 (FAT1). Uptake of 13C-myristic acid (14:0) and, to a lesser extent, 13C-palmitic acid (16:0) was enhanced after expression of RiFAT1 or RiFAT2 in S. cerevisiae Δfat1 cells. The uptake of 2H-labeled fatty acids from 2H-myristoylglycerol or 2H-palmitoylglycerol was also increased after RiFAT1 and RiFAT2 expression in Δfat, but intact 2H-MAGs were not detected. RiFAT1 and RiFAT2 expression was induced in colonized roots compared with extraradical mycelium. 13C-label in the AMF-specific palmitvaccenic acid (16:1Δ11) and eicosatrienoic acid (20:3) were detected in colonized roots only when 13C2-acetate was supplemented but not 13C-fatty acids, demonstrating that de novo synthesized, host-derived fatty acids are rapidly taken up by R. irregularis from the roots. The results show that RiFAT1 and RiFAT2 are involved in the uptake of myristic acid (14:0) and palmitic acid (16:0), while fatty acids from MAGs are only taken up after hydrolysis. Therefore, the two proteins might be involved in fatty acid import into the fungal arbuscules in colonized roots.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Myristoylation alone is sufficient for PKA catalytic subunits to associate with the plasma membrane to regulate neuronal functions.

Myristoylation is a posttranslational modification that plays diverse functional roles in many protein species. The myristate moiety is considered insufficient for protein-membrane associations unless additional membrane-affinity motifs, such as a stretch of positively charged residues, are present. Here, we report that the electrically neutral N-terminal fragment of the protein kinase A catalytic subunit (PKA-C), in which myristoylation is the only functional motif, is sufficient for membrane association. This myristoylation can associate a fraction of PKA-C molecules or fluorescent proteins (FPs) to the plasma membrane in neuronal dendrites. The net neutral charge of the PKA-C N terminus is evolutionally conserved, even though its membrane affinity can be readily tuned by changing charges near the myristoylation site. The observed membrane association, while moderate, is sufficient to concentrate PKA activity at the membrane by nearly 20-fold and is required for PKA regulation of AMPA receptors at neuronal synapses. Our results indicate that myristoylation may be sufficient to drive functionally significant membrane association in the absence of canonical assisting motifs. This provides a revised conceptual base for the understanding of how myristoylation regulates protein functions.

Requirement of the acyl-CoA carrier ACBD6 in myristoylation of proteins: Activation by ligand binding and protein interaction.

Glycine N-myristoylation is an essential acylation modification modulating the functions, stability, and membrane association of diverse cytosolic proteins in human cells. Myristoyl-CoA is the 14-carbon acyl donor of the acyltransferase reaction. Acyl-CoAs of a chain length compatible with the binding site of the N-myristoyltransferase enzymes (NMT) are competitive inhibitors, and the mechanism protecting these enzymes from unwanted acyl-CoA species requires the acyl-CoA binding protein ACBD6. The acyl-CoA binding domain (ACB) and the ankyrin-repeat motifs (ANK) of ACBD6 can perform their functions independently. Interaction of ANK with human NMT2 was necessary and sufficient to provide protection. Fusion of the ANK module to the acyl-CoA binding protein ACBD1 was sufficient to confer the NMT-stimulatory property of ACBD6 to the chimera. The ACB domain is dispensable and sequestration of the competitor was not the basis for NMT2 protection. Acyl-CoAs bound to ACB modulate the function of the ANK module and act as positive effector of the allosteric activation of the enzyme. The functional relevance of homozygous mutations in ACBD6 gene, which have not been associated with a disease so far, is presented. Skin-derived fibroblasts of two unrelated individuals with neurodevelopmental disorder and carrying loss of function mutations in the ACBD6 gene were deficient in protein N-myristoylation. These cells were sensitive to substrate analog competing for myristoyl-CoA binding to NMT. These findings account for the requirement of an ANK-containing acyl-CoA binding protein in the cellular mechanism protecting the NMT enzymes and establish that in human cells, ACBD6 supports the N-myristoylation of proteins.

Analytical Method for Diacylglycerol Kinase ζ Activity in Cells Using Protein Myristoylation and Liquid Chromatography-Tandem Mass Spectrometry.

Specific inhibitors of diacylglycerol kinase (DGK) ζ can be promising anticancer medications via the activation of cancer immunity. Although the detection of cellular activities of target enzymes is essential for drug screening in addition to in vitro assays, it is difficult to detect the activity of DGKζ in cells. In the present study, we generated AcGFP-DGKζ cDNA with a consensus N-myristoylation sequence at the 5' end (Myr-AcGFP-DGKζ) to target DGKζ to membranes. Using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) (LC-MS/MS), we showed that Myr-AcGFP-DGKζ, but not AcGFP-DGKζ without the myristoylation sequence, substantially augmented the levels of several phosphatidic acid (PtdOH) species. In contrast to Myr-AcGFP-DGKζ, its inactive mutant did not exhibit an increase in PtdOH production, indicating that the increase in PtdOH production was DGK activity-dependent. This method will be useful in chemical compound selection for the development of drugs targeting DGKζ and can be applicable to various soluble (nonmembrane bound) lipid-metabolizing enzymes, including other DGK isozymes.

Signal Production and Response Specificity in the phc Quorum Sensing Systems of Ralstonia solanacearum Species Complex.

Ralstonia solanacearum strains are devastating plant pathogens with global distribution, a wide host range, and genetic diversity, and they are now also referred to as the R. solanacearum species complex (RSSC). RSSC strains employ the quorum sensing (QS) system composed of the phcBSR operon to regulate their virulence on plants. The RSSC strains previously examined produce either (R)-methyl 3-hydroxymyristate (3-OH MAME) or (R)-methyl 3-hydroxypalmitate (3-OH PAME) as their QS signals. Analogously, the phylogenetic analyses of the signal synthase PhcB and the signal receptor PhcS from 15 RSSC strains revealed that these proteins have two clades dependent on their QS signal types. However, the biochemical mechanism underlying this selectivity of QS signal production remains to be elucidated. We demonstrated that the PhcB methyltransferases synthesize QS signals from the cognate fatty acids (R)-3-hydroxymyristic acid or (R)-3-hydroxypalmitic acid. The RSSC strains used here produced both fatty acids, and thus the selectivity of QS signal production depends on the activity of PhcB enzymes. On the other hand, the enantioselective supply of the precursors functioned in the production of enantiopure QS signals. The opposite QS signals weakly induced the production of virulence factors in the RSSC strains. Furthermore, the complementation of the phcB gene encoding the 3-OH PAME-type synthase to the phcB-deletion mutant of the 3-OH MAME-producing strain did not rescue its virulence on tomato plants. Taken together, we propose that the specific production of 3-OH MAME/3-OH PAME ensures full virulence of the RSSC strains.

Functional characterization and mechanistic modeling of the human cytochrome P450 enzyme CYP4A22.

The human cytochrome P450 (CYP) enzyme CYP4A22 is an orphan CYP with unknown function. Here, through functional expression in fission yeast, we show that CYP4A22 catalyzes fatty acid hydroxylation as well as aliphatic or aromatic hydroxylations of luciferin-based probe substrates. Mechanistic molecular modeling of CYP4A22 suggests that its ω-hydroxylation activity is hampered by a more spacious active site compared to CYP4B1. Substrate recognition via side-chains R96 and R233 is indicated by dynamic three-dimensional pharmacophores (dynophores) derived from molecular dynamics simulations. CYP4A22 activity is inhibited by three unspecific CYP inhibitors. A comparison of CYP4A22*1 (the reference standard sequence) with CYP4A22-WT (the most common allele) revealed that for the four substrates tested the WT-enzyme always had lower activity.

Structural modification of Escherichia coli lipid A by myristoyltransferase gene from Klebsiella pneumoniae.

Lipid A in lipopolysaccharide (LPS) of Escherichia coli mutant strains was modified by the introduction of myristoyltransferase gene cloned from Klebsiella pneumoniae. When the gene was introduced into the mutant having lipid A containing only 3-hydroxymyristic acids, it produced lipid A with two additional myristic acids (C14:0 ). When the same gene was introduced into the mutant with pentaacylated lipid A containing one lauric acid (C12:0 ), C12:0 was replaced by C14:0 . IL-6-inducing activity of LPS with modified lipid A structure suggested that C12:0 in lipid A could be replaced by C14:0 without changing the immunostimulating activity.

Myristoylated rhinovirus VP4 protein activates TLR2-dependent proinflammatory gene expression.

Asthma exacerbations are often caused by rhinovirus (RV). We and others have shown that Toll-like receptor 2 (TLR2), a membrane surface receptor that recognizes bacterial lipopeptides and lipoteichoic acid, is required and sufficient for RV-induced proinflammatory responses in vitro and in vivo. We hypothesized that viral protein-4 (VP4), an internal capsid protein that is myristoylated upon viral replication and externalized upon viral binding, is a ligand for TLR2. Recombinant VP4 and myristoylated VP4 (MyrVP4) were purified by Ni-affinity chromatography. MyrVP4 was also purified from RV-A1B-infected HeLa cells by urea solubilization and anti-VP4 affinity chromatography. Finally, synthetic MyrVP4 was produced by chemical peptide synthesis. MyrVP4-TLR2 interactions were assessed by confocal fluorescence microscopy, fluorescence resonance energy transfer (FRET), and monitoring VP4-induced cytokine mRNA expression in the presence of anti-TLR2 and anti-VP4. MyrVP4 and TLR2 colocalized in TLR2-expressing HEK-293 cells, mouse bone marrow-derived macrophages, human bronchoalveolar macrophages, and human airway epithelial cells. Colocalization was absent in TLR2-null HEK-293 cells and blocked by anti-TLR2 and anti-VP4. Cy3-labeled MyrVP4 and Cy5-labeled anti-TLR2 showed an average fractional FRET efficiency of 0.24 ± 0.05, and Cy5-labeled anti-TLR2 increased and unlabeled MyrVP4 decreased FRET efficiency. MyrVP4-induced chemokine mRNA expression was higher than that elicited by VP4 alone and was attenuated by anti-TLR2 and anti-VP4. Cytokine expression was similarly increased by MyrVP4 purified from RV-infected HeLa cells and synthetic MyrVP4. We conclude that, during RV infection, MyrVP4 and TLR2 interact to generate a proinflammatory response.

Protein Lipidation: Occurrence, Mechanisms, Biological Functions, and Enabling Technologies.

Protein lipidation, including cysteine prenylation, N-terminal glycine myristoylation, cysteine palmitoylation, and serine and lysine fatty acylation, occurs in many proteins in eukaryotic cells and regulates numerous biological pathways, such as membrane trafficking, protein secretion, signal transduction, and apoptosis. We provide a comprehensive review of protein lipidation, including descriptions of proteins known to be modified and the functions of the modifications, the enzymes that control them, and the tools and technologies developed to study them. We also highlight key questions about protein lipidation that remain to be answered, the challenges associated with answering such questions, and possible solutions to overcome these challenges.

Discovery and Characterization of the 3-Hydroxyacyl-ACP Dehydratase Component of the Plant Mitochondrial Fatty Acid Synthase System.

We report the characterization of the Arabidopsis (Arabidopsis thaliana) 3-hydroxyacyl-acyl carrier protein dehydratase (mtHD) component of the mitochondrial fatty acid synthase (mtFAS) system, encoded by AT5G60335. The mitochondrial localization and catalytic capability of mtHD were demonstrated with a green fluorescent protein transgenesis experiment and by in vivo complementation and in vitro enzymatic assays. RNA interference (RNAi) knockdown lines with reduced mtHD expression exhibit traits typically associated with mtFAS mutants, namely a miniaturized morphological appearance, reduced lipoylation of lipoylated proteins, and altered metabolomes consistent with the reduced catalytic activity of lipoylated enzymes. These alterations are reversed when mthd-rnai mutant plants are grown in a 1% CO2 atmosphere, indicating the link between mtFAS and photorespiratory deficiency due to the reduced lipoylation of glycine decarboxylase. In vivo biochemical feeding experiments illustrate that sucrose and glycolate are the metabolic modulators that mediate the alterations in morphology and lipid accumulation. In addition, both mthd-rnai and mtkas mutants exhibit reduced accumulation of 3-hydroxytetradecanoic acid (i.e. a hallmark of lipid A-like molecules) and abnormal chloroplastic starch granules; these changes are not reversible by the 1% CO2 atmosphere, demonstrating two novel mtFAS functions that are independent of photorespiration. Finally, RNA sequencing analysis revealed that mthd-rnai and mtkas mutants are nearly equivalent to each other in altering the transcriptome, and these analyses further identified genes whose expression is affected by a functional mtFAS system but independent of photorespiratory deficiency. These data demonstrate the nonredundant nature of the mtFAS system, which contributes unique lipid components needed to support plant cell structure and metabolism.

Lipids Cooperate with the Reovirus Membrane Penetration Peptide to Facilitate Particle Uncoating.

Virus-host interactions play a role in many stages of the viral lifecycle, including entry. Reovirus, a model system for studying the entry mechanisms of nonenveloped viruses, undergoes a series of regulated structural transitions that culminate in delivery of the viral genetic material. Lipids can trigger one of these conformational changes, infectious subviral particle (ISVP)-to-ISVP* conversion. ISVP* formation releases two virally encoded peptides, myristoylated µ1N (myr-µ1N) and Φ. Among these, myr-µ1N is sufficient to form pores within membranes. Released myr-µ1N can also promote ISVP* formation in trans Using thermal inactivation as a readout for ISVP-to-ISVP* conversion, we demonstrate that lipids render ISVPs less thermostable in a virus concentration-dependent manner. Under conditions in which neither lipids alone nor myr-µ1N alone promotes ISVP-to-ISVP* conversion, myr-µ1N induces particle uncoating when lipids are present. These data suggest that the pore-forming activity and the ISVP*-promoting activity of myr-µ1N are linked. Lipid-associated myr-µ1N interacts with ISVPs and triggers efficient ISVP* formation. The cooperativity between a reovirus component and lipids reveals a distinct virus-host interaction in which membranes can facilitate nonenveloped virus entry.

High Fat Diet Administration during Specific Periods of Pregnancy Alters Maternal Fatty Acid Profiles in the Near-Term Rat.

Excessive fat intake is a global health concern as women of childbearing age increasingly ingest high fat diets (HFDs). We therefore determined the maternal fatty acid (FA) profiles in metabolic organs after HFD administration during specific periods of gestation. Rats were fed a HFD for the first (HF1), second (HF2), or third (HF3) week, or for all three weeks (HFG) of gestation. Total maternal plasma non-esterified fatty acid (NEFA) concentrations were monitored throughout pregnancy. At day 20 of gestation, maternal plasma, liver, adipose tissue, and placenta FA profiles were determined. In HF3 mothers, plasma myristic and stearic acid concentrations were elevated, whereas docosahexaenoic acid (DHA) was reduced in both HF3 and HFG mothers. In HF3 and HFG mothers, hepatic stearic and oleic acid proportions were elevated; conversely, DHA and linoleic acid (LA) proportions were reduced. In adipose tissue, myristic acid was elevated, whereas DHA and LA proportions were reduced in all mothers. Further, adipose tissue stearic acid proportions were elevated in HF2, HF3, and HFG mothers; with oleic acid increased in HF1 and HFG mothers. In HF3 and HFG mothers, placental neutral myristic acid proportions were elevated, whereas DHA was reduced. Further, placental phospholipid DHA proportions were reduced in HF3 and HFG mothers. Maintenance on a diet, high in saturated fat, but low in DHA and LA proportions, during late or throughout gestation, perpetuated reduced DHA across metabolic organs that adapt during pregnancy. Therefore a diet, with normal DHA proportions during gestation, may be important for balancing maternal FA status.

Branched phospholipids render lipid vesicles more susceptible to membrane-active peptides.

Iso- and anteiso-branched lipids are abundant in the cytoplasmic membranes of bacteria. Their function is assumed to be similar to that of unsaturated lipids in other organisms - to maintain the membrane in a fluid state. However, the presence of terminally branched membrane lipids is likely to impact other membrane properties as well. For instance, lipid acyl chain structure has been shown to influence the activity of antimicrobial peptides. Moreover, the development of resistance to antimicrobial agents in Staphylococcus aureus is accompanied by a shift in the fatty acid composition toward a higher fraction of anteiso-branched lipids. Little is known about how branched lipids and the location of the branch point affect the activity of membrane-active peptides. We hypothesized that bilayers containing lipids with low phase transition temperatures would tend to exclude peptides and be less susceptible to peptide-induced perturbation than those made from higher temperature melting lipids. To test this hypothesis, we synthesized a series of asymmetric phospholipids that only differ in the type of fatty acid esterified at the sn-2 position of the lipid glycerol backbone. We tested the influence of acyl chain structure on peptide activity by measuring the kinetics of release from dye-encapsulated lipid vesicles made from these synthetic lipids. The results were compared to those obtained using vesicles made from S. aureus and Staphylococcus sciuri membrane lipid extracts. Anteiso-branched phospholipids, which melt at very low temperatures, produced lipid vesicles that were only slightly less susceptible to peptide-induced dye release than those made from the iso-branched isomer. However, liposomes made from bacterial phospholipid extracts were generally much more resistant to peptide-induced perturbation than those made from any of the synthetic lipids. The results suggest that the increase in the fraction of anteiso-branched fatty acids in antibiotic-resistant strains of S. aureus is unlikely to be the sole factor responsible for the observed increased antibiotic resistance. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Deregulation of mitochondrial functions provoked by long-chain fatty acid accumulating in long-chain 3-hydroxyacyl-CoA dehydrogenase and mitochondrial permeability transition deficiencies in rat heart--mitochondrial permeability transition pore opening as a potential contributing pathomechanism of cardiac alterations in these disorders.

Mitochondrial trifunctional protein and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies are fatty acid oxidation disorders biochemically characterized by tissue accumulation of long-chain fatty acids and derivatives, including the monocarboxylic long-chain 3-hydroxy fatty acids (LCHFAs) 3-hydroxytetradecanoic acid (3HTA) and 3-hydroxypalmitic acid (3HPA). Patients commonly present severe cardiomyopathy for which the pathogenesis is still poorly established. We investigated the effects of 3HTA and 3HPA, the major metabolites accumulating in these disorders, on important parameters of mitochondrial homeostasis in Ca(2+) -loaded heart mitochondria. 3HTA and 3HPA significantly decreased mitochondrial membrane potential, the matrix NAD(P)H pool and Ca(2+) retention capacity, and also induced mitochondrial swelling. These fatty acids also provoked a marked decrease of ATP production reflecting severe energy dysfunction. Furthermore, 3HTA-induced mitochondrial alterations were completely prevented by the classical mitochondrial permeability transition (mPT) inhibitors cyclosporin A and ADP, as well as by ruthenium red, a Ca(2+) uptake blocker, indicating that LCHFAs induced Ca(2+)-dependent mPT pore opening. Milder effects only achieved at higher doses of LCHFAs were observed in brain mitochondria, implying a higher vulnerability of heart to these fatty acids. By contrast, 3HTA and docosanoic acids did not change mitochondrial homeostasis, indicating selective effects for monocarboxylic LCHFAs. The present data indicate that the major LCHFAs accumulating in mitochondrial trifunctional protein and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies induce mPT pore opening, compromising Ca(2+) homeostasis and oxidative phosphorylation more intensely in the heart. It is proposed that these pathomechanisms may contribute at least in part to the severe cardiac alterations characteristic of patients affected by these diseases.

G protein-membrane interactions I: Gαi1 myristoyl and palmitoyl modifications in protein-lipid interactions and its implications in membrane microdomain localization.

G proteins are fundamental elements in signal transduction involved in key cell responses, and their interactions with cell membrane lipids are critical events whose nature is not fully understood. Here, we have studied how the presence of myristic and palmitic acid moieties affects the interaction of the Gαi1 protein with model and biological membranes. For this purpose, we quantified the binding of purified Gαi1 protein and Gαi1 protein acylation mutants to model membranes, with lipid compositions that resemble different membrane microdomains. We observed that myristic and palmitic acids not only act as membrane anchors but also regulate Gαi1 subunit interaction with lipids characteristics of certain membrane microdomains. Thus, when the Gαi1 subunit contains both fatty acids it prefers raft-like lamellar membranes, with a high sphingomyelin and cholesterol content and little phosphatidylserine and phosphatidylethanolamine. By contrast, the myristoylated and non-palmitoylated Gαi1 subunit prefers other types of ordered lipid microdomains with higher phosphatidylserine content. These results in part explain the mobility of Gαi1 protein upon reversible palmitoylation to meet one or another type of signaling protein partner. These results also serve as an example of how membrane lipid alterations can change membrane signaling or how membrane lipid therapy can regulate the cell's physiology.

Pheromones mediating copulation and attraction in Drosophila.

Intraspecific olfactory signals known as pheromones play important roles in insect mating systems. In the model Drosophila melanogaster, a key part of the pheromone-detecting system has remained enigmatic through many years of research in terms of both its behavioral significance and its activating ligands. Here we show that Or47b-and Or88a-expressing olfactory sensory neurons (OSNs) detect the fly-produced odorants methyl laurate (ML), methyl myristate, and methyl palmitate. Fruitless (fru(M))-positive Or47b-expressing OSNs detect ML exclusively, and Or47b- and Or47b-expressing OSNs are required for optimal male copulation behavior. In addition, activation of Or47b-expressing OSNs in the male is sufficient to provide a competitive mating advantage. We further find that the vigorous male courtship displayed toward oenocyte-less flies is attributed to an oenocyte-independent sustained production of the Or47b ligand, ML. In addition, we reveal that Or88a-expressing OSNs respond to all three compounds, and that these neurons are necessary and sufficient for attraction behavior in both males and females. Beyond the OSN level, information regarding the three fly odorants is transferred from the antennal lobe to higher brain centers in two dedicated neural lines. Finally, we find that both Or47b- and Or88a-based systems and their ligands are remarkably conserved over a number of drosophilid species. Taken together, our results close a significant gap in the understanding of the olfactory background to Drosophila mating and attraction behavior; while reproductive isolation barriers between species are created mainly by species-specific signals, the mating enhancing signal in several Drosophila species is conserved.

Alteration of Wax Ester Content and Composition in Euglena gracilis with Gene Silencing of 3-ketoacyl-CoA Thiolase Isozymes.

Euglena gracilis produces wax ester under hypoxic and anaerobic culture conditions with a net synthesis of ATP. In wax ester fermentation, fatty acids are synthesized by reversing beta-oxidation in mitochondria. A major species of wax ester produced by E. gracilis is myristyl myristate (14:0-14:0Alc). Because of its shorter carbon chain length with saturated compounds, biodiesel produced from E. gracilis wax ester may have good cold flow properties with high oxidative stability. We reasoned that a slight metabolic modification would enable E. gracilis to produce a biofuel of ideal composition. In order to produce wax ester with shorter acyl chain length, we focused on isozymes of the enzyme 3-ketoacyl-CoA thiolase (KAT), a condensing enzyme of the mitochondrial fatty acid synthesis pathway in E. gracilis. We performed a gene silencing study of KAT isozymes in E. gracilis. Six KAT isozymes were identified in the E. gracilis EST database, and silencing any three of them (EgKAT1-3) altered the wax ester amount and composition. In particular, silencing EgKAT1 induced a significant compositional shift to shorter carbon chain lengths in wax ester. A model fuel mixture inferred from the composition of wax ester in EgKAT1-silenced cells showed a significant decrease in melting point compared to that of the control cells.