Postexposure Liponucleotide Prophylaxis and Treatment Attenuates Acute Respiratory Distress Syndrome in Influenza-infected Mice.

There is an urgent need for new drugs for patients with acute respiratory distress syndrome (ARDS), including those with coronavirus disease (COVID-19). ARDS in influenza-infected mice is associated with reduced concentrations of liponucleotides (essential precursors for de novo phospholipid synthesis) in alveolar type II (ATII) epithelial cells. Because surfactant phospholipid synthesis is a primary function of ATII cells, we hypothesized that disrupting this process could contribute significantly to the pathogenesis of influenza-induced ARDS. The goal of this study was to determine whether parenteral liponucleotide supplementation can attenuate ARDS. C57BL/6 mice inoculated intranasally with 10,000 plaque-forming units/mouse of H1N1 influenza A/WSN/33 virus were treated with CDP (cytidine 5'-diphospho)-choline (100 µg/mouse i.p.) ± CDP -diacylglycerol 16:0/16:0 (10 µg/mouse i.p.) once daily from 1 to 5 days after inoculation (to model postexposure influenza prophylaxis) or as a single dose on Day 5 (to model treatment of patients with ongoing influenza-induced ARDS). Daily postexposure prophylaxis with CDP-choline attenuated influenza-induced hypoxemia, pulmonary edema, alterations in lung mechanics, impairment of alveolar fluid clearance, and pulmonary inflammation without altering viral replication. These effects were not recapitulated by the daily administration of CTP (cytidine triphosphate) and/or choline. Daily coadministration of CDP-diacylglycerol significantly enhanced the beneficial effects of CDP-choline and also modified the ATII cell lipidome, reversing the infection-induced decrease in phosphatidylcholine and increasing concentrations of most other lipid classes in ATII cells. Single-dose treatment with both liponucleotides at 5 days after inoculation also attenuated hypoxemia, altered lung mechanics, and inflammation. Overall, our data show that liponucleotides act rapidly to reduce disease severity in mice with severe influenza-induced ARDS.

Phosphatidylserine synthesis is essential for viability of the human fungal pathogen Cryptococcus neoformans.

Phospholipids are an integral part of the cellular membrane structure and can be produced by a de novo biosynthetic pathway and, alternatively, by the Kennedy pathway. Studies in several yeast species have shown that the phospholipid phosphatidylserine (PS) is synthesized from CDP-diacylglycerol and serine, a route that is different from its synthesis in mammalian cells, involving a base-exchange reaction from preexisting phospholipids. Fungal-specific PS synthesis has been shown to play an important role in fungal virulence and has been proposed as an attractive drug target. However, PS synthase, which catalyzes this reaction, has not been studied in the human fungal pathogen Cryptococcus neoformans Here, we identified and characterized the PS synthase homolog (Cn Cho1) in this fungus. Heterologous expression of Cn CHO1 in a Saccharomyces cerevisiae cho1Δ mutant rescued the mutant's growth defect in the absence of ethanolamine supplementation. Moreover, an Sc cho1Δ mutant expressing Cn CHO1 had PS synthase activity, confirming that the Cn CHO1 encodes PS synthase. We also found that PS synthase in C. neoformans is localized to the endoplasmic reticulum and that it is essential for mitochondrial function and cell viability. Of note, its deficiency could not be complemented by ethanolamine or choline supplementation for the synthesis of phosphatidylethanolamine (PE) or phosphatidylcholine (PC) via the Kennedy pathway. These findings improve our understanding of phospholipid synthesis in a pathogenic fungus and indicate that PS synthase may be a useful target for antifungal drugs.

Cytidinediphosphate-diacylglycerol synthase 5 is required for phospholipid homeostasis and is negatively involved in hyperosmotic stress tolerance.

Cytidinediphosphate diacylglycerol synthase (CDS) uses phosphatidic acid (PA) and cytidinetriphosphate to produce cytidinediphosphate-diacylglycerol, an intermediate for phosphatidylglycerol (PG) and phosphatidylinositol (PI) synthesis. This study shows that CDS5, one of the five CDSs of the Oryza sativa (rice) genome, has multifaceted effects on plant growth and stress responses. The loss of CDS5 resulted in a decrease in PG and PI levels, defective thylakoid membranes, pale leaves in seedlings and growth retardation. In addition, the loss of CDS5 led to an elevated PA level and enhanced hyperosmotic tolerance. The inhibition of phospholipase D (PLD)-derived PA formation in cds5 restored the hyperosmotic stress tolerance of the mutant phenotype to that of the wild type, suggesting that CDS5 functions as a suppressor in PLD-derived PA signaling and negatively affects hyperosmotic stress tolerance.

Molecular and functional evidence of phosphatidylserine synthase in Vibrio parahaemolyticus.

Phosphatidylserine synthase (Pss) catalyzes phosphatidylserine synthesis, which is critical to synthesizing the component of cell membrane. However, few putative pss genes of bacteria have been studied. In this study, it was found that Vibrio parahaemolyticus, a common foodborne pathogen that causes human gastroenteritis, has a type I Pss with two HKD motifs and is a phospholipase D superfamily member. The transcriptional start site of pss was mapped through sequencing and was identified at -37 nucleotides upstream of the start codon. Pss mRNA was found to be expressed mainly during the exponential phase. In addition, the promoter was identified using a lux reporter assay and gel shift assay with an RNA polymerase. To analyze the catalytic activity, a soluble form of His6 -tagged recombinant Pss was overexpressed and purified from Escherichia coli. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry, it was found that Pss can catalyze cytidine diphosphate diacylglycerol and L-serine to form phosphatidylserine. Since Pss is conserved in vibrios, the current study can promote understanding the biosynthesis of phospholipid in Vibrio bacteria that might cause vibriosis. This is the first report of molecular characterization of the pss gene and identification of Pss enzyme activity in V. parahaemolyticus using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Lipid topogenesis--35years on.

Glycerophospholipids are the principal fabric of cellular membranes. The pathways by which these lipids are synthesized were elucidated mainly through the work of Kennedy and colleagues in the late 1950s and early 1960s. Subsequently, attention turned to cell biological aspects of lipids: Where in the cell are lipids synthesized? How are lipids integrated into membranes to form a bilayer? How are they sorted and transported from their site of synthesis to other cellular destinations? These topics, collectively termed 'lipid topogenesis', were the subject of a review article in 1981 by Bell, Ballas and Coleman. We now assess what has been learned about early events of lipid topogenesis, i.e. "lipid synthesis, the integration of lipids into membranes, and lipid translocation across membranes", in the 35 years since the publication of this important review. We highlight the recent elucidation of the X-ray structures of key membrane enzymes of glycerophospholipid synthesis, progress on identifying lipid scramblase proteins needed to equilibrate lipids across membranes, and new complexities in the subcellular location and membrane topology of phosphatidylinositol synthesis revealed through a comparison of two unicellular model eukaryotes. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Streptococcus mitis and S. oralis Lack a Requirement for CdsA, the Enzyme Required for Synthesis of Major Membrane Phospholipids in Bacteria.

Synthesis and integrity of the cytoplasmic membrane are fundamental to cellular life. Experimental evolution studies have hinted at unique physiology in the Gram-positive bacteria Streptococcus mitis and S. oralis These organisms commonly cause bacteremia and infectious endocarditis (IE) but are rarely investigated in mechanistic studies of physiology and evolution. Unlike in other Gram-positive pathogens, high-level (MIC ≥ 256 µg/ml) daptomycin resistance rapidly emerges in S. mitis and S. oralis after a single drug exposure. In this study, we found that inactivating mutations in cdsA are associated with high-level daptomycin resistance in S. mitis and S. oralis IE isolates. This is surprising given that cdsA is an essential gene for life in commonly studied model organisms. CdsA is the enzyme responsible for the synthesis of CDP-diacylglycerol, a key intermediate for the biosynthesis of all major phospholipids in prokaryotes and most anionic phospholipids in eukaryotes. Lipidomic analysis by liquid chromatography-mass spectrometry (LC-MS) showed that daptomycin-resistant strains have an accumulation of phosphatidic acid and completely lack phosphatidylglycerol and cardiolipin, two major anionic phospholipids in wild-type strains, confirming the loss of function of CdsA in the daptomycin-resistant strains. To our knowledge, these daptomycin-resistant streptococci represent the first model organisms whose viability is CdsA independent. The distinct membrane compositions resulting from the inactivation of cdsA not only provide novel insights into the mechanisms of daptomycin resistance but also offer unique opportunities to study the physiological functions of major anionic phospholipids in bacteria.

Topological organisation of the phosphatidylinositol 4,5-bisphosphate-phospholipase C resynthesis cycle: PITPs bridge the ER-PM gap.

Phospholipase C (PLC) is a receptor-regulated enzyme that hydrolyses phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the plasma membrane (PM) triggering three biochemical consequences, the generation of soluble inositol 1,4,5-trisphosphate (IP3), membrane-associated diacylglycerol (DG) and the consumption of PM PI(4,5)P2 Each of these three signals triggers multiple molecular processes impacting key cellular properties. The activation of PLC also triggers a sequence of biochemical reactions, collectively referred to as the PI(4,5)P2 cycle that culminates in the resynthesis of this lipid. The biochemical intermediates of this cycle and the enzymes that mediate these reactions are topologically distributed across two membrane compartments, the PM and the endoplasmic reticulum (ER). At the PM, the DG formed during PLC activation is rapidly converted into phosphatidic acid (PA) that needs to be transported to the ER where the machinery for its conversion into PI is localised. Conversely, PI from the ER needs to be rapidly transferred to the PM where it can be phosphorylated by lipid kinases to regenerate PI(4,5)P2 Thus, two lipid transport steps between membrane compartments through the cytosol are required for the replenishment of PI(4,5)P2 at the PM. Here, we review the topological constraints in the PI(4,5)P2 cycle and current understanding how these constraints are overcome during PLC signalling. In particular, we discuss the role of lipid transfer proteins in this process. Recent findings on the biochemical properties of a membrane-associated lipid transfer protein of the PITP family, PITPNM proteins (alternative name RdgBα/Nir proteins) that localise to membrane contact sites are discussed. Studies in both Drosophila and mammalian cells converge to provide a resolution to the conundrum of reciprocal transfer of PA and PI during PLC signalling.

The active site of yeast phosphatidylinositol synthase Pis1 is facing the cytosol.

Five yeast enzymes synthesizing various glycerophospholipids belong to the CDP-alcohol phosphatidyltransferase (CAPT) superfamily. They only share the so-called CAPT motif, which forms the active site of all these enzymes. Bioinformatic tools predict the CAPT motif of phosphatidylinositol synthase Pis1 as either ER luminal or cytosolic. To investigate the membrane topology of Pis1, unique cysteine residues were introduced into either native or a Cys-free form of Pis1 and their accessibility to the small, membrane permeating alkylating reagent N-ethylmaleimide (NEM) and mass tagged, non-permeating maleimides, in the presence and absence of non-denaturing detergents, was monitored. The results clearly point to a cytosolic location of the CAPT motif. Pis1 is highly sensitive to non-denaturing detergent, and low concentrations (0.05%) of dodecylmaltoside change the accessibility of single substituted Cys in the active site of an otherwise cysteine free version of Pis1. Slightly higher detergent concentrations inactivate the enzyme. Removal of the ER retrieval sequence from (wt) Pis1 enhances its activity, again suggesting an influence of the lipid environment. The central 84% of the Pis1 sequence can be aligned and fitted onto the 6 transmembrane helices of two recently crystallized archaeal members of the CAPT family. Results delineate the accessibility of different parts of Pis1 in their natural context and allow to critically evaluate the performance of different cysteine accessibility methods. Overall the results show that cytosolically made inositol and CDP-diacylglycerol can access the active site of the yeast PI synthase Pis1 from the cytosolic side and that Pis1 structure is strongly affected by mild detergents.

Biosynthesis and roles of phospholipids in mitochondrial fusion, division and mitophagy.

Mitochondria move, fuse and divide in cells. The dynamic behavior of mitochondria is central to the control of their structure and function. Three conserved mitochondrial dynamin-related GTPases (i.e., mitofusin, Opa1 and Drp1 in mammals and Fzo1, Mgm1 and Dnm1 in yeast) mediate mitochondrial fusion and division. In addition to dynamins, recent studies demonstrated that phospholipids in mitochondria also play key roles in mitochondrial dynamics by interacting with dynamin GTPases and by directly changing the biophysical properties of the mitochondrial membranes. Changes in phospholipid composition also promote mitophagy, which is a selective mitochondrial degradation process that is mechanistically coupled to mitochondrial division. In this review, we will discuss the biogenesis and function of mitochondrial phospholipids.

Distinct properties of the two isoforms of CDP-diacylglycerol synthase.

CDP-diacylglycerol synthases (CDS) are critical enzymes that catalyze the formation of CDP-diacylglycerol (CDP-DAG) from phosphatidic acid (PA). Here we show in vitro that the two isoforms of human CDS, CDS1 and CDS2, show different acyl chain specificities for its lipid substrate. CDS2 is selective for the acyl chains at the sn-1 and sn-2 positions, the most preferred species being 1-stearoyl-2-arachidonoyl-sn-phosphatidic acid. CDS1, conversely, shows no particular substrate specificity, displaying similar activities for almost all substrates tested. Additionally, we show that inhibition of CDS2 by phosphatidylinositol is also acyl chain-dependent, with the strongest inhibition seen with the 1-stearoyl-2-arachidonoyl species. CDS1 shows no acyl chain-dependent inhibition. Both CDS1 and CDS2 are inhibited by their anionic phospholipid end products, with phosphatidylinositol-(4,5)-bisphosphate showing the strongest inhibition. Our results indicate that CDS1 and CDS2 could create different CDP-DAG pools that may serve to enrich different phospholipid species with specific acyl chains.

Phosphatidylcholine biosynthesis and function in bacteria.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of the domain Bacteria. Usually, PC can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation (Pmt) pathway or the phosphatidylcholine synthase (Pcs) pathway. The three subsequent enzymatic methylations of phosphatidylethanolamine are performed by a single phospholipid N-methyltransferase in some bacteria whereas other bacteria possess multiple phospholipid N-methyltransferases each one performing one or several distinct methylation steps. Phosphatidylcholine synthase condenses choline directly with CDP-diacylglycerol to form CMP and PC. Like in eukaryotes, bacterial PC also functions as a biosynthetic intermediate during the formation of other biomolecules such as choline, diacylglycerol, or diacylglycerol-based phosphorus-free membrane lipids. Bacterial PC may serve as a specific recognition molecule but it affects the physicochemical properties of bacterial membranes as well. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

CDP-DAG synthesis by peripheral membrane-bound Tam41-type enzymes.

Cytidine diphosphate diacylglycerol (CDP-DAG) is a critical intermediate that is converted to multiple phospholipids in prokaryotes and eukaryotes. In budding yeast, CDP-DAG synthesis from cytidine triphosphate (CTP) and phosphatidic acid (PA) is catalyzed by the membrane-integrated protein Cds1 in the endoplasmic reticulum and the peripheral membrane-bound protein Tam41 in mitochondria. Although a recent study revealed that the fission yeast SpTam41 consists of a nucleotidyltransferase domain and a winged helix domain, forming an active-site pocket for CTP binding between the two domains together with a C-terminal amphipathic helix for membrane association, how CTP and Mg 2+, a most-favoured divalent cation, are accommodated with PA remains obscure. A more recent report by Kimura et al. (J. Biochem. 2022; 171:429-441) solved the crystal structure of FbTam41, a functional ortholog from a Firmicutes bacterium, with CTP-Mg 2+, successfully providing a detailed molecular view of CDP-DAG synthesis. In this commentary, our current understanding of Tam41-mediated reaction is discussed.

Coordinated upregulation of two CDP-diacylglycerol synthases, YnbB and CdsA, is essential for cell growth and membrane protein export in the cold.

YnbB is a paralogue of CdsA, a CDP-diacylglycerol synthase. While the cdsA gene is essential, the ynbB gene is dispensable. So far, no phenotype of ynbB knockout has been observed. We found that a ynbB knockout strain acquired cold-sensitivity on growth under CdsA-limited conditions. We found that MPIase, a glycolipid involved in protein export, is cold-upregulated to facilitate protein export in the cold, by increasing the mRNA levels of not only CdsA but also that of YnbB. Under non-permissive conditions, phospholipid biosynthesis proceeded normally, however, MPIase upregulation was inhibited with accumulation of precursors of membrane and secretory proteins such as M13 procoat and proOmpA, indicating that YnbB is dedicated to MPIase biosynthesis, complementing the CdsA function.

RNA Sequencing of Intestinal Enterocytes Pre- and Post-Roux-en-Y Gastric Bypass Reveals Alteration in Gene Expression Related to Enterocyte Differentiation, Restitution, and Obesity with Regulation by Schlafen 12.

BACKGROUND: The intestinal lining renews itself in a programmed fashion that can be affected by adaptation to surgical procedures such as gastric bypass. METHODS: To assess adaptive mechanisms in the human intestine after Roux-en-Y gastric bypass (RYGB), we biopsied proximal jejunum at the anastomotic site during surgery to establish a baseline and endoscopically re-biopsied the same area 6-9 months after bypass for comparison. Laser microdissection was performed on pre- and post-RYGB biopsies to isolate enterocytes for RNA sequencing. RESULTS: RNA sequencing suggested significant decreases in gene expression associated with G2/M DNA damage checkpoint regulation of the cell cycle pathway, and significant increases in gene expression associated with the CDP-diacylglycerol biosynthesis pathway TCA cycle II pathway, and pyrimidine ribonucleotide salvage pathway after RYGB. Since Schlafen 12 (SLFN12) is reported to influence enterocytic differentiation, we stained mucosa for SLFN12 and observed increased SLFN12 immunoreactivity. We investigated SLFN12 overexpression in HIEC-6 and FHs 74 Int intestinal epithelial cells and observed similar increased expression of the following genes that were also increased after RYGB: HES2, CARD9, SLC19A2, FBXW7, STXBP4, SPARCL1, and UTS. CONCLUSIONS: Our data suggest that RYGB promotes SLFN12 protein expression, cellular mechanism and replication pathways, and genes associated with differentiation and restitution (HES2, CARD9, SLC19A2), as well as obesity-related genes (FBXW7, STXBP4, SPARCL1, UTS).

The Anticancer Drug Bleomycin Shows Potent Antifungal Activity by Altering Phospholipid Biosynthesis.

Invasive fungal infections are difficult to treat with limited drug options, mainly because fungi are eukaryotes and share many cellular mechanisms with the human host. Most current antifungal drugs are either fungistatic or highly toxic. Therefore, there is a critical need to identify important fungal specific drug targets for novel antifungal development. Numerous studies have shown the fungal phosphatidylserine (PS) biosynthetic pathway to be a potential target. It is synthesized from CDP-diacylglycerol and serine, and the fungal PS synthesis route is different from that in mammalian cells, in which preexisting phospholipids are utilized to produce PS in a base-exchange reaction. In this study, we utilized a Saccharomyces cerevisiae heterologous expression system to screen for inhibitors of Cryptococcus PS synthase Cho1, a fungi-specific enzyme essential for cell viability. We identified an anticancer compound, bleomycin, as a positive candidate that showed a phospholipid-dependent antifungal effect. Its inhibition on fungal growth can be restored by ethanolamine supplementation. Further exploration of the mechanism of action showed that bleomycin treatment damaged the mitochondrial membrane in yeast cells, leading to increased generation of reactive oxygen species (ROS), whereas supplementation with ethanolamine helped to rescue bleomycin-induced damage. Our results indicate that bleomycin does not specifically inhibit the PS synthase enzyme; however, it may affect phospholipid biosynthesis through disruption of mitochondrial function, namely, the synthesis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), which helps cells maintain membrane composition and functionality. IMPORTANCE Invasive fungal pathogens cause significant morbidity and mortality, with over 1.5 million deaths annually. Because fungi are eukaryotes that share much of their cellular machinery with the host, our armamentarium of antifungal drugs is highly limited, with only three classes of antifungal drugs available. Drug toxicity and emerging resistance have limited their use. Hence, targeting fungi-specific enzymes that are important for fungal survival, growth, or virulence poses a strategy for novel antifungal development. In this study, we developed a heterologous expression system to screen for chemical compounds with activity against Cryptococcus phosphatidylserine synthase, Cho1, a fungi-specific enzyme that is essential for viability in C. neoformans. We confirmed the feasibility of this screen method and identified a previously unexplored role of the anticancer compound bleomycin in disrupting mitochondrial function and inhibiting phospholipid synthesis.

CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum.

Phosphatidylinositol (PI) is essential for numerous cell functions and is generated by consecutive reactions catalyzed by CDP-diacylglycerol synthase (CDS) and PI synthase. In this study, we investigated the membrane organization of CDP-diacylglycerol synthesis. Separation of mildly disrupted A431 cell membranes on sucrose density gradients revealed cofractionation of CDS and PI synthase activities with cholesterol-poor, endoplasmic reticulum (ER) membranes and partial overlap with plasma membrane caveolae. Cofractionation of CDS activity with caveolae was also observed when low-buoyant density caveolin-enriched membranes were prepared using a carbonate-based method. However, immunoisolation studies determined that CDS activity localized to ER membrane fragments containing calnexin and type III inositol (1,4,5)-trisphosphate receptors but not to caveolae. Membrane fragmentation in neutral pH buffer established that CDP-diacylglycerol and PI syntheses were restricted to a subfraction of the calnexin-positive ER. In contrast to lipid rafts enriched for caveolin, cholesterol, and GM1 glycosphingolipids, the CDS-containing ER membranes were detergent soluble. In cell imaging studies, CDS and calnexin colocalized in microdomain-sized patches of the ER and also unexpectedly at the plasma membrane. These results demonstrate that key components of the PI pathway localize to nonraft, phospholipid-synthesizing microdomains of the ER that are also enriched for calnexin.

AGPAT2 interaction with CDP-diacylglycerol synthases promotes the flux of fatty acids through the CDP-diacylglycerol pathway.

AGPATs (1-acylglycerol-3-phosphate O-acyltransferases) catalyze the acylation of lysophosphatidic acid to form phosphatidic acid (PA), a key step in the glycerol-3-phosphate pathway for the synthesis of phospholipids and triacylglycerols. AGPAT2 is the only AGPAT isoform whose loss-of-function mutations cause a severe form of human congenital generalized lipodystrophy. Paradoxically, AGPAT2 deficiency is known to dramatically increase the level of its product, PA. Here, we find that AGPAT2 deficiency impairs the biogenesis and growth of lipid droplets. We show that AGPAT2 deficiency compromises the stability of CDP-diacylglycerol (DAG) synthases (CDSs) and decreases CDS activity in both cell lines and mouse liver. Moreover, AGPAT2 and CDS1/2 can directly interact and form functional complexes, which promote the metabolism of PA along the CDP-DAG pathway of phospholipid synthesis. Our results provide key insights into the regulation of metabolic flux during lipid synthesis and suggest substrate channelling at a major branch point of the glycerol-3-phosphate pathway.

Cardiolipin molecular species with shorter acyl chains accumulate in Saccharomyces cerevisiae mutants lacking the acyl coenzyme A-binding protein Acb1p: new insights into acyl chain remodeling of cardiolipin.

The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition. The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content. Despite the presence of an intact CL remodeling system, acyl chains shorter than 16 carbon atoms (C16) were found to accumulate in CL in cells lacking Acb1p. Further experiments revealed that Taz1p, a key CL remodeling enzyme, was not responsible for the shortening of CL in the absence of Acb1p. This left de novo CL synthesis as the only possible source of acyl chains shorter than C16 in CL. Experiments in which the substrate specificity of the yeast cardiolipin synthase Crd1p and the acyl chain composition of individual short CL species were investigated, indicated that both CL precursors (i.e. phosphatidylglycerol and CDP-diacylglycerol) contribute to comparable extents to the shorter acyl chains in CL in acb1 mutants. Based on the findings, we conclude that the fatty acid composition of mature CL in yeast is governed by the substrate specificity of the CL-specific lipase Cld1p and the fatty acid composition of the Taz1p substrates.

Antidepressant stimulation of CDP-diacylglycerol synthesis does not require monoamine reuptake inhibition.

BACKGROUND: Recent studies demonstrate that diverse antidepressant agents increase the cellular production of the nucleolipid CDP-diacylglycerol and its synthetic derivative, phosphatidylinositol, in depression-relevant brain regions. Pharmacological blockade of downstream phosphatidylinositide signaling disrupted the behavioral antidepressant effects in rats. However, the nucleolipid responses were resistant to inhibition by serotonin receptor antagonists, even though antidepressant-facilitated inositol phosphate accumulation was blocked. Could the neurochemical effects be additional to the known effects of the drugs on monoamine transmitter transporters? To examine this question, we tested selected agents in serotonin-depleted brain tissues, in PC12 cells devoid of serotonin transporters, and on the enzymatic activity of brain CDP-diacylglycerol synthase - the enzyme that catalyzes the physiological synthesis of CDP-diacylglycerol. RESULTS: Imipramine, paroxetine, and maprotiline concentration-dependently increased the levels of CDP-diacylglycerol and phosphatidylinositides in PC12 cells. Rat forebrain tissues depleted of serotonin by pretreatment with p-chlorophenylalanine showed responses to imipramine or maprotiline that were comparable to respective responses from saline-injected controls. With fluoxetine, nucleolipid responses in the serotonin-depleted cortex or hippocampus were significantly reduced, but not abolished. Each drug significantly increased the enzymatic activity of CDP-diacylglycerol synthase following incubations with cortical or hippocampal brain tissues. CONCLUSION: Antidepressants probably induce the activity of CDP-diacylglycerol synthase leading to increased production of CDP-diacylglycerol and facilitation of downstream phosphatidylinositol synthesis. Phosphatidylinositol-dependent signaling cascades exert diverse salutary effects in neural cells, including facilitation of BDNF signaling and neurogenesis. Hence, the present findings should strengthen the notion that modulation of brain phosphatidylinositide signaling probably contributes to the molecular mechanism of diverse antidepressant medications.

CDP-diacylglycerol, a critical intermediate in lipid metabolism.

The roles of lipids expand beyond the basic building blocks of biological membranes. In addition to forming complex and dynamic barriers, the thousands of different lipid species in the cell contribute to essentially all the processes of life. Specific lipids are increasingly identified in cellular processes, including signal transduction, membrane trafficking, metabolic control and protein regulation. Tight control of their synthesis and degradation is essential for homeostasis. Most of the lipid molecules in the cell originate from a small number of critical intermediates. Thus, regulating the synthesis of intermediates is essential for lipid homeostasis and optimal biological functions. Cytidine diphosphate diacylglycerol (CDP-DAG) is an intermediate which occupies a branch point in lipid metabolism. CDP-DAG is incorporated into different synthetic pathways to form distinct phospholipid end-products depending on its location of synthesis. Identification and characterization of CDP-DAG synthases which catalyze the synthesis of CDP-DAG has been hampered by difficulties extracting these membrane-bound enzymes for purification. Recent developments have clarified the cellular localization of the CDP-DAG synthases and identified a new unrelated CDP-DAG synthase enzyme. These findings have contributed to a deeper understanding of the extensive synthetic and signaling networks stemming from this key lipid intermediate.