Phosphatidylserine Synthesis Research Articles

Effect of Cobalamin Deficiency on the Biosynthesis of Phosphatidylcholine in Euglena gracilis

Euglena gracilis requires cobalamin (Cbl) as an essential growth factor. Phosphatidylcholine (PC) synthesis was greatly reduced by Cbl deficiency. Rapid cell division occurred after Cbl was replenished, and PC was actively synthesized during the cell divisions. When the deficient cells were given methionine (a precursor for the choline moiety), active synthesis of PC occurred even without the Cbl supplement, although cell division was not induced. As methionine synthase in Euglena requires methylcobalamin as a coenzyme, decrease in methionine synthesis may account for reduced PC synthesis under Cbl‐deficient conditions. Phosphatidyleth‐anolamine and phosphatidylserine synthesis were also suppressed, commensurate with decrease of PC synthesis, under Cbl deficiency, even though Cbl is not thought to participate in their synthesis. In contrast, a lot of triglyceride and wax ester accumulated in Cbl‐deficient cells. Moreover, Cbl depletion altered fatty acid composition, notably due to increased proportion of odd‐numbered fatty acids

Effect of membrane potential on phosphatidylserine synthesis and calcium movements in control and CD3-activated Jurkat T cells

CD3 mAb induced calcium movements are unaffected by hyperpolarization of the membrane in Jurkat T cells treated with valinomycin. By contrast, the CD3 induced Ca 2+ influx was impaired by depolarization of the membrane with either gramicidin or by equimolar substitution of KCl for NaCl in the medium. In depolarized cells, the synthesis of phosphatidylserine was strongly diminished as a result of impaired transport of the [ 3H]serine substrate. In depolarized cells, the CD3-induced release of Ca 2+ from intracellular stores (endoplasmic reticulum) was unaffected. Emptying of the Ca 2+ stores by CD3 was shown by the lack of effect of additional treatment of the cells with the Ca 2+ ionophore, ionomycin. The empty status of the calcium stores was also confirmed by measurements of phosphatyidylserine synthesis through the Ca 2+-dependent base exchange enzyme system that was found to be significantly decreased despite the low amount synthesized in the presence of a defective [ 3H]serine transport in depolarized cells.

Submicromolar La3+ concentrations block the calcium release-activated channel, and impair CD69 and CD25 expression in CD3- or thapsigargin-activated Jurkat cells.

The calcium release-activated channel (CRAC) opened in Jurkat cells activated either with CD3 monoclonal antibody or the endoplasmic reticulum Ca2(+)-ATPase blocker, thapsigargin, is blocked by La3+ with an IC50 of 20 nM. Similarly, the entry of Mn2+, used as a surrogate for Ca2+, is also blocked by submicromolar La3+ concentrations. La3+ seems to play its role simply by plugging the CRAC because this ion does not penetrate the cells, as demonstrated by chelation experiments with EGTA. Blocking the Ca2+ influx in activated Jurkat cells results in a lack of expression of CD25, a chain of the interleukin-2 receptor and of CD69, a marker of T-cell activation. By contrast, the very early steps of the T-cell signalling pathway such as the release of Ca2+ from intracellular stores and the subsequent inhibition of phosphatidylserine synthesis are not affected by La3+.

A regulatory mechanism for the balanced synthesis of membrane phospholipid species in Escherichia coli.

The mechanism that assures the balanced synthesis of zwitterionic (phosphatidylethanolamine) and acidic phospholipids (phosphatidylglycerol and cardiolipin) in Escherichia coli has been examined by genetically manipulating the two enzymes at the biosynthetic branch point, i.e., phosphatidylglycerophosphate synthase, encoded by pgsA, and phosphatidylserine synthase, encoded by pssA. A mutant in which the most part of the pssA gene was replaced with a drug resistance gene lacked phosphatidylserine synthase and phosphatidylethanolamine and required divalent metal ions for growth, as did a previously reported insertion-inactivated pssA mutant. When this mutant harbored a plasmid containing a Bacillus subtilis gene that encodes membrane-bound phosphatidylserine synthase, the phosphatidylethanolamine content was dependent on its activity, in contrast to that with the soluble E. coli counterpart. A defective mutation, pgsA3, caused reductions not only in acidic-phospholipid synthesis but also in phosphatidylethanolamine synthesis, despite the normal level of phosphatidylserine synthase activity. These results, together with previous observations, indicate that phosphatidylserine synthesis requires the membrane-associated form of phosphatidylserine synthase, which is related to the membrane-levels of acidic phospholipids, thus yielding balanced compositions of zwitterionic and acidic phospholipids.

Oleylamine and stearylamine increase phosphatidylserine synthesis in T cells by synergy with calcium ions

Oleylamine and stearylamine, two cationic amphiphilic drugs, strongly increase phosphatidylserine synthesis in human T cells. The two compounds had little effect on either phosphatidylcholine or phosphatidylethanolamine synthesis. A decrease in the formation of phosphatidylethanolamine through decarboxylation of phosphatidylserine was observed, but this effect is only marginally involved in increased phosphatidylserine synthesis. The high incorporation of [3H]-serine into phosphatidylserine is a protein kinase C independent process and is due to a synergy of either oleyamine or stearylamine with calcium ions.

Calmodulin, a Junction between Two Independent Immunosuppressive Pathways in Jurkat T Cells

Calmodulin (CaM) antagonists chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide HCl inhibit Jurkat T cell activation, as monitored by measuring interleukin-2 synthesis in cells treated by a combination of CD3 monoclonal antibody and phorbol myristate acetate. T cell activation with CD3 monoclonal antibody is accompanied by a decreased synthesis of phosphatidylserine due to the release of Ca2+ from the endoplasmic reticulum. CaM antagonists reverse the phosphatidylserine (PtdSer) inhibition induced by CD3. This increase of PtdSer synthesis was observed in the absence of any modification of CD3-induced Ca2+ movements. Both in intact cells and in an acellular system, the increase of PtdSer synthesis induced by CaM antagonists was abolished in the presence of EGTA, indicating that the base exchange enzyme system responsible for PtdSer synthesis is regulated by CaM provided that Ca2+ is present. By contrast, cyclosporin A that inhibits T cell activation through the interaction of cyclophilin-cyclosporin A complexes with the calmodulin-activated phosphatase, calcineurin, had no effect on PtdSer synthesis. Calmodulin thus appears as a junction leading to at least two independent pathways of regulation of T cell activation, one involving the calcineurin phosphatase and the other the base exchange enzyme system responsible for PtdSer synthesis.

Inhibition by Glucose and Deoxyglucose of Phosphatidylserine Synthesis in Ehrlich Ascites Tumor Cells: A Possible Relation to the Crabtree Effect and Depletion of Endoplasmic Reticulum Ca 2+ Stores

Incorporation of [ 14C]serine into phosphatidylserine by the base exchange reaction in Ehrlich ascites tumor cells is inhibited by 30% by glucose and by 60% by 2-deoxyglucose (10 mM each). The inhibition by thapsigargin (0.2 μM) amounts to 80%. This inhibition is interpreted as being due to depletion of calcium stores in the endoplasmic reticulum and is compatible with the previous observation [Teplova, V.V., Bogucka, K., Czyż, A., Evtodienko, Yu. V., Duszyński, J., and Wojtczak, L. (1993) Biochem. Biophys. Res. Commun. 196, 1148-1154] that glucose and deoxyglucose elicit an increase of cytoplasmic [Ca 2+] at the expense of intracellular Ca 2+ stores and with the finding [Barańska, J. (1989) FEES Lett. 256, 33-37] that the base exchange reaction requires high concentration of Ca 2+ within the endoplasmic reticulum lumen.

Loss of phosphatidylserine synthesis results in aberrant solute sequestration and vacuolar morphology in Saccharomyces cerevisiae

Null cho1 mutants of Saccharomyces cerevisiae are incapable of phosphatidyl-serine synthesis. They were more susceptible than wild-type strains to 100 mM CaCl 2, 3 mM ZnCl 2 or 1 mM MnCl 2, but not to MgCl 2 nor KCl. They were also susceptible to high concentrations of basic amino acids, l-lysine and l-arginine, and to an l-lysine analog, S-2-aminoethyl- l-cysteine. Their vacuolar pools of amino acids, especially those of basic ones, were decreased. Pigmentation of chol ade2 double mutants was obscured and vacuoles of cho1 mutants were considerably fragmented. These indicate that phosphatidylserine plays vital roles in normal vacuolar function and morphogenesis.

The selective use of stearoyl-polyunsaturated molecular species of phosphatidylcholine and phosphatidylethanolamine for the synthesis of phosphatidylserine

In rat liver microsomes, [ 3H]serine was incorporated primarily into the two most abundant molecular species of microsomal phosphatidylserine (PS), 18:0 20:4 and 18:0 22:6 , by Ca 2+-dependent base exchange. The pattern of PS molecular species synthesized was very similar to the species composition of PS and markedly different from the species composition of either microsomal precursor, phosphatidylcholine (PC) or phosphatidylethanolamine (PE). The data indicated that the enrichment of rat liver PS in mainly three fatty acids — stearic, arachidonic, and docosahexaenoic acids, occurred by (1) the preference by PS synthases for the stearoyl-polyunsaturated molecular species, 18:0 20:4 and 18:0 22:6 , of PC and PE and (2) a discrimination against the use of the palmitoyl-polyunsaturated species, 16:0 20:4 and 16:0 22:6 , and the stearoyl-diunsaturated species, 18:0 18:2 . The preferential use of the two species of PC and PE, based on their acyl chain content and not on their relative abundance, demonstrates that an individual molecular species can be selected out of the total pool for a defined function.

Regulation of T Cell Activation by Cytochrome P450 Inhibitors

Cytochrome P450 inhibitors such as α-naphthoflavone, the imidazole antimycotics, econazole, clotrimazole, and miconazole and the lipoxygenase inhibitors, nordihydroguaiaretic acid and eicosatetraynoic acid, strongly diminished CD3-induced human T cell proliferation. This effect is due to a marked inhibition of IL-2 synthesis. The mechanism leading to the in vitro immunosuppressive effect of cytochrome P450 inhibitors appears to be a consequence of a blockade of Ca 2+ influx induced by CD3 mAb. The drugs tested did not interfere with the release of Ca 2+ from the endoplasmic reticulum as demonstrated by Ca 2+ measurements in the presence of the Ca 2+ chelator, EGTA. Measurements of CD3-induced changes in phosphatidylserine synthesis, which reflect the status (full/empty) of intracellular Ca 2+ stores confirmed that CD3 mAb remained able to empty the Ca 2+ stores either alone or in the presence of cytochrome P450 inhibitors. Altogether, our results support the hypothesis that a cytochrome P450 regulates Ca 2+ influx in T cells and are consistent with the proposal that impairing Ca 2+ influx leads to the inhibition of IL-2 synthesis and subsequent T cell proliferation.

Modulation of phosphatidylserine synthesis by a muscarinic receptor occupancy in human neuroblastoma cell line LA-N-1.

The incorporation of [3H]serine into lipids, water-soluble metabolites and proteins by the human neuroblastoma cell line LA-N-1 exposed to oxotremorine-M, a muscarinic agonist, was investigated. Oxotremorine-M increased the incorporation of this labelled precursor into phosphatidylserine and proteins in a concentration-dependent manner, with the maximal stimulation at 250 microM. This activation was blunted by 100 microM atropine. There were no detectable changes of the radioactivity in the water-soluble metabolites. Acetylcholine, another muscarinic agonist, slightly decreased the serine incorporation into lipids, but did not affect the protein or water-soluble compartments. Several other muscarinic agonists, including 250 microM pilocarpine, 100 microM McN-A-343 and 1 mM carbachol, did not effect these [3H]serine incorporations. Preincubation of cells with 1 mM oxotremorine M, or 1 mM carbachol, or 1 mM McN-A-343, for 4 h prevented the oxotremorine-M-induced increase of serine incorporation. These observations are consistent with the oxotremorine-M action being mediated by muscarinic-receptor occupancy. The G-protein inhibitor guanosine 5'-[beta-thio]diphosphate (1 mM) and the G-protein activators, guanosine 5'-[gamma-thio]triphosphate (100 microM) and A1F3, prevented the oxotremorine stimulation. The muscarinic agonists, 250 microM oxotremorine-M, 1 mM carbamoylcholine and 500 microM acetylcholine, triggered the accumulation of inositol mono- and di-phosphates by cells that had been prelabelled with myo-[3H]inositol, and this phospholipase C activation was blunted by 100 microM atropine. The protein kinase C inhibitor H7 prevented the oxotremorine-M stimulation of serine incorporation. Over-night exposure of LA-N-1 cells to 100 nM phorbol 12-myristate 13-acetate resulted in a decrease of cytosolic protein kinase C activity, and prevented the oxotremorine-M stimulation of serine incorporation. Neither oxotremorine-M nor acetylcholine caused a redistribution of protein kinase C activity between the cytosol and membrane compartments. In addition, oxotremorine-M did not activate phospholipase D of the LA-N-1 cells.

Phenylarsine oxide and phorbol myristate acetate inhibit the CD3-induced rise of cytosolic Ca2+ in Jurkat cells by refilling internal Ca2+ stores

Phenylarsine oxide (PAO), an inhibitor of tyrosine phosphatases, has been found to inhibit the early elevation in cytosolic Ca2+ concentration ([Ca2+]i), related to the CD3 activation pathway in Jurkat T cells. This inhibition was dose-dependent, consistent with previously reported effects of PAO on tyrosine phosphatases, and reversed by dimercaptopropanol. By contrast, okadaic acid, an inhibitor of serine/threonine phosphatases, had no effect on CD3-induced Ca2+ flux. PAO was compared with phorbol 12-myristate 13-acetate (PMA), which caused a similar, although less potent, inhibition as previously described. The two reagents produced additive inhibition of the CD3-induced [Ca2+]i rise, but did not affect thapsigargin- or ionomycin-driven Ca2+ flux in Jurkat cells. PAO and PMA prevented cells from complete depletion of intracellular Ca2+ stores by an anti-CD3 monoclonal antibody (mAb) and restored, at least partially, the ionomycin-sensitive pool, when added after anti-CD3 mAb. Moreover, the CD3-induced inhibition of phosphatidylserine synthesis, due to depletion of internal Ca2+ stores, is reversed by PAO and PMA. Anti-phosphotyrosine immunoblot analysis show that these effects cannot be accounted for by an inhibition of CD3-induced tyrosine phosphorylations. We propose that PAO and, to a lesser extent, PMA allow the refilling of internal compartments by Ca2+, which consequently abrogates a capacitative entry of external Ca2+.

Effect of thimerosal on cytosolic calcium and phosphatidylserine synthesis in Jurkat T cells

1. 1. We investigated the effect of the thiol reagent, thimerosal on calcium movements in the Jurkat T cell line. 2. 2. Thimerosal induced a rise in cytosolic Ca 2+ concentration due both to a release of Ca 2+ from intracellular stores and a Ca 2+ influx. 3. 3. Thimerosal, released Ca 2+ from the same intracellular stores than CD3 mAb and ionomycin. 4. 4. Emptying the Ca 2+ intracellular stores was accompanied by a marked decrease of phosphatidylserine synthesis indicating that phosphatidylserine synthesis occurs within or close to the endoplasmic reticulum Ca 2+-stores as previously described in CD3-, ionomycin- or Ca 2+-ATPase inhibitor-treated lymphocytes.

Involvement of mitochondrial contact sites in the subcellular compartmentalization of phospholipid biosynthetic enzymes.

The concerted synthesis of phospholipids derived from serine involving two microsomal enzymes (phosphatidylserine synthase and phosphatidylethanolamine N-methyltransferase) and a mitochondrial one (phosphatidylserine decarboxylase) occurs in reconstituted cell-free systems. Subfractionation of crude mitochondria after swelling and separating on a sucrose density gradient resulted in the isolation of two contact site-enriched fractions from total outer membranes and inner membranes, respectively. Estimation of marker enzyme activities shows a high recovery of glucose-6-phosphate phosphatase (a marker for the endoplasmic reticulum) associated with contact site-enriched fractions. Accordingly, the linked synthesis of phosphatidylserine, phosphatidylethanolamine, and at a lesser extent phosphatidylcholine can occur. This biosynthetic pathway was absent from purified contact site-enriched fractions correlative with the absence of glucose-6-phosphate phosphatase activity. Reconstitution experiments, including contact site-enriched fractions incubated with endoplasmic reticulum-rich fraction, led to the restoration of the linked synthesis of phospholipids, thereby demonstrating that a reversible association between these two fractions can occur. These functional interactions between the endoplasmic reticulum and mitochondria are confirmed at the ultrastructural level using either chemical or physical fixation before resin embedding. These results show that the interorganelle trafficking of lipids may involve only highly specialized microdomains of both membranes, thereby allowing the maintenance of a specific lipid composition and distribution within membranes.

Effect of the Ionophore A23187, Thapsigargin, Caffeine and Heparin on Phosphatidylserine Synthesis in Rat Liver Microsomal Fraction

It has been shown that the incorporation of [ 14C]serine into phosphatidylserine in rat liver microsomal fractions is distinctly stimulated by ATP and Mg 2+ at low (10 μM) Ca 2+ concentration. This stimulation occurred in untreated microsomes, but did not occur in fully disrupted microsomal vesicles. The stimulatory effect of ATP and Mg 2+ on phosphatidylserine synthesis was reduced by calcium ionophore A23187 and by thapsigargin, which is a specific blocker of Ca 2+-ATPase. It was also diminished by caffeine and enhanced by heparin, agents known to modulate Ca 2+ release by receptor channels. It is therefore postulated that the synthesis of phosphatidylserine occurs on the luminal side of the endoplasmic reticulum and can be regulated by events responsible for Ca 2+ release and entry into these structures.

Ca2+-ATPase Inhibitors Induce Interleukin-2 Synthesis and T Cell Proliferation

In Jurkat cells, the three Ca2+-ATPase blockers, thapsigargin, cyclopiazonic acid, and di-teributylhydroquinone (DtBuHQ) induced both a release of Ca2+ from intracellular stores and a Ca2+ influx. In contrast to CD3 mAb, the Ca2+-ATPase inhibitors did not induce the formation of inositol trisphosphate from the hydrolysis of phosphatidylinositides. Emptying intracellular Ca2+ stores was accompanied by a decrease of phosphatidylserine (PtdSer) synthesis as previously observed in PHA- or CD3 mAb-treated Jurkat cells. In the presence of a phorbol ester able to activate protein kinase C, TPA, the three Ca2+-ATPase inhibitors induced Jurkat cells to synthesize large amounts of interleukin-2 demonstrating that early signal transduction mechanisms can be bypassed by Ca2+-ATPase inhibitors. In purified human peripheral blood T lymphocytes, the same inhibitors induced moderate if any cytosolic Ca2+ rise, in the absence of external calcium. Nevertheless analysis of PtdSer synthesis suggested that intracellular stores were efficiently depleted by DtBuHQ and cyclopiazonic acid but not by thapsigargin. In contrast, the three compounds induced similar Ca2+ influx. However, in the presence of TPA, cyclopiazonic acid and DtBuHQ induce highly purified T cells to proliferate while thapsigargin did not, suggesting that the status of internal Ca2+ store may have a decisive role in T cell activation.

The ATP-dependent translocation of phosphatidylserine to the mitochondria is a process that is restricted to the autologous organelle.

The general properties of phosphatidylserine synthesis and translocation to the inner mitochondrial membrane which occur in both intact and permeabilized cells are now shown to occur in cells that are disrupted by shearing. In disrupted cells the synthesis of phosphatidylserine requires ATP and is blocked by preventing Ca2+ sequestration. The translocation of nascent phosphatidylserine to the mitochondria also requires ATP in disrupted cells. Using disrupted cells it is possible to test whether the translocation of nascent phosphatidylserine to the mitochondria is restricted to autologous mitochondrial acceptors or is possible with heterologous mitochondrial acceptors. To examine the nature of the transport process, a donor and acceptor population of cells were prepared. The donor cells were poisoned with hydroxylamine which irreversibly inactivates phosphatidylserine decarboxylase and will prevent the conversion of nascent phosphatidylserine to phosphatidylethanolamine. The acceptor cells were not treated with hydroxylamine so that any phosphatidylserine generated in the donor cell that reached the inner mitochondrial membrane of the acceptor cells would be decarboxylated to form phosphatidylethanolamine. Disrupted donor cells were pulse labeled with [3H]serine for 40 min to permit the synthesis of [3H] phosphatidylserine, and then further incorporation of radiolabel was arrested by chelating Ca2+. When the prelabeled disrupted donor cells were mixed with disrupted acceptor cells the latter failed to decarboxylate the nascent phosphatidylserine. In control experiments the addition of hydroxylamine-poisoned cells did not alter the transfer of nascent phosphatidylserine to autologous mitochondria present in unpoisoned cells. These results indicate that the ATP-dependent translocation of phosphatidylserine to the mitochondria is a process that is restricted to the autologous organelle.

Possible involvement of the base exchange enzymes in the phospholipid metabolism in LAN-2 cells

The incorporation of [14C] choline, [14C] ethanolamine, and [14C] serine by LAN-2 cells into their corresponding phospholipids was investigated in the presence or absence of TPA. The presence of TPA increased the amount of radioactivity incorporated into the phospholipids with a corresponding decrease in the amount of radioactivity in the cytosolic compartment compared to control cultures. There were no differences between TPA-exposed and control cells in the distribution of radioactivity in free choline, phosphorylcholine or CDP-choline of [14C] choline labeled cells. This indicates that the increased lipid labeling was not accompanied by enhanced labeling of the intermediates of the de novo pathway. These results suggest that a choline base exchange enzyme was stimulated in TPA exposed cells. In addition, the enhanced incorporation of serine by TPA into its corresponding phospholipid implies the stimulation of the serine base exchange enzyme which is responsible for phosphatidylserine synthesis in mammals. These observations suggest a

Imidazole antimycotics inhibitors of cytochrome P 450 increase phosphatidylserine synthesis similarly to K +-channel blockers in Jurkat T cells

The imidazole antimycotics, miconazole, econazole and triclomazole as well as α-naphtoflavone, known as powerful inhibitors of cytochrome P 450 and previously recognized as K + channel blockers are shown to be potent activators of the base exchange enzyme system responsible for the biosynthesis of phosphatidylserine in Jurkat T cells. The inhibition of CD3-induced Ca 2+ influx by antimycotics but not by K + channel blockers, demonstrated that the rise in phosphatidylserine synthesis caused by the two classes of drugs, was independent of Ca 2+ influx in the cells. In addition, we show that the action of these drugs on phosphatidylserine synthesis was not mimicked by modifications of membrane potential. The regulation of both K + channels and the base exchange enzyme system thus occurs through a similar (or common) pathway that is independent of Ca 2+-influx and membrane potential.

Phosphatidylserine synthesis in rat cerebral cortex: effects of hypoxia, hypocapnia and development.

Phosphatidylserine, which is necessary for protein kinase C activity, is synthesized in mammalian tissues by the Ca(2+)-dependent base exchange enzyme. The synthesis of phosphatidylserine is greater in slices or homogenates of rat cerebral cortex subjected to hypoxia by N2 treatment when compared with O2 plus 5% CO2. An intermediate effect was observed when the treatment was done with N2 plus 5% CO2. Incorporation rates were dependent on Ca2+ in Krebs-Henseleit Ringer bicarbonate medium, being greater with 2 mM Ca2+ than with the same medium prepared without Ca2+. The increase of phosphatidylserine synthesis, due to hypoxia, was, on the contrary, more evident in the medium lacking added Ca2+. Similar results were obtained with the homogenates. This suggests that elevation of intracellular Ca2+, caused by hypocapnia and hypoxia, may be responsible for the greater incorporation of serine into phosphatidylserine. In both cerebrocortical slices and homogenate, [14C]serine incorporation decreased with development both in O2 plus 5% CO2 and N2-treated preparations. However, in younger rats (14-18 days) hypoxia induced a lesser increase of phosphatidylserine than in 40 day old animals. We suggest that a regulatory mechanism for phosphatidylserine synthesis is established during development and that N2-treatment can increase phosphatidylserine synthesis by interfering with this regulatory mechanism.