Phosphatidic acid

SUMMARYHarpin HrpZ is one of the most abundant proteins secreted through the pathogenesis‐associated type III secretion system of the plant pathogen Pseudomonas syringae. HrpZ shows membrane‐binding and pore‐forming activities in vitro, suggesting that it could be targeted to the host cell plasma membrane. We studied the native molecular forms of HrpZ and found that it forms dimers and higher order oligomers. Lipid binding by HrpZ was tested with 15 different membrane lipids, with HrpZ interacting only with phosphatidic acid. Pore formation by HrpZ in artificial lipid vesicles was found to be dependent on the presence of phosphatidic acid. In addition, HrpZ was able to form pores in vesicles prepared from Arabidopsis thaliana plasma membrane, providing evidence for the suggested target of HrpZ in the host. To map the functions associated with HrpZ, we constructed a comprehensive series of deletions in the hrpZ gene derived from P. syringae pv. phaseolicola, and studied the mutant pr...

The purpose of this study was to determine whether phospholipids (PL) play a role in the adaptation to metabolic acidosis by toad urinary bladder epithelium. Toads were placed in an NH,CI acidosis for 48 hr. Quarter bladders were removed and incubated with [32P]orthophosphate or [3H]arachidonic acid for 1 hr at 25OC. PL were detected by thin layer chromatography, autoradiography, and quantitated by liquid scintillation counting or fractional amounts were determined from phosphate content and expressed as counts per minute per micromolar of total phosphate or as percentage of fraction of total PL. Incorporation of [3H]arachidonic acid into urinary bladder PL was measured in acidotic and normal toads. There was a higher rate of arachidonic acid incorporation into several PL in acidotic animals. Phosphatidic acid and phosphatidylserine fraction in acidosis was 37,705 f 6,821 and in normal bladders was 9,254 f 2,652 (P c 0.005); phosphatidylcholine fraction in acidotic toads was 80,462 f 16,862 and in normal bladders was 26,892 f 5,198 (P c 0.025); and the phosphatidylethanolamine (PE) fraction in acidotic was 48,665 f 10,998 and in normal animals was 17,441 f 3,905 (P c 0.025). 32P labeling revealed a higher rate of incorporation in bladders from acidotic toads compared with normal toads. In the acidotic bladders, the phosphatidic acid and phosphatidylserine fraction was 19,754 2 3,597 and in normal bladders was 12,980 f 1,394 (P < 0.05) and for PE acidotic bladders was 9,129 f 1,304 and in normal bladders was 3,285 f 416 (P c 0.001). Fractional PL (reported as percentage of fraction of total PL based on total lipid phosphorus) analysis in normal toads revealed phosphatidylinositol = 8.1 f 0.6% and PE = 27 f 1.2Y0, whereas for acidotic toads phosphatidylinositol = 11 f 0.6% and PE = 32 f 1.0% (P c 0.01 for both). Aldosterone, a known stimulator of acidification, had no effect on 32P incorporation into PL fractions of the bladder. The increase in PL turnover following induction of acidosis is consistent with increased membrane synthesis or turnover during metabolic acidosis and this may reflect an increased transport of vesicular H+-ATPase into the apical membrane or the result of a proliferation of acid-secreting mitochondria-rich cells or both.

During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain. Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transpor...

CIN85 (Cbl-interacting protein of 85 kDa) is an important molecule involved in receptor tyrosine kinase endocytosis. Here we report that through its positively charged C-terminus, CIN85 associates with a fusogenic lipid -phosphatidic acid. Its coiled-coil domain plays an important role in mediating this protein-lipid interaction. Deletion of the coiled-coil domain results in loss of membrane association, and reduced interaction with c-cbl, finally causing the blockage of epidermal growth factor receptor downregulation. In addition, a significant portion of CIN85 is located on the endosomal compartment and is related to endocytic cargo sorting, characterized by CIN85's localization on the "E class" compartment and EGF degradation blockage in CIN85 knockdown cells. Taken together, our results suggest that CIN85 may function as a scaffold molecule in both the internalization and endocytic cargo sorting processes through its association with the endosomal membrane.

Herein, an instrument free facile acid-base titrimetric methodology is reported for lysophosphatidic acid (LPA) measurement in serum and plasma samples for ovarian cancer detection. The concept is based on the titrimetric method in which alkaline solution was titrated with free fatty acid. Free fatty acid is generated due to action of the lysophospholipase to LPA. A phospholipid derivative known as LPA can function as a signaling molecule. A glycerol backbone serves as the foundation for phosphatidic acid, which also has bonds to an unsaturated fatty acid at carbon-1, a hydroxyl group at carbon-2, and a phosphate molecule at carbon-3. Free fatty acid and glycerol-3-phosphate are formed when LPA reacts with lysophospholipase. The formation of free fatty acid depends on the concentration of LPA. The standard graph of known concentrations of LPA, LPA spiked serum and LPA spiked plasma was plotted. The concentration of LPA in unknown serum and plasma were calculated from the standard graph. The limit of detection of LPA in spiked serum and plasma samples via titrimetric assay was calculated as 0.156 μmol/L. A patient's chance of survival may be outweighed by an early diagnosis of ovarian cancer.

DGKζ-deficiency in mice results in larger numbers of CD69-positive T cells in bone marrow, with enhanced expression of IFNγ and lytic enzymes. • DGKζ loss recapitulates many clinical aspects of human aplastic anemia, identifying a critical hub for immune system-dependent bone marrow failure.

Despite the fact that phosphatidic acid (PtdOH) has been implicated as a lipid second messenger for nearly a decade, its intracellular targets have remained unclear. We sought to investigate how an increase in the level of PtdOH could modulate phosphatidylinositol 4-phosphate 5-kinase (PIPkin), an enzyme involved in phosphatidylinositol 4,5-bisphosphate synthesis. Transfection of porcine aortic endothelial (PAE) cells with haemagglutinin (HA)-tagged type IK K PIPkin followed by immunofluorescence confocal microscopy revealed the enzyme to be localised to the plasma membrane. When the transfected PAE cells were stimulated with lyso-PtdOH, increased PIPkin activity was found to be associated with HA immunoprecipitates in an in vitro assay. This PIPkin activation was found to be greatly reduced by prior treatment of the cells with 1-butanol, thereby implicating phospholipase D (PLD) as the in vivo generator of PtdOH. In order to determine if the PtdOHdependent activation of type IK K PIPkin was dictated by a specific molecular composition of PtdOH, the wild type murine and porcine K K isoforms of diacylglycerol kinase (DGK) were individually co-transfected along with type IK K PIPkin. Under these conditions an increase in type IK K PIPkin lipid kinase activity was found in HA immunoprecipitates in an in vitro assay. No increases in lipid kinase activity were observed when type IK K PIPkin was co-transfected with either the human DGKO O isoform or a kinase-dead mutant of the murine DGKK K isoform. These results provide the first direct evidence for the unification of the production of saturated/monounsaturated PtdOH (through two different routes, PLD and DGK) and the in vivo activation of type IK K PIPkin by this lipid second messenger.

Diacylglycerol kinase (DGK) is suggested to attenuate diacylglycerol-induced cell responses through the phosphorylation of this second messenger to phosphatidic acid. Here, we show that DGK ␣ , an isoform highly expressed in T lymphocytes, translocates from cytosol to the plasma membrane in response to two different receptors known to elicit T cell activation responses: an ectopically expressed muscarinic type I receptor and the endogenous T cell receptor. Translocation in response to receptor stimulation is rapid, transient, and requires calcium and tyrosine kinase activation. DGK ␣ -mediated phosphatidic acid generation allows dissociation of the enzyme from the plasma membrane and return to the cytosol, as demonstrated using a pharmacological inhibitor and a catalytically inactive version of the enzyme. The NH 2 -terminal domain of the protein is shown to be responsible for receptor-induced translocation and phosphatidic acid-mediated membrane dissociation. After examining induction of the T cell activation marker CD69 in cells expressing a constitutively active form of the enzyme, we present evidence of the negative regulation that DGK ␣ exerts on diacylglycerolderived cell responses. This study is the first to describe DGK ␣ as an integral component of the signaling cascades that link plasma membrane receptors to nuclear responses.

Lipid binding sites and properties are compared in two families of hetero-oligomeric membrane protein complexes known to have similar functions in order to gain further understanding of the role of lipid in the function, dynamics, and assembly of these complexes. Using the crystal structure information for both complexes, lipid binding properties were compared for the cytochrome b 6 f and bc 1 complexes that function in photosynthetic and respiratory membrane energy transduction. Comparison of lipid and detergent binding sites in the b 6 f complex with those in bc 1 shows significant conservation of lipid positions. Seven lipid binding sites in the cyanobacterial b 6 f complex overlap three natural sites in the C. reinhardtii algal complex, and four sites in the yeast mitochondrial bc 1 complex. The specific identity of lipids is different in b 6 f and bc 1 complexes: b 6 f contains SDG, PG, MGDG, and DGDG, whereas cardiolipin, PE, and PA are present in the yeast bc 1 complex. The lipidic chlorophyll a and β-carotene in cyanobacterial b 6 f, as well as eicosane in C. reinhardtii, are unique to the photosynthetic b 6 f complex. The inferences of lipid binding sites and functions were supported by sequence, intermolecular distance, and Bfactor information on interacting lipid groups and coordinating amino acid residues. The lipid functions inferred in the b 6 f complex are: (i) substitution of a trans-membrane helix (TMH) by a lipid and chlorin ring; (ii) lipid and β-carotene connection of peripheral and core domains; (iii) stabilization of iron-sulfur protein TMH; (iv) n-side charge and polarity compensation; (v) βcarotene-mediated super-complex with photosystem I complex.

Glycerophospholipid (GPL) homeostasis in eukaryotic cells is thought to be maintained via biosynthesis, degradation and acyl chain remodeling. Here we provide evidence for an additional process termed "head-group remodeling" where other GPLs, when in excess, are rapidly converted to phosphatidylcholine and triacylglycerol. Mass spectrometric studies showed the formation of diacylglycerol, but not phosphatidic acid, from the exogenous GPL thus indicating that the first step is catalyzed by a phospholipase C-type enzyme. Consistently, triacylglycerol formation was significantly inhibited by the knock-down of several PLCs, but not phospholipase Ds. Second, we found that each exogenous GPL strongly inhibited the synthesis of the corresponding endogenous GPL class. Based on these and previous data we hypothesize how mammalian cells could coordinate the multiple processes contributing to GPL homeostasis in mammalian cells. In conclusion, this study provides the first evidence that head group remodeling plays an important role in GPL homeostasis in mammalian cells.

SummaryGlycerophospholipid (GPL) homeostasis in eukaryotic cells is thought to be maintained via biosynthesis, degradation and acyl chain remodeling. Here we provide evidence for an additional process termed “head-group remodeling” where other GPLs, when in excess, are rapidly converted to phosphatidylcholine and triacylglycerol. Mass spectrometric studies showed the formation of diacylglycerol, but not phosphatidic acid, from the exogenous GPL thus indicating that the first step is catalyzed by a phospholipase C-type enzyme. Consistently, triacylglycerol formation was significantly inhibited by the knock-down of several PLCs, but not phospholipase Ds. Second, we found that each exogenous GPL strongly inhibited the synthesis of the corresponding endogenous GPL class. Based on these and previous data we hypothesize how mammalian cells could coordinate the multiple processes contributing to GPL homeostasis in mammalian cells. In conclusion, this study provides the first evidence that ...

The diacylglycerol kinase (DGK) enzymes function as regulators of intracellular signaling by altering the levels of the second messengers, diacylglycerol and phosphatidic acid. The DGK delta and eta isozymes possess a common protein-protein interaction module known as a sterile alpha-motif (SAM) domain. In DGK delta, SAM domain self-association inhibits the translocation of DGK delta to the plasma membrane. Here we show that DGK delta SAM forms a polymer and map the polymeric interface by a genetic selection for soluble mutants. A crystal structure reveals that DGKSAM forms helical polymers through a head-to-tail interaction similar to other SAM domain polymers. Disrupting polymerization by polymer interface mutations constitutively localizes DGK delta to the plasma membrane. Thus, polymerization of DGK delta regulates the activity of the enzyme by sequestering DGK delta in an inactive cellular location. Regulation by dynamic polymerization is an emerging theme in signal transduction.

Hyperosmolarity is a controversial signal for renal cells. It can induce cell stress or differentiation and both require an active lipid metabolism. We showed that hyperosmolarity upregulates phospholipid (PL) de novo synthesis in renal cells. PL synthesis requires fatty acids (FA), usually stored as triglycerides (TAG). PL and TAG de novo synthesis utilize the same initial biosynthetic route: sn-glycerol 3P (G3P)  phosphatidic acid (PA)  diacylglycerol (DAG). In the present work, we evaluate how such pathway contributes to PL and TAG synthesis in renal cells subjected to hyperosmolarity. Our results show an increase in PA and DAG formation under hyperosmotic conditions; augmented DAG production, due to lipin enzyme activity, le ad to the increase of both TAG and PL. However, at early stages (24 and 48 h), most of the de novo synthesized DAG was directed to PL synthesis; longer treatments downregulated PL synthesis and the DAG formed was mainly driven to TAG synthesis. Hyperosmolarity induced ACC and FASN transcription which mediated FA de novo synthesis. New FA molecules were stored in TAG. Silencing experiments revealed that hyperosmotic-induction of lipin-1 and -2 was mediated by SREBP1. Interestingly, SREBP1 knockdown also dropped SREBP2, indicating a modulatory action between both isoforms. Impairing SREBP activity leads to a decline in TAG levels but not PL. Membrane homeostasis is maintained through the adequate PL synthesis and renewal and constitute a protective mechanism against hyperosmolarity. The present data reveal the relevance of TAG synthesis and storage for PL synthesis in renal cells.

Phosphatidic acid (PA) is a central precursor for membrane phospholipid biosynthesis. Lipin family is a Mg-dependent type I PA phosphatase, involved in de novo synthesis of neutral lipids and of phospholipids. The regulation of Lipin activity may govern the pathways by which these lipids are synthesized and control the cellular levels of important signaling lipids. On the other hand, the proto-oncoprotein c-Fos has an emerging role in glycerolipid synthesis regulation: by interacting with key synthetizing enzymes it is able to increase overall phopho- and glyco- lipid synthesis. We studied the Lipin 1β enzyme activity in a cell-free system using PA/Triton X-100 mixed micelles as substrate, analyzing it in the presence/absence of c-Fos. We found that Lipin 1β kcat increases around 40% in the presence of c-Fos, with no change in the Lipin 1β affinity for the PA/Triton X-100 mixed micelles. We also probed a physical interaction between both proteins. While the c-Fos domain involved in ...

This paper explores the way ceramide, sphingosine, ceramide 1-phosphate, and sphingosine 1-phosphate modulate the generation of second lipid messengers from phosphatidic acid in two experimental models of the central nervous system: in vertebrate rod outer segments prepared from dark-adapted retinas as well as in rod outer segments prepared from light-adapted retinas and in rat cerebral cortex synaptosomes under physiological aging conditions. Particular attention is paid to lipid phosphate phosphatase, diacylglycerol lipase, and monoacylglycerol lipase. Based on the findings reported in this paper, it can be concluded that proteins related to phototransduction phenomena are involved in the effects derived from sphingosine 1-phosphate/sphingosine or ceramide 1-phosphate/ceramide and that age-related changes occur in the metabolism of phosphatidic acid from cerebral cortex synaptosomes in the presence of either sphingosine 1-phosphate/sphingosine or ceramide 1-phosphate/ceramide. The...

The aim of the present research was to analyze the pathways for phosphatidic acid metabolism in purified nuclei from liver. Lipid phosphate phosphatase, diacylglycerol lipase, monoacylglycerol lipase and PA‐phospholipase type A activities were detected. The presence of lysophosphatidic acid significantly reduced DAG production while sphingosine 1‐phoshate and ceramide 1‐phosphate reduced MAG formation from PA. Using different enzymatic modulators (detergents and ions) an increase in the PA metabolism by phospholipase type A was observed. Our findings evidence an active PA metabolism in purified liver nuclei which generates important lipid second messengers, and which could thus be involved in nuclear processes such as gene transcription.

The product of the open reading frame YPL206c, Pgc1p, of the yeast Saccharomyces cerevisiae displays homology to bacterial and mammalian glycerophosphodiester phosphodiesterases. Deletion of PGC1 causes an accumulation of the anionic phospholipid, phosphatidylglycerol (PG), especially under conditions of inositol limitation. This PG accumulation was not caused by increased production of phosphatidylglycerol phosphate or by decreased consumption of PG in the formation of cardiolipin, the end product of the pathway. PG accumulation in the pgc1⌬ strain was caused rather by inactivation of the PG degradation pathway. Our data demonstrate an existence of a novel regulatory mechanism in the cardiolipin biosynthetic pathway in which Pgc1p is required for the removal of excess PG via a phospholipase C-type degradation mechanism.

The product of the open reading frame YPL206c, Pgc1p, of the yeast Saccharomyces cerevisiae displays homology to bacterial and mammalian glycerophosphodiester phosphodiesterases. Deletion of PGC1 causes an accumulation of the anionic phospholipid, phosphatidylglycerol (PG), especially under conditions of inositol limitation. This PG accumulation was not caused by increased production of phosphatidylglycerol phosphate or by decreased consumption of PG in the formation of cardiolipin, the end product of the pathway. PG accumulation in the pgc1⌬ strain was caused rather by inactivation of the PG degradation pathway. Our data demonstrate an existence of a novel regulatory mechanism in the cardiolipin biosynthetic pathway in which Pgc1p is required for the removal of excess PG via a phospholipase C-type degradation mechanism.

Background: Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgB␤) promotes metastatic colonization and angiogenesis in humans. We demonstrate that RdgB␤ is a phosphatidic acid (PA)-and phosphatidylinositol-binding protein and binds PA derived from the phospholipase D pathway. Conclusion: RdgB␤ is the first lipid-binding protein identified that can bind and transfer PA. Significance: PA bound to RdgB␤ is a likely effector downstream of phospholipase D.

199 ethanolamine PE have been investigated from the metal viewpoint by solution Hg-NMR and from the membrane side 31 Ž . by diphenylhexatriene fluorescence polarization and solid state P-NMR. Results can be summarized as follows: 1 Ž . 199 CH Hg II strong binding to membranes results in a progressive decrease of the free CH HgOH Hg-NMR isotropic 3 3 signal and because of a slow exchange, in the NMR time scale, between free and bound methylmercury pools the Ž . lipidrwater partition coefficients, K , of the CH HgOH species can be determined in the lamellar gel fluid phase. It is lw 3 Ž . therefore electrostatic in nature and the phosphate moiety is proposed as a potential binding site. 2 The presence of CH HgOH stabilizes the PG gel phase and destabilizes that of PS. No effect is observed on PC, PA and PE thermotropism. 3 Ž . 31 3 methylmercury promotes the formation of isotropic P-NMR lines with PG, PA and PE systems suggesting the presence of non-bilayer phases and hence membrane reorganization. The above effects are compared to those of inorganic Ž . mercury Hg II and discussed in the context of cell toxicity.

Cadmium(II) interactions with multilamellar vesicles of dimyristoyl (DM)-and dipalmitoyl (DP)-phosphatidylcholine (PC), -phosphatidylserine (PS), -phosphatidic acid (PA), -phosphatidylglycerol (PG) and -phosphatidylethanolamine (PE) have been investigated both from the metal and the membrane viewpoints, respectively, by solution I13Cd-NMR and diphenylhexatriene fluorescence polarization coupled with solid-state 31p-NMR. Results can be summarized as follows. (1) Strong cadmium binding to membrane phospholipids results in a decrease of the free Cd(II) 113Cd-NMR isotropic signal and because of slow exchange, in the NMR time scale, between free and bound cadmium pools, the lipid/water partition coefficients, Klw, of the Cd(II) species can be determined in the lamellar gel (fluid) phase. It is found K~w(DMPC)=K~w(EggPE)=2+2 (2+2); Kjw(DMPA)=392+20 (505+25); K~w(DMPG)=428+21 (352+17); KIw(DMPS) = 544 + 27 (672 + 34). Cadmium interactions with membrane phospholipids are therefore electrostatic in nature and the phosphate moiety is proposed as a potential binding site. (2) The presence of Cd(II) stabilizes the gel phases of PG, PA and PS lipids and leads to suppression of the main phase transition for PA and PS. These effects are reduced upon increasing salinity to 0.5 M C1-and abolished at 1.8 M CI-, Cd(II) being removed from the membranes due to formation of soluble CdCln species. Moving the pH from 7 to 6 also decreases Cd(II) binding to PA, because of surface charge reduction. (3) Cadmium promotes the formation of isotropic 31p-NMR lines with PG systems and of a hexagonal phase on egg PE bilayers at 24°C, suggesting dramatic membrane reorganization. Properties of cadmium and calcium interacting with phospholipid model membranes are compared, and the potential roles of these interactions in the molecular mechanisms of cadmium uptake and toxicity in cells are discussed.

Phosphatidylcholine (PC) hydrolysis has been shown to occur in hormone-stimulated cells and represents a potential metabolic source, in addition to phosphoinositides, for the generation of diradylglycerols (DG). We performed studies in order to quantify the importance of this pathway in DG formation. We incubated murine peritoneal macrophages with platelet-activating factor (PAF), ionomycin, phorbol myristate acetate (PMA) or no stimulus in a series of timed incubations ranging from 15 s to 20 min. We quantified the profiles of the molecular species in the accumulated DG after extraction, specific radiolabelling to give [32P]phosphatidic acid by DG kinase, and conversion to the dimethyl derivative. We used two independent methods for molecular species analysis: (1) reversed-phase h.p.l.c. separation with in-line fi- radiation detection of peaks, and (2) an argentation-t.l.c. separation with scintillation counting of bands. Our results showed a clearly biphasic sequence in the composition of accumulated DG. The molecular species composition of early DG (up to 1 min stimulation time) was very similar to that of unstimulated DG, whereas the proportions of the species present in later DG were substantially altered. In the same experiments, we extracted native phospholipids from unstimulated macrophages, separated phosphatidylinositol (PI), PC, phosphatidylethanolamine (PE) and phosphatidyl- serine (PS), converted them to the corresponding DGs by using phospholipase C, and determined their molecular species compositions as above. In comparison with the diradyl compositions of stimulated DG, the diradyl composition of PI closely matched that of early DG, the differences between the PC and PI compositions matched the differences between early and late DG very closely, and the compositions of PE and PS were unique and unrelated. We quantified these relationships more precisely by multilinear regression analysis to calculate the theoretical best mix of five molecular species compositions (PI, PC, PE, PS and unstimulated DG) that would most closely replicate the early and late accumulated DG compositions. We found that by both h.p.l.c. and t.l.c. analyses, 15-30% (PAF) or 25-50% (ionomycin and PMA) of the later DG could be accounted for by PC hydrolysis. These results represent quantifications of phospholipid class contributions to stimulated DG formation, and demonstrate the potential importance of PC hydrolysis in phagocytic leucocytes.

Phosphatidylcholine (PC) hydrolysis has been shown to occur in hormone-stimulated cells and represents a potential metabolic source, in addition to phosphoinositides, for the generation of diradylglycerols (DG). We performed studies in order to quantify the importance of this pathway in DG formation. We incubated murine peritoneal macrophages with platelet-activating factor (PAF), ionomycin, phorbol myristate acetate (PMA) or no stimulus in a series of timed incubations ranging from 15 s to 20 min. We quantified the profiles of the molecular species in the accumulated DG after extraction, specific radiolabelling to give [32P]phosphatidic acid by DG kinase, and conversion to the dimethyl derivative. We used two independent methods for molecular species analysis: (1) reversed-phase h.p.l.c. separation with in-line beta-radiation detection of peaks, and (2) an argentation-t.l.c. separation with scintillation counting of bands. Our results showed a clearly biphasic sequence in the compo...

The objective of the present work was to develop a simple and sensitive radioenzymatic assay to quantify lysophosphatidic acid (LPA). For that, a recombinant rat LPA acid acyltransferase (LPAAT) produced in Escherichia coli was used. In the presence of [ 14 C]oleoyl-CoA, LPAAT selectively catalyzes the transformation of LPA and alkyl-LPA into [ 14 C]phosphatidic acid. Acylation of LPA was complete and linear from 0 to 200 pmol with a minimal detection of 0.2 pmol. This method was used to quantify LPA in butanolextracted lipids from bovine sera, as well as from human and mouse plasma. This radioenzymatic assay represents a new, simple, and highly sensitive method to quantify LPA in various biological fluids.

The aim of the present work was to depict the metabolic pathways involved in extra-cellular production of lysophosphatidic acid (LPA) by adipocytes. LPA was followed by quantifying the accumulation of LPA in the incubation medium (conditioned medium: CM) of 3T3F442A adipocytes, or human adipose tissue explants, using a radioenzymatic assay. Surprisingly, after separation from the cells, the amount of LPA present in CM could significantly be increased by further incubation at 37°C. This suggested the presence of a LPA-synthesizing activity (LPA-SA) in CM. LPA-SA appeared as a soluble activity which was inhibited by divalent ion chelators: EDTA and phenanthrolin. The effect of EDTA was preferentially reverted by CoCl2, as described for a lysophospholipase D-(lyso-PLD) activity previously identified in rat plasma. LPA concentration could also be increased by treatment with a bacterial PLD, demonstrating the presence of PLDsensitive LPA-precursors (mainly lysophosphatidylcholine) in adipocyte CM. LPA-SA could be increased by addition of exogenous lysophosphatidylcholine, lysophosphatidylglycerol, or lyso-platelet activating factor, demonstrating that LPA-SA resulted from the action of a lyso-PLD. LPA-SA was not inhibited, but rather activated, by primary alcohol (ethanol and 1-butanol), suggesting that adipocyte lyso-PLD was not a classical PLD. Finally, LPA-SA was found to be weaker in CM of undifferentiated adipocyte (preadipocytes) as compared to CM of differentiated adipocytes. In conclusion, our results reveal the existence of a secreted lyso-PLD activity regulated during adipocyte-differentiation and involved in extra-cellular production of synthesis of LPA by adipocytes.

The objective of the present work was to develop a simple and sensitive radioenzymatic assay to quantify lysophosphatidic acid (LPA). For that, a recombinant rat LPA acid acyltransferase (LPAAT) produced in Escherichia coli was used. In the presence of [ 14 C]oleoyl-CoA, LPAAT selectively catalyzes the transformation of LPA and alkyl-LPA into [ 14 C]phosphatidic acid. Acylation of LPA was complete and linear from 0 to 200 pmol with a minimal detection of 0.2 pmol. This method was used to quantify LPA in butanolextracted lipids from bovine sera, as well as from human and mouse plasma. This radioenzymatic assay represents a new, simple, and highly sensitive method to quantify LPA in various biological fluids.

The aim of the present work was to depict the metabolic pathways involved in extra-cellular production of lysophosphatidic acid (LPA) by adipocytes. LPA was followed by quantifying the accumulation of LPA in the incubation medium (conditioned medium: CM) of 3T3F442A adipocytes, or human adipose tissue explants, using a radioenzymatic assay. Surprisingly, after separation from the cells, the amount of LPA present in CM could significantly be increased by further incubation at 37°C. This suggested the presence of a LPA-synthesizing activity (LPA-SA) in CM. LPA-SA appeared as a soluble activity which was inhibited by divalent ion chelators: EDTA and phenanthrolin. The effect of EDTA was preferentially reverted by CoCl2, as described for a lysophospholipase D-(lyso-PLD) activity previously identified in rat plasma. LPA concentration could also be increased by treatment with a bacterial PLD, demonstrating the presence of PLDsensitive LPA-precursors (mainly lysophosphatidylcholine) in adipocyte CM. LPA-SA could be increased by addition of exogenous lysophosphatidylcholine, lysophosphatidylglycerol, or lyso-platelet activating factor, demonstrating that LPA-SA resulted from the action of a lyso-PLD. LPA-SA was not inhibited, but rather activated, by primary alcohol (ethanol and 1-butanol), suggesting that adipocyte lyso-PLD was not a classical PLD. Finally, LPA-SA was found to be weaker in CM of undifferentiated adipocyte (preadipocytes) as compared to CM of differentiated adipocytes. In conclusion, our results reveal the existence of a secreted lyso-PLD activity regulated during adipocyte-differentiation and involved in extra-cellular production of synthesis of LPA by adipocytes.

Glycerophospholipids (GPLs) play important roles in cellular compartmentalization and signaling. Among them, phosphatidic acids (PA) exist as many distinct species depending on acyl chain composition, each one potentially displaying unique signaling function. Although the signaling functions of PA have already been demonstrated in multiple cellular processes, the specific roles of individual PA species remain obscure due to a lack of appropriate tools. Indeed, current synthetic PA analogues fail to preserve all the functions of natural PA. To circumvent these limitations, we developed a novel synthetic approach to produce PA analogues without compromising structural integrity of acyl chains. Moreover, addition of a clickable moiety allowed flexible grafting of different molecules to PA analogues for various biological applications. Hence, this innovation also provides powerful tools to investigate specific biological activities of individual PA species, with potential applications in unraveling complex GPL-mediated signaling pathways.

Small 10-kD acyl-coenzyme A-binding proteins (ACBPs) are highly conserved proteins that are prevalent in eukaryotes. In Arabidopsis (Arabidopsis thaliana), other than the 10-kD ACBP homolog (designated Arabidopsis ACBP6), there are five larger forms of ACBPs ranging from 37.5 to 73.1 kD. In this study, the cytosolic subcellular localization of Arabidopsis ACBP6 was confirmed by analyses of transgenic Arabidopsis expressing autofluorescence-tagged ACBP6 and western-blot analysis of subcellular fractions using ACBP6-specific antibodies. The expression of Arabidopsis ACBP6 was noticeably induced at 48 h after 4°C treatment by northern-blot analysis and western-blot analysis. Furthermore, an acbp6 T-DNA insertional mutant that lacked ACBP6 mRNA and protein displayed increased sensitivity to freezing temperature (28°C), while ACBP6-overexpressing transgenic Arabidopsis plants were conferred enhanced freezing tolerance. Northern-blot analysis indicated that ACBP6-associated freezing tolerance was not dependent on the induction of cold-regulated COLD-RESPONSIVE gene expression. Instead, ACBP6 overexpressors showed increased expression of mRNA encoding phospholipase Dd. Lipid profiling analyses of rosettes from cold-acclimated, freezingtreated (28°C) transgenic Arabidopsis plants overexpressing ACBP6 showed a decline in phosphatidylcholine (236% and 246%) and an elevation of phosphatidic acid (73% and 67%) in comparison with wild-type plants. From our comparison, the gain in freezing tolerance in ACBP6 overexpressors that was accompanied by decreases in phosphatidylcholine and an accumulation of phosphatidic acid is consistent with previous findings on phospholipase Dd-overexpressing transgenic Arabidopsis. In vitro filterbinding assays indicating that histidine-tagged ACBP6 binds phosphatidylcholine, but not phosphatidic acid or lysophosphatidylcholine, further imply a role for ACBP6 in phospholipid metabolism in Arabidopsis, including the possibility of ACBP6 in the cytosolic trafficking of phosphatidylcholine.

The use of natural substances in health applications may be hampered by the difficulties in establishing the mechanisms of action, especially at molecular-level. The protein-polysaccharide complex extracted from the mushroom Agaricus blazei Murill, referred to as CAb, has been considered for treating various diseases with probable interaction with cell membranes. In this study, we investigate the interaction between CAb and a cell membrane model represented by a Langmuir monolayer of dimyristoyl phosphatidic acid (DMPA). CAb affects the structural properties of DMPA monolayers causing expansion and increasing compressibility. In addition, interaction with DMPA polar heads led to neutralization of the electrical double layer, yielding a zero surface potential at large areas per molecule. CAb remained at the interface even at high surface pressures, which allowed transfer of Langmuir-Blodgett (LB) films onto solid supports with the CAb-DMPA mixture. The mass transferred, according to quartz crystal microbalance (QCM) measurements, increased linearly with the number of deposited layers. With UV-vis absorption, fluorescence and FTIR spectroscopies, we confirmed that the LB films contain polysaccharides, proteins and DMPA. Therefore, the CAb biological action must be attributed not only to polysaccharides but also to proteins in the complex.

Macrophages are pivotal in mounting liver inflammatory and tissue repair reactions upon hepatic injury showing remarkable functional plasticity. Nevertheless, the molecular mechanisms determining macrophage transition from inflammatory to restorative phenotypes in the damaged liver remain unclear. Using mouse models of acute (APAP) or chronic (CCl4) drug-induced hepatotoxic injury we show that the immune receptor Trem-2 controls phenotypic shifts in liver macrophages and impacts endothelial cell differentiation during tissue recovery.Trem-2 gene ablation led to delayed re-population of Kupffer cells correlating with deterred resolution of hepatic damage following acute and chronic injury. We found that during tissue recovery macrophages in transition to the Kupffer cell compartment expressed high levels of Trem-2. Acquisition of the transition phenotype was associated with an unique transcriptomic profile denoting strong responsiveness to oxidative stress and downmodulation of the p...

The anti-inflammatory properties of omega-3 fatty acids contained in fish oil may help alleviate symptoms of delayed onset of muscle soreness (DOMS) and improve recovery from exercise. PURPOSE: To examine the effects of fish oil supplementation on the time-course of post-resistance exercise muscle soreness in young untrained females. METHODS: Seventeen non-resistance trained females (age 23.6 ± 3.0 y, % fat 27.4 ± 3.2, weight 60.2 ± 9.6 kg) were randomized into one of two groups: fish oil (6 g/d; 5:1 EPA:DHA) or placebo (6 g/d corn/soy oil). After consuming the supplements for one week, participants underwent a single bout of resistance exercise designed to induce muscle damage. Subjects performed 10 sets to failure of biceps curl machine and leg extensions using 50% of the previously measured 1-repetition maximum. Over the next week, subjective muscle soreness of the upper and lower body was measured via a grounded 10-cm visual analog scale. At 48 hours and 1 week post-exercise, subjects performed a test to determine soreness during functional movements. The comparison-wise error rate was set at p < 0.10. RESULTS: Muscle soreness increased significantly in both groups and peaked at 48 hours post-exercise. The fish oil group perceived less muscle soreness in the lower body than the placebo group (p= 0.06), but there was no difference in the upper body (p= 0.27). The fish oil group reported less perceived soreness during functional movements (p= 0.07 for upper and lower body soreness). Effect sizes, indicating the reduction in muscle soreness that may be attributed to fish oil (∆ effect size between groups), was 0.75 (95% CI: -0.70 -2.20) for the arms and 0.77 (-0.76 -2.33) for the legs. The effect size for the functional tests was 0.63 (-0.71 -1.97) for the arms and 0.57 (-0.85 -1.99) for the legs. CONCLUSION: Supplementing the diet with 6 grams per day of fish oil may alleviate the muscle soreness experienced after resistance training in young untrained females, but additional studies with larger sample sizes should be conducted to confirm these findings.

Lipid kinases have largely been neglected as targets in cancer, and an increasing number of reports suggest diacylglycerol kinase alpha (DGKα) may be one with promising therapeutic potential. DGKα is one of 10 DGK family members that convert diacylglycerol (DAG) to phosphatidic acid (PA), and both DAG and PA are critical lipid second messengers in the plasma membrane. A host of important oncogenic proteins and pathways affect cancer cells in part through DGKα, including the c-Met and VEGF receptors. Others partially mediate the effects of DGKα inhibition in cancer, such as mTOR and HIF-1α. DGKα inhibition can directly impair cancer cell viability, inhibits angiogenesis, and notably may also boost T-cell activation and enhance cancer immunotherapies. Although two structurally similar inhibitors of DGKα were established decades ago, they have seen minimal in vivo usage, and it is unlikely that either of these older DGKα inhibitors will have utility for cancer. An abandoned compound th...

Sphingosylphosphorylcholine (SPC), or lysophingomyelin, a wide-spectrum growth promoting agent for a variety of cell types (Desai, N. N., and S. Spiegel. 1991. Biochem. Biophys. Res. Comm. 181: 361-366), stimulates cellular proliferation of quiescent Swiss 3T3 fibroblasts to a greater extent than other known growth factors or than the structurally related molecules, sphingosine and sphingosine-l-phosphate. SPC potentiated the mitogenic effect of an activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate, and did not compete with phorbol esters for binding to protein kinase C in intact Swiss 3T3 fibroblasts. However, downregulation of protein kinase C, by prolonged treatment with phorbol ester, reduced, but did not eliminate, the ability of SPC to stimulate DNA synthesis, indicating that SPC may act via both protein kinase C-dependent and -independent signaling path-ways. SPC induced a rapid rise in intracellular free calcium ([Ca2+]i) in viable 3T3 fibroblasts determined with a digital imaging system. Although the increases in [Ca2+]i were observed even in the absence of calcium in the external medium, no increase in the levels of inositol phosphates could be detected in response to mitogenic concentrations of SPC. Furthermore, in contrast to sphingosine or sphingosine-l-phosphate, the mitogenic effect of SPC was not accompanied by increases in phosphatidic acid levels or changes in cAMP levels. SPC, but not sphingosine or sphingosine-l-phosphate, stimulates the release of arachidonic acid. Therefore, the ability of SPC to act as an extremely potent mitogen may be due to activation of signaling pathway(s) distinct from those used by sphingosine or sphingosine-l-phosphate.

We have studied phospholipase D activation in [32P]orthophosphoric acid‐prelabeled rat pancreatic acini by measuring the formation of 32P‐phosphatidylalcohols as stimulated in the presence of ethanol or butanol. A small but significant and time‐dependent basal accumulation of [32P]phosphatidylethanol and [32P]phosphatidylbutanol was detected, which was further stimulated by phorbol myristate acetate, orthovanadate and pervanadate. However, the secretagogues cholecystokinin octapeptide and carbachol did not enhance basal accumulation of 32P‐phosphatidylalcohol, yet they decreased [32P]phosphatidylcholine content and stimulated the generation of [32P]phosphatidic acid. Our results stress the need to examine the transphosphatidylation reaction as well as agonist effects on the synthesis of phosphatidylcholine in order to assess unambiguously phospholipase D activity.

Three members of the obscurin protein family that contain tandem kinase domains with important signaling functions for cardiac and striated muscles are the giant protein obscurin, its obscurin-associated kinase splice isoform, and the striated muscle enriched protein kinase (SPEG). While there is increasing evidence for the specific roles that each individual kinase domain plays in cross-striated muscles, their biology and regulation remains enigmatic. Our present study focuses on kinase domain 1 and the adjacent low sequence complexity inter-kinase domain linker in obscurin and SPEG. Using Phos-tag gels, we show that the linker in obscurin contains several phosphorylation sites, while the same region in SPEG remained unphosphorylated. Our homology modeling, mutational analysis and molecular docking demonstrate that kinase 1 in obscurin harbors all key amino acids important for its catalytic function and that actions of this domain result in autophosphorylation of the protein. Our bioinformatics analyses also assign a list of putative substrates for kinase domain 1 in obscurin and SPEG, based on the known and our newly proposed phosphorylation sites in muscle proteins, including obscurin itself.

Three members of the obscurin protein family that contain tandem kinase domains with important signaling functions for cardiac and striated muscles are the giant protein obscurin, its obscurin-associated kinase splice isoform, and the striated muscle enriched protein kinase (SPEG). While there is increasing evidence for the specific roles that each individual kinase domain plays in cross-striated muscles, their biology and regulation remains enigmatic. Our present study focuses on kinase domain 1 and the adjacent low sequence complexity inter-kinase domain linker in obscurin and SPEG. Using Phos-tag gels, we show that the linker in obscurin contains several phosphorylation sites, while the same region in SPEG remained unphosphorylated. Our homology modeling, mutational analysis and molecular docking demonstrate that kinase 1 in obscurin harbors all key amino acids important for its catalytic function and that actions of this domain result in autophosphorylation of the protein. Our b...

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Copyright Activation of the chloroplast monogalactosyldiacylglycerol synthase MGD1 by phosphatidic acid and phosphatidylglycerol.

Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine-and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cellfree extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.

The cytokine interleukin (IL)-1␤ induces a biphasic effect in rat pancreatic islets, with an early and transitory stimulation of insulin release followed by progressive functional suppression. To clarify the mechanisms involved in these effects, we have recently performed a differential display of messenger RNA (mRNA) by RT-PCR (DDRT-PCR) on rat ␤-cells exposed for 6 or 24 h to IL-1␤. Among the different IL-1␤-induced genes, there was an early and transient increase in phospholipase D-1 (PLD1) expression. PLD1 can induce phosphatidic acid formation and subsequent activation of protein kinase C, a process which stimulates insulin release. In the present study, we characterized the regulation of PLD isoforms by IL-1␤ in pancreatic ␤-cells. By using different combinations of primers and RT-PCR, we observed that IL-1␤ induces an early increase (2 and 6 h) in the expression of both alternatively spliced isoforms of PLD1 (PLD1a and 1b). Prolonged exposure to IL-1␤ (12 and 24 h) caused a decrease of PLD1a mRNA expression compared with control ␤-cells, and lead to a return of PLD1b mRNA to basal level. N G -methyl-L-arginine (L-MA), a blocker of the inducible form of nitric oxide synthase (iNOS), prevented this late inhibitory effect of IL-1␤, suggesting that IL-1␤induced decrease in PLD1a expression is NO-mediated. IL-1␤ induced an early (2-6 h) and sustained (16 -24 h) increase in PLD1a mRNA expression in insulin-producing RINm5F cells. This was paralleled by a cytokine-induced increase in PLD1 protein expression and enzyme activity. RINm5F cells, but not primary ␤-cells, expressed PLD2, and the expression of this gene was not affected by IL-1␤. In conclusion, we have shown that the cytokine IL-1␤ regulates PLD1 expression in primary and clonal ␤-cells. The early induction of PLD1 probably contributes to the early stimulatory effects of IL-1␤ on islet insulin release. (Endocrinology 141: [2822][2823][2824][2825][2826][2827][2828] 2000)

It has been postulated that the diacylglycerol lipase pathway is a predominant source of the free arachidonic acid which is released from phospholipids upon the exposure of human platelets to thrombin. The amount of released arachidonic acid and other fatty acids in thrombin-stimulated platelets was determined in the presence of BW755C, the cyclooxygenase/ lipoxygenase inhibitor, and in relation to phosphatidylinositol degradation Copyright

PTEN (phosphatase and tensin homologue deleted on chromosome ten) proteins are dual phosphatases with both protein and phosphoinositide phosphatase activity. They modulate signalling pathways controlling growth, metabolism and apoptosis in animals and are implied in several human diseases. In the present paper we describe a novel class of PTEN pro-teins in plants, termed PTEN2, which comprises the AtPTEN (Arabidopsis PTEN) 2a and AtPTEN2b proteins in Arabidopsis. Both display low in vitro tyrosine phosphatase activity. In addition, AtPTEN2a actively dephosphorylates in vitro the 3′ phosphate group of PI3P (phosphatidylinositol 3-phosphate), PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate) and PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate). In contrast with animal PTENs, PI(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) is a poor substrate. Site-directed mutagenesis of AtPTEN2a and molecular modelling of protein–phosphoinositide interactions indicated that substitutions at t...

Phospholipids are the crucial components of biological membranes and signal transduction. Among different tissues, flower phospholipids are one of the least characterized features of plant lipidome. Here, we report that floral reproductive organs of Arabidopsis thaliana contain high levels of phosphatidic acid (PA), a known lipid second messenger. By using floral homeotic mutants enriched with specific floral organs, lipidomics study showed increased levels of PA species in ap3-3 mutant with enriched pistils. Accompanied gene expression study for 7 diacylglycerol kinases and 11 PA phosphatases revealed distinct floral organ specificity, suggesting an active phosphorylation/ dephosphorylation between PA and diacylglycerol in flowers. Our results suggest that PA is a major phospholipid class in floral reproductive organs of A. thaliana.

Phospholipids are the crucial components of biological membranes and signal transduction. Among different tissues, flower phospholipids are one of the least characterized features of plant lipidome. Here, we report that floral reproductive organs of Arabidopsis thaliana contain high levels of phosphatidic acid (PA), a known lipid second messenger. By using floral homeotic mutants enriched with specific floral organs, lipidomics study showed increased levels of PA species in ap3-3 mutant with enriched pistils. Accompanied gene expression study for 7 diacylglycerol kinases and 11 PA phosphatases revealed distinct floral organ specificity, suggesting an active phosphorylation/ dephosphorylation between PA and diacylglycerol in flowers. Our results suggest that PA is a major phospholipid class in floral reproductive organs of A. thaliana.