Generation Of Phosphatidic Acid Research Articles

Phospholipids in Salt Stress Response.

High salinity threatens crop production by harming plants and interfering with their development. Plant cells respond to salt stress in various ways, all of which involve multiple components such as proteins, peptides, lipids, sugars, and phytohormones. Phospholipids, important components of bio-membranes, are small amphoteric molecular compounds. These have attracted significant attention in recent years due to the regulatory effect they have on cellular activity. Over the past few decades, genetic and biochemical analyses have partly revealed that phospholipids regulate salt stress response by participating in salt stress signal transduction. In this review, we summarize the generation and metabolism of phospholipid phosphatidic acid (PA), phosphoinositides (PIs), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), as well as the regulatory role each phospholipid plays in the salt stress response. We also discuss the possible regulatory role based on how they act during other cellular activities.

Thrombin Stimulation of Human Endothelial Cell Phospholipase D Activity. Regulation by Phospholipase C, Protein Kinase C, and Cyclic Adenosine 3' 5’ -Monophosphate

The activation of membrane-bound phospholipase D (PLD) resulting in the generation of phosphatidic acid (PA) is increasingly recognized as an integral event in the initiation of a variety of cellular responses. We explored whether alpha-thrombin is a physiologic agonist for PLD activation in human umbilical vein endothelial cells (HUVEC). HUVEC monolayers were labeled with [32Pi] and PLD activity determined by formation of the PLD metabolite [32P] phosphatidylethanol (PEt) in the presence of 5 g/L ethanol by thin-layer chromatography. alpha-Thrombin rapidly (1 minute) increased PA and PEt formation in a dose-dependent manner (10(-6) to 10(-10)) with maximal PLD stimulation achieved with 10 nmol/L alpha-thrombin producing a threefold to fourfold increase in PA and a sixfold to eightfold increase in PEt over controls at 15 minutes. Esterolytically active zeta-thrombin (10 nmol/L) and gamma-thrombin (1 mumol/L), but not inactive DIP-alpha-thrombin (1 mumol/L) also increased PLD activity. The role of Ca2+ flux in human endothelial cell PLD activation was investigated and PEt formation was significantly enhanced by Ca2+ ionophores A23187 and ionomycin (1 mumol/L, three-fold to fourfold increase in PEt). Alpha-Thrombin-stimulated PEt formation was abolished (greater than 90% inhibition) with chelation of intracellular calcium (Ca2+i) by pretreatment with BAPTA-AM (25 mumol/L, 30 minutes) but only mildly attenuated (30% inhibition) by removal of extracellular calcium (Ca2+E) with EGTA (5 mmol/L). The protein kinase C (PKC) inhibitor staurosporine reduced alpha-thrombin-induced PEt formation in a dose-dependent manner (10 mumol/L, 78% inhibition) and PKC downregulation with chronic PMA treatment (18 hours) also resulted in marked inhibition of alpha-thrombin-induced PEt formation. Neither pertussis nor botulinum C bacterial toxins significantly altered alpha-thrombin-induced PLD responses. In contrast, similar pretreatment with cholera toxin (1 microgram/mL, 60 minutes) consistently augmented alpha-thrombin-stimulated PLD activity by 50% to 90%. Comparable results were observed with agents which increased cAMP such as forskolin, 8-bromo cAMP, or dibutyryl cAMP and cholera toxin augmentation was abolished by 2-dideoxyadenosine, a competitive inhibitor of adenylyl cyclase activity. These studies demonstrate that alpha-thrombin is a potent stimulus for human PLD-mediated PA formation and that cyclic adenosine nucleotides modulate agonist-induced cellular PLD activity. In this model of PLD activation, alpha-thrombin receptor occupancy leads to the breakdown of phosphatidylinositol 4,5-bisphosphate catalyzed by phospholipase C producing the Ca2+ secretagogue IP3 and DAG.(ABSTRACT TRUNCATED AT 400 WORDS)

Phospholipase D2 in prostate cancer: protein expression changes with Gleason score

BackgroundPhospholipases D1 and D2 (PLD1/2) are implicated in tumorigenesis through their generation of the signalling lipid phosphatidic acid and its downstream effects. Inhibition of PLD1 blocks prostate cell growth and colony formation. Here a role for PLD2 in prostate cancer (PCa), the major cancer of men in the western world, is examined.MethodsPLD2 expression was analysed by immunohistochemistry and western blotting. The effects of PLD2 inhibition on PCa cell viability and cell motility were measured using MTS, colony forming and wound-healing assays.ResultsPLD2 protein is expressed about equally in luminal and basal prostate epithelial cells. In cells from different Gleason-scored PCa tissue PLD2 protein expression is generally higher than in non-tumorigenic cells and increases in PCa tissue scored Gleason 6–8. PLD2 protein is detected in the cytosol and nucleus and had a punctate appearance. In BPH tissue stromal cells as well as basal and luminal cells express PLD2. PLD2 protein co-expresses with chromogranin A in castrate-resistant PCa tissue. PLD2 inhibition reduces PCa cell viability, colony forming ability and directional cell movement.ConclusionsPLD2 expression correlates with increasing Gleason score to GS8. PLD2 inhibition has the potential to reduce PCa progression.

A polydiacetylenes-based sensor for the efficient detection of phospholipase D activity

Phospholipase D (PLD), as an enzyme that catalyses the hydrolysis of L-α-phosphatidylcholine to produce phosphatidic acid, plays an important role in regulating biological processes. In this work, polydiacetylenes (PDAs) bearing imidazolium as head group are reported for the efficient detection of PLD activity. Owing to the strong interaction between negatively charged phosphatidic acid and positively charged imidazolium moieties of PDAs, the PDAs were found to produce a colour transition from blue to red, and an increase in fluorescence. No obvious changes to PDAs were observed when PLD was absent in the reaction, or when the activity of PLD was inhibited by halopemide. The colour and fluorescence changes of PDAs are attributed to the generation of phosphatidic acid in the PLD-catalysed reaction. Furthermore, a paper sensor based on PDAs was constructed and exhibited convenient and sensitive detection of PLD activity.

Inhibition of tomato fruit ripening by 1-MCP, wortmannin and hexanal is associated with a decrease in transcript levels of phospholipase D and other ripening related genes

Membrane deterioration is an inherent aspect of the advancement in senescence and loss in fruit quality during storage. Postharvest technologies used for extending shelf life and quality are targeted to reduce membrane damage through downregulating or blocking ethylene action. In this study, mature green tomato fruit were treated with inhibitors of ethylene receptor (ETR), phosphatidylinositol 3-kinase (PI3K) and phospholipase D (PLD), all recognized to be targets of regulation of fruit ripening. The inhibitors used included 1-methylcyclopropene (1-MCP, an ethylene receptor blocker), wortmannin (an inhibitor of PI3K), and hexanal (a PLD inhibitor). Fruit were treated at optimal levels of the inhibitors and were stored at 21 °C for 10 days. Color development was strongly delayed in wortmannin treated tomatoes just as in 1-MCP treated fruit; while, changes in respiration, firmness and ethylene evolution were very similar to that of control fruit. Hexanal delayed the initiation of these changes; while 1-MCP and wortmannin blocked the ripening process. Changes in expression levels of key genes involved in ethylene signalling, phosphoinositide metabolism, and lycopene synthesis that occurred in response to inhibitors, suggested potential roles for PI3K and PLD in ethylene signalling. Furthermore, fruit treated with all the three inhibitors showed a marked reduction in PLD transcript levels; suggesting that, regulation of PLD gene expression is a common critical regulatory point that regulates ripening. Lowered PLD levels may reduce membrane lipid catabolism and the generation of phosphatidic acid (PA), an intermediate in ethylene signalling regulation through downstream components.

Targeting phospholipase D in cancer, infection and neurodegenerative disorders.

Lipid second messengers have essential roles in cellular function and contribute to the molecular mechanisms that underlie inflammation, malignant transformation, invasiveness, neurodegenerative disorders, and infectious and other pathophysiological processes. The phospholipase D (PLD) isoenzymes PLD1 and PLD2 are one of the major sources of signal-activated phosphatidic acid (PtdOH) generation downstream of a variety of cell-surface receptors, including G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and integrins. Recent advances in the development of isoenzyme-selective PLD inhibitors and in molecular genetics have suggested that PLD isoenzymes in mammalian cells and pathogenic organisms may be valuable targets for the treatment of several human diseases. Isoenzyme-selective inhibitors have revealed complex inter-relationships between PtdOH biosynthetic pathways and the role of PtdOH in pathophysiology. PLD enzymes were once thought to be undruggable owing to the ubiquitous nature of PtdOH in cell signalling and concerns that inhibitors would be too toxic for use in humans. However, recent promising discoveries suggest that small-molecule isoenzyme-selective inhibitors may provide novel compounds for a unique approach to the treatment of cancers, neurodegenerative disorders and other afflictions of the central nervous system, and potentially serve as broad-spectrum antiviral and antimicrobial therapeutics.

Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells

Caveolae are the primary route for internalization and transendothelial transport of macromolecules, such as insulin and albumin. Caveolae-mediated endocytosis is activated by Src-dependent caveolin-1 (Cav-1) phosphorylation and subsequent recruitment of dynamin-2 and filamin A (FilA), which facilitate vesicle fission and trafficking, respectively. Here, we tested the role of RalA and phospholipase D (PLD) signaling in the regulation of caveolae-mediated endocytosis and trafficking. The addition of albumin to human lung microvascular endothelial cells induced the activation of RalA within minutes, and siRNA-mediated down-regulation of RalA abolished fluorescent BSA uptake. Co-immunoprecipitation studies revealed that albumin induced the association between RalA, Cav-1, and FilA; however, RalA knockdown with siRNA did not affect FilA recruitment to Cav-1, suggesting that RalA was not required for FilA and Cav-1 complex formation. Rather, RalA probably facilitates caveolae-mediated endocytosis by activating downstream effectors. PLD2 was shown to be activated by RalA, and inhibition of PLD2 abolished Alexa-488-BSA uptake, indicating that phosphatidic acid (PA) generated by PLD2 may facilitate caveolae-mediated endocytosis. Furthermore, using a PA biosensor, GFP-PASS, we observed that BSA induced an increase in PA co-localization with Cav-1-RFP, which could be blocked by a dominant negative PLD2 mutant. Total internal reflection fluorescence microscopy studies of Cav-1-RFP also showed that fusion of caveolae with the basal plasma membrane was dependent on PLD2 activity. Thus, our results suggest that the small GTPase RalA plays an important role in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA but by stimulating PLD2-mediated generation of phosphatidic acid.

EHD3 Protein Is Required for Tubular Recycling Endosome Stabilization, and an Asparagine-Glutamic Acid Residue Pair within Its Eps15 Homology (EH) Domain Dictates Its Selective Binding to NPF Peptides

An elaborate network of dynamic lipid membranes, termed tubular recycling endosomes (TRE), coordinates the process of endocytic recycling in mammalian cells. The C-terminal Eps15 homology domain (EHD)-containing proteins have been implicated in the bending and fission of TRE, thus regulating endocytic recycling. EHD proteins have an EH domain that interacts with proteins containing an NPF motif. We found that NPF-containing EHD1 interaction partners such as molecules interacting with CasL-like1 (MICAL-L1) and Syndapin2 are essential for TRE biogenesis. Also crucial for TRE biogenesis is the generation of phosphatidic acid, an essential lipid component of TRE that serves as a docking point for MICAL-L1 and Syndapin2. EHD1 and EHD3 have 86% amino acid identity; they homo- and heterodimerize and partially co-localize to TRE. Despite their remarkable identity, they have distinct mechanistic functions. EHD1 induces membrane vesiculation, whereas EHD3 supports TRE biogenesis and/or stabilization by an unknown mechanism. While using phospholipase D inhibitors (which block the conversion of glycerophospholipids to phosphatidic acid) to deplete cellular TRE, we observed that, upon inhibitor washout, there was a rapid and dramatic regeneration of MICAL-L1-marked TRE. Using this "synchronized" TRE biogenesis system, we determined that EHD3 is involved in the stabilization of TRE rather than in their biogenesis. Moreover, we identify the residues Ala-519/Asp-520 of EHD1 and Asn-519/Glu-520 of EHD3 as defining the selectivity of these two paralogs for NPF-containing binding partners, and we present a model to explain the atomic mechanism and provide new insight for their differential roles in vesiculation and tubulation, respectively.

Loss of Diacylglycerol Kinase-Ζ Inhibits Cell Proliferation and Survival in Human Gliomas.

Diacylglycerol kinases ζ (DGKζ) is a critical lipid kinase which is involved in phosphatidic acid (PA) generation via diacylglycerol (DAG) phosphorylation. DGKζ is highly expressed in central nervous system and essential for brain development. Studies have indicated that DGKζ is associated with colon cancer invasion and metastasis. However, the involvement of DGKζ in human glioma development remains elusive. Here, we explored the impact and possible mechanisms of DGKζ knockdown on the proliferation and survival of glioma cells. The relationship between DGKζ expression status and human glioma stages was explored in 111 specimens of human gliomas via immunohistochemistry technology. Then the impact of DGKζ on cell proliferation, cell cycle, survival, and colony formation ability was determined in U-87 MG glioma cell lines via lentiviral-mediated small interfering (shRNA) strategy. The influence of DGKζ knockdown on global gene expression in U-87 MGglioma cell lines was further analyzed by microarray platform to reveal the possible molecular mechanisms underlying DGKζ-mediated glioma development and progression. Immunohistochemistry analysis revealed that DGKζ expression is positively correlated with human gliomagrade. Lentiviral-mediated small interfering (shRNA) strategyefficiently reduced DGKζ expression and DGKζ knockdown impaired cell proliferation, inhibited colony formation ability, and induced cell cycle arrest and cell apoptosis in U-87 MG glioma cells. Finally, microarray analysis revealed that multiple cancer-associated pathways and oncogenes were regulated by DGKζ knockdown, which provides insights into underlying mechansims of DGKζ-associated glioma development and progression. Our results established the positive correlation between DGKζ expression and gliomagrade. Furthermore, DGKζ knockdown in human glioma cell lines U-87 MG impaired cell proliferation, inhibited colony formation ability, and induced cell cycle arrest and apoptosis which microarray analysis showed that DGKζ knockdown interrupted multiple oncogenes and cancer-associated pathways. Taken together, we provided confidential evidence for the causal role of DGKζ in glioma development and progression.

Composition Effect of the Outer Layer on the Vesicle Fusion Catalyzed by Phospholipase D

Phospholipase D (PLD) catalyzed the generation of phosphatidic acid (PA) from phosphatidylcholine (PC) at the outer layer of the vesicles prepared through layer by layer via a double emulsion technique. The generation induced a curvature change in the vesicles, which eventually led them to fuse each other. The ratio of two-fattyacid-tail ethanolamine (PE) to one-fatty-acid-tail ethanolamine (PE) was found to acquire the condition where the mixed-phospholipid vesicles were stable identically with pure two-fatty-acid-tail PC. The effect of the outer-layer mixture on the PLD-induced vesicle fusion was investigated using the fluorescence intensity change. 8-Aminonaph- thalene-1,3,6-trisulfonic acid disodium salt (ANTS) and p-Xylene-bis(N-pyridinium bromide) (DPX) were encapsulated in the vesicles, respectively, for the quantification of the fusion. The fluorescence scale was calibrated with the fluorescence of a 1/1 mixture of ANTS and DPX vesicles in NaCl buffer taken as 100% fluorescence (0% fusion) and the vesicles containing both ANTS and DPX as 0% fluorescence (100% fusion), considering the leakage into the medium studied directly in a separate experiment using vesicles containing both ANTS and DPX. The fusion data for each composition were acquired with the subtraction of the leakage from the quenching. From the monitoring, the vesicle fusion caused by the PLD reaction seems dominantly to occur rather than the vesicle lysis, because the composition effect on the fusion was observed identically with that on the change in the vesicle structure. Furthermore, the diameter measurements also support the fusion dominancy.

Diacylglycerol Kinase α Regulates Tubular Recycling Endosome Biogenesis and Major Histocompatibility Complex Class I Recycling

Major histocompatibility complex class I (MHC I) presents intracellular-derived peptides to cytotoxic T lymphocytes and its subcellular itinerary is important in regulating the immune response. While a number of diacylglycerol kinase isoforms have been implicated in clathrin-dependent internalization, MHC I lacks the typical motifs known to mediate clathrin-dependent endocytosis. Here we show that depletion of diacylglycerol kinase α (DGKα), a kinase devoid of a clathrin-dependent adaptor protein complex 2 binding site, caused a delay in MHC I recycling to the plasma membrane without affecting the rate of MHC I internalization. We demonstrate that DGKα knock-down causes accumulation of intracellular and surface MHC I, resulting from decreased degradation. Furthermore, we provide evidence that DGKα is required for the generation of phosphatidic acid required for tubular recycling endosome (TRE) biogenesis. Moreover, we show that DGKα forms a complex with the TRE hub protein, MICAL-L1. Given that MICAL-L1 and the F-BAR-containing membrane-tubulating protein Syndapin2 associate selectively with phosphatidic acid, we propose a positive feedback loop in which DGKα generates phosphatidic acid to drive its own recruitment to TRE via its interaction with MICAL-L1. Our data support a novel role for the involvement of DGKα in TRE biogenesis and MHC I recycling.

Phospholipase D is a central regulator of collagen I‐induced cytoskeletal rearrangement and podosome formation in megakaryocytes

Blood platelets are small anucleated cell fragments generated from bone marrow megakaryocytes (MKs) by a cytoskeleton-driven process. Thereby, mature MKs form long cytoplasmic protrusions (pro-platelets), which extend into the sinusoids within the bone marrow and finally release platelets. Podosomes are F-actin rich matrix contacts that have been suggested to play an important role in cell migration, but also in pro-platelet formation by MKs. Phospholipase D (PLD) has been proposed to contribute to the regulation of actin dynamics through the local generation of phosphatidic acid but its role in platelet formation is unknown. We sought to investigate the significance of PLD in MK podosome formation and thrombocytopoiesis. Podosome formation, spreading and ultra-structure of PLD single- and double-deficient MKs were analyzed using confocal and transmission electron microscopy. Phospholipase D-deficient MKs displayed a highly altered ultra-structure in vivo and abnormal actin rearrangement, with almost abolished formation of podosomes upon spreading on collagen I in vitro. However, MK endomitosis and platelet production were not altered by PLD deficiency. Together, our findings point to a specific function of PLD in actin dynamics as well as podosome formation and size determination in MKs on a collagen I matrix. The normal platelet number in PLD-deficient mice, however, suggests the existence of compensatory mechanisms in vivo that overcome the defective podosome formation observed in vitro.

Phosphatidic acid: a new therapeutic lead to suppress hepatic glucose production.

Phosphatidic acid: a new therapeutic lead to suppress hepatic glucose production.

Metabolome Response to Glucose in the β-Cell Line INS-1 832/13

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is triggered by metabolism of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liquid chromatography with mass spectrometry was used to determine levels of 87 metabolites temporally following a change in glucose from 3 to 10 mM glucose and in response to increasing concentrations of glucose in the INS-1 832/13 β-cell line. U-[(13)C]Glucose was used to probe flux in specific metabolic pathways. Results include a rapid increase in ATP/ADP, anaplerotic tricarboxylic acid cycle flux, and increases in the malonyl CoA pathway, support prevailing theories of GSIS. Novel findings include that aspartate used for anaplerosis does not derive from the glucose fuel added to stimulate insulin secretion, glucose flux into glycerol-3-phosphate, and esterification of long chain CoAs resulting in rapid consumption of long chain CoAs and de novo generation of phosphatidic acid and diacylglycerol. Further, novel metabolites with potential roles in GSIS such as 5-aminoimidazole-4-carboxamide ribotide (ZMP), GDP-mannose, and farnesyl pyrophosphate were found to be rapidly altered following glucose exposure.

Alteration of Plasma Membrane Organization by an Anticancer Lysophosphatidylcholine Analogue Induces Intracellular Acidification and Internalization of Plasma Membrane Transporters in Yeast

The lysophosphatidylcholine analogue edelfosine is a potent antitumor lipid that targets cellular membranes. The underlying mechanisms leading to cell death remain controversial, although two cellular membranes have emerged as primary targets of edelfosine, the plasma membrane (PM) and the endoplasmic reticulum. In an effort to identify conditions that enhance or prevent the cytotoxic effect of edelfosine, we have conducted genome-wide surveys of edelfosine sensitivity and resistance in Saccharomyces cerevisiae presented in this work and the accompanying paper (Cuesta-Marbán, Á., Botet, J., Czyz, O., Cacharro, L. M., Gajate, C., Hornillos, V., Delgado, J., Zhang, H., Amat-Guerri, F., Acuña, A. U., McMaster, C. R., Revuelta, J. L., Zaremberg, V., and Mollinedo, F. (January 23, 2013) J. Biol. Chem. 288,), respectively. Our results point to maintenance of pH homeostasis as a major player in modulating susceptibility to edelfosine with the PM proton pump Pma1p playing a main role. We demonstrate that edelfosine alters PM organization and induces intracellular acidification. Significantly, we show that edelfosine selectively reduces lateral segregation of PM proteins like Pma1p and nutrient H(+)-symporters inducing their ubiquitination and internalization. The biology associated to the mode of action of edelfosine we have unveiled includes selective modification of lipid raft integrity altering pH homeostasis, which in turn regulates cell growth.

Identification and characterization of a gene encoding a putative lysophosphatidyl acyltransferase from Arachis hypogaea

Lysophosphatidyl acyltransferase (LPAT) is the important enzyme responsible for the acylation of lysophosphatidic acid (LPA), leading to the generation of phosphatidic acid (PA) in plant. Its encoding gene is an essential candidate for oil crops to improve oil composition and increase seed oil content through genetic engineering. In this study, a full length AhLPAT4 gene was isolated via cDNA library screening and rapid amplification of cDNA ends (RACE); our data demonstrated that AhLPAT4 had 1631 nucleotides, encoding a putative 43.8 kDa protein with 383 amino acid residues. The deduced protein included a conserved acyltransferase domain and four motifs (I–IV) with putative LPA and acyl-CoA catalytic and binding sites. Bioinformatic analysis indicated that AhLPAT4 contained four transmembrane domains (TMDs), localized to the endoplasmic reticulum (ER) membrane; detailed analysis indicated that motif I and motifs II–III in AhLPAT4 were separated by the third TMD, which located on cytosolic and ER luminal side respectively, and hydrophobic residues on the surface of AhLPAT4 protein fold to form a hydrophobic tunnel to accommodate the acyl chain. Subcellular localization analysis confirmed that AhLPAT4 was a cytoplasm protein.Phylogenetic analysis revealed that AhLPAT4 had a high homology (63.7–78.3%) with putative LPAT4 proteins from Glycine max, Arabidopsis thaliana and Ricinus communis. AhLPAT4 was ubiquitously expressed in diverse tissues except in flower, which is almost undetectable. The expression analysis in different developmental stages in peanut seeds indicated that AhLPAT4 did not coincide with oil accumulation.

A salt stress-activated mitogen-activated protein kinase in soybean is regulated by phosphatidic acid in early stages of the stress response

Salt stress inhibits plant growth and development and plants activate kinase-dependent survival pathways in response to salt stress. However, the role of soybean mitogenactivated protein kinases (MAPKs) in salt stress response has yet to be characterized. In this study, we found that salt stress activates Glycine max MAP kinase 1 (GMK1), a soybean MAPK. The activity of GMK1 induced with increasing salt concentrations, up to 300 mM NaCl, after 5 min of the treatment and was regulated by post-translational modification. We found that mastoparan, a heteromeric G-protein activator, also activated GMK1, and that n-butanol, a phospholipase D inhibitor, and neomycin, a phospholipase C inhibitor, inhibited its activity. Moreover, GMK1 activity was reduced by suramin, a heteromeric G-protein inhibitor, and by two inhibitors of phosphatidic acid (PA) generation after 5 min of 300 mM NaCl treatment. Endogenous PA levels were highest 5 min after induction of salt stress, and exogenous PA directly activated GMK1. From these data, we propose that salt stress signaling is transduced from heteromeric G-protein to GMK1 via phospholipases in the early stages of the response to salt stress in soybean.

The Synaptic Ribbon Is a Site of Phosphatidic Acid Generation in Ribbon Synapses

Ribbon synapses continuously transmit graded membrane potential changes into changes of synaptic vesicle exocytosis and rely on intense synaptic membrane trafficking. The synaptic ribbon is considered central to this process. In the present study we asked whether tonically active ribbon synapses are associated with the generation of certain lipids, specifically the highly active signaling phospholipid phosphatidic acid (PA). Using PA-sensor proteins, we demonstrate that PA is enriched at mouse retinal ribbon synapses in close vicinity to the synaptic ribbon in situ. As shown by heterologous expression, RIBEYE, a main component of synaptic ribbons, is responsible for PA binding at synaptic ribbons. Furthermore, RIBEYE is directly involved in the synthesis of PA. Using various independent substrate binding and enzyme assays, we demonstrate that the B domain of RIBEYE possesses lysophosphatidic acid (LPA) acyltransferase (LPAAT) activity, which leads to the generation of PA from LPA. Since an LPAAT-deficient RIBEYE mutant does not recruit PA-binding proteins to artificial synaptic ribbons, whereas wild-type RIBEYE supports PA binding, we conclude that the LPAAT activity of the RIBEYE(B) domain is a physiologically relevant source of PA generation at the synaptic ribbon. We propose that PA generated at synaptic ribbons likely facilitates synaptic vesicle trafficking.

Phospholipase D Mediates Nutrient Input to Mammalian Target of Rapamycin Complex 1 (mTORC1)

The mammalian target of rapamycin (mTOR) is a critical sensor of nutritional sufficiency. Although much is known about the regulation of mTOR in response to growth factors, much less is known about the regulation of mTOR in response to nutrients. Amino acids have no impact on the signals that regulate Rheb, a GTPase required for the activation of mTOR complex 1 (mTORC1). Phospholipase D (PLD) generates a metabolite, phosphatidic acid, that facilitates association between mTOR and the mTORC1 co-factor Raptor. We report here that elevated PLD activity in human cancer cells is dependent on both amino acids and glucose and that amino acid- and glucose-induced increases in mTORC1 activity are dependent on PLD. Amino acid- and glucose-induced PLD and mTORC1 activity were also dependent on the GTPases RalA and ARF6 and the type III phosphatidylinositol-3-kinase hVps34. Thus, a key stimulatory event for mTORC1 activation in response to nutrients is the generation of phosphatidic acid by PLD.

Interfacial enzyme kinetics of a membrane bound kinase analyzed by real-time MAS-NMR

The simultaneous observation of interdependent reactions within different phases as catalyzed by membrane-bound enzymes is still a challenging task. One such enzyme, the Escherichia coli integral membrane protein diacylglycerol kinase (DGK), is a key player in lipid regulation. It catalyzes the generation of phosphatidic acid within the membrane through the transfer of the γ-phosphate from soluble MgATP to membrane-bound diacylglycerol. We demonstrate that time-resolved (31)P magic angle spinning NMR offers a unique opportunity to simultaneously and directly detect both ATP hydrolysis and diacylglycerol phosphorylation. This experiment demonstrates that solid-state NMR provides a general approach for the kinetic analysis of coupled reactions at the membrane interface regardless of their compartmentalization. The enzymatic activity of DGK was probed with different lipid substrates as well as ATP analogs. Our data yield conclusions about intersubunit cooperativity, reaction stoichiometries and phosphoryl transfer mechanism and are discussed in the context of known structural data.