RESUMEN
Latent tuberculosis, caused by dormant Mycobacterium tuberculosis (Mtb), poses a threat to global health through the incubation of undiagnosed infections within the community. Dormant Mtb, which is phenotypically tolerant to antibiotics, accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. TAG is vital to mycobacteria, serving as a cell envelope component and energy reservoir during latency. TAG synthesis occurs by sequential acylation of glycerol-3-phosphate, wherein the second acylation step is catalyzed by acylglycerol-3-phosphate acyltransferase (AGPAT), resulting in the production of phosphatidic acid (PA), a precursor for the synthesis of TAG and various phospholipids. Here, we have characterized a putative acyltransferase of Mtb encoded by Rv3816c. We found that Rv3816c has all four characteristic motifs of AGPAT, exists as a membrane-bound enzyme, and functions as 1-acylglycerol-3-phosphate acyltransferase. The enzyme could transfer the acyl group to acylglycerol-3-phosphate (LPA) from monounsaturated fatty acyl-coenzyme A of chain length 16 or 18 to produce PA. Complementation of Escherichia coli PlsC mutant in vivo by Rv3816c confirmed that it functions as AGPAT. Its active site mutants, H43A and D48A, were incapable of transferring the acyl group to LPA in vitro and were not able to rescue the growth defect of E. coli PlsC mutant in vivo. Identifying Rv3816c as AGPAT and comparing its properties with other AGPAT homologs is not only a step toward understanding the TAG biosynthesis in mycobacteria but has the potential to explore it as a drug target.
Asunto(s)
Mycobacterium tuberculosis , Triglicéridos , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Triglicéridos/biosíntesis , Triglicéridos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Glicerol-3-Fosfato O-Aciltransferasa/genética , Aciltransferasas/metabolismo , Aciltransferasas/genética , Acilación , Ácidos Grasos/metabolismo , Ácidos Grasos/biosíntesis , Ácidos Fosfatidicos/metabolismo , Ácidos Fosfatidicos/biosíntesis , Acilcoenzima A/metabolismoRESUMEN
Phosphatidic acid biosynthesis represents the initial part of de novo formation of all glycerophospholipids (membrane lipids) as well as triacylglycerols (storage lipids), and is thus the centerpiece of glycerolipid metabolism. The universal route of phosphatidic acid biosynthesis starts from the precursor glycerol-3-phosphate and comprises two consecutive acylation reactions which are catalyzed by a glycerol-3-phosphate acyltransferase and a 1-acyl glycerol-3-phosphate acyltransferase. In addition, yeast and mammals harbor a set of enzymes which can synthesize phosphatidic acid from the precursor dihydroxyacetone phosphate. In the present review our current knowledge about enzymes contributing to phosphatidic acid biosynthesis in the invaluable model organism yeast, Saccharomyces cerevisiae, is summarized. A special focus is laid upon the regulation and the localization of these enzymes. Furthermore, research needs for a deeper insight into the high complexity of phosphatidic acid biosynthesis and consequently the entire lipid metabolic network is presented.
Asunto(s)
Ácidos Fosfatidicos/biosíntesis , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimologíaRESUMEN
Phosphatidic acid (PA) is a glycerophospholipid intermediate in the triglyceride synthesis pathway that has incredibly important structural functions as a component of cell membranes and dynamic effects on intracellular and intercellular signaling pathways. Although there are many pathways to synthesize and degrade PA, a family of PA phosphohydrolases (lipin family proteins) that generate diacylglycerol constitute the primary pathway for PA incorporation into triglycerides. Previously, it was believed that the pool of PA used to synthesize triglyceride was distinct, compartmentalized, and did not widely intersect with signaling pathways. However, we now know that modulating the activity of lipin 1 has profound effects on signaling in a variety of cell types. Indeed, in most tissues except adipose tissue, lipin-mediated PA phosphohydrolase activity is far from limiting for normal rates of triglyceride synthesis, but rather impacts critical signaling cascades that control cellular homeostasis. In this review, we will discuss how lipin-mediated control of PA concentrations regulates metabolism and signaling in mammalian organisms.
Asunto(s)
Glicerofosfolípidos/genética , Fosfatidato Fosfatasa/genética , Triglicéridos/biosíntesis , Tejido Adiposo/metabolismo , Glicerofosfolípidos/metabolismo , Humanos , Redes y Vías Metabólicas/genética , Músculo Esquelético/metabolismo , Fosfatidato Fosfatasa/ultraestructura , Ácidos Fosfatidicos/biosíntesis , Ácidos Fosfatidicos/metabolismo , Conformación Proteica , Triglicéridos/metabolismoRESUMEN
Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid (PA). Mammalian DGK consists of ten isozymes (α-κ) and governs a wide range of physiological and pathological events, including immune responses, neuronal networking, bipolar disorder, obsessive-compulsive disorder, fragile X syndrome, cancer, and type 2 diabetes. DG and PA comprise diverse molecular species that have different acyl chains at the sn-1 and sn-2 positions. Because the DGK activity is essential for phosphatidylinositol turnover, which exclusively produces 1-stearoyl-2-arachidonoyl-DG, it has been generally thought that all DGK isozymes utilize the DG species derived from the turnover. However, it was recently revealed that DGK isozymes, except for DGKε, phosphorylate diverse DG species, which are not derived from phosphatidylinositol turnover. In addition, various PA-binding proteins (PABPs), which have different selectivities for PA species, were recently found. These results suggest that DGK-PA-PABP axes can potentially construct a large and complex signaling network and play physiologically and pathologically important roles in addition to DGK-dependent attenuation of DG-DG-binding protein axes. For example, 1-stearoyl-2-docosahexaenoyl-PA produced by DGKδ interacts with and activates Praja-1, the E3 ubiquitin ligase acting on the serotonin transporter, which is a target of drugs for obsessive-compulsive and major depressive disorders, in the brain. This article reviews recent research progress on PA species produced by DGK isozymes, the selective binding of PABPs to PA species and a phosphatidylinositol turnover-independent DG supply pathway.
Asunto(s)
Proteínas Portadoras/metabolismo , Diacilglicerol Quinasa/metabolismo , Ácidos Fosfatidicos/biosíntesis , Fosfatidilinositoles/metabolismo , Animales , Humanos , Isoenzimas/metabolismo , Mamíferos/metabolismo , Sistemas de Mensajero Secundario , Especificidad por SustratoRESUMEN
Lipids function not only as the major structural components of cell membranes, but also as molecular messengers that transduce signals to trigger downstream signaling events in the cell. Phosphatidic acid (PA), the simplest and a minor class of glycerophospholipids, is a key intermediate for the synthesis of membrane and storage lipids, and also plays important roles in mediating diverse cellular and physiological processes in eukaryotes ranging from microbes to mammals and higher plants. PA comprises different molecular species that can act differently, and is found in virtually all organisms, tissues, and organellar membranes, with variations in total content and molecular species composition. The cellular levels of PA are highly dynamic in response to stimuli and multiple enzymatic reactions can mediate its production and degradation. Moreover, its unique physicochemical properties compared with other glycerophospholipids allow PA to influence membrane structure and dynamics, and interact with various proteins. PA has emerged as a class of new lipid mediators modulating various signaling and cellular processes via its versatile effects, such as membrane tethering, conformational changes, and enzymatic activities of target proteins, and vesicular trafficking.
Asunto(s)
Membrana Celular/metabolismo , Ácidos Fosfatidicos/biosíntesis , Transducción de Señal/fisiología , Animales , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Escherichia coli/metabolismo , Humanos , Ácidos Fosfatidicos/química , Fosfolipasa D/metabolismo , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Phospholipases D (PLDs) catalyze hydrolysis of the diester bond of phospholipids to generate phosphatidic acid and the free lipid headgroup. In mammals, PLD enzymes comprise the intracellular enzymes PLD1 and PLD2 and possibly the proteins encoded by related genes, as well as a class of cell surface and secreted enzymes with structural homology to ectonucleotide phosphatases/phosphodiesterases as typified by autotaxin (ENPP2) that have lysoPLD activities. Genetic and pharmacological loss-of-function approaches implicate these enzymes in intra- and intercellular signaling mediated by the lipid products phosphatidic acid, lysophosphatidic acid, and their metabolites, while the possibility that the water-soluble product of their reactions is biologically relevant has received far less attention. PLD1 and PLD2 are highly selective for phosphatidylcholine (PC), whereas autotaxin has broader substrate specificity for lysophospholipids but by virtue of the high abundance of lysophosphatidylcholine (LPC) in extracellular fluids predominantly hydrolyses this substrate. In all cases, the water-soluble product of these PLD activities is choline. Although choline can be formed de novo by methylation of phosphatidylethanolamine, this activity is absent in most tissues, so mammals are effectively auxotrophic for choline. Dietary consumption of choline in both free and esterified forms is substantial. Choline is necessary for synthesis of the neurotransmitter acetylcholine and of the choline-containing phospholipids PC and sphingomyelin (SM) and also plays a recently appreciated important role as a methyl donor in the pathways of "one-carbon (1C)" metabolism. This review discusses emerging evidence that some of the biological functions of these intra- and extracellular PLD enzymes involve generation of choline with a particular focus on the possibility that these choline and PLD dependent processes are dysregulated in cancer.
Asunto(s)
Colina/metabolismo , Fosfolipasa D/metabolismo , Animales , Humanos , Hidrólisis , Neoplasias , Ácidos Fosfatidicos/biosíntesis , Transducción de SeñalRESUMEN
Lipids play a vital role in numerous cellular functions starting from a structural role as major constituents of membranes to acting as signaling intracellular or extracellular entities. Accordingly, it has been known for decades that lipids, especially those coming from diet, are important to maintain normal physiological functions and good health. On the other side, the exact molecular nature of these beneficial or deleterious lipids, as well as their precise mode of action, is only starting to be unraveled. This recent improvement in our knowledge is largely resulting from novel pharmacological, molecular, cellular, and genetic tools to study lipids in vitro and in vivo. Among these important lipids, phosphatidic acid plays a unique and central role in a great variety of cellular functions. This review will focus on the proposed functions of phosphatidic acid generated by phospholipase D in the last steps of regulated exocytosis with a specific emphasis on hormonal and neurotransmitter release and its potential impact on different neurological diseases.
Asunto(s)
Exocitosis , Enfermedades del Sistema Nervioso/enzimología , Ácidos Fosfatidicos/fisiología , Fosfolipasa D/metabolismo , Humanos , Ácidos Fosfatidicos/biosíntesis , Transducción de SeñalRESUMEN
Morusin is a prenylated flavonoid found in mulberry that shows antimicrobial activity against foodborne pathogens. The MIC values of morusin toward S. aureus ATCC 6538 and S. aureus ATCC 25923 were both 14.9 µmol L-1. This study further investigated the antimicrobial mechanism of morusin in inhibiting the growth of Staphylococcus aureus ATCC 6538. Scanning electron microscopy and transmission electron microscopy revealed that morusin disrupted the integrity of the bacterial cell membrane. Morusin may also affect the phospholipid-repair system of bacteria, which repairs membrane structures. To test this hypothesis, quantitative real-time PCR was used to examine the effect of morusin treatment of S. aureus on the regulation of genes associated with the cell phosphatidic acid biosynthesis pathway. Gas chromatography-mass spectrometry was used to investigate the fatty acid components, which are used to synthesize bacterial phosphatidic acids. In summary, the results revealed that morusin showed a potent antibacterial effect by disrupting the cell membrane architecture and inhibiting the phosphatidic acid biosynthesis pathway of S. aureus.
Asunto(s)
Antibacterianos/farmacología , Membrana Celular/efectos de los fármacos , Flavonoides/farmacología , Ácidos Fosfatidicos/biosíntesis , Extractos Vegetales/farmacología , Staphylococcus aureus/efectos de los fármacos , Membrana Celular/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Pruebas de Sensibilidad Microbiana , Morus/química , Ácidos Fosfatidicos/química , Staphylococcus aureus/metabolismoRESUMEN
Acute cardiac rupture and adverse left ventricular (LV) remodeling causing heart failure are serious complications of acute myocardial infarction (MI). While cardio-hepatic interactions have been recognized, their role in MI remains unknown. We treated cultured cardiomyocytes with conditioned media from various cell types and analyzed the media by mass spectrometry to identify α1-microglobulin (AM) as an Akt-activating hepatokine. In mouse MI model, AM protein transiently distributed in the infarct and border zones during the acute phase, reflecting infiltration of AM-bound macrophages. AM stimulation activated Akt, NFκB, and ERK signaling and enhanced inflammation as well as macrophage migration and polarization, while inhibited fibrogenesis-related mRNA expression in cultured macrophages and cardiac fibroblasts. Intramyocardial AM administration exacerbated macrophage infiltration, inflammation, and matrix metalloproteinase 9 mRNA expression in the infarct and border zones, whereas disturbed fibrotic repair, then provoked acute cardiac rupture in MI. Shotgun proteomics and lipid pull-down analysis found that AM partly binds to phosphatidic acid (PA) for its signaling and function. Furthermore, systemic delivery of a selective inhibitor of diacylglycerol kinase α-mediated PA synthesis notably reduced macrophage infiltration, inflammation, matrix metalloproteinase activity, and adverse LV remodeling in MI. Therefore, targeting AM signaling could be a novel pharmacological option to mitigate adverse LV remodeling in MI.
Asunto(s)
alfa-Globulinas/metabolismo , Hormonas/metabolismo , Infarto del Miocardio/patología , Transducción de Señal , Animales , Membrana Celular/metabolismo , Movimiento Celular , Activación Enzimática , Fibrosis , Inflamación/metabolismo , Hígado/metabolismo , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ácidos Fosfatidicos/biosíntesis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Remodelación VentricularRESUMEN
Plants use phospholipase D (PLD, EC 3.1.4.4)/phosphatidic acid (PtdOH) for the transduction of environmental signals including those coming from wounding. Based on our previous findings suggesting that wound-induced PLDα-derived PtdOH can act as a local signaling molecule in cotton (Gossypium hirsutum), we show that wounding immediately increases local NADPH oxidase (NADPHox) and cellulose synthase A (CeSA) gene expression. After developing a novel fluorimetric assay for the investigation of n-butanol inhibitory effect on PLD activity, we show that only NADPHox gene upregulation is reduced when n-butanol is applied prior to wounding. This suggests that NADPHox is a possible downstream target of PLD function, while a different CeSA-involving response system may exist in cotton. Overall, this study provides new knowledge on signal-transduction mechanisms following wounding of cotton leaves.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/genética , Glucosiltransferasas/genética , Gossypium/genética , NADPH Oxidasas/genética , Ácidos Fosfatidicos/biosíntesis , Glucosiltransferasas/metabolismo , Gossypium/metabolismo , NADPH Oxidasas/metabolismo , Ácidos Fosfatidicos/química , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Transducción de Señal/genéticaRESUMEN
MAIN CONCLUSION: The phytotoxin botrydial triggers PA production in tomato cell suspensions via PLD and PLC/DGK activation. PLC/DGK-derived PA is partially required for botrydial-induced ROS generation. Phosphatidic acid (PA) is a phospholipid second messenger involved in the induction of plant defense responses. It is generated via two distinct enzymatic pathways, either via phospholipase D (PLD) or by the sequential action of phospholipase C and diacylglycerol kinase (PLC/DGK). Botrydial is a phytotoxic sesquiterpene generated by the necrotrophic fungus Botrytis cinerea that induces diverse plant defense responses, such as the production of reactive oxygen species (ROS). Here, we analyzed PA and ROS production and their interplay upon botrydial treatments, employing tomato (Solanum lycopersicum) cell suspensions as a model system. Botrydial induces PA production within minutes via PLD and PLC/DGK. Either inhibition of PLC or DGK diminishes ROS generation triggered by botrydial. This indicates that PLC/DGK is upstream of ROS production. In tomato, PLC is encoded by a multigene family constituted by SlPLC1-SlPLC6 and the pseudogene SlPLC7. We have shown that SlPLC2-silenced plants have reduced susceptibility to B. cinerea. In this work, we studied the role of SlPLC2 on botrydial-induced PA production by silencing the expression of SlPLC2 via a specific artificial microRNA. Upon botrydial treatments, SlPLC2-silenced-cell suspensions produce PA levels similar to wild-type cells. It can be concluded that PA is a novel component of the plant responses triggered by botrydial.
Asunto(s)
Aldehídos/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Ácidos Fosfatidicos/biosíntesis , Solanum lycopersicum/efectos de los fármacos , Botrytis/metabolismo , Células Cultivadas , Diacilglicerol Quinasa/metabolismo , Solanum lycopersicum/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Imaging approaches that track biological molecules within cells are essential tools in modern biochemistry. Lipids are particularly challenging to visualize, as they are not directly genetically encoded. Phospholipids, the most abundant subgroup of lipids, are structurally diverse and accomplish many cellular functions, acting as major structural components of membranes and as signaling molecules that regulate cell growth, division, apoptosis, cytoskeletal dynamics, and numerous other physiological processes. Cells regulate the abundance, and therefore bioactivity, of phospholipids by modulating the activities of their biosynthetic enzymes. Thus, techniques that enable monitoring of flux through individual lipid biosynthetic pathways can provide key functional information. For example, the choline analogue propargylcholine (ProCho) can report on de novo biosynthesis of phosphatidylcholine by conversion to an alkynyl lipid that can be imaged following click chemistry tagging with an azido fluorophore. We report that ProCho is also a substrate of phospholipase D enzymes-which normally hydrolyze phosphatidylcholine to generate the lipid second messenger phosphatidic acid-in a transphosphatidylation reaction, generating the identical alkynyl lipid. By controlling the activities of phosphatidylcholine biosynthesis and phospholipase D enzymes, we establish labeling conditions that enable this single probe to selectively report on two different biosynthetic pathways. Just as nature exploits the economy of common metabolic intermediates to efficiently diversify biosynthesis, so can biochemists in interrogating such pathways with careful probe design. We envision that ProCho's ability to report on multiple metabolic pathways will enable studies of membrane dynamics and improve our understanding of the myriad roles that lipids play in cellular homeostasis.
Asunto(s)
Alquinos/metabolismo , Colina/análogos & derivados , Ácidos Fosfatidicos/biosíntesis , Fosfatidilcolinas/biosíntesis , Fosfolipasa D/metabolismo , Alquinos/química , Arachis/enzimología , Biotinilación , Colina/química , Colina/metabolismo , Cromatografía Líquida de Alta Presión , Química Clic , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Células HeLa , Humanos , Lípidos de la Membrana/biosíntesis , Fosfolipasa D/antagonistas & inhibidores , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Especificidad por Sustrato , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
mTOR, the mammalian target of rapamycin, integrates growth factor and nutrient signals to promote a transformation from catabolic to anabolic metabolism, cell growth, and cell cycle progression. Phosphatidic acid (PA) interacts with the FK506-binding protein-12-rapamycin-binding (FRB) domain of mTOR, which stabilizes both mTOR complexes: mTORC1 and mTORC2. We report here that mTORC1 and mTORC2 are activated in response to exogenously supplied fatty acids via the de novo synthesis of PA, a central metabolite for membrane phospholipid biosynthesis. We examined the impact of exogenously supplied fatty acids on mTOR in KRas-driven cancer cells, which are programmed to utilize exogenous lipids. The induction of mTOR by oleic acid was dependent upon the enzymes responsible for de novo synthesis of PA. Suppression of the de novo synthesis of PA resulted in G1 cell cycle arrest. Although it has long been appreciated that mTOR is a sensor of amino acids and glucose, this study reveals that mTOR also senses the presence of lipids via production of PA.
Asunto(s)
Complejos Multiproteicos/metabolismo , Ácidos Fosfatidicos/biosíntesis , Serina-Treonina Quinasas TOR/metabolismo , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Células Hep G2 , Humanos , Células MCF-7 , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/genética , Ácido Oléico/farmacología , Ácidos Fosfatidicos/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Serina-Treonina Quinasas TOR/genéticaRESUMEN
In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1-77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1-77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Ácidos Fosfatidicos/biosíntesis , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Sustitución de Aminoácidos , Quinasa de la Caseína II/química , Quinasa de la Caseína II/genética , Mutación Missense , Ácidos Fosfatidicos/química , Ácidos Fosfatidicos/genética , Fosforilación/fisiología , Proteínas Represoras/química , Proteínas Represoras/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Serina/química , Serina/genética , Serina/metabolismoRESUMEN
Phosphatidic acid (PA) is a potent lipid secondary messenger, the synthesis of which is tightly regulated in both space and time. Established tools for detecting PA involve exâ vivo analysis and do not provide information on the subcellular locations where this lipid is synthesized. Here, a chemoenzymatic strategy for imaging sites of cellular PA synthesis by phospholipaseâ D (PLD) enzymes is reported. PLDs were found to be able to catalyze phospholipid head-group exchange with alkynols to generate alkyne-labeled PA analogues within cells. Subsequent fluorophore tagging through Cu-catalyzed azide-alkyne cycloaddition enabled both visualization by fluorescence microscopy and quantification by HPLC. Our studies revealed several intracellular sites of PLD-mediated PA synthesis. We envision applications of this approach to dissect PA-dependent signaling pathways, image PLD activity in disease, and remodel intracellular membranes with new functionality.
Asunto(s)
Ácidos Fosfatidicos/biosíntesis , Fosfolipasa D/metabolismo , Células HeLa , Humanos , Microscopía Confocal , Estructura Molecular , Imagen Óptica , Ácidos Fosfatidicos/química , Fosfolipasa D/químicaRESUMEN
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.
Asunto(s)
Caveolas/metabolismo , Endocitosis/fisiología , Células Endoteliales/metabolismo , Ácidos Fosfatidicos/biosíntesis , Fosfolipasa D/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Transporte Biológico Activo/fisiología , Membrana Celular/genética , Membrana Celular/metabolismo , Células Endoteliales/citología , Humanos , Mutación , Ácidos Fosfatidicos/genética , Fosfolipasa D/genética , Proteínas de Unión al GTP ral/genéticaRESUMEN
Timely activation of Aurora kinase A (AURA, also known as AURKA) is vital for centrosome formation and the progression of mitosis. Nonetheless, it is still unclear if and when other cellular functions are activated by AURA. We report here that Src phosphorylates and activates AURA at T288, and AURA also activates focal adhesion kinase (FAK, also known as PTK2), leading to initiation of cell movement. An additional and new way by which AURA is regulated, is by phospholipase D2 (PLD2), which causes AURA activation. In addition, AURA phosphorylates PLD, so both proteins engage in a positive reinforcement loop. AURA and PLD2 form a proteinprotein complex and colocalize to cytoplasmic regions in cells. The reason why PLD activates AURA is because of the production of phosphatidic acid by the lipase, which binds directly to AURA, with the region E171E211 projected to be a phosphatidic-acid-binding pocket. Furthermore, this direct interaction with phosphatidic acid enhances tubulin polymerization and cooperates synergistically with AURA, FAK and Src in yielding a fully effectual cellular migration. Thus, Src and FAK, and PLD and phosphatidic acid are new upstream regulators of AURA that mediate its role in the non-mitotic cellular function of cell migration.
Asunto(s)
Aurora Quinasa A/metabolismo , Movimiento Celular/fisiología , Quinasa 2 de Adhesión Focal/metabolismo , Fosfolipasa D/metabolismo , Familia-src Quinasas/metabolismo , Animales , Aurora Quinasa A/genética , Células COS , Chlorocebus aethiops , Activación Enzimática , Células Epiteliales/metabolismo , Mitosis/fisiología , Simulación del Acoplamiento Molecular , Ácidos Fosfatidicos/biosíntesis , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de Señal , Tubulina (Proteína)/metabolismoRESUMEN
It is difficult to derive all qualitative proteomic and metabolomic experimental data in male (pollen tube) and female (pistil) reproductive tissues during pollination because of the limited sensitivity of current technology. In this study, genome-scale enzyme correlation network models for plants (Arabidopsis/maize) were constructed by analyzing the enzymes and metabolic routes from a global perspective. Then, we developed a data-driven computational pipeline using the "guilt by association" principle to analyze the transcriptional coexpression profiles of enzymatic genes in the consecutive steps for metabolic routes in the fast-growing pollen tube and stigma during pollination. The analysis identified an inferred pattern of pollen tube-stigma ethanol coupling. When the pollen tube elongates in the transmitting tissue (TT) of the pistil, this elongation triggers the mobilization of energy from glycolysis in the TT cells of the pistil. Energy-rich metabolites (ethanol) are secreted that can be taken up by the pollen tube, where these metabolites are incorporated into the pollen tube's tricarboxylic acid (TCA) cycle, which leads to enhanced ATP production for facilitating pollen tube growth. In addition, our analysis also provided evidence for the cooperation of kaempferol, dTDP-alpha-L-rhamnose and cell-wall-related proteins; phosphatidic-acid-mediated Ca2+ oscillations and cytoskeleton; and glutamate degradation IV for γ-aminobutyric acid (GABA) signaling activation in Arabidopsis and maize stigmas to provide the signals and materials required for pollen tube tip growth. In particular, the "guilt by association" computational pipeline and the genome-scale enzyme correlation network models (GECN) developed in this study was initiated with experimental "omics" data, followed by data analysis and data integration to determine correlations, and could provide a new platform to assist inachieving a deeper understanding of the co-regulation and inter-regulation model in plant research.
Asunto(s)
Arabidopsis/genética , Tubo Polínico/genética , Tubo Polínico/metabolismo , Polinización/genética , Transcripción Genética , Zea mays/genética , Arabidopsis/metabolismo , Pared Celular/genética , Etanol/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Ácido Glutámico/metabolismo , Quempferoles/biosíntesis , Redes y Vías Metabólicas/genética , Ácidos Fosfatidicos/biosíntesis , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubo Polínico/enzimología , Ramnosa/biosíntesis , Zea mays/metabolismoRESUMEN
Malaria parasites scavenge nutrients from their host but also harbour enzymatic pathways for de novo macromolecule synthesis. One such pathway is apicoplast-targeted type II fatty acid synthesis, which is essential for late liver-stage development in rodent malaria. It is likely that fatty acids synthesized in the apicoplast are ultimately incorporated into membrane phospholipids necessary for exoerythrocytic merozoite formation. We hypothesized that these synthesized fatty acids are being utilized for apicoplast-targeted phosphatidic acid synthesis, the phospholipid precursor. Phosphatidic acid is typically synthesized in a three-step reaction utilizing three enzymes: glycerol 3-phosphate dehydrogenase, glycerol 3-phosphate acyltransferase and lysophosphatidic acid acyltransferase. The Plasmodium genome is predicted to harbour genes for both apicoplast- and cytosol/endoplasmic reticulum-targeted phosphatidic acid synthesis. Our research shows that apicoplast-targeted Plasmodium yoelii glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate acyltransferase are expressed only during liver-stage development and deletion of the encoding genes resulted in late liver-stage growth arrest and lack of merozoite differentiation. However, the predicted apicoplast-targeted lysophosphatidic acid acyltransferase gene was refractory to deletion and was expressed solely in the endoplasmic reticulum throughout the parasite life cycle. Our results suggest that P. yoelii has an incomplete apicoplast-targeted phosphatidic acid synthesis pathway that is essential for liver-stage maturation.
Asunto(s)
Hígado/parasitología , Ácidos Fosfatidicos/biosíntesis , Plasmodium yoelii/enzimología , Plasmodium yoelii/fisiología , Plastidios/enzimología , Plastidios/metabolismo , Aciltransferasas/metabolismo , Animales , Glicerol-3-Fosfato Deshidrogenasa (NAD+)/metabolismo , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Ratones , Plasmodium yoelii/metabolismo , Transporte de ProteínasRESUMEN
During cultivation of the filamentous fungus Lentinus tigrinus on a medium containing lignin, a high oxygen content stimulated the growth of the fungus and contributed to the yield of lipids. A high content of phosphatidic acid and a reduction in the level of phosphatidylethanolamine and phosphatidylserine were first detected in the composition of phospholipids. Changes in the composition of neutral lipids, such as variation in the ratio of esterified and free sterols, have occurred; thus, the amount of sterol esters reduced simultaneously with a decrease in the content of free fatty acids. Based on the obtained results, the possible role of phosphatidic acid as a second messenger in the process of the consumption of lignin by the fungus Lentinus tigrinus is discussed.