RESUMEN
Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.
Asunto(s)
Aterosclerosis/inmunología , Colesterol/biosíntesis , Desmosterol/metabolismo , Células Espumosas/metabolismo , Metabolismo de los Lípidos , Transcriptoma , Animales , Aterosclerosis/metabolismo , Colesterol/análogos & derivados , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Células Espumosas/inmunología , Técnicas de Silenciamiento del Gen , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
Lenz-Majewski syndrome (LMS) is a rare disease characterized by complex craniofacial, dental, cutaneous, and limb abnormalities combined with intellectual disability. Mutations in thePTDSS1gene coding one of the phosphatidylserine (PS) synthase enzymes, PSS1, were described as causative in LMS patients. Such mutations render PSS1 insensitive to feedback inhibition by PS levels. Here we show that expression of mutant PSS1 enzymes decreased phosphatidylinositol 4-phosphate (PI4P) levels both in the Golgi and the plasma membrane (PM) by activating the Sac1 phosphatase and altered PI4P cycling at the PM. Conversely, inhibitors of PI4KA, the enzyme that makes PI4P in the PM, blocked PS synthesis and reduced PS levels by 50% in normal cells. However, mutant PSS1 enzymes alleviated the PI4P dependence of PS synthesis. Oxysterol-binding protein-related protein 8, which was recently identified as a PI4P-PS exchanger between the ER and PM, showed PI4P-dependent membrane association that was significantly decreased by expression of PSS1 mutant enzymes. Our studies reveal that PS synthesis is tightly coupled to PI4P-dependent PS transport from the ER. Consequently, PSS1 mutations not only affect cellular PS levels and distribution but also lead to a more complex imbalance in lipid homeostasis by disturbing PI4P metabolism.
Asunto(s)
Anomalías Múltiples/enzimología , Enfermedades del Desarrollo Óseo/enzimología , Membrana Celular/enzimología , Retículo Endoplásmico/enzimología , Aparato de Golgi/enzimología , Discapacidad Intelectual/enzimología , Mutación , Transferasas de Grupos Nitrogenados/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Anomalías Múltiples/genética , Enfermedades del Desarrollo Óseo/genética , Membrana Celular/genética , Retículo Endoplásmico/genética , Aparato de Golgi/genética , Células HEK293 , Humanos , Discapacidad Intelectual/genética , Antígenos de Histocompatibilidad Menor , Transferasas de Grupos Nitrogenados/genética , Fosfatos de Fosfatidilinositol/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW264.7 macrophages uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of 2-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of 2-AG. An unbiased systems-based mechanistic model of the kinetic data revealed that binding of AA or 2-AG at the allosteric site of COX-2 results in a decreased catalytic efficiency of the enzyme toward 2-AG, whereas 2-AG binding at the allosteric site increases COX-2's efficiency toward AA. The results suggest that substrates interact with COX-2 via multiple potential complexes involving binding to both the catalytic and allosteric sites. Competition between AA and 2-AG for these sites, combined with differential allosteric modulation, gives rise to a complex interplay between the substrates, leading to preferential oxygenation of AA.
Asunto(s)
Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/metabolismo , Ciclooxigenasa 2/metabolismo , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Prostaglandinas/metabolismo , Algoritmos , Regulación Alostérica , Sitio Alostérico , Animales , Unión Competitiva , Dominio Catalítico , Línea Celular , Simulación por Computador , Ciclooxigenasa 2/química , Cinética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Oxidación-Reducción , Unión Proteica , Multimerización de Proteína , Células Sf9 , Spodoptera , Especificidad por Sustrato , Zimosan/farmacologíaRESUMEN
Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their de novo synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for de novo dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibitors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Desoxirribonucleótidos/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Fosfolipasa D/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Replicación Viral , Apoptosis , Western Blotting , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular , Células Cultivadas , Replicación del ADN , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Humanos , Activación de Linfocitos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Phospholipase D is a ubiquitous class of enzymes that generates phosphatidic acid as an intracellular signaling species. The phospholipase D superfamily plays a central role in a variety of functions in prokaryotes, viruses, yeast, fungi, plants, and eukaryotic species. In mammalian cells, the pathways modulating catalytic activity involve a variety of cellular signaling components, including G protein-coupled receptors, receptor tyrosine kinases, polyphosphatidylinositol lipids, Ras/Rho/ADP-ribosylation factor GTPases, and conventional isoforms of protein kinase C, among others. Recent findings have shown that phosphatidic acid generated by phospholipase D plays roles in numerous essential cellular functions, such as vesicular trafficking, exocytosis, autophagy, regulation of cellular metabolism, and tumorigenesis. Many of these cellular events are modulated by the actions of phosphatidic acid, and identification of two targets (mammalian target of rapamycin and Akt kinase) has especially highlighted a role for phospholipase D in the regulation of cellular metabolism. Phospholipase D is a regulator of intercellular signaling and metabolic pathways, particularly in cells that are under stress conditions. This review provides a comprehensive overview of the regulation of phospholipase D activity and its modulation of cellular signaling pathways and functions.
Asunto(s)
Neoplasias/patología , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/metabolismo , Animales , Humanos , Terapia Molecular Dirigida , Neoplasias/terapia , Transducción de Señal/fisiologíaRESUMEN
A reliable method for purifying envelope-stripped nuclei from immortalized murine embryonic fibroblasts (iMEFs) was established. Quantitative profiling of the glycerophospholipids (GPLs) in envelope-free iMEF nuclei yields several conclusions. First, we find the endonuclear glycerophospholipidome differs from that of bulk membranes, and phosphatidylcholine (PtdCho) and phosphatidylethanolamine species are the most abundant endonuclear GPLs by mass. By contrast, phosphatidylinositol (PtdIns) represents a minor species. We also find only a slight enrichment of saturated versus unsaturated GPL species in iMEF endonuclear fractions. Moreover, much lower values for GPL mass were measured in the iMEF nuclear matrix than those reported for envelope-stripped IMF-32 nuclei. The collective results indicate that the nuclear matrix in these cells is a GPL-poor environment where GPL occupies only approximately 0.1% of the total nuclear matrix volume. This value suggests GPL accommodation in this compartment can be satisfied by binding to resident proteins. Finally, we find no significant role for the PtdIns/PtdCho-transfer protein, PITPα, in shuttling PtdIns into the iMEF nuclear matrix.
Asunto(s)
Fibroblastos/metabolismo , Membrana Nuclear/metabolismo , Fosfolípidos/metabolismo , Animales , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Células Cultivadas , Embrión de Mamíferos/química , Fibroblastos/ultraestructura , Ratones , Proteínas de Transferencia de Fosfolípidos/metabolismoRESUMEN
Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxa(vu463) ) that has an inactivating mutation in the etfa gene. dxa(vu463) recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxa(vu463) zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxa(vu463) zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.
Asunto(s)
Flavoproteínas Transportadoras de Electrones/genética , Enfermedades Mitocondriales/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Complejos Multiproteicos/genética , Serina-Treonina Quinasas TOR/genética , Animales , Modelos Animales de Enfermedad , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Mitocondrias/genética , Mitocondrias/patología , Enfermedades Mitocondriales/fisiopatología , Terapia Molecular Dirigida , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/fisiopatología , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/terapia , Complejos Multiproteicos/antagonistas & inhibidores , Transducción de Señal , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Phospholipase D is a ubiquitous protein in eukaryotes that hydrolyzes phospholipids to generate the signaling lipid phosphatidic acid (PtdOH). PldA, a Pseudomonas aeruginosa PLD, is a secreted protein that targets bacterial and eukaryotic cells. Here we have characterized the in vitro factors that modulate enzymatic activity of PldA, including divalent cations and phosphoinositides. We have identified several similarities between the eukaryotic-like PldA and the human PLD isoforms, as well as several properties in which the enzymes diverge. Notable differences include the substrate preference and transphosphatidylation efficiency for PldA. These findings offer new insights into potential regulatory mechanisms of PldA and its role in pathogenesis.
Asunto(s)
Proteínas Bacterianas/química , Fosfolipasa D/química , Pseudomonas aeruginosa/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Humanos , Fosfolipasa D/genética , Fosfolipasa D/metabolismo , Pseudomonas aeruginosa/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Especificidad por Sustrato/fisiologíaRESUMEN
The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an "omics" approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.
Asunto(s)
Lípidos/sangre , Lípidos/orina , Enfermedad del Hígado Graso no Alcohólico , Polimorfismo de Nucleótido Simple , Adulto , Biomarcadores/metabolismo , Biomarcadores/orina , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/epidemiología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/orinaRESUMEN
The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipaseD(PLD) is a novel regulator of Akt inGBM.Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase.
Asunto(s)
Autofagia/fisiología , Fosfolipasa D/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/efectos de los fármacos , Autofagia/genética , Proteínas Relacionadas con la Autofagia , Beclina-1 , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Medio de Cultivo Libre de Suero/farmacología , Inhibidores Enzimáticos/farmacología , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Células HEK293 , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/antagonistas & inhibidores , Fosfolipasa D/genética , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Lipid metabolism plays a fundamental role during influenza virus replication, although key regulators of lipid-dependent trafficking and virus production remain inadequately defined. This report demonstrates that infection by influenza virus stimulates phospholipase D (PLD) activity and that PLD co-localizes with influenza during infection. Both chemical inhibition and RNA interference of PLD delayed viral entry and reduced viral titers in vitro. Although there may be contributions by both major isoenzymes, the effects on viral infectivity appear to be more dependent on the PLD2 isoenzyme. In vivo, PLD2 inhibition reduced virus titer and correlated with significant increases in transcription of innate antiviral effectors. The reduction in viral titer downstream of PLD2 inhibition was dependent on Rig-I (retinoic acid-inducible gene-1), IRF3, and MxA (myxovirus resistance gene A) but not IRF7. Inhibition of PLD2 accelerated the accumulation of MxA in foci as early as 30 min postinfection. Together these data suggest that PLD facilitates the rapid endocytosis of influenza virus, permitting viral escape from innate immune detection and effectors that are capable of limiting lethal infection.
Asunto(s)
Inmunidad Innata/genética , Gripe Humana/virología , Orthomyxoviridae/genética , Fosfolipasa D/biosíntesis , Línea Celular , Endocitosis/genética , Regulación Viral de la Expresión Génica , Humanos , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana/genética , Gripe Humana/patología , Orthomyxoviridae/patogenicidad , Fosfolipasa D/antagonistas & inhibidores , Fosfolipasa D/genética , Fosfolípidos , Interferencia de ARN , Internalización del Virus , Replicación Viral/genéticaRESUMEN
Thirty years ago, glycerolipids captured the attention of biochemical researchers as novel cellular signaling entities. We now recognize that these biomolecules occupy signaling nodes critical to a number of physiological and pathological processes. Thus, glycerolipid-metabolizing enzymes present attractive targets for new therapies. A number of fields-ranging from neuroscience and cancer to diabetes and obesity-have elucidated the signaling properties of glycerolipids. The biochemical literature teems with newly emerging small molecule inhibitors capable of manipulating glycerolipid metabolism and signaling. This ever-expanding pool of chemical modulators appears daunting to those interested in exploiting glycerolipid-signaling pathways in their model system of choice. This review distills the current body of literature surrounding glycerolipid metabolism into a more approachable format, facilitating the application of small molecule inhibitors to novel systems. This article is part of a Special Issue entitled Tools to study lipid functions.
Asunto(s)
Lípidos/fisiología , Transducción de Señal , Aciltransferasas/metabolismo , Glicerol/química , Lipasa/metabolismo , Lípidos/química , Fosfolipasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas/metabolismoRESUMEN
High levels of saturated FAs (SFAs) are acutely toxic to a variety of cell types, including hepatocytes, and have been associated with diseases such as type 2 diabetes and nonalcoholic fatty liver disease. SFA accumulation has been previously shown to degrade endoplasmic reticulum (ER) function leading to other manifestations of the lipoapoptotic cascade. We hypothesized that dysfunctional phospholipid (PL) metabolism is an initiating factor in this ER stress response. Treatment of either primary hepatocytes or H4IIEC3 cells with the SFA palmitate resulted in dramatic dilation of the ER membrane, coinciding with other markers of organelle dysfunction. This was accompanied by increased de novo glycerolipid synthesis, significant elevation of dipalmitoyl phosphatidic acid, diacylglycerol, and total PL content in H4IIEC3 cells. Supplementation with oleate (OA) reversed these markers of palmitate (PA)-induced lipotoxicity. OA/PA cotreatment modulated the distribution of PA between lipid classes, increasing the flux toward triacylglycerols while reducing its incorporation into PLs. Similar trends were demonstrated in both primary hepatocytes and the H4IIEC3 hepatoma cell line. Overall, these findings suggest that modifying the FA composition of structural PLs can protect hepatocytes from PA-induced ER stress and associated lipotoxicity.
Asunto(s)
Estrés del Retículo Endoplásmico/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/metabolismo , Ácido Palmítico/toxicidad , Fosfolípidos/farmacología , Animales , Línea Celular Tumoral , Hepatocitos/patología , Hígado/patología , Ratas , Ratas Sprague-DawleyRESUMEN
Phosphatidic acid (PA) is a lipid second messenger located at the intersection of several lipid metabolism and cell signaling events including membrane trafficking, survival, and proliferation. Generation of signaling PA has long been primarily attributed to the activation of phospholipase D (PLD). PLD catalyzes the hydrolysis of phosphatidylcholine into PA. A variety of both receptor-tyrosine kinase and G-protein-coupled receptor stimulations have been shown to lead to PLD activation and PA generation. This study focuses on profiling the PA pool upon P2Y6 receptor signaling manipulation to determine the major PA producing enzymes. Here we show that PLD, although highly active, is not responsible for the majority of stable PA being produced upon UDP stimulation of the P2Y6 receptor and that PA levels are tightly regulated. By following PA flux in the cell we show that PLD is involved in an initial increase in PA upon receptor stimulation; however, when PLD is blocked, the cell compensates by increasing PA production from other sources. We further delineate the P2Y6 signaling pathway showing that phospholipase Cß3 (PLCß3), PLCδ1, DGKζ and PLD are all downstream of receptor activation. We also show that DGKζ is a novel negative regulator of PLD activity in this system that occurs through an inhibitory mechanism with PKCα. These results further define the downstream events resulting in PA production in the P2Y6 receptor signaling pathway.
Asunto(s)
Ácidos Fosfatidicos/biosíntesis , Fosfatidilcolinas/metabolismo , Fosfolipasa D/metabolismo , Receptores Purinérgicos P2/metabolismo , 1-Butanol/farmacología , Western Blotting , Línea Celular Tumoral , Diacilglicerol Quinasa/genética , Diacilglicerol Quinasa/metabolismo , Diglicéridos/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Hidrólisis , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Espectrometría de Masas , Modelos Biológicos , Fosfolipasa C delta/genética , Fosfolipasa C delta/metabolismo , Fosfolipasa D/antagonistas & inhibidores , Fosfolipasa D/genética , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Interferencia de ARN , Receptores Purinérgicos P2/genética , Transducción de Señal/efectos de los fármacos , Uridina Difosfato/farmacologíaRESUMEN
25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.
Asunto(s)
Hidroxicolesteroles/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Ésteres del Colesterol/metabolismo , Perfilación de la Expresión Génica , Hidroxicolesteroles/metabolismo , Receptores X del Hígado , Macrófagos/citología , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Muromegalovirus/fisiología , Receptores Nucleares Huérfanos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Esfingolípidos/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidoresRESUMEN
This Letter describes the on-going SAR efforts based on two scaffolds, a PLD1-biased piperidinyl benzimidazolone and a PLD2-biased piperidinyl triazaspirone, with the goal of enhancing PLD inhibitory potency and isoform selectivity. Here, we found that addition of an α-methyl moiety within the PLD2-biased piperidinyl triazaspirone scaffold abolished PLD2 preference, while the incorporation of substituents onto the piperidine moiety of the PLD1-biased piperidinyl benzimidazolone, or replacement with a bioisosteric [3.3.0] core, generally retained PLD1 preference, but at diminished significance. The SAR uncovered within these two allosteric PLD inhibitor series further highlights the inherent challenges of developing isoform selective PLD inhibitors.
Asunto(s)
Inhibidores Enzimáticos/química , Fosfolipasa D/antagonistas & inhibidores , Animales , Bencimidazoles/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Cinética , Microsomas/metabolismo , Fosfolipasa D/metabolismo , Piperidinas/química , Unión Proteica , Ratas , Relación Estructura-ActividadRESUMEN
Human cytomegalovirus induces and requires fatty acid synthesis. This suggests an essential role for lipidome remodeling in viral replication. We used mass spectrometry to quantify glycerophospholipids in mock-infected and virus-infected fibroblasts, as well as in virions. Although the lipid composition of mock-infected and virus-infected fibroblasts was similar, virions were markedly different. The virion envelope contained twofold more phosphatidylethanolamines and threefold less phosphatidylserines than the host cell. This indicates that the virus buds from a membrane with a different lipid composition from the host cell as a whole. Compared with published datasets, the virion envelope showed the greatest similarity to the synaptic vesicle lipidome. Synaptosome-associated protein of 25 kDa (SNAP-25) is a component of the complex that mediates exocytosis of synaptic vesicles in neurons; and its homolog, SNAP-23, functions in exocytosis in many other cell types. Infection induced the relocation of SNAP-23 to the cytoplasmic viral assembly zone, and knockdown of SNAP-23 inhibited the production of virus. We propose that cytomegalovirus capsids acquire their envelope by budding into vesicles with a lipid composition similar to that of synaptic vesicles, which subsequently fuse with the plasma membrane to release virions from the cell.
Asunto(s)
Citomegalovirus/química , Lípidos/química , Proteínas SNARE/metabolismo , Virión/química , Western Blotting , Línea Celular , Células Cultivadas , Cromatografía Liquida , Citomegalovirus/fisiología , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/virología , Técnica del Anticuerpo Fluorescente , Glicerofosfolípidos/química , Glicerofosfolípidos/metabolismo , Interacciones Huésped-Patógeno , Humanos , Masculino , Espectrometría de Masas , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Interferencia de ARN , Proteínas SNARE/genética , Vesículas Sinápticas/química , Virión/fisiología , Replicación ViralRESUMEN
Metabolomics is a rapidly growing field of research used in the identification and quantification of the small molecule metabolites within an organism, thereby providing insights into cell metabolism and bioenergetics as well as processes important in clinical medicine, such as disposition of pharmaceutical compounds. It offers comprehensive information about thousands of low-molecular mass compounds (<1500 Da) that represent a wide range of pathways and intermediary metabolism. Because of its vast expansion in the past two decades, mass spectrometry has become an indispensable tool in "omic" analyses. The use of different ionization techniques such as the more traditional electrospray and matrix-assisted laser desorption, as well as recently popular desorption electrospray ionization, has allowed the analysis of a wide range of biomolecules (e.g., peptides, proteins, lipids, and sugars), and their imaging and analysis in the original sample environment in a workup free fashion. An overview of the current state of the methodology is given, as well as examples of application.
Asunto(s)
Espectrometría de Masas/métodos , Metabolómica/métodos , Isótopos de Carbono , Cromatografía Liquida , Ciclo del Ácido Cítrico , Cromatografía de Gases y Espectrometría de Masas/métodos , Glucólisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Monitoring lipid distribution and metabolism in cells and biological fluids poses many challenges because of the many molecular species and metabolic pathways that exist. This study describes the synthesis and study of molecules that contain an alkyne functional group as surrogates for natural lipids in cultured cells. Thus, hexadec-15-ynoic and hexadec-7-ynoic acids were readily incorporated into RAW 264.7 cells, principally as phosphocholine esters; the alkyne was used as a "tag" that could be transformed to a stable dicobalt-hexacarbonyl complex; and the complex could then be detected by HPLC/MS or HPLC/UV(349nm). The 349 nm absorbance of the cobalt complexes was used to provide qualitative and quantitative information about the distribution and cellular concentrations of the alkyne lipids. The alkyne group could also be used as an affinity tag for the lipids by a catch-and-release strategy on phosphine-coated silica beads. Lipid extracts were enriched in the tagged lipids in this way, making the approach of potential utility to study lipid transformations in cell culture. Both terminal alkynes and internal alkynes were used in this affinity "pull-down" strategy. This method facilitates measuring lipid species that might otherwise fall below limits of detection.
Asunto(s)
Alquinos/metabolismo , Cobalto/metabolismo , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Ácidos Grasos/metabolismo , Espectrometría de Masas , RatonesRESUMEN
Studies of macrophage biology have been significantly advanced by the availability of cell lines such as RAW264.7 cells. However, it is unclear how these cell lines differ from primary macrophages such as thioglycolate-elicited peritoneal macrophages (TGEMs). We used the inflammatory stimulus Kdo2-lipid A (KLA) to stimulate RAW264.7 and TGEM cells. Temporal changes of lipid and gene expression levels were concomitantly measured and a systems-level analysis was performed on the fold-change data. Here we present a comprehensive comparison between the two cell types. Upon KLA treatment, both RAW264.7 and TGEM cells show a strong inflammatory response. TGEM (primary) cells show a more rapid and intense inflammatory response relative to RAW264.7 cells. DNA levels (fold-change relative to control) are reduced in RAW264.7 cells, correlating with greater downregulation of cell cycle genes. The transcriptional response suggests that the cholesterol de novo synthesis increases considerably in RAW264.7 cells, but 25-hydroxycholesterol increases considerably in TGEM cells. Overall, while RAW264.7 cells behave similarly to TGEM cells in some ways and can be used as a good model for inflammation- and immune function-related kinetic studies, they behave differently than TGEM cells in other aspects of lipid metabolism and phenotypes used as models for various disorders such as atherosclerosis.