RESUMEN
Platelet-activating factor (PAF), a proinflammatory lipid mediator, plays a crucial role in the formation of the atherosclerotic plaque. Therefore, the inhibition of endothelium inflammation by nutraceuticals, such as PAF inhibitors, is a promising alternative for preventing cardiovascular diseases. The aim of the present study was to evaluate the impact of a new functional yogurt enriched with PAF inhibitors of natural origin from olive oil by-products on PAF metabolism. Ninety-two apparently healthy, but mainly overweight volunteers (35-65 years) were randomly allocated into three groups by block-randomization. The activities of PAF's biosynthetic and catabolic enzymes were measured, specifically two isoforms of acetyl-CoA:lyso-PAF acetyltransferase (LPCATs), cytidine 5'-diphospho-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT) and two isoforms of platelet activating factor acetylhydrolase in leucocytes (PAF-AH) and plasma (lipoprotein associated phospholipase-A2, LpPLA2). The intake of the enriched yogurt resulted in reduced PAF-CPT and LpPLA2 activities. No difference was observed in the activities of the two isoforms of lyso PAF-AT. In conclusion, intake of yogurt enriched in PAF inhibitors could favorably modulate PAF biosynthetic and catabolic pathways.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , 1-Alquil-2-acetilglicerofosfocolina Esterasa/antagonistas & inhibidores , Suplementos Dietéticos , Inhibidores Enzimáticos/administración & dosificación , Olea , Factor de Activación Plaquetaria , Yogur , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , Adulto , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factor de Activación Plaquetaria/antagonistas & inhibidores , Factor de Activación Plaquetaria/metabolismoRESUMEN
Radiation induces the generation of platelet-activating factor receptor (PAF-R) ligands, including PAF and oxidized phospholipids. Alternatively, PAF is also synthesized by the biosynthetic enzymes lysophosphatidylcholine acyltransferases (LPCATs) which are expressed by tumor cells including melanoma. The activation of PAF-R by PAF and oxidized lipids triggers a survival response protecting tumor cells from radiation-induced cell death, suggesting the involvement of the PAF/PAF-R axis in radioresistance. Here, we investigated the role of LPCATs in the melanoma cell radiotherapy response. LPCAT is a family of four enzymes, LPCAT1-4, and modular nucleic acid nanoparticles (NANPs) allowed for the simultaneous silencing of all four LPCATs. We found that the in vitro simultaneous silencing of all four LPCAT transcripts by NANPs enhanced the therapeutic effects of radiation in melanoma cells by increasing cell death, reducing long-term cell survival, and activating apoptosis. Thus, we propose that NANPs are an effective strategy for improving radiotherapy efficacy in melanomas.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa , Silenciador del Gen , Melanoma , Nanopartículas , Proteínas de Neoplasias , Ácidos Nucleicos , 1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , 1-Acilglicerofosfocolina O-Aciltransferasa/biosíntesis , Línea Celular Tumoral , Humanos , Melanoma/tratamiento farmacológico , Melanoma/enzimología , Nanopartículas/química , Nanopartículas/uso terapéutico , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/biosíntesis , Ácidos Nucleicos/química , Ácidos Nucleicos/farmacologíaRESUMEN
Poor prognosis of esophageal cancer is associated with limited clinical treatment efficacy and lack of targeted therapies. With advances in nanomedicine, nanoparticle drug delivery systems play increasingly important roles in tumor treatment by enabling the simultaneous delivery of multiple therapeutic agents. We here propose a novel nanovector for targeted combination gene therapy and chemotherapy in esophageal cancer. A novel lipid nanovector (EYLN) was designed to carry the chemotherapy drug doxorubicin (Dox) and small interfering RNA against the lipid anabolic metabolism gene LPCAT1, which we previously showed to be significantly overexpressed in esophageal cancer tissues, and its interference inhibited the proliferation, invasion, and metastasis of esophageal cancer cells. This vector, EYLN-Dox/siLPCAT1, was further coated with leukocyte membranes to obtain mEYLNs-Dox/siLPCAT1. The particle size of the coated nanovector was approximately 136 nm, and the surface zeta potential was -21.18 mV. Compared with EYLNs-Dox/siLPCAT1, mEYLNs-Dox/siLPCAT1 were more easily internalized by esophageal cancer cells due to the LFA-1 highly expressed leukocyte membrane coating and showed significant inhibition of the proliferation, migration, and metastasis of esophageal cancer cells, along with their LPCAT1 expression, through more effective delivery of the drugs. Moreover, the nanovectors showed improved blood circulation time, tissue distribution, tumor targeting, and tumor suppression in a mouse model. Thus, combining chemo and gene therapy with this new nanodelivery system achieved greater therapeutic efficacy, providing a new strategy for the treatment of esophageal cancer.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Antibióticos Antineoplásicos/farmacología , Doxorrubicina/farmacología , Neoplasias Esofágicas/tratamiento farmacológico , Terapia Genética , Leucocitos/efectos de los fármacos , ARN Interferente Pequeño/farmacología , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Animales , Antibióticos Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Doxorrubicina/química , Portadores de Fármacos/química , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Esofágicas/diagnóstico por imagen , Neoplasias Esofágicas/metabolismo , Femenino , Humanos , Leucocitos/patología , Lípidos/química , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Nanopartículas/química , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Tamaño de la Partícula , ARN Interferente Pequeño/química , Propiedades de Superficie , Células Tumorales CultivadasRESUMEN
Biophysical properties of membranes are dependent on their glycerophospholipid compositions. Lysophospholipid acyltransferases (LPLATs) selectively incorporate fatty chains into lysophospholipids to affect the fatty acid composition of membrane glycerophospholipids. Lysophosphatidic acid acyltransferases (LPAATs) of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family incorporate fatty chains into phosphatidic acid during the de novo glycerophospholipid synthesis in the Kennedy pathway. Other LPLATs of both the AGPAT and the membrane bound O-acyltransferase (MBOAT) families further modify the fatty chain compositions of membrane glycerophospholipids in the remodeling pathway known as the Lands' cycle. The LPLATs functioning in these pathways possess unique characteristics in terms of their biochemical activities, regulation of expressions, and functions in various biological contexts. Essential physiological functions for LPLATs have been revealed in studies using gene-deficient mice, and important roles for several enzymes are also indicated in human diseases where their mutation or dysregulation causes or contributes to the pathological condition. Now several LPLATs are emerging as attractive therapeutic targets, and further understanding of the mechanisms underlying their physiological and pathological roles will aid in the development of novel therapies to treat several diseases that involve altered glycerophospholipid metabolism.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Aciltransferasas/antagonistas & inhibidores , Membrana Celular/metabolismo , Desarrollo de Medicamentos , Glicerofosfolípidos/biosíntesis , Glicerofosfolípidos/química , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Aciltransferasas/metabolismo , Animales , Membrana Celular/química , Membrana Celular/enzimología , Ácidos Grasos/química , Ácidos Grasos/metabolismo , HumanosRESUMEN
Lysophosphatidylcholine acyltransferase 3 (LPCAT3) is an important enzyme in phospholipid remodeling, a process that influences the biophysical properties of cell membranes and thus cell function. Multiple lines of evidence suggest that LPCAT3 is involved in several diseases, including atherosclerosis, non-alcoholic steatohepatitis, and carcinoma. Thus, LPCAT3 may have potential as a therapeutic target for these diseases. In the present study, we devised an assay based on reversed-phase HPLC to measure LPCAT3 activity, which may facilitate the identification of LPCAT3 inhibitors and activators. We found that optimal pH and temperature of recombinant human LPCAT3 are 6.0 and 30 °C, respectively. The enzyme Km values for substrates NBD-labelled lysophosphatidylcholine and arachidonoyl CoA were 266.84 ± 3.65 and 11.03 ± 0.51 µmol·L-1 , respectively, and the Vmax was 39.76 ± 1.86 pmol·min-1 ·U-1 . Moreover, we used our new method to determine the IC50 of a known LPCAT inhibitor, TSI-10. In conclusion, this novel assay can be used to measure the effects of compounds on LPCAT3 activity.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/análisis , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Pruebas de Enzimas/métodos , 1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos/farmacología , Humanos , Concentración de Iones de Hidrógeno , Cinética , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/análisis , Proteínas Recombinantes/metabolismo , TemperaturaRESUMEN
Eicosanoids derived from the enzymatic oxidation of arachidonic acid play important roles in a large number of physiological and pathological processes in humans. Many animal and cellular models have been used to investigate the intricate mechanisms regulating their biosynthesis and actions. Zebrafish is a widely used model to study the embryonic development of vertebrates. It expresses homologs of the key enzymes involved in eicosanoid production, and eicosanoids have been detected in extracts from adult or embryonic fish. In this study we prepared cell suspensions from kidney marrow, the main hematopoietic organ in fish. Upon stimulation with calcium ionophore, these cells produced eicosanoids including PGE2, LTB4, 5-HETE and, most abundantly, 12-HETE. They also produced small amounts of LTB5 derived from eicosapentaenoic acid. These eicosanoids were also produced in kidney marrow cells stimulated with ATP, and this production was greatly enhanced by preincubation with thimerosal, an inhibitor of arachidonate reacylation into phospholipids. Microsomes from these cells exhibited acyltransferase activities consistent with expression of MBOAT5/LPCAT3 and MBOAT7/LPIAT1, the main arachidonoyl-CoA:lysophospholipid acyltransferases. In summary, this work introduces a new cellular model to study the regulation of eicosanoid production through a phospholipid deacylation-reacylation cycle from a well-established, versatile vertebrate model species.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Eicosanoides/biosíntesis , Células Mieloides/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Animales , Calcimicina/administración & dosificación , Ionóforos de Calcio/administración & dosificación , Lisofosfolípidos/metabolismo , Modelos Animales , Células Mieloides/enzimología , Espectrometría de Masa por Ionización de Electrospray , Pez CebraRESUMEN
Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Macrófagos Peritoneales/enzimología , Factor de Activación Plaquetaria/biosíntesis , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/metabolismo , Animales , Ratones , Células RAW 264.7RESUMEN
Cholestasis is one of the major causes of liver diseases. A chronic accumulation of toxic bile acids in the liver, which occurs in this condition, can induce fibrosis and cirrhosis. Inflammation is a fundamental component of acute and chronic cholestatic liver injury. Platelet-activating factor (PAF) is a proinflammatory lipid which may be generated by two independent pathways called the de novo and remodeling pathway being the last responsible for the synthesis of PAF during inflammation. In recent years a key role in PAF remodeling has been attributed to lysophosphatidylcholine acyltransferase (LPCAT) enzymes. Although the knowledge on their characteristic is growing, the exact mechanism of LPCAT in pathological conditions remains still unknown. Here, we reported that the level of lyso-PAF and PAF significantly increased in the liver of cirrhotic vs. control rats together with a significant decrease in both mRNA abundance and protein level of both LPCAT1 and LPCAT2. Acyltransferase activities of both LPCAT1 and LPCAT2 were parallel decreased in the liver of cirrhotic animals. Interestingly, treatment with silybin strongly decreased the level of both pro-inflammatory lipids and restored the activity and expression of both LPCAT1 and LPCAT2 of cirrhotic liver. Silybin effect was specific for LPCAT1 and LPCAT2 since it did not affect LPCAT3 mRNA abundance of cirrhotic liver.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Cromatografía en Capa Delgada , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/prevención & control , Cirrosis Hepática/complicaciones , Factor de Activación Plaquetaria/metabolismo , Silimarina/farmacología , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Animales , Antioxidantes/farmacología , Western Blotting , Regulación hacia Abajo , Inflamación/etiología , Inflamación/metabolismo , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Fosfolipasas A2/metabolismo , Factor de Activación Plaquetaria/genética , ARN Mensajero/genética , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SilibinaRESUMEN
BACKGROUND & AIMS: Several lipid synthesis pathways play important roles in the development and progression of hepatocellular carcinoma (HCC), although the precise molecular mechanisms remain to be elucidated. Here, we show the relationship between HCC progression and alteration of phospholipid composition regulated by lysophosphatidylcholine acyltransferase (LPCAT). METHODS: Molecular lipidomic screening was performed by imaging mass spectrometry (IMS) in 37 resected HCC specimens. RT-PCR and Western blotting were carried out to examine the mRNA and protein levels of LPCATs, which catalyze the conversion of lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) and have substrate specificity for some kinds of fatty acids. We examined the effect of LPCAT1 overexpression or knockdown on cell proliferation, migration, and invasion in HCC cell lines. RESULTS: IMS revealed the increase of PC species with palmitoleic acid or oleic acid at the sn-2-position and the reduction of LPC with palmitic acid at the sn-1-position in HCC tissues. mRNA and protein of LPCAT1, responsible for LPC to PC conversion, were more abundant in HCCs than in the surrounding parenchyma. In cell line experiments, LPCAT1 overexpression enriched PCs observed in IMS and promoted cell proliferation, migration, and invasion. LPCAT1 knockdown did viceversa. CONCLUSIONS: Enrichment or depletion of some specific PCs, was found in HCC by IMS. Alteration of phospholipid composition in HCC would affect tumor character. LPCAT1 modulates phospholipid composition to create favorable conditions to HCC cells. LPCAT1 is a potent target molecule to inhibit HCC progression.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfolípidos/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Fosfolípidos/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Especificidad por Sustrato , Regulación hacia ArribaRESUMEN
The lipid modifier phospholipase A2 catalyzes the hydrolysis of phospholipids to inverted-cone-shaped lysophospholipids that contribute to membrane curvature and/or tubulation. Conflicting findings exist regarding the function of cytosolic phospholipase A2 (cPLA2) and its role in membrane regulation at the Golgi and early endosomes. However, no studies addressed the role of cPLA2 in the regulation of cholesterol-rich membranes that contain glycosylphosphatidylinositol-anchored proteins (GPI-APs). Our studies support a role for cPLA2α in the vesiculation of GPI-AP-containing membranes, using endogenous CD59 as a model for GPI-APs. On cPLA2α depletion, CD59-containing endosomes became hypertubular. Moreover, accumulation of lysophospholipids induced by a lysophospholipid acyltransferase inhibitor extensively vesiculated CD59-containing endosomes. However, overexpression of cPLA2α did not increase the endosomal vesiculation, implying a requirement for additional factors. Indeed, depletion of the "pinchase" EHD1, a C-terminal Eps15 homology domain (EHD) ATPase, also induced hypertubulation of CD59-containing endosomes. Furthermore, EHD1 and cPLA2α demonstrated in situ proximity (<40 nm) and interacted in vivo. The results presented here provide evidence that the lipid modifier cPLA2α and EHD1 are involved in the vesiculation of CD59-containing endosomes. We speculate that cPLA2α induces membrane curvature and allows EHD1, possibly in the context of a complex, to sever the curved membranes into vesicles.
Asunto(s)
Antígenos CD59/metabolismo , Colesterol/metabolismo , Endosomas/metabolismo , Proteínas Ligadas a GPI/metabolismo , Fosfolipasas A2 Grupo IV/metabolismo , Proteínas de Transporte Vesicular/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anilidas , Transporte Biológico , Proteínas del Citoesqueleto/metabolismo , Dinamina II/metabolismo , Endocitosis , Endosomas/ultraestructura , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/metabolismo , Fosfolipasas A2 Grupo IV/genética , Células HeLa , Humanos , Proteínas con Dominio LIM/metabolismo , Proteínas de Microfilamentos , Microscopía Fluorescente , Microtúbulos/metabolismo , Oxigenasas de Función Mixta , Unión Proteica , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
Molecular and biochemical analyses of membrane phospholipids have revealed that, in addition to their physico-chemical properties, the metabolites of phospholipids play a crucial role in the recognition, signalling and responses of cells to a variety of stimuli. Such responses are mediated in large part by the removal and/or addition of different acyl chains to provide different phospholipid molecular species. The reacylation reactions, catalysed by specific acyltransferases control phospholipid composition and the availability of the important mediators free arachidonic acid and lysophospholipids. Lysophospholipid acyltransferases are therefore key control points for cellular responses to a variety of stimuli including inflammation. Regulation or manipulation of lysophospholipid acyltransferases may thus provide important mechanisms for novel anti-inflammatory therapies. This review will highlight mammalian lysophospholipid acyltransferases with particular reference to the potential role of lysophosphatidylcholine acyltransferase and its substrates in sepsis and other inflammatory conditions and as a potential target for novel anti-inflammatory therapies.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/fisiología , Inhibidores Enzimáticos/uso terapéutico , Inflamación/etiología , Sepsis/tratamiento farmacológico , 1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Apoptosis , Lesiones Encefálicas/metabolismo , Descubrimiento de Drogas , Humanos , Inflamación/enzimología , Interferón gamma/farmacología , Lipopolisacáridos/toxicidad , Lisofosfatidilcolinas/metabolismo , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Fosfolípidos/metabolismo , Sepsis/metabolismoRESUMEN
Sepsis, an overwhelming inflammatory response to infection, is a major cause of morbidity and mortality worldwide and has no specific therapy. Phospholipid metabolites, such as lysophospholipids, have been shown to regulate inflammatory responses in sepsis, although their mechanism of action is not well understood. The phospholipid-metabolizing enzymes, lysophospholipid acyltransferases, control membrane phospholipid composition, function, and the inflammatory responses of innate immune cells. Here, we show that lysophosphatidylcholine acyltransferase (LPCAT) regulates inflammatory responses to LPS and other microbial stimuli. Specific inhibition of LPCAT down-regulated inflammatory cytokine production in monocytes and epithelial cells by preventing translocation of TLR4 into membrane lipid raft domains. Our observations demonstrate a new regulatory mechanism that facilitates the innate immune responses to microbial molecular patterns and provide a basis for the anti-inflammatory activity observed in many phospholipid metabolites. This provides the possibility of the development of new classes of anti-inflammatory and antisepsis agents.
Asunto(s)
Membrana Celular/metabolismo , Inflamación/metabolismo , Lisofosfolípidos/metabolismo , Receptor Toll-Like 4/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Lipopolisacáridos/metabolismo , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Nistatina/farmacología , Transporte de Proteínas/efectos de los fármacos , Piridinas/farmacología , Receptor Toll-Like 2/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The mechanism of coat protein (COP)II vesicle fission from the endoplasmic reticulum (ER) remains unclear. Lysophospholipid acyltransferases (LPATs) catalyze the conversion of various lysophospholipids to phospholipids, a process that can promote spontaneous changes in membrane curvature. Here, we show that 2,2-methyl-N-(2,4,6,-trimethoxyphenyl)dodecanamide (CI-976), a potent LPAT inhibitor, reversibly inhibited export from the ER in vivo and the formation of COPII vesicles in vitro. Moreover, CI-976 caused the rapid and reversible accumulation of cargo at ER exit sites (ERESs) containing the COPII coat components Sec23/24 and Sec13/31 and a marked enhancement of Sar1p-mediated tubule formation from ERESs, suggesting that CI-976 inhibits the fission of assembled COPII budding elements. These results identify a small molecule inhibitor of a very late step in COPII vesicle formation, consistent with fission inhibition, and demonstrate that this step is likely facilitated by an ER-associated LPAT.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Anilidas/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Animales , Línea Celular , Guanosina Trifosfato/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Transporte de Proteínas/fisiología , Ratas , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Previous studies have shown that inhibition of a Golgi-complex-associated lysophospholipid acyltransferase (LPAT) activity by the drug CI-976 stimulates Golgi tubule formation and subsequent redistribution of resident Golgi proteins to the endoplasmic reticulum (ER). Here, we show that CI-976 stimulates tubule formation from all subcompartments of the Golgi complex, and often these tubules formed independently, i.e. individual tubules usually did not contain markers from different subcompartments. Whereas the cis, medial and trans Golgi membranes redistributed to the ER, the trans Golgi network (TGN) collapsed back to a compact juxtanuclear position similar to that seen with brefeldin A (BFA) treatment. Also similar to BFA, CI-976 induced the formation of endosome tubules, but unlike BFA, these tubules did not fuse with TGN tubules. Finally, CI-976 produced an apparently irreversible block in the endocytic recycling pathway of transferrin (Tf) and Tf receptors (TfRs) but had no direct effect on Tf uptake from the cell surface. Tf and TfRs accumulated in centrally located, Rab11-positive vesicles indicating that CI-976 inhibits export of cargo from the central endocytic recycling compartment. These results, together with previous studies, demonstrate that CI-976 inhibits multiple membrane trafficking steps, including ones found in the endocytic and secretory pathways, and imply a wider role for lysophospholipid acyltransferases in membrane trafficking.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Anilidas/farmacología , Inhibidores Enzimáticos/farmacología , Membranas Intracelulares/efectos de los fármacos , Vesículas Secretoras/efectos de los fármacos , Vesículas Transportadoras/efectos de los fármacos , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Animales , Células Cultivadas , Endosomas/efectos de los fármacos , Endosomas/enzimología , Endosomas/metabolismo , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Membranas Intracelulares/enzimología , Membranas Intracelulares/metabolismo , Microscopía Fluorescente , Ratas , Receptores de Transferrina/antagonistas & inhibidores , Receptores de Transferrina/metabolismo , Vesículas Secretoras/metabolismo , Transfección , Transferrina/antagonistas & inhibidores , Transferrina/metabolismo , Vesículas Transportadoras/fisiología , Proteínas de Unión al GTP rab/metabolismo , Red trans-Golgi/efectos de los fármacos , Red trans-Golgi/enzimología , Red trans-Golgi/metabolismoRESUMEN
Recent studies have suggested that formation of Golgi membrane tubules involves the generation of membrane-associated lysophospholipids by a cytoplasmic Ca2+-independent phospholipase A2 (PLA2). Herein, we provide additional support for this idea by showing that inhibition of lysophospholipid reacylation by a novel Golgi-associated lysophosphatidylcholine acyltransferase (LPAT) induces the rapid tubulation of Golgi membranes, leading in their retrograde movement to the endoplasmic reticulum. Inhibition of the Golgi LPAT was achieved by 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976), a previously characterized antagonist of acyl-CoA cholesterol acyltransferase. The effect of CI-976 was similar to that of brefeldin A, except that the coatomer subunit beta-COP remained on Golgi-derived membrane tubules. CI-976 also enhanced the cytosol-dependent formation of tubules from Golgi complexes in vitro and increased the levels of lysophosphatidylcholine in Golgi membranes. Moreover, preincubation of cells with PLA2 antagonists inhibited the ability of CI-976 to induce tubules. These results suggest that Golgi membrane tubule formation can result from increasing the content of lysophospholipids in membranes, either by stimulation of a PLA2 or by inhibition of an LPAT. These two opposing enzyme activities may help to coordinately regulate Golgi membrane shape and tubule formation.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Aparato de Golgi/enzimología , Fosfolipasas A/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Acilación , Anilidas/farmacología , Brefeldino A/farmacología , Células HeLa , Humanos , Lisofosfatidilcolinas/metabolismo , Fosfolipasas A2 , Fracciones SubcelularesRESUMEN
Mononuclear phagocytes play a pivotal role in the progression of septic shock by producing tumor necrosis factor-alpha (TNF-alpha) and other inflammatory mediators in response to lipopolysaccharide (LPS) from Gram-negative bacteria. Our previous studies have shown monocyte and macrophage activation correlate with changes in membrane phospholipid composition, mediated by acyltransferases. Interferon-gamma (IFN-gamma), which activates and primes these cells for enhanced inflammatory responses to LPS, was found to selectively activate lysophosphatidylcholine acyltransferase (LPCAT) (P < 0.05) but not lysophosphatidic acid acyltransferase (LPAAT) activity. When used to prime the human monocytic cell line MonoMac 6, the production of TNF-alpha and interleukin-6 (IL-6) was approximately five times greater in cells primed with IFN-gamma than unprimed cells. Two LPCAT inhibitors SK&F 98625 (diethyl 7-(3,4,5-triphenyl-2-oxo2,3-dihydro-imidazole-1-yl)heptane phosphonate) and YM 50201 (3-hydroxyethyl 5,3'-thiophenyl pyridine) strongly inhibited (up to 90%) TNF-alpha and IL-6 production in response to LPS in both unprimed MonoMac-6 cells and in cells primed with IFN-gamma. In similar experiments, these inhibitors also substantially decreased the response of both primed and unprimed peripheral blood mononuclear cells to LPS. Sequence-based amplification methods showed that SK&F 98625 inhibited TNF-alpha production by decreasing TNF-alpha mRNA levels in MonoMac-6 cells. Taken together, the data from these studies suggest that LPCAT is a key enzyme in both the pathways of activation (priming) and the inflammatory response to LPS in monocytes.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Lipopolisacáridos/farmacología , Lisofosfatidilcolinas/metabolismo , Monocitos/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Acilación , Línea Celular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Imidazoles/metabolismo , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-6/metabolismo , Lisofosfatidilcolinas/química , Microsomas/metabolismo , Estructura Molecular , Monocitos/citología , Monocitos/inmunología , Compuestos Organofosforados/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The supply of free arachidonic acid from phospholipids is generally regarded as the rate-limiting step for prostaglandin (PG) synthesis by tissues. Two enzymes involved in arachidonic acid uptake into, and release from, phospholipids are acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase A2 (PLA2), respectively. PGF2 alpha produced by the endometrium induces luteolysis in several species including guinea-pigs. Thimerosal, an inhibitor of ACLAT, and aristolochic acid, an inhibitor of PLA2, both reduced, in a concentration-dependent manner, the output of PGF2 alpha from guinea-pig endometrium cultured for 24 h on days 7 and 15 of the oestrous cycle. This study showed that the continual production of PGF 2 alpha by guinea-pig endometrium is not only dependent upon the activity of PLA2 for releasing free arachidonic acid for PGF2 alpha synthesis, but also on the incorporation of arachidonic acid into the phospholipid pool by the activity of ACLAT. The inhibitory effects of thimerosal and aristolochic acid on the outputs of PGE2 and 6-keto-PGF1 alpha were less marked, particularly on day 7 when the low output of PGE2 was unaffected and the output of 6-keto-PGF1 alpha was increased at the lower concentrations of thimerosal. This finding indicates that there are different pools of arachidonic acid bound as phospholipid for the syntheses of PGF2 alpha and 6-keto-PGF1 alpha by guinea-pig endometrium.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Ácido Araquidónico/metabolismo , Ácidos Aristolóquicos , Endometrio/metabolismo , Oxidorreductasas/antagonistas & inhibidores , Prostaglandinas/biosíntesis , Timerosal/farmacología , 6-Cetoprostaglandina F1 alfa/biosíntesis , Animales , Técnicas de Cultivo , Dinoprost/biosíntesis , Dinoprostona/biosíntesis , Relación Dosis-Respuesta a Droga , Endometrio/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Cobayas , Fenantrenos/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2RESUMEN
The filamentous fungus, Mortierella alpina, accumulates complex lipids relatively rich in arachidonic acid (C(20:4) Delta(5,8,11,14)). The lignan, sesamin, has been used to reduce arachidonic acid production by specifically inhibiting Delta(5)-desaturation [Shimizu, Akimoto, Shinmen, Kawashima, Sugano and Yamada (1991) Lipids 26, 512-516]. Microsomal membrane preparations from M. alpina exhibit acyl-CoA:1-acyl lysophosphatidylcholine acyltransferase (LPCAT) activity. LPCAT is an enzyme involved in channelling fatty acid substrates to phosphatidylcholine for subsequent desaturation. Sesamin was found to inhibit this enzyme as measured in both spectrophotometric and radioactive assays. The inhibitory effect of sesamin on LPCAT was only evident in species of Mortierella and could not be demonstrated in other organisms.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Dioxoles/farmacología , Lignanos/farmacología , Microsomas/enzimología , Mucorales/enzimología , 1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , 1-Acilglicerofosfocolina O-Aciltransferasa/aislamiento & purificación , Antioxidantes/farmacología , Membranas Intracelulares/enzimología , Cinética , Aceite de SésamoRESUMEN
A photoreactive substrate analog of lysophosphatidylcholine (LPC), 1-([(4-azidosalicyl)-12-amino)]dodecanoyl-sn-glycerol-3-phospho cholin e (azido-LPC) was synthesized. Fast atom bombardment mass spectrometry was employed to confirm the structures of azido-LPC and its intermediates. Azido-LPC was used to label putative acyl-CoA:LPC acyltransferase from microsomal membranes of developing soybean cotyledons. The synthesized substrate analog acts as a substrate for the target acyltransferases and phospholipases in the dark. When the microsomal membranes were incubated with the acyl acceptor analog and immediately photolyzed, LPC acyltransferase was irreversibly inhibited. Photoinactivation of the enzyme by the photoprobe decreased in the presence of LPC. Microsomal membranes were photolyzed with 125I-labeled azido-LPC and analyzed by SDS-PAGE followed by autoradiography. These revealed that the analog preferentially labeled 54- and 114-kDa polypeptides. Substrate protected the labeling of both the polypeptides. In our earlier report, the same polypeptides were also labeled with photoreactive acyl-CoA analogs, suggesting that these polypeptides could be putative LPC acyltransferase(s). These results demonstrated that the photoreactive phospholipid analog could be a powerful tool to label acyltransferases involved in lipid biosynthesis.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/química , Glicerol/síntesis química , Glycine max/enzimología , Fosforilcolina/síntesis química , 1-Acilglicerofosfocolina O-Aciltransferasa/antagonistas & inhibidores , Marcadores de Afinidad , Azidas/síntesis química , Membranas Intracelulares/enzimología , Espectrometría de Masas , FotólisisRESUMEN
Spur cell anemia may occur in severe liver disease including alcoholic cirrhosis. Spur cell anemia red blood cells (RBCs) have a characteristic morphology, with irregular projections, an increased ratio of membrane cholesterol (Ch) to phospholipid, evidence of oxidative damage, and shortened survival resulting in hemolytic anemia. Normal RBCs may acquire many of the features of spur cells either by transfusion into a spur cell patient or in an in vitro model system that loads the RBC membrane with Ch relative to phospholipid by means of Ch-rich, phospholipid-Ch sonicates. We found evidence of abnormal phospholipid repair metabolism in spur cell anemia RBCs characterized by decreased arachidonate (Ar) uptake into phospholipids and by increased uptake into a fatty acid membrane repair intermediate, acylcarnitine (AcylCn). To study the possible modulation of phospholipid repair metabolism in spur cells by Ch-loading, we compared the Ar metabolism of RBCs loaded with Ch in vitro with that of control cells incubated in autologous serum. Ar, a polyunsaturated fatty acid, is especially sensitive to peroxidation and, thus, is likely to be involved in phospholipid repair. Ch-loading decreased the incorporation of [14C]Ar into total lipids (Ch-loaded, 1,113 +/- 48 pmol/10(10) RBCs; control, 1,525 +/- 48 pmol/10(10) RBCs) including phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. Uptake of [14C]Ar into AcylCn increased (control AcylCn, 169 +/- 31 pmol/10(10) RBCs; Ch-loaded AcylCn, 196 +/- 35 pmol/10(10) RBCs; P = .0012). Thimerosal, an inhibitor of arachidonoyl- CoA:l-palmitoyl-sn-glycero-3-phosphocholine acyl transferase or lysophosphocholine acyl transferase (LAT), produced a similar pattern of metabolic abnormality, with decreased incorporation into phospholipid but relative increase into AcylCn. We assayed LAT in RBC membranes from Ch-loaded RBCs, using [14C]arachidonoyl CoA as precursor, and found similar decreased LAT activity at concentrations of 1-palmitoyllysophosphatidylcholine (LPC) from 1 to 30 micromol/L. Similar LAT assay results were obtained using [14C]palmitoyl LPC as the precursor. We conclude that Ch-loading of RBC membranes results in inhibition of LAT in the cell-free system in vitro and may account for the inhibited phospholipid repair in Ch-loaded intact RBCs in vitro and in spur cell anemia RBCs in vivo. Decreased ability to replace peroxidized membrane fatty acid by this metabolic pathway may contribute to the hemolytic process in spur cell anemia.