RESUMEN
N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.
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Conducta Animal/efectos de los fármacos , Inhibidores Enzimáticos/química , Metabolismo de los Lípidos/efectos de los fármacos , Fosfatidiletanolaminas/metabolismo , Fosfolipasa D/antagonistas & inhibidores , Amidohidrolasas/metabolismo , Animales , Proteínas Sanguíneas/metabolismo , Encéfalo/metabolismo , Antagonistas de Receptores de Cannabinoides/metabolismo , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Miedo/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Receptores de Cannabinoides/metabolismo , Transducción de SeñalRESUMEN
The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Fosfolipasa D/metabolismo , Línea Celular Tumoral , Diacilglicerol Quinasa/antagonistas & inhibidores , Diacilglicerol Quinasa/genética , Diglicéridos/metabolismo , Endocitosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Lipidómica/métodos , Lípidos/análisis , Fosfolipasa D/antagonistas & inhibidores , Fosfolipasa D/genética , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Pirimidinonas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ricina/metabolismo , Tiazoles/farmacologíaRESUMEN
N-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides. Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and N-acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC50 of 68 µm), but LCA is also a highly potent ligand for TGR5 (EC50 0.52 µm). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC50 of 34 µm). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse Nape-pld to screen a panel of bile acids and a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited Nape-pld with potency similar to that of LCA. We identified 14 potent Nape-pld inhibitors in the Spectrum Collection, with the two most potent (IC50 = â¼2 µm) being symmetrically substituted dichlorophenes, i.e. hexachlorophene and bithionol. Structure-activity relationship assays using additional substituted dichlorophenes identified key moieties needed for Nape-pld inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for Nape-pld compared with nontarget lipase activities such as Streptomyces chromofuscus PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD versus other tissue-associated lipases.
Asunto(s)
Diclorofeno , Inhibidores Enzimáticos , Fosfolipasa D , Animales , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bitionol/química , Bitionol/farmacología , Diclorofeno/química , Diclorofeno/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Células HEK293 , Hexaclorofeno/química , Hexaclorofeno/farmacología , Humanos , Ratones , Fosfolipasa D/antagonistas & inhibidores , Fosfolipasa D/química , Fosfolipasa D/metabolismo , Quinazolinas/química , Quinazolinas/farmacología , Streptomyces/enzimología , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
BACKGROUND: Phosphotyrosine kinase (PTK)-mediated phospholipase C-γ1 (PLC-γ1) signaling plays a crucial role in the release of the universal second messenger calcium from intracellular stores, which is mandatory for several cellular processes, including cell migration. However, PLC-γ1 could also be activated in a PTK-independent manner by phospholipase D (PLD)-derived phosphatidic acid (PA). Because both higher PLD expression levels and PLD activity have also been associated with breast cancer cell invasion and migration, we wondered whether there might be a link between PLD and PLC-γ1, which was investigated in this study. MATERIALS: MDA-MB-468-NEO (EGFR positive) and MDA-MB-468-HER2 (EGFR and HER2 positive) human breast cancer cells were used in this study. The migratory behavior of the cells in the presence of epidermal growth factor (EGF) and the PLD inhibitor 5-fluoro-2-indolyl-des-chlorohalopemide (FIPI) was analyzed using the 3D collagen matrix migration assay. Changes in cytosolic calcium levels in the presence of EGF, FIPI and Sig-1R agonists and antagonists as well as in PLD1 siRNA knockdown cells were determined by flow cytometry. Western blot analyses were performed to determine the basal expression levels and phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44, PLC-γ1 and Sig-1R. RESULTS: The EGF-induced migration of MDA-MB-468-NEO and MDA-MB-468-HER2 cells was significantly impaired by FIPI. Likewise, FIPI also significantly abolished EGF-induced calcium release in both cell lines. However, neither the expression levels nor the phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44 and PLC-γ1 were markedly changed by FIPI. Knockdown of PLD1 expression by siRNA also significantly impaired EGF-induced calcium release in both cell lines. Targeting Sig-1R, which interacts with IP3R, with the antagonist BD1047 also abrogated EGF-induced calcium release. However, EGF-induced calcium release was also impaired if cells were treated with the Sig-1R agonists PRE084 and PPBP maleate. CONCLUSION: In summary, blocking PLD activity with the specific inhibitor FIPI or knocking down PDL1 expression by siRNA significantly impaired EGF-induced calcium release in MDA-MB-468-NEO and MDA-MB-468-HER2 cells, likely indicating a connection between PLD activity and PLC-γ1-mediated calcium signaling. However, how PLD activity interferes with the release of calcium from intracellular stores remains unclear. Video Abstract.
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Neoplasias de la Mama/metabolismo , Señalización del Calcio , Domperidona/análogos & derivados , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Indoles/farmacología , Fosfolipasa D/antagonistas & inhibidores , Neoplasias de la Mama/patología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Domperidona/farmacología , Receptores ErbB/metabolismo , Femenino , Humanos , Fosfolipasa D/metabolismo , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Receptores sigma/metabolismo , Receptor Sigma-1RESUMEN
PLD3 and PLD4 have recently been revealed to be endosomal exonucleases that regulate the innate immune response by digesting the ligands of nucleic acid sensors. These enzymes can suppress RNA and DNA innate immune sensors like toll-like receptor 9, and PLD4-deficent mice exhibit inflammatory disease. Targeting these immunoregulatory enzymes presents an opportunity to indirectly regulate innate immune nucleic acid sensors that could yield immunotherapies, adjuvants, and nucleic acid drug stabilizers. To aid in delineating the therapeutic potential of these targets, we have developed a high-throughput fluorescence enzymatic assay to identify modulators of PLD3 and PLD4. Screening of a diversity library (N = 17952) yielded preferential inhibitors of PLD3 and PLD4 in addition to a PLD3 selective activator. The modulation models of these compounds were delineated by kinetic analysis. This work presents an inexpensive and simple method to identify modulators of these immunoregulatory exonucleases.
Asunto(s)
Activadores de Enzimas/química , Inhibidores Enzimáticos/química , Exodesoxirribonucleasas/antagonistas & inhibidores , Fosfolipasa D/antagonistas & inhibidores , Pruebas de Enzimas , Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Nitrofenoles/química , Nucleótidos de Timina/química , Umbeliferonas/químicaRESUMEN
Resistance of glioblastoma to the chemotherapeutic compound temozolomide is associated with the presence of glioblastoma stem cells in glioblastoma and is a key obstacle for the poor prognosis of glioblastoma. Here, we show that phospholipase D1 is elevated in CD44High glioblastoma stem cells and in glioblastoma, especially recurring glioblastoma. Phospholipase D1 elevation positively correlated with the level of CD44 and poor prognosis in glioblastoma patients. Temozolomide significantly upregulated the expression of phospholipase D1 in the low and moderate CD44 populations of glioblastoma stem cells, but not in the CD44High population in which phospholipase D1 is highly expressed. Phospholipase D1 conferred resistance to temozolomide in CD44High glioblastoma stem cells and increased their self-renewal capacity and maintenance. Phospholipase D1 expression significantly correlated with levels of temozolomide resistance factors, which were suppressed by microRNA-320a and -4496 induced by phospholipase D1 inhibition. Genetic and pharmacological targeting of phospholipase D1 attenuated glioblastoma stem cell-derived intracranial tumors of glioblastoma using the microRNAs, and improved survival. Treatment solely with temozolomide produced no benefits on the glioblastoma, whereas in combination, phospholipase D1 inhibition sensitized glioblastoma stem cells to temozolomide and reduced glioblastoma tumorigenesis. Together, these findings indicate that phospholipase D1 inhibition might overcome resistance to temozolomide and represents a potential treatment strategy for glioblastoma. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Antineoplásicos Alquilantes/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , MicroARNs/farmacología , Fosfolipasa D/antagonistas & inhibidores , Temozolomida/uso terapéutico , Animales , Biomarcadores de Tumor/antagonistas & inhibidores , Neoplasias Encefálicas/metabolismo , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Regulación hacia Abajo , Glioblastoma/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , MicroARNs/uso terapéutico , Trasplante de Neoplasias , Regulación hacia ArribaRESUMEN
Lipids are key building blocks of biological membranes and are involved in complex signaling processes such as metabolism, proliferation, migration, and apoptosis. Extracellular signaling by growth factors, stress, and nutrients is transmitted through receptors that activate lipid-modifying enzymes such as the phospholipases, sphingosine kinase, or phosphoinositide 3-kinase, which then modify phospholipids, sphingolipids, and phosphoinositides. One such important enzyme is phospholipase D (PLD), which cleaves phosphatidylcholine to yield phosphatidic acid and choline. PLD isoforms have dual role in cells. The first involves maintaining cell membrane integrity and cell signaling, including cell proliferation, migration, cytoskeletal alterations, and invasion through the PLD product PA, and the second involves protein-protein interactions with a variety of binding partners. Increased evidence of elevated PLD expression and activity linked to many pathological conditions, including cancer, neurological and inflammatory diseases, and infection, has motivated the development of dual- and isoform-specific PLD inhibitors. Many of these inhibitors are reported to be efficacious and safe in cells and mouse disease models, suggesting the potential for PLD inhibitors as therapeutics for cancer and other diseases. Current knowledge and ongoing research of PLD signaling networks will help to evolve inhibitors with increased efficacy and safety for clinical studies.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Neoplasias/enzimología , Ácidos Fosfatidicos , Fosfolipasa D/antagonistas & inhibidores , Animales , Humanos , Ratones , Transducción de SeñalRESUMEN
Functions for phospholipase D1 and D2 (PLD1 and PLD2), the canonical isoforms of the PLD superfamily in mammals, have been explored using cell biological and animal disease models for two decades. PLD1 and PLD2, which are activated as a consequence of extracellular signaling events and generate the second messenger signaling lipid phosphatidic acid (PA), have been reported to play roles in settings ranging from platelet activation to the response to cardiac ischemia, viral infection, neurodegenerative disease, and cancer. Of these, the most tractable as therapeutic targets may be thrombotic disease and cancer, as will be discussed here in the context of ongoing efforts to develop small molecule PLD inhibitors.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Neoplasias/enzimología , Fosfolipasa D/antagonistas & inhibidores , Trombosis/enzimología , Animales , Humanos , Isoenzimas/antagonistas & inhibidores , Transducción de SeñalRESUMEN
Dendritic spines on the dendrites of pyramidal neurons are one of the most important components for excitatory synapses, where excitatory information exchanges and integrates. The defects of dendritic spine development have been closely connected with many nervous system diseases including autism, intellectual disability and so forth. Based on our previous studies, we here report a new functional signaling link between phospholipase D1 (PLD1) and protein kinase D1 (PKD1) in dendritic spine morphogenesis. Coimmunoprecipitation assays showed that PLD1 associates with PKD1. A series of knocking down and rescuing experiments demonstrated that PLD1 acts upstream of PKD1 in positively regulating dendritic spine morphogenesis. Using PLD1 inhibitor, we found that PLD1 activates PKD1 to promote dendritic spine morphogenesis. Thus, we further reveal the roles of the two different enzymes in neuronal development.
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Espinas Dendríticas/metabolismo , Neurogénesis , Fosfolipasa D/metabolismo , Canales Catiónicos TRPP/metabolismo , Animales , Línea Celular , Células Cultivadas , Espinas Dendríticas/fisiología , Ratones , Fosfolipasa D/antagonistas & inhibidores , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
Phospholipase D1 (PLD1) plays a crucial role in various inflammatory and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease. However, the role of PLD1 in the pathogenesis of RA remains unknown. Here, we first investigated the role and effects of PLD1 in collagen-induced arthritis (CIA) and found that genetic and pharmacological inhibition of PLD1 in DBA1/J mice with CIA reduced the incidence of CIA, decreased the clinical score, and abrogated disease symptoms including infiltration of leukocytes, synovial inflammation, bone erosion, and cartilage destruction. Moreover, ablation and inhibition of PLD1 suppressed the production of type II collagen-specific IgG2a autoantibody and proinflammatory cytokines, accompanied by an increase in the regulatory T (Treg) cell population and a decrease in the Th17 cell population in CIA mice. The PLD1 inhibitor also promoted differentiation of Treg cells and suppressed differentiation of Th17 cells in vitro. Furthermore, the PLD1 inhibitor attenuated pathologic bone destruction in CIA mice by suppressing osteoclastogenesis and bone resorption. Thus, our findings indicate that the targeting of PLD1 can ameliorate CIA by modulating the imbalance of Treg and Th17 cells and suppressing osteoclastogenesis, which might be a novel strategy to treat autoimmune diseases, such as RA.
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Artritis Experimental/prevención & control , Bencimidazoles/farmacología , Osteogénesis/efectos de los fármacos , Fosfolipasa D/antagonistas & inhibidores , Piperidinas/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Células Th17/efectos de los fármacos , Animales , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/prevención & control , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Citocinas/sangre , Modelos Animales de Enfermedad , Articulación de la Rodilla/efectos de los fármacos , Articulación de la Rodilla/metabolismo , Articulación de la Rodilla/patología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Osteogénesis/genética , Fosfolipasa D/genética , Fosfolipasa D/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Microtomografía por Rayos XRESUMEN
Vascular calcification (VC) is the pathological accumulation of calcium phosphate crystals in one of the layers of blood vessels, leading to loss of elasticity and causing severe calcification in vessels. Medial calcification is mostly seen in patients with chronic kidney disease (CKD) and diabetes. Identification of key enzymes and their actions during calcification will contribute to understand the onset of pathological calcification. Phospholipase D (PLD1, PLD2) is active at the earlier steps of mineralization in osteoblasts and chondrocytes. In this study, we aimed to determine their effects during high-phosphate treatment in mouse vascular smooth muscle cell line MOVAS, in the ex vivo model of the rat aorta, and in the in vivo model of adenine-induced CKD. We observed an early increase in PLD1 gene and protein expression along with the increase in the PLD activity in vascular muscle cell line, during calcification induced by ascorbic acid and ß-glycerophosphate. Inhibition of PLD1 by the selective inhibitor VU0155069, or the pan-PLD inhibitor, halopemide, prevented calcification. The mechanism of PLD activation is likely to be protein kinase C (PKC)-independent since bisindolylmaleimide X hydrochloride, a pan-PKC inhibitor, did not affect the PLD activity. In agreement, we found an increase in Pld1 gene expression and PLD activity in aortic explant cultures treated with high phosphate, whereas PLD inhibition by halopemide decreased calcification. Finally, an increase in both Pld1 and Pld2 expression occurred simultaneously with the appearance of VC in a rat model of CKD. Thus, PLD, especially PLD1, promotes VC in the context of CKD and could be an important target for preventing onset or progression of VC.
Asunto(s)
Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Fosfolipasa D/metabolismo , Fósforo Dietético , Insuficiencia Renal Crónica/complicaciones , Calcificación Vascular/etiología , Animales , Calcio de la Dieta , Línea Celular , Transdiferenciación Celular , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Inhibidores de Fosfodiesterasa/farmacología , Fosfolipasa D/antagonistas & inhibidores , Fosfolipasa D/genética , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/enzimología , Transducción de Señal , Técnicas de Cultivo de Tejidos , Calcificación Vascular/enzimología , Calcificación Vascular/patología , Calcificación Vascular/prevención & controlRESUMEN
We sought to determine the mechanism by which angiotensin II (ANGII) stimulates NADPH oxidase-mediated superoxide (O2 .- ) production in bovine pulmonary artery smooth muscle cells (BPASMCs). ANGII-induced increase in phospholipase D (PLD) and NADPH oxidase activities were inhibited upon pretreatment of the cells with chemical and genetic inhibitors of PLD2, but not PLD1. Immunoblot study revealed that ANGII treatment of the cells markedly increases protein kinase C-α (PKC-α), -δ, -ε, and -ζ levels in the cell membrane. Pretreatment of the cells with chemical and genetic inhibitors of PKC-ζ, but not PKC-α, -δ, and -ε, attenuated ANGII-induced increase in NADPH oxidase activity without a discernible change in PLD activity. Transfection of the cells with p47phox small interfering RNA inhibited ANGII-induced increase in NADPH oxidase activity without a significant change in PLD activity. Pretreatment of the cells with the chemical and genetic inhibitors of PLD2 and PKC-ζ inhibited ANGII-induced p47phox phosphorylation and subsequently translocation from cytosol to the cell membrane, and also inhibited its association with p22phox (a component of membrane-associated NADPH oxidase). Overall, PLD-PKCζ-p47phox signaling axis plays a crucial role in ANGII-induced increase in NADPH oxidase-mediated O2 .- production in the cells.
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Angiotensina II/farmacología , NADPH Oxidasas/metabolismo , Fosfolipasa D/metabolismo , Angiotensina II/metabolismo , Angiotensina II/fisiología , Animales , Bovinos , Técnicas de Cultivo de Célula/métodos , Membrana Celular/metabolismo , Miocitos del Músculo Liso/metabolismo , NADPH Oxidasas/fisiología , Oxidación-Reducción , Fosfolipasa D/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Proteína Quinasa C-alfa/metabolismo , Arteria Pulmonar/metabolismo , Transducción de Señal/efectos de los fármacos , Superóxidos/metabolismoRESUMEN
BACKGROUND: Local anesthetics cause reversible block of pain and robustly inhibit TWIK-related K channel (TREK-1) currents. Before local anesthesia onset, injection of local anesthetics can cause unwanted transient pain. TREK-1 is an anesthetic-sensitive potassium channel that when inhibited produces pain. A disordered C-terminal loop of TREK-1 is thought to contribute to anesthetic sensitivity, but the molecular basis for TREK-1 inhibition by local anesthetics is unknown. Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA) required for TREK-1 activation and also binds to the channel's C terminus. METHODS: Here, we use biophysical and cellular techniques to characterize direct and indirect lipid-mediated mechanism for TREK-1 inhibition (respectively). We characterized direct binding of local anesthetic to TREK-1 by reconstituting the purified channel into artificial membranes and measuring ion flux. We characterized indirect PA-mediated inhibition of TREK-1 by monitoring lipid production in live whole cells using a fluorescent PLD2 product release assay and ion channel current using live whole-cell patch-clamp electrophysiology. We monitored anesthetic-induced nanoscale translocation of PLD2 to TREK-1 channels with super-resolution direct stochastic reconstruction microscopy (dSTORM). RESULTS: We find local anesthetics tetracaine, lidocaine, and bupivacaine directly bind to and inhibit PLD2 enzymatic activity. The lack of PLD2 activity indirectly inhibited TREK-1 currents. Select local anesthetics also partially blocked the open pore of TREK-1 through direct binding. The amount of pore block was variable with tetracaine greater than bupivacaine and lidocaine exhibiting a minor effect. Local anesthetics also disrupt lipid rafts, a mechanism that would normally activate PLD2 were it not for their direct inhibition of enzyme catalysis. CONCLUSIONS: We propose a mechanism of TREK-1 inhibition comprised of (1) primarily indirect PLD2-dependent inhibition of lipid catalysis and (2) limited direct inhibition for select local anesthetics through partial open pore block. The inhibition through PLD2 explains how the C terminus can regulate the channel despite being devoid of structure and putative binding sites for local anesthetics.
Asunto(s)
Anestésicos Locales/farmacología , Bupivacaína/farmacología , Lidocaína/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Fosfolipasa D/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Tetracaína/farmacología , Animales , Células CHO , Línea Celular Tumoral , Cricetulus , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/genética , Fosfolipasa D/metabolismo , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Dominios y Motivos de Interacción de ProteínasRESUMEN
Humans are exposed to the antimicrobial agent triclosan (TCS) through use of TCS-containing products. Exposed tissues contain mast cells, which are involved in numerous biological functions and diseases by secreting various chemical mediators through a process termed degranulation. We previously demonstrated that TCS inhibits both Ca2+ influx into antigen-stimulated mast cells and subsequent degranulation. To determine the mechanism linking the TCS cytosolic Ca2+ depression to inhibited degranulation, we investigated the effects of TCS on crucial signaling enzymes activated downstream of the Ca2+ rise: protein kinase C (PKC; activated by Ca2+ and reactive oxygen species [ROS]) and phospholipase D (PLD). We found that TCS strongly inhibits PLD activity within 15 minutes post-antigen, a key mechanism of TCS mast cell inhibition. In addition, experiments using fluorescent constructs and confocal microscopy indicate that TCS delays antigen-induced translocations of PKCßII, PKCδ and PKC substrate myristoylated alanine-rich C-kinase. Surprisingly, TCS does not inhibit PKC activity or overall ability to translocate, and TCS actually increases PKC activity by 45 minutes post-antigen; these results are explained by the timing of both TCS inhibition of cytosolic Ca2+ (~15+ minutes post-antigen) and TCS stimulation of ROS (~45 minutes post-antigen). These findings demonstrate that it is incorrect to assume that all Ca2+ -dependent processes will be synchronously inhibited when cytosolic Ca2+ is inhibited by a toxicant or drug. The results offer molecular predictions of the effects of TCS on other mammalian cell types, which share these crucial signal transduction elements and provide biochemical information that may underlie recent epidemiological findings implicating TCS in human health problems.
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Antiinfecciosos/toxicidad , Calcio/metabolismo , Degranulación de la Célula/efectos de los fármacos , Mastocitos/efectos de los fármacos , Fosfolipasa D/antagonistas & inhibidores , Triclosán/toxicidad , Línea Celular , Humanos , Mastocitos/metabolismo , Mastocitos/patología , Mastocitos/fisiología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Proteína Quinasa C/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
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.
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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
BACKGROUND: Phospholipases D1 and D2 (PLD1/2) hydrolyse cell membrane glycerophospholipids to generate phosphatidic acid, a signalling lipid, which regulates cell growth and cancer progression through effects on mTOR and PKB/Akt. PLD expression and/or activity is raised in breast, colorectal, gastric, kidney and thyroid carcinomas but its role in prostate cancer (PCa), the major cancer of men in the western world, is unclear. METHODS: PLD1 protein expression in cultured PNT2C2, PNT1A, P4E6, LNCaP, PC3, PC3M, VCaP, 22RV1 cell lines and patient-derived PCa cells was analysed by western blotting. PLD1 protein localisation in normal, benign prostatic hyperplasia (BPH), and castrate-resistant prostate cancer (CRPC) tissue sections and in a PCa tissue microarray (TMA) was examined by immunohistochemistry. PLD activity in PCa tissue was assayed using an Amplex Red method. The effect of PLD inhibitors on PCa cell viability was measured using MTS and colony forming assays. RESULTS: PLD1 protein expression was low in the luminal prostate cell lines (LNCaP, VCaP, 22RV1) compared with basal lines (PC3 and PC3M). PLD1 protein expression was elevated in BPH biopsy tissue relative to normal and PCa samples. In normal and BPH tissue, PLD1 was predominantly detected in basal cells as well in some stromal cells, rather than in luminal cells. In PCa tissue, luminal cells expressed PLD1. In a PCa TMA, the mean peroxidase intensity per DAB-stained Gleason 6 and 7 tissue section was significantly higher than in sections graded Gleason 9. In CRPC tissue, PLD1 was expressed prominently in the stromal compartment, in luminal cells in occasional glands and in an expanding population of cells that co-expressed chromogranin A and neurone-specific enolase. Levels of PLD activity in normal and PCa tissue samples were similar. A specific PLD1 inhibitor markedly reduced the survival of both prostate cell lines and patient-derived PCa cells compared with two dual PLD1/PLD2 inhibitors. Short-term exposure of PCa cells to the same specific PLD1 inhibitor significantly reduced colony formation. CONCLUSIONS: A new specific inhibitor of PLD1, which is well tolerated in mice, reduces PCa cell survival and thus has potential as a novel therapeutic agent to reduce prostate cancer progression. Increased PLD1 expression may contribute to the hyperplasia characteristic of BPH and in the progression of castrate-resistant PCa, where an expanding population of neuroendocrine-like cells express PLD1.
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Inhibidores Enzimáticos/farmacología , Fosfolipasa D/antagonistas & inhibidores , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/enzimología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Bencimidazoles/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Domperidona/análogos & derivados , Domperidona/farmacología , Humanos , Inmunohistoquímica , Indoles/farmacología , Masculino , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Fosfolipasa D/biosíntesis , Fosfolipasa D/metabolismo , Piperidinas/farmacología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/patología , Análisis de Matrices Tisulares , Células Tumorales CultivadasRESUMEN
This letter describes the on-going SAR efforts to develop PLD1, PLD2 and dual PLD1/2 inhibitors with improved physiochemical and disposition properties as well as securing intellectual property position. Previous PLD inhibitors, based on a triazaspiro[4.5]decanone core proved to be highly selective PLD2 inhibitors, but with low plasma free fraction (rat, human fuâ¯<â¯0.03), high predicted hepatic clearance (rat CLhepâ¯>â¯65â¯mL/min/kg) and very short half-lives in vivo (t1/2â¯<â¯0.15â¯h). Removal of a nitrogen atom from this core generated a 2,8-diazaspiro[4.5]decanone core, harboring a new chiral center, as well as increased sp3 character. This new core demonstrated enantioselective inhibition of the individual PLD isoforms, enhanced free fraction (rat, human fuâ¯<â¯0.13), engendered moderate predicted hepatic clearance (rat CLhepâ¯â¼â¯43â¯mL/min/kg), improved half-lives in vivo (t1/2â¯>â¯3â¯h), and led to the first issued US patent claiming composition of matter for small molecule PLD inhibitors.
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Inhibidores Enzimáticos/química , Fosfolipasa D/metabolismo , Compuestos de Espiro/química , Animales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Células HEK293 , Semivida , Humanos , Concentración 50 Inhibidora , Fosfolipasa D/antagonistas & inhibidores , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Ratas , Compuestos de Espiro/síntesis química , Compuestos de Espiro/farmacocinética , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
In order to interact with the egg and undergo acrosomal exocytosis or the acrosome reaction (AR), mammalian spermatozoa must undergo a series of biochemical changes in the female reproductive tract, collectively called capacitation. We showed that F-actin is formed during sperm capacitation and fast depolymerization occurs prior to the AR. We hypothesized that F-actin protects the sperm from undergoing spontaneous-AR (sAR) which decreases fertilization rate. We show that activation of the actin-severing protein gelsolin induces a significant increase in sAR. Moreover, inhibition of CaMKII or PLD during sperm capacitation, caused an increase in sAR and inhibition of F-actin formation. Spermine, which leads to PLD activation, was able to reverse the effects of CaMKII inhibition on sAR-increase and F-actin-decrease. Furthermore, the increase in sAR and the decrease in F-actin caused by the inactivation of the PLD-pathway, were reversed by activation of CaMKII using H2O2 or by inhibiting protein phosphatase 1 which enhance the phosphorylation and oxidation states of CaMKII. These results indicate that two distinct pathways lead to F-actin formation in the sperm capacitation process which prevents the occurrence of sAR.
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Reacción Acrosómica/fisiología , Acrosoma/enzimología , Actinas/metabolismo , Citoesqueleto de Actina/ultraestructura , Animales , Calcimicina/farmacología , Señalización del Calcio/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Bovinos , Activación Enzimática/efectos de los fármacos , Exocitosis/fisiología , Gelsolina/metabolismo , Gelsolina/farmacología , Peróxido de Hidrógeno/farmacología , Masculino , Toxinas Marinas , Oxazoles/farmacología , Fragmentos de Péptidos/farmacología , Fosfolipasa D/antagonistas & inhibidores , Fosfolipasa D/fisiología , Polimerizacion , Capacitación Espermática/fisiología , Espermatozoides/efectos de los fármacos , Espermatozoides/ultraestructuraRESUMEN
Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 118: 726-738, 2017. © 2016 Wiley Periodicals, Inc.
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Proteínas de Artrópodos/antagonistas & inhibidores , Araña Reclusa Parda/enzimología , Diseño de Fármacos , Fosfolipasa D/antagonistas & inhibidores , Venenos de Araña/antagonistas & inhibidores , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Bencimidazoles/farmacología , Araña Reclusa Parda/genética , Araña Reclusa Parda/patogenicidad , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hemólisis/efectos de los fármacos , Humanos , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Necrosis , Fosfolipasa D/química , Fosfolipasa D/genética , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Piperidinas/farmacología , Conejos , Proteínas Recombinantes/genética , Piel/efectos de los fármacos , Piel/patología , Picaduras de Arañas/tratamiento farmacológico , Picaduras de Arañas/enzimología , Venenos de Araña/química , Venenos de Araña/genética , Suramina/farmacologíaRESUMEN
Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDß. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.