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1.
Food Chem ; 297: 124955, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253342

RESUMO

This study aimed to investigate the effect of hydrogen peroxide (H2O2) on membrane lipids metabolism and its relation to pulp breakdown development of longan fruit during postharvest storage. Compared to the control longans, H2O2-treated longans showed higher pulp breakdown index, cell membrane permeability, and activities of phospholipase D (PLD), lipase and lipoxygenase (LOX). Moreover, H2O2-treated longans maintained higher levels of pulp phosphatidic acid (PA) and saturated fatty acids (SFA). However, H2O2-treated longans exhibited lower levels of pulp phosphatidylcholine (PC), phosphatidylinositol (PI) and unsaturated fatty acids (USFA), lower index of unsaturated fatty acids (IUFA), and lower ratio of USFA to SFA (U/S). These findings demonstrated that H2O2 caused the increased activities of enzymes involving in membrane lipids degradation and the accelerated decompositions of membrane USFA and phospholipids in longan pulp, which eventually triggered the destruction of the pulp cell membrane structure and the development of pulp breakdown in longans during storage.


Assuntos
Enzimas/metabolismo , Frutas/química , Peróxido de Hidrogênio/farmacologia , Lipídeos de Membrana/metabolismo , Sapindaceae/química , Enzimas/química , Ácidos Graxos/análise , Ácidos Graxos/química , Armazenamento de Alimentos , Frutas/efeitos dos fármacos , Frutas/metabolismo , Peróxido de Hidrogênio/química , Lipase/química , Lipase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoxigenase/química , Lipoxigenase/metabolismo , Lipídeos de Membrana/química , Fosfolipase D/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sapindaceae/efeitos dos fármacos , Sapindaceae/metabolismo
2.
Plant Cell Physiol ; 60(7): 1556-1566, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31073607

RESUMO

Oil crop Brassica napus is subjected to environmental stresses such as drought, cold and salt. Phospholipase Ds (PLDs) have vital roles in regulation of plant growth, development and stress tolerance. In this study, 32 BnaPLD genes were identified and classified into six subgroups depending on the conserved protein structures. High similarity in gene and protein structures exists between BnaPLDs and AtPLDs. Gene expression analysis showed that BnaPLDα1s and BnaPLDδs had higher expression than other PLDs. BnaPLDα1 and BnaPLDδ were significantly induced by abiotic stresses including dehydration, NaCl, abscisic acid (ABA) and 4�C. Lipidomic analysis showed that the content of main membrane phospholipids decreased gradually under stresses, except phosphatidylglycerol increased under the treatment of ABA and phosphatidylethanolamine increased under 4�C. Correspondingly, their product of phosphatidic acid increased often with a transient peak at 8 h. The plant height of mutants of PLDα1 was significantly reduced. Agronomic traits such as yield, seed number, silique number and branches were significantly impaired in PLDα1 mutants. These results indicate that there is a large family of PLD genes in B. napus, especially BnaPLDα1s and BnaPLDδs may play important roles in membrane lipids remodeling and maintaining of the growth and stress tolerance of B. napus.


Assuntos
Brassica napus/genética , Genes de Plantas/genética , Fosfolipase D/genética , Fosfolipídeos/metabolismo , Brassica napus/enzimologia , Brassica napus/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/fisiologia , Estudo de Associação Genômica Ampla , Metabolismo dos Lipídeos , Lipídeos/fisiologia , Fosfolipase D/metabolismo , Fosfolipídeos/fisiologia , Filogenia , Folhas de Planta/metabolismo , Estresse Fisiológico , Transcriptoma
3.
Cell Biol Int ; 43(6): 678-694, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30977575

RESUMO

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.


Assuntos
Angiotensina II/farmacologia , NADPH Oxidases/metabolismo , Fosfolipase D/metabolismo , Angiotensina II/metabolismo , Angiotensina II/fisiologia , Animais , Bovinos , Técnicas de Cultura de Células/métodos , Membrana Celular/metabolismo , Miócitos de Músculo Liso/metabolismo , NADPH Oxidases/fisiologia , Oxirredução , Fosfolipase D/antagonistas & inibidores , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa/metabolismo , Artéria Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismo
4.
Mol Biol Cell ; 30(10): 1198-1213, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30865555

RESUMO

Mitochondria are essential and dynamic organelles undergoing constant fission and fusion. The primary players in mitochondrial morphology (MFN1/2, OPA1, DRP1) have been identified, but their mechanism(s) of regulation are still being elucidated. ARL2 is a regulatory GTPase that has previously been shown to play a role in the regulation of mitochondrial morphology. Here we demonstrate that ELMOD2, an ARL2 GTPase-activating protein (GAP), is necessary for ARL2 to promote mitochondrial elongation. We show that loss of ELMOD2 causes mitochondrial fragmentation and a lower rate of mitochondrial fusion, while ELMOD2 overexpression promotes mitochondrial tubulation and increases the rate of fusion in a mitofusin-dependent manner. We also show that a mutant of ELMOD2 lacking GAP activity is capable of promoting fusion, suggesting that ELMOD2 does not need GAP activity to influence mitochondrial morphology. Finally, we show that ELMOD2, ARL2, Mitofusins 1 and 2, Miros 1 and 2, and mitochondrial phospholipase D (mitoPLD) all localize to discrete, regularly spaced puncta along mitochondria. These results suggest that ELMOD2 is functioning as an effector downstream of ARL2 and upstream of the mitofusins to promote mitochondrial fusion. Our data provide insights into the pathway by which mitochondrial fusion is regulated in the cell.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Dinâmica Mitocondrial/fisiologia , Animais , Células COS , Linhagem Celular , Cercopithecus aethiops , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Inativação de Genes/métodos , Humanos , Fusão de Membrana/fisiologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo
5.
Microbiol Immunol ; 63(3-4): 119-129, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30854712

RESUMO

Phosphatidylserine synthase (Pss) catalyzes phosphatidylserine synthesis, which is critical to synthesizing the component of cell membrane. However, few putative pss genes of bacteria have been studied. In this study, it was found that Vibrio parahaemolyticus, a common foodborne pathogen that causes human gastroenteritis, has a type I Pss with two HKD motifs and is a phospholipase D superfamily member. The transcriptional start site of pss was mapped through sequencing and was identified at -37 nucleotides upstream of the start codon. Pss mRNA was found to be expressed mainly during the exponential phase. In addition, the promoter was identified using a lux reporter assay and gel shift assay with an RNA polymerase. To analyze the catalytic activity, a soluble form of His6 -tagged recombinant Pss was overexpressed and purified from Escherichia coli. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry, it was found that Pss can catalyze cytidine diphosphate diacylglycerol and L-serine to form phosphatidylserine. Since Pss is conserved in vibrios, the current study can promote understanding the biosynthesis of phospholipid in Vibrio bacteria that might cause vibriosis. This is the first report of molecular characterization of the pss gene and identification of Pss enzyme activity in V. parahaemolyticus using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.


Assuntos
CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , Membrana Celular/metabolismo , Vibrio parahaemolyticus/enzimologia , Vibrio parahaemolyticus/metabolismo , Diglicerídeos de Citidina Difosfato/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Fosfatidilserinas/biossíntese , Fosfolipase D/metabolismo , Serina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vibrio parahaemolyticus/genética
6.
Mol Cell Biochem ; 457(1-2): 119-132, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30877512

RESUMO

Linoleic acid (LA) is an essential and omega-6 polyunsaturated fatty acid that mediates a variety of biological processes, including migration and invasion in breast cancer cells. Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. Increases of expression and activity of PLD are reported in several human cancers, including gastric, colorectal, renal, stomach, lung and breast. In this article, we demonstrate that LA induces an increase of PLD activity in MDA-MB-231 breast cancer cells. Particularly, PLD1 and/or PLD2 mediate migration and invasion induced by LA. Moreover, LA induces increases in number and size of spheroids via PLD activity. FFAR1 also mediates migration and invasion, whereas PLD activation induced by LA requires the activities of FFAR1, FFAR4 and EGFR in MDA-MB-231 cells. In summary, PLD plays a pivotal role in migration and invasion induced by LA in MDA-MB-231 breast cancer cells.


Assuntos
Neoplasias da Mama/enzimologia , Movimento Celular/efeitos dos fármacos , Ácido Linoleico/farmacologia , Proteínas de Neoplasias/metabolismo , Fosfolipase D/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Células MCF-7 , Invasividade Neoplásica
7.
Infect Immun ; 87(5)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30745329

RESUMO

Arcanobacterium haemolyticum is an emerging human pathogen that causes pharyngitis and wound infections. A few studies have suggested that A. haemolyticum is able to induce its uptake into nonphagocytic epithelial cells, but the bacterial factors associated with host cell invasion and the host cell processes involved have yet to be studied. We investigated how two A. haemolyticum virulence factors, arcanolysin (ALN) and phospholipase D (PLD), affect the ability of the bacteria to adhere to and subsequently invade Detroit 562 pharyngeal epithelial cells. The sphingomyelinase activity of phospholipase D was necessary to increase bacterial adherence, while the absence of a functional arcanolysin had no effect on A. haemolyticum adherence but did lead to a decrease in A. haemolyticum invasion into Detroit 562 cells. Because of the known roles of cholesterol-dependent cytolysins in disrupting calcium gradients and inducing F-actin-mediated bacterial internalization, we sought to determine whether ALN and PLD played a similar role in the ability of A. haemolyticum to invade nonphagocytic cells. Elimination of extracellular calcium and inhibition of the Arp2/3 complex or F-actin polymerization also caused a decrease in the ability of A. haemolyticum to invade Detroit 562 cells. Overall, our findings suggest that A. haemolyticum utilizes phospholipase D primarily for adherence and utilizes arcanolysin primarily for invasion into Detroit 562 cells in a process dependent on extracellular calcium and F-actin polymerization. Our work marks the first insight into how the individual activities of arcanolysin and phospholipase D affect A. haemolyticum host-pathogen interactions using the biologically relevant Detroit 562 cell line.


Assuntos
Infecções por Actinomycetales/patologia , Arcanobacterium/enzimologia , Arcanobacterium/patogenicidade , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Infecção/metabolismo , Fosfolipase D/metabolismo , Humanos
8.
Plant Sci ; 279: 45-50, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30709492

RESUMO

Phospholipase D (PLD) hydrolyzes membrane phospholipids to generate phosphatidic acid (PA). Both PLD and its lipid product PA are involved in various physiological processes, including plant response to pathogens. The PLD family is comprised of multiple members in higher plants, and PLDs have been reported to play positive and/or negative roles in plant immunity, depending on the types of pathogens and specific PLDs involved. Individual PLDs have distinguishable biochemical properties, such as Ca2+ and phosphatidylinositide requirements. In addition, PLDs and PA are found to interact with various proteins in hormone and stress signaling. The different biochemical and regulatory properties of PLDs and PA shed light on the mechanisms for the functional diversity of PLDs in plant defense signaling and response.


Assuntos
Ácidos Fosfatídicos/metabolismo , Fosfolipase D/metabolismo , Imunidade Vegetal , Interações Hospedeiro-Patógeno , Ácidos Fosfatídicos/fisiologia , Fosfolipase D/fisiologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia
9.
Antonie Van Leeuwenhoek ; 112(7): 1055-1065, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30771116

RESUMO

Diphtheria by Corynebacterium ulcerans is increasingly occurring in children, adolescents and adults. In addition to diphtheria toxin (DT), phospholipase D (PLD) is considered a virulence factor of C. ulcerans. In the present study, a first case of concurrent diphtheria by a PLD-negative C. ulcerans and infectious mononucleosis (IM) was verified. Clinical and microbiological profiles and binding properties to human Fibrinogen (Fbg), Fibronectin (Fn) and type I collagen (col I) biotinylated proteins and virulence to Caenorhabditis elegans were investigated for C. ulcerans strain 2590 (clinical isolate) and two control strains, including PLD-positive BR-AD22 wild type and PLD-negative ELHA-1 PLD mutant strains. MALDI-TOF assays and a multiplex PCR of genes coding for potentially toxigenic corynebacteria identified strain 2590 as non-DT producing. Interestingly, strain 2590 did not express PLD activity in the CAMP test although the presence of the pld gene was verified. PLD-negative 2590 and a PLD-positive 210932 strains showed similar affinity to Fbg, Fn and type I collagen. C. elegans were able to escape from C. ulcerans strains, independent of PLD and DT production. Higher mortality of nematodes was verified for PLD-negative strains. Additional studies concerning multifactorial virulence potential of C. ulcerans, including environmental conditions remain necessary.


Assuntos
Infecções por Corynebacterium/microbiologia , Corynebacterium/isolamento & purificação , Difteria/microbiologia , Mononucleose Infecciosa/microbiologia , Adolescente , Animais , Antibacterianos/farmacologia , Caenorhabditis elegans , Corynebacterium/classificação , Corynebacterium/efeitos dos fármacos , Corynebacterium/genética , Humanos , Masculino , Fosfolipase D/análise , Fosfolipase D/metabolismo , Fatores de Virulência/análise , Fatores de Virulência/metabolismo
10.
Nat Commun ; 10(1): 457, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30692526

RESUMO

Variations in N-acylethanolamines (NAE) levels are associated with obesity and metabolic comorbidities. Their role in the gut remains unclear. Therefore, we generated a mouse model of inducible intestinal epithelial cell (IEC)-specific deletion of N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD), a key enzyme involved in NAE biosynthesis (Napepld∆IEC). We discovered that Napepld∆IEC mice are hyperphagic upon first high-fat diet (HFD) exposure, and develop exacerbated obesity and steatosis. These mice display hypothalamic Pomc neurons dysfunctions and alterations in intestinal and plasma NAE and 2-acylglycerols. After long-term HFD, Napepld∆IEC mice present reduced energy expenditure. The increased steatosis is associated with higher gut and liver lipid absorption. Napepld∆IEC mice display altered gut microbiota. Akkermansia muciniphila administration partly counteracts the IEC NAPE-PLD deletion effects. In conclusion, intestinal NAPE-PLD is a key sensor in nutritional adaptation to fat intake, gut-to-brain axis and energy homeostasis and thereby constitutes a novel target to tackle obesity and related disorders.


Assuntos
Gorduras na Dieta/metabolismo , Fígado Gorduroso/metabolismo , Mucosa Intestinal/metabolismo , Obesidade/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolipase D/metabolismo , Adaptação Fisiológica , Animais , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/etiologia , Microbioma Gastrointestinal/fisiologia , Homeostase , Mucosa Intestinal/microbiologia , Metabolismo dos Lipídeos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Obesidade/etiologia
11.
Appl Microbiol Biotechnol ; 103(6): 2635-2648, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30685815

RESUMO

Bacterial non-specific nucleases of the phospholipase D family are widely distributed among the members of the Enterobacteriaceae. Each genome mainly contains a single copy of a gene encoding a phospholipase D family protein. However, two distantly related isozymes (< 40% identity at the protein level) were identified by BLAST-analyses in the plant pathogenic competitor enterobacterium Pantoea agglomerans. The two nucleases PaNuc-1 and PaNuc-2 were produced in Escherichia coli. Identical gene constructs and expression conditions resulted in the production of PaNuc-1 in soluble form, while PaNuc-2 remained insoluble in inclusion bodies. PaNuc-2 was refolded and both proteins were purified by a combination of affinity and ion exchange chromatography. Proteolytic removal of the HIS-tag allowed the characterization of pure and mature tag-less proteins. Enzymatic properties of both isozymes revealed that they are non-specific nucleases, displaying activities against RNA, single- and double-stranded genomic DNA as well as circular plasmids. However, their biochemical activity profiles were clearly different, with PaNuc-1 being optimally active at 70 °C and pH 7.0, while PaNuc-2 was most active at 45 °C and pH 7.0. The enzymes retained > 90% nuclease activity at EDTA concentrations of 4 mM (PaNuc-2) and 20 mM (PaNuc-1), respectively. Different enzymatic properties suggest that the roles of PaNuc-1 and PaNuc-2 differ in the cell and might be the result of functional diversification after an ancient gene duplication event took place. The fact that both enzymes could be easily produced in recombinant form and their tolerance against metal ion chelators in combination with a broad substrate promiscuity might pave the way to versatile biotechnological applications.


Assuntos
Proteínas de Bactérias/metabolismo , Pantoea/enzimologia , Fosfolipase D/metabolismo , Proteínas de Bactérias/genética , Cromatografia por Troca Iônica , Endonucleases , Escherichia coli/genética , Duplicação Gênica , Concentração de Íons de Hidrogênio , Isoenzimas/genética , Isoenzimas/metabolismo , Pantoea/genética , Fosfolipase D/genética , Plantas/microbiologia , Plasmídeos , RNA/metabolismo , Proteínas Recombinantes/biossíntese , Temperatura Ambiente
12.
Food Chem ; 274: 535-542, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30372975

RESUMO

This work was aimed to develop a novel phosphatidylserine (PS) production process for the food industry. The pldsh gene, encoding phospholipase D from Streptomyces halstedii (PLDsh) was cloned, and the codon optimized pldmsh gene was freely expressed by Pichia pastoris GS115 and successfully overexpressed on the cell surface of P. pastoris GS115 as displayed PLDMsh (dPLDMsh) - a whole-cell biocatalyst for PS synthesis from phosphatidylcholine and l-serine. dPLDMsh was stable over a broad range of temperatures (20-60 °C) and pH values (4.0-8.0), indicating significant improvement in stability compared with its free counterpart expressed by P. pastoris GS115. Under the optimum conditions, the conversion yield of PS was 53%, and the relative yield remained above 40% after 4 repeated batch cycles of dPLDMsh catalysis in an aqueous system. Thus, dPLDMsh and the associated reaction system provided a novel strategy for efficient PS production for the food industry.


Assuntos
Proteínas de Bactérias/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipase D/metabolismo , Pichia/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Biocatálise , Fosfolipase D/genética , Estabilidade Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Água/química
13.
Toxicol Lett ; 302: 35-41, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30553937

RESUMO

Environmental tobacco smoke (ETS) exposure during brain development has been associated with several disorders, such as depression, anxiety, sudden infant death syndrome, and the predisposition to addiction. The endocannabinoid system plays an essential role in neuronal development. We investigated the effects of early postnatal ETS exposure on the endocannabinoid system in different brain regions. C57BL/6 J mice were exposed to ETS that was generated from 3R4F cigarettes from postnatal day 3 (P3) to P14. Receptors and enzymes of the endocannabinoid system were assessed in infancy, adolescence, and adulthood by Western blot. In the brainstem, ETS exposure decreased cannabinoid 1 (CB1) receptor, CB2 receptor, N-arachidonoyl phosphatidyl ethanol-specific phospholipase D (NAPE-PLD), and fatty acid amino hydrolase (FAAH) levels and increased in diacylglycerol lipase (DAGL) and monoacylglycerol lipase (MAGL) levels during infancy and decreased CB2 and FAAH levels during adulthood. In the striatum, ETS decreased in the NAPE-PLD and MAGL levels and increased FAAH levels during infancy, increased FAAH levels during adolescence, and decreased NAPE-PLD levels during adulthood. The present findings indicate that exposure to ETS during a critical period of brain development can disturb the endocannabinoid system in the brainstem and striatum, regions that are involved in the pathogenesis of sudden infant death syndrome and the susceptibility to addiction.


Assuntos
Tronco Encefálico/efeitos dos fármacos , Fumar Cigarros/efeitos adversos , Corpo Estriado/efeitos dos fármacos , Endocanabinoides/metabolismo , Poluição por Fumaça de Tabaco/efeitos adversos , Amidoidrolases/metabolismo , Animais , Animais Recém-Nascidos , Tronco Encefálico/crescimento & desenvolvimento , Tronco Encefálico/metabolismo , Corpo Estriado/crescimento & desenvolvimento , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Lipase Lipoproteica/metabolismo , Camundongos Endogâmicos C57BL , Monoacilglicerol Lipases/metabolismo , Fosfolipase D/metabolismo , Receptor CB1 de Canabinoide/efeitos dos fármacos , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/efeitos dos fármacos , Receptor CB2 de Canabinoide/metabolismo
14.
Int J Biol Macromol ; 129: 1140-1147, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30550823

RESUMO

A novel phospholipase D that originate from marine Vibrio harveyi (VhPLD) was recombinant expressed and biochemically characterized. Moreover, effects of C-terminal peptides on catalytic and interfacial binding properties of VhPLD were investigated by constructing two truncated mutants (VhPLD-Δ(472-483) and VhPLD-Δ(437-483)). Optimal reaction temperature and pH value for wild-type VhPLD (VhPLD-WT) was 45 °C and pH 8.0. However, optimal reaction temperature of VhPLD-Δ(437-483) increased to 50 °C. Meanwhile, catalytic efficiency (kcat/KM) of VhPLD-Δ(472-483) and VhPLD-Δ(437-483) to the 1,2-Dioctanoyl-sn-glycero-3-phosphatidyl-p-nitrophenol (PpNP) was 12.9 and 14.2 times higher than that of VhPLD-WT. However, when compare the catalytic efficiency between VhPLD-Δ(472-483) and VhPLD-Δ(437-483), no significant change can be found between the two mutants. These results strongly indicated that the C-terminal 12 amino acids (472-483) have important role on the activity of VhPLD. Effects of C-terminal peptides on the interfacial binding properties of VhPLD to different phospholipid monolayers were also investigated by using monolayer film technology. Results of the maximum insertion pressure (MIP) indicated that deletion the C-terminal segment of VhPLD improved its interfacial binding properties to different phospholipid monolayers.


Assuntos
Fragmentos de Peptídeos/genética , Fosfolipase D/genética , Fosfolipase D/metabolismo , Deleção de Sequência , Vibrio/enzimologia , Sequência de Aminoácidos , Biocatálise , Química Bioinorgânica , Concentração de Íons de Hidrogênio , Fosfolipase D/química , Fosfolipídeos/metabolismo , Tensoativos/farmacologia , Temperatura Ambiente , Vibrio/genética
15.
BMC Plant Biol ; 18(1): 355, 2018 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-30547756

RESUMO

BACKGROUND: Plant phospholipase D (PLD), which can hydrolyze membrane phospholipids to produce phosphatidic acid (PA), a secondary signaling molecule, has been proposed to function in diverse plant stress responses. Both PLD and PA play key roles in plant growth, development, and cellular processes. PLD was suggested to mediate the regulation of stomatal movements by abscisic acid (ABA) as a response to water deficit. In this research, we characterized the roles of the cucumber phospholipase D alpha gene (CsPLDα, GenBank accession number EF363796) in the growth and tolerance of transgenic tobacco (Nicotiana tabacum) to drought stress. RESULTS: The CsPLDα overexpression in tobacco lines correlated with the ABA synthesis and metabolism, regulated the rapid stomatal closure in drought stress, and reduced the water loss. The NtNCED1 expression levels in the transgenic lines and wild type (WT) were sharply up-regulated after 16 days of drought stress compared with those before treatment, and the expression level in the transgenic lines was significantly higher than that in the WT. The NtAOG expression level evidently improved after 8 and 16 days compared with that at 0 day of treatment and was significantly lower in the transgenic lines than in the WT. The ABA content in the transgenic lines was significantly higher than that in the WT. The CsPLDα overexpression could increase the osmolyte content and reduce the ion leakage. The proline, soluble sugar, and soluble protein contents significantly increased. By contrast, the electrolytic leakage and malondialdehyde accumulation in leaves significantly decreased. The shoot and root fresh and dry weights of the overexpression lines significantly increased. These results indicated that a significant correlation between CsPLDα overexpression and improved resistance to water deficit. CONCLUSIONS: The plants with overexpressed CsPLDα exhibited lower water loss, higher leaf relative water content, and heavier fresh and dry matter accumulation than the WT. We proposed that CsPLDα was involved in the ABA-dependent pathway in mediating the stomatal closure and preventing the elevation of intracellular solute potential.


Assuntos
Cucumis sativus/genética , Ácidos Fosfatídicos/genética , Fosfolipase D/genética , Estresse Fisiológico/genética , Tabaco/fisiologia , Ácido Abscísico/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Peroxidação de Lipídeos/genética , Peroxidação de Lipídeos/fisiologia , Malondialdeído/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estômatos de Plantas/fisiologia , Plantas Geneticamente Modificadas , Prolina/metabolismo , Tabaco/genética , Água/metabolismo
16.
Bioorg Med Chem Lett ; 28(23-24): 3670-3673, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30528979

RESUMO

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 fu < 0.03), high predicted hepatic clearance (rat CLhep > 65 mL/min/kg) and very short half-lives in vivo (t1/2 < 0.15 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 fu < 0.13), engendered moderate predicted hepatic clearance (rat CLhep ∼ 43 mL/min/kg), improved half-lives in vivo (t1/2 > 3 h), and led to the first issued US patent claiming composition of matter for small molecule PLD inhibitors.


Assuntos
Inibidores Enzimáticos/química , Fosfolipase D/metabolismo , Compostos de Espiro/química , Animais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Células HEK293 , Meia-Vida , Humanos , Concentração Inibidora 50 , Fosfolipase D/antagonistas & inibidores , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Ratos , Compostos de Espiro/síntese química , Compostos de Espiro/farmacocinética , Estereoisomerismo , Relação Estrutura-Atividade
17.
Int J Mol Sci ; 20(1)2018 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-30587782

RESUMO

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. Here, we present a shot-gun differential proteomic analysis on roots of two Arabidopsis pldα1 mutants compared to the wild type. Interestingly, PLDα1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLDα1 may be involved in the stability of attachment sites of endoplasmic reticulum to the plasma membrane as suggested by increased abundance of synaptotagmin 1, which was validated by immunoblotting and whole-mount immunolabelling analyses. Moreover, we noticed a robust abundance alterations of proteins involved in mitochondrial import and electron transport chain. Notably, the abundances of numerous proteins implicated in glucosinolate biosynthesis were also affected in pldα1 mutants. Our results suggest a broader biological involvement of PLDα1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glucosinolatos/biossíntese , Proteínas Mitocondriais/metabolismo , Fosfolipase D/metabolismo , Proteoma/metabolismo , Proteômica , Proteínas de Arabidopsis/genética , Cromatografia Líquida de Alta Pressão , Endocitose , Ontologia Genética , Fosfolipase D/genética , Raízes de Plantas/metabolismo , Transporte Proteico , Sinaptotagmina I/metabolismo , Espectrometria de Massas em Tandem , Proteína Desacopladora 1/metabolismo
18.
Int J Mol Sci ; 19(8)2018 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-30126228

RESUMO

The effects of N-terminal (1⁻34 amino acids) and C-terminal (434⁻487 amino acids) amino acid sequences on the interfacial binding properties of Phospholipase D from Vibrio parahaemolyticus (VpPLD) were characterized by using monomolecular film technology. Online tools allowed the prediction of the secondary structure of the target N- and C-terminal VpPLD sequences. Various truncated forms of VpPLD with different N- or C-terminal deletions were designed, based on their secondary structure, and their membrane binding properties were examined. The analysis of the maximum insertion pressure (MIP) and synergy factor "a" indicated that the loop structure (1⁻25 amino acids) in the N-terminal segment of VpPLD had a positive effect on the binding of VpPLD to phospholipid monolayers, especially to 1,2-dimyristoyl-sn-glycero-3-phosphoserine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The deletion affecting the N-terminus loop structure caused a significant decrease of the MIP and synergy factor a of the protein for these phospholipid monolayers. Conversely, the deletion of the helix structure (26⁻34 amino acids) basically had no influence on the binding of VpPLD to phospholipid monolayers. The deletion of the C-terminal amino acids 434⁻487 did not significantly change the binding selectivity of VpPLD for the various phospholipid monolayer tested here. However, a significant increase of the MIP value for all the phospholipid monolayers strongly indicated that the three-strand segment (434⁻469 amino acids) had a great negative effect on the interfacial binding to these phospholipid monolayers. The deletion of this peptide caused a significantly greater insertion of the protein into the phospholipid monolayers examined. The present study provides detailed information on the effect of the N- and C-terminal segments of VpPLD on the interfacial binding properties of the enzyme and improves our understanding of the interactions between this enzyme and cell membranes.


Assuntos
Fosfolipase D/metabolismo , Fosfolipídeos/metabolismo , Vibrio parahaemolyticus/enzimologia , Sequência de Aminoácidos , Humanos , Fosfolipase D/química , Ligação Proteica , Estrutura Secundária de Proteína , Vibrioses/microbiologia , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/metabolismo
19.
J Plant Physiol ; 229: 158-163, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30096586

RESUMO

2,4-dichlorophenoxyacetic acid (2,4-D) is among the most commonly used herbicides applied for weed control during wheat cultivation. However, its application could affect wheat growth. The present study investigates the effect of the ascomycetous fungus Trichoderma harzianum on lipid peroxidation, phospholipids, signaling lipids and phospholipase D in the seedlings of wheat (Triticum aestivum L.) treated with 2,4-D (2.5 mg L-1). In the group of 4-day-old seedlings exposed to the herbicide, increased lipid peroxidation and inhibition of growth were observed in shoots and roots. Moreover, elevated levels of oxylipins were noted. Among them, the amount of 13-HOTrE oxygenated from linolenic acid (18:3) increased the most significantly. Concurrently, in the seedlings inoculated with T. harzianum, growth was stimulated when the level of phosphatidylcholine (PC) increased. Moreover, in wheat seedlings treated with 2,4-D and T. harzianum, the level of lipid peroxidation was similar to that in the control and there was no increase observed in oxylipins and phospholipase D activity. T. harzianum might have partly alleviated the toxic effect of 2,4-D on wheat seedlings.


Assuntos
Ácido 2,4-Diclorofenoxiacético/farmacologia , Trichoderma/patogenicidade , Triticum/metabolismo , Triticum/microbiologia , Estresse Oxidativo/fisiologia , Fosfatidilcolinas/metabolismo , Fosfolipase D/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Brotos de Planta/microbiologia , Plântula/efeitos dos fármacos , Plântula/metabolismo , Plântula/microbiologia , Triticum/efeitos dos fármacos
20.
Oncol Rep ; 40(2): 1026-1034, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29989648

RESUMO

The DNA mismatch repair (MMR) system plays an important role in the initiation, diagnosis and treatments of colorectal cancer (CRC). Compared to CRC patients deficient in DNA MMR (dMMR), CRC patients proficient in DNA MMR (pMMR) have higher metastasis, short survival and poor response to chemotherapy and immunotherapy. It is well­known that a high­fat diet can cause CRC, and lipid metabolism is closely related to the development and metastasis of CRC. However, there have been few studies that address the difference in lipid metabolism between dMMR and pMMR CRC. Liquid chromatography­tandem mass spectrometry (LC/MS) is an advanced technique that can perform the analysis of lipid metabolites and the roles of lipids present in low abundance in cell signaling and membrane stability. In the present study, we used the LC/MS technique to analyze the difference in the lipid metabolic profiles between dMMR cell lines (HCT116, DLD1, LoVo and HCT15) and pMMR cell lines (SW480, SW620, HT29 and NCM460). The results revealed that, among the 19 classes and 157 intact lipid species identified by the LC/MS analysis, the levels of most phospholipids were lower in dMMR cells than pMMR cells. Higher levels of phosphatidylcholine (PC; 16:0/18:1) and phosphatidic acid (PA; 18:0/18:0) were observed in pMMR cells than in dMMR cells. Furthermore, our results revealed that SCD1 and PLD1, the key enzymes involved in lipid metabolism associated with metastasis, are higher in pMMR cells than dMMR cells. To the best of our knowledge, we are the first to reveal that the levels of metastasis­associated lipids and key enzymes in lipid metabolism were higher in the CRC patients with pMMR compared with the CRC patients with dMMR. This study identified potential anti­metastatic targets in the therapy of patients with pMMR, and also personalized therapy for the patients with pMMR.


Assuntos
Neoplasias Colorretais/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Metabolismo dos Lipídeos/genética , Metaboloma/genética , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/métodos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Metabolômica/métodos , Fosfolipase D/genética , Fosfolipase D/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Espectrometria de Massas em Tandem/métodos
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