RESUMO
Native Americans domesticated maize (Zea mays ssp. mays) from lowland teosinte parviglumis (Zea mays ssp. parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 (HPC1), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern United States, Canada, and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time.
Assuntos
Adaptação Fisiológica , Flores , Interação Gene-Ambiente , Fosfatidilcolinas , Fosfolipases A1 , Proteínas de Plantas , Zea mays , Alelos , Mapeamento Cromossômico , Flores/genética , Flores/metabolismo , Genes de Plantas , Ligação Genética , Fosfatidilcolinas/metabolismo , Fosfolipases A1/classificação , Fosfolipases A1/genética , Fosfolipases A1/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Zea mays/genética , Zea mays/crescimento & desenvolvimentoRESUMO
Leishmaniasis is caused by several species of protozoan parasites of the genus Leishmania and represents an important global health problem. Leishmania braziliensis in particular is responsible of cutaneous and mucocutaneous forms of this parasitosis, with prevalence in Latin America. In the present work, we describe in L. braziliensis promastigotes and amastigotes the presence of a Phospholipase A1 (PLA1) activity, an enzyme that catalyses extensive deacylation of phospholipids like phosphatidylcholine. In order to deepen the knowledge about L. braziliensis PLA1, the cloning and expression of the gene that codifies for this enzyme was carried out in a baculovirus expression system with the obtaintion of a purified recombinant protein that displayed PLA1 activity. Given that this is the first molecular and functional protein characterization of a PLA1 in the Leishmania genus, we also performed a phylogenetic analysis of this gene throughout 12 species whose genome sequences were available. The results presented here will contribute to increase the knowledge about trypanosome phospholipases, which could be novel and valuable as potential targets to fight neglected diseases like Leishmaniasis.
Assuntos
Leishmania braziliensis , Fosfolipases A1 , Animais , Baculoviridae/genética , Clonagem Molecular/métodos , Expressão Gênica , Genes de Protozoários , América Latina , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Leishmaniose Cutânea/parasitologia , Fosfolipases A1/genética , Fosfolipases A1/isolamento & purificação , Fosfolipases A1/metabolismo , Filogenia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9RESUMO
Insect venom phospholipases have been identified in nearly all clinically relevant social Hymenoptera, including bees, wasps and ants. Among other biological roles, during the envenoming process these enzymes cause the disruption of cellular membranes and induce hypersensitive reactions, including life threatening anaphylaxis. While phospholipase A2 (PLA2) is a predominant component of bee venoms, phospholipase A1 (PLA1) is highly abundant in wasps and ants. The pronounced prevalence of IgE-mediated reactivity to these allergens in sensitized patients emphasizes their important role as major elicitors of Hymenoptera venom allergy (HVA). PLA1 and -A2 represent valuable marker allergens for differentiation of genuine sensitizations to bee and/or wasp venoms from cross-reactivity. Moreover, in massive attacks, insect venom phospholipases often cause several pathologies that can lead to fatalities. This review summarizes the available data related to structure, model of enzymatic activity and pathophysiological roles during envenoming process of insect venom phospholipases A1 and -A2.
Assuntos
Venenos de Artrópodes/enzimologia , Himenópteros/enzimologia , Mordeduras e Picadas de Insetos/imunologia , Fosfolipases A1/imunologia , Fosfolipases A2/imunologia , Sequência de Aminoácidos , Animais , Venenos de Artrópodes/imunologia , Humanos , Mordeduras e Picadas de Insetos/enzimologia , Fosfolipases A1/química , Fosfolipases A1/metabolismo , Fosfolipases A2/química , Fosfolipases A2/metabolismoRESUMO
Lipids from microorganisms are ligands of Toll like receptors (TLRs) and modulate the innate immune response. Herein, we analyze in vitro the effect of total lipid extracts from Trypanosoma cruzi amastigotes of RA and K98 strains (with polar biological behavior) on the induction of the inflammatory response and the involvement of TLRs in this process. We demonstrated that total lipid extracts from both strains induced lipid body formation, cyclooxygenase-2 expression and TNF-α and nitric oxide release in macrophages, as well as NF-κB activation and IL-8 release in HEK cells specifically through a TLR2/6 dependent pathway. We also evaluated the inflammatory response induced by total lipid extracts obtained from lysed parasites that were overnight incubated to allow the action of parasite hydrolytic enzymes, such as Phospholipase A1, over endogenous phospholipids. After incubation, these total lipid extracts showed a significantly reduced pro-inflammatory response, which could be attributed to the changes in the content of known bioactive lipid molecules like lysophospholipids and fatty acids, here reported. Moreover, analyses of total fatty acids in each lipid extract were performed by gas chromatography-mass spectrometry. Our results indicate a relevant role of T. cruzi lipids in the induction of a pro-inflammatory response through the TLR2/6 pathway that could contribute to the modulation of the immune response and host survival.
Assuntos
Lipídeos/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 6 Toll-Like/imunologia , Receptores Toll-Like/imunologia , Trypanosoma cruzi/metabolismo , Animais , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Ácidos Graxos/imunologia , Células HEK293 , Humanos , Imunidade Inata , Interleucina-8/metabolismo , Gotículas Lipídicas , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Fosfolipases A1/genética , Fosfolipases A1/metabolismo , Proteínas Recombinantes , Trypanosoma cruzi/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Lecitase Ultra has been immobilized on cyanogen bromide agarose (via covalent attachment) and on octyl agarose (via physical adsorption on the hydrophobic support by interfacial activation). Both immobilized preparations have been incubated in dextran sulfate (DS) or polyethylenimine (PEI) solutions to coat the enzyme surface. Then, the activity versus different substrates and under different experimental conditions was evaluated. The PEI coating generally produced a significant increase in enzyme activity, in some cases even by more than a 30-fold factor (using the octyl-Lecitase at pH 5 in the hydrolysis of methyl phenyl acetate). In opposition, the DS coating usually produced some negative effects on the enzyme activity. The rate of irreversible inhibition of the covalent preparation using diethyl p-nitrophenylphosphate did not increase after PEI coating suggesting that the increase in Lecitase activity is not a consequence of the stabilization of the open form of Lecitase. Moreover, the coating greatly increased the stability of the immobilized Lecitase, for example using DS and the covalent preparation, the half-life was increased by a 30-fold factor in 30% acetonitrile. The stabilizing effect was not found in all cases, in certain cases even a certain destabilization is found (e.g., octyl-Lecitase-DS at pH 7). Thus, the effects of the ionic polymer coating strongly depend on the substrate, experimental conditions and immobilization technique employed.
Assuntos
Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Fosfolipases A1/química , Fosfolipases A1/metabolismo , Biotecnologia , Catálise , Materiais Revestidos Biocompatíveis/química , Brometo de Cianogênio , Sulfato de Dextrana , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Polietilenoimina , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , SefaroseRESUMO
Phospholipase A1 (PLA1) has been described in the infective stages of Trypanosoma cruzi as a membrane-bound/secreted enzyme that significantly modified host cell lipid profile with generation of second lipid messengers and concomitant activation of protein kinase C. In the present work we determined higher levels of PLA1 expression in the infective amastigotes and trypomastigotes than in the non-infective epimastigotes of lethal RA strain. In addition, we found similar expression patterns but distinct PLA1 activity levels in bloodstream trypomastigotes from Cvd and RA (lethal) and K98 (non-lethal) T. cruzi strains, obtained at their corresponding parasitemia peaks. This fact was likely due to the presence of different levels of anti-T. cruzi PLA1 antibodies in sera of infected mice, that modulated the enzyme activity. Moreover, these antibodies significantly reduced in vitro parasite invasion indicating the participation of T. cruzi PLA1 in the early events of parasite-host cell interaction. We also demonstrated the presence of lysophospholipase activity in live infective stages that could account for self-protection against the toxic lysophospholipids generated by T. cruzi PLA1 action. At the genome level, we identified at least eight putative genes that codify for T. cruzi PLA1 with high amino acid sequence variability in their amino and carboxy-terminal regions; a putative PLA1 selected gene was cloned and expressed as a recombinant protein that possessed PLA1 activity. Collectively, the results presented here point out at T. cruzi PLA1 as a novel virulence factor implicated in parasite invasion.
Assuntos
Fosfolipases A1/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/patogenicidade , Fatores de Virulência/metabolismo , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Chlorocebus aethiops , Clonagem Molecular , DNA de Protozoário/química , DNA de Protozoário/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita , Camundongos , Dados de Sequência Molecular , Parasitemia/imunologia , Parasitemia/parasitologia , Fosfolipases A1/genética , Análise de Sequência de DNA , Trypanosoma cruzi/genética , Células Vero , Fatores de Virulência/genéticaRESUMO
The aim of the present research was to analyse the pathways for phosphatidic acid metabolism in purified nuclei from cerebellar cells. Lipid phosphate phosphatase and diacylglyceride lipase activities were detected in nuclei from cerebellar cells. It was observed that DAGL activity makes up 50% of LPP activity and that PtdOH can also be metabolised to lysophosphatidic acid. With a nuclear protein content of approximately 40 µg, the production of diacylglycerol and monoacylglycerol was linear for 30 min and 5 min, respectively, whereas it increased with PtdOH concentrations of up to 250 µM. LysoPtdOH, sphingosine 1-phosphate and ceramide 1-phosphate, which are alternative substrates for LPP, significantly reduced DAG production from PA. DAG and MAG production increased in the presence of Triton X-100 (1 mM) whereas no modifications were observed in the presence of ionic detergent sodium deoxycholate. Ca²+ and Mg²+ stimulated MAG production without affecting DAG formation whereas fluoride and vanadate inhibited the generation of both products. Specific PtdOH-phospholipase A1 and PtdOH-phospholipase A2 were also detected in nuclei. Our findings constitute the first reported evidence of active PtdOH metabolism involving LPP, DAGL and PtdOH-selective PLA activities in purified nuclei prepared from cerebellar cells.
Assuntos
Núcleo Celular/metabolismo , Cerebelo/citologia , Redes e Vias Metabólicas , Ácidos Fosfatídicos/metabolismo , Animais , Cálcio/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Ceramidas/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/enzimologia , Cerebelo/metabolismo , Detergentes/farmacologia , Diglicerídeos/biossíntese , Diglicerídeos/metabolismo , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Lisofosfolipídeos/metabolismo , Magnésio/farmacologia , Masculino , Monoglicerídeos/biossíntese , Monoglicerídeos/metabolismo , Fosfolipases A1/metabolismo , Fosfolipases A2/metabolismo , Ratos , Ratos Wistar , Fluoreto de Sódio/farmacologia , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fatores de Tempo , Vanadatos/farmacologiaRESUMO
The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40 Da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients.