RESUMO
Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.
Assuntos
Antifúngicos , Biofilmes , Cryptococcus neoformans , Proteínas Fúngicas , Lisofosfolipase , Macrófagos Alveolares , Própole , Humanos , Biofilmes/efeitos dos fármacos , Linhagem Celular Tumoral , Criptococose/prevenção & controle , Criptococose/terapia , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/patogenicidade , Etanol/química , Proteínas Fúngicas/antagonistas & inibidores , Lipossomos , Pneumopatias Fúngicas/prevenção & controle , Pneumopatias Fúngicas/terapia , Lisofosfolipase/antagonistas & inibidores , Macrófagos Alveolares/microbiologia , Própole/química , Própole/farmacologia , Virulência/efeitos dos fármacos , Fatores de Virulência/antagonistas & inibidores , Antifúngicos/química , Antifúngicos/farmacologiaRESUMO
The autotaxin-lysophosphatidic acid (ATX-LPA) axis has been implicated in several disease conditions including inflammation, fibrosis and cancer. This makes ATX an attractive drug target and its inhibition may lead to useful therapeutic agents. Through a high throughput screen (HTS) we identified a series of small molecule inhibitors of ATX which have subsequently been optimized for potency, selectivity and developability properties. This has delivered drug-like compounds such as 9v (CRT0273750) which modulate LPA levels in plasma and are suitable for in vivo studies. X-ray crystallography has revealed that these compounds have an unexpected binding mode in that they do not interact with the active site zinc ions but instead occupy the hydrophobic LPC pocket extending from the active site of ATX together with occupying the LPA 'exit' channel.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Lisofosfolipase/antagonistas & inibidores , Lisofosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Cristalografia por Raios X , Inibidores Enzimáticos/farmacocinética , Humanos , Lisofosfolipase/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacologiaRESUMO
Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.
Assuntos
Hidrolases/metabolismo , Lisofosfolipase/antagonistas & inibidores , Serina/metabolismo , Animais , Cristalização , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Humanos , Lisofosfolipase/químicaRESUMO
Sequence homology indicates the existence of three human cytosolic acyl protein thioesterases, including APT1 that is known to depalmitoylate H- and N-Ras. One of them is the lysophospholipase-like 1 (LYPLAL1) protein that on the one hand is predicted to be closely related to APT1 but on the other hand might also function as a potential triacylglycerol lipase involved in obesity. However, its role remained unclear. The 1.7 Å crystal structure of LYPLAL1 reveals a fold very similar to APT1, as expected, but features a shape of the active site that precludes binding of long-chain substrates. Biochemical data demonstrate that LYPLAL1 exhibits neither phospholipase nor triacylglycerol lipase activity, but rather accepts short-chain substrates. Furthermore, extensive screening efforts using chemical array technique revealed a first small molecule inhibitor of LYPLAL1.
Assuntos
Lisofosfolipase/química , Cristalografia por Raios X , Humanos , Lipase/metabolismo , Lisofosfolipase/antagonistas & inibidores , Lisofosfolipase/metabolismo , Modelos Moleculares , Fosfolipases/metabolismo , Especificidade por SubstratoRESUMO
Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide and phospholipid metabolism. Among the members of the NPP family, NPP1 and -3 act on nucleotides such as ATP, while NPP2, -6, and -7 act on phospholipids such as lysophosphatidylcholine and sphingomyelin. NPP6, a recently characterized NPP family member, is a choline-specific glycerophosphodiester phosphodiesterase, but its functions remain to be analyzed, partly due to the lack of highly sensitive activity assay systems and practical inhibitors. Here we report synthesis of novel NPP6 fluorescence probes, TG-mPC and its analogues TG-mPC(3)C, TG-mPC(5)C, TG-mPENE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC(3)C, and TG-mPENE. TG-mPC was hydrolyzed in the cell lysate from NPP6-transfected cells, but not control cells, showing that it is suitable for use in cell-based NPP6 assays. We also examined the usefulness of TG-mPC as a fluorescence imaging probe. We further applied TG-mPC to carry out high-throughput NPP6 inhibitor screening and found several NPP6-selective inhibitors in a library of about 80,000 compounds. Through structure-activity relationship (SAR) analysis, we identified a potent and selective NPP6 inhibitor with an IC(50) value of 0.21 µM. Our NPP6-selective fluorescence probe, TG-mPC, and the inhibitor are expected to be useful to elucidate the biological function of NPP6.
Assuntos
Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/farmacologia , Lisofosfolipase/antagonistas & inibidores , Lisofosfolipase/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Animais , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Células HEK293 , Células HeLa , Humanos , Lisofosfolipase/química , Camundongos , Estrutura Molecular , Células NIH 3T3 , Diester Fosfórico Hidrolases/química , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the sn-2 hydroxy group forms a five-membered ring with the sn-3 phosphate. Here, we describe the synthesis of R-3-CCPA and S-3-CCPA along with their pharmacological properties as inhibitors of lysophospholipase D/autotaxin, agonists of the LPA(5) GPCR, and blockers of lung metastasis of B16-F10 melanoma cells in a C57BL/6 mouse model. S-3CCPA was significantly more efficacious in the activation of LPA(5) compared to the R-stereoisomer. In contrast, no stereoselective differences were found between the two isomers toward the inhibition of autotaxin or lung metastasis of B16-F10 melanoma cells in vivo. These results extend the potential utility of these compounds as potential lead compounds warranting evaluation as cancer therapeutics.
Assuntos
Ácidos Fosfatídicos/química , Animais , Modelos Animais de Doenças , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Lisofosfolipase/antagonistas & inibidores , Lisofosfolipase/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Ácidos Fosfatídicos/síntese química , Ácidos Fosfatídicos/farmacologia , Fosfodiesterase I/antagonistas & inibidores , Fosfodiesterase I/metabolismo , Diester Fosfórico Hidrolases , Pirofosfatases/antagonistas & inibidores , Pirofosfatases/metabolismo , Receptores de Ácidos Lisofosfatídicos/agonistas , Receptores de Ácidos Lisofosfatídicos/metabolismo , EstereoisomerismoRESUMO
A series of bis(alkylpyridinium)alkanes with a twelve carbon spacer between the positive charges was synthesised and their antifungal activity has been investigated. Compounds with 2-pentyl, 4-pentyl, 4-hexyl, 4-octyl, 4-propylbenzene, 3,4-dipentyl, 4-(5'-nonyl) and 3-methyl,4-pentyl head groups were the most potent antifungal agents with MICs in the range of 1.4-2.7 microM against reference strains of both Cryptococcus neoformans and Candida albicans.
Assuntos
Antifúngicos/síntese química , Compostos de Piridínio/síntese química , Animais , Antifúngicos/química , Antifúngicos/toxicidade , Candida albicans/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Hemólise , Lisofosfolipase/antagonistas & inibidores , Lisofosfolipase/metabolismo , Testes de Sensibilidade Microbiana , Inibidores de Fosfolipase A2 , Fosfolipases A2/metabolismo , Compostos de Piridínio/química , Compostos de Piridínio/toxicidade , SuínosRESUMO
Cyclooctatin, a diterpene characterized by a 5-8-5 fused ring system, is a potent inhibitor of lysophospholipase. Here we report the cloning and characterization of a complete cyclooctatin biosynthetic gene cluster from Streptomyces melanosporofaciens MI614-43F2 and heterologous production of cyclooctatin in S. albus. Sequence analysis coupled with subcloning and gene deletion revealed that the minimal cyclooctatin biosynthetic gene cluster consists of four genes, cotB1 to cotB4, encoding geranylgeranyl diphosphate (GGDP) synthase, terpene cyclase (CotB2), and two cytochromes P450, respectively. Incubation of the recombinant CotB2 with GGDP resulted in the formation of cyclooctat-9-en-7-ol, an unprecedented tricyclic diterpene alcohol. The present study establishes the complete biosynthetic pathway of cyclooctatin and provides insights into both the stereospecific diterpene cyclization mechanism of the GGDP cyclase and the molecular bases for the stereospecific and regiospecific hydroxylation.
Assuntos
Compostos Bicíclicos com Pontes/metabolismo , Clonagem Molecular/métodos , Sistema Enzimático do Citocromo P-450/genética , Liases Intramoleculares/genética , Redes e Vias Metabólicas/genética , Família Multigênica/genética , Diterpenos , Hidroxilação , Lisofosfolipase/antagonistas & inibidores , Streptomyces/genéticaRESUMO
The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.
Assuntos
Lisofosfolipase/metabolismo , Lipídeos de Membrana/metabolismo , Mycobacterium tuberculosis/enzimologia , Sequência de Bases , Catálise , Primers do DNA , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Lisofosfolipase/antagonistas & inibidores , Lisofosfolipase/química , Lisofosfolipase/genética , Lisofosfolipídeos/metabolismo , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/genética , Especificidade por Substrato , TemperaturaRESUMO
A series of bisquaternary ammonium salts with a 12-carbon spacer between the positive charges were synthesized, and their antifungal activity has been investigated. Compounds with butyl, pentyl, and isopentyl headgroups were the most potent antifungal agents with MICs in the range of 2.2-5.5 microM against both Cryptococcus neoformans and Candida albicans. The antifungal activity of these compounds correlated with their inhibition of cryptococcal phospholipase B1 (PLB1), a newly identified virulence factor. This indicates that the mode of action of these compounds may be inhibition of the fungal PLB1 enzyme, further validating this enzyme as a target for the development of novel antifungal therapies.
Assuntos
Antifúngicos/síntese química , Compostos de Amônio Quaternário/síntese química , Animais , Antifúngicos/química , Antifúngicos/farmacologia , Bovinos , Cryptococcus neoformans/enzimologia , Hemólise , Humanos , Técnicas In Vitro , Lisofosfolipase/antagonistas & inibidores , Testes de Sensibilidade Microbiana , Fosfolipases A/antagonistas & inibidores , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/farmacologia , Relação Estrutura-Atividade , Fatores de Virulência/antagonistas & inibidoresRESUMO
The alkyl phosphocholine drug miltefosine is structurally similar to natural substrates of the fungal virulence determinant phospholipase B1 (PLB1), which is a potential drug target. We determined the MICs of miltefosine against key fungal pathogens, correlated antifungal activity with inhibition of the PLB1 activities (PLB, lysophospholipase [LPL], and lysophospholipase-transacylase [LPTA]), and investigated its efficacy in a mouse model of disseminated cryptococcosis. Miltefosine inhibited secreted cryptococcal LPTA activity by 35% at the subhemolytic concentration of 25 microM (10.2 microg/ml) and was inactive against mammalian pancreatic phospholipase A2 (PLA2). At 250 microM, cytosolic PLB, LPL, and LPTA activities were inhibited by 25%, 51%, and 77%, respectively. The MICs at which 90% of isolates were inhibited (MIC90s) against Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, Cryptococcus gattii, Aspergillus fumigatus, Fusarium solani, Scedosporium prolificans, and Scedosporium apiospermum were 2 to 4 microg/ml. The MICs of miltefosine against Candida tropicalis (n = 8) were 2 to 4 microg/ml, those against Aspergillus terreus and Candida parapsilosis were 8 microg/ml (MIC90), and those against Aspergillus flavus (n = 8) were 2 to 16 microg/ml. Miltefosine was fungicidal for C. neoformans, with rates of killing of 2 log units within 4 h at 7.0 microM (2.8 microg/ml). Miltefosine given orally to mice on days 1 to 5 after intravenous infection with C. neoformans delayed the development of illness and mortality and significantly reduced the brain cryptococcal burden. We conclude that miltefosine has broad-spectrum antifungal activity and is active in vivo in a mouse model of disseminated cryptococcosis. The relatively small inhibitory effect on PLB1 enzyme activities at concentrations exceeding the MIC by 2 to 20 times suggests that PLB1 inhibition is not the only mechanism of the antifungal effect.
Assuntos
Antifúngicos/farmacologia , Criptococose/tratamento farmacológico , Fosforilcolina/análogos & derivados , Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Fungos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Lisofosfolipase/antagonistas & inibidores , Lisofosfolipase/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêuticoRESUMO
Secreted phospholipase B is a proven virulence factor for the pathogenic fungus Cryptococcus neoformans and exhibits three phospholipase activities in the one protein. These are phospholipase B (PLB), lysophospholipase (LPL), and lysophospholipase transacylase (LPTA). Our aim was to investigate the feasibility of using this enzyme as a target for antifungal therapy. We determined in C. neoformans var. grubii strain H99 that 82% of PLB activity was secreted but that 64% of LPL activity and 70% of LPTA activity were cell associated. Cell-associated activities (cytosolic and membrane) were further characterized, since it is likely that any fungicidal effect would depend on inhibition of these enzymes. Four commercially available compounds with structural similarities to phospholipid substrates were tested as inhibitors. These were alexidine dihydrochloride (compound A), dioctadecyldimethylammonium bromide (compound O), 1,12 bis-(tributylphosphonium)dodecane dibromide (compound P), and decamethonium dibromide (compound D). The best phospholipase inhibitors (compounds A and P) were also the most potent antifungal agents by the standard broth microdilution test. Compound A was highly selective for secreted and cell-associated PLB activities and showed no inhibition of mammalian phospholipase A(2) at 0.25 micro M. Compound O, which was specific for secretory and cytosolic LPL and LPTA and membrane-associated PLB, was not antifungal. We conclude that inhibitors of cryptococcal phospholipases can be selective for fungal enzymes and intrinsically antifungal. They also provide tools for assessing the relative importance of the various enzyme activities in virulence. Our results enable further rational structure-function studies to validate the use of phospholipases as antifungal targets.
Assuntos
Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fosfolipases/antagonistas & inibidores , 1,2-Dipalmitoilfosfatidilcolina/farmacologia , Aciltransferases/antagonistas & inibidores , Animais , Criptococose/microbiologia , Proteínas Fúngicas/biossíntese , Fungos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Lisofosfolipase/antagonistas & inibidores , Testes de Sensibilidade Microbiana , Complexos Multienzimáticos/antagonistas & inibidores , Pâncreas/enzimologia , SuínosRESUMO
Lysophospholipases (LysoPLAs) are a large family of enzymes for removing lysophospholipids from cell membranes. Potent inhibitors are needed to define the importance of LysoPLAs as targets for toxicants and potential therapeutics. This study considers organophosphorus (OP) inhibitors with emphasis on mouse brain total LysoPLA activity relative to the mipafox-sensitive neuropathy target esterase (NTE)-LysoPLA recently established as 17% of the total activity and important in the action of OP delayed toxicants. The most potent inhibitors of total LysoPLA in mouse brain are isopropyl dodecylphosphonofluoridate (also for LysoPLA of Vibrio bacteria), ethyl octylphosphonofluoridate (EOPF), and two alkyl-benzodioxaphosphorin 2-oxides (BDPOs)[(S)-octyl and dodecyl] (IC50 2-8 nM). OP inhibitors acting in vitro and in vivo differentiate a more sensitive portion but not a distinct NTE-LysoPLA compared with total LysoPLA activity. For 10 active inhibitors, NTE-LysoPLA is 17-fold more sensitive than total LysoPLA, but structure-activity comparisons give a good correlation (r(2) = 0.94) of IC50 values, suggesting active site structural similarity or identity. In mice 4 h after intraperitoneal treatment with discriminating doses, EOPF, tribufos (a plant defoliant), and dodecanesulfonyl fluoride inhibit 41-57% of the total brain LysoPLA and 85-99% of the NTE-LysoPLA activity. Total LysoPLA as well as NTE-LysoPLA is decreased in activity in Nte(+/-)-haploinsufficient mice compared to their Nte(+/+) littermates. The lysolecithin level of spinal cord but not brain is elevated significantly following EOPF treatment (3 mg/kg), thereby focusing attention on localized rather than general alterations in lysophospholipid metabolism in OP-induced hyperactivity and toxicity.
Assuntos
Encéfalo/efeitos dos fármacos , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Isoflurofato/análogos & derivados , Isoflurofato/farmacologia , Fígado/efeitos dos fármacos , Lisofosfolipase/antagonistas & inibidores , Compostos Organofosforados/farmacologia , Animais , Encéfalo/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Humanos , Fígado/enzimologia , Lisofosfolipase/metabolismo , Masculino , CamundongosRESUMO
OBJECTIVE: Acute lung injury in Pseudomonas aeruginosa pneumonia depends primarily on ExoU toxin being delivered directly into the eukaryotic cell cytosol through the type III secretion system. The amino-acid sequence of ExoU has a potato patatin-like phospholipase domain, similar to the sequence of mammalian Ca-independent phospholipase A2. We examined whether the acute lung injury caused by cytotoxic P. aeruginosa was dependent on the patatin-like phospholipase domain of ExoU. DESIGN: Laboratory investigation using an established mouse model for P. aeruginosa pneumonia with quantitative measurements of acute lung injury and mortality. SETTING: University experimental research laboratory. SUBJECTS: Balb/c mice. INTERVENTIONS: First, a site-directional mutation was introduced in the predicted catalytically active site of the patatin-like phospholipase domain of recombinant ExoU protein. The effect of the mutation on the catalytic activity of ExoU was tested by the in vitro lysophospholipase A assay. Second, the same site-directional mutation was introduced into the exoU gene of P. aeruginosa PA103. Mice were intratracheally infected with either a wild-type P. aeruginosa strain PA103 or an isogenic mutant containing the mutation in exoU. Acute epithelial lung injury, lung edema, bacteremia, and mortality were evaluated quantitatively. MEASUREMENTS AND MAIN RESULTS: Recombinant ExoU had lysophospholipase A activity. Site-directional mutations in the predicted catalytic site of ExoU caused a loss of the lysophospholipase A activity. Whereas the airspace instillation of PA103 caused acute lung injury and death of the infected mice, the airspace instillation of isogenic mutants secreting catalytically inactive ExoU were noncytotoxic and did not cause acute lung injury or death of the infected mice. CONCLUSION: Virulent P. aeruginosa causes acute lung injury and death by the cytotoxic activity derived from the patatin-like phospholipase domain of ExoU.
Assuntos
Proteínas de Bactérias/fisiologia , Modelos Animais de Doenças , Lisofosfolipase/fisiologia , Pneumonia Bacteriana/complicações , Infecções por Pseudomonas/complicações , Pseudomonas aeruginosa/fisiologia , Síndrome do Desconforto Respiratório/microbiologia , Doença Aguda , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/antagonistas & inibidores , Domínio Catalítico/fisiologia , Citosol/microbiologia , Citosol/fisiologia , Células Eucarióticas/microbiologia , Células Eucarióticas/fisiologia , Immunoblotting , Inflamação , Lisofosfolipase/análise , Lisofosfolipase/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/fisiologia , Pneumonia Bacteriana/enzimologia , Mutação Puntual , Infecções por Pseudomonas/enzimologia , Pseudomonas aeruginosa/patogenicidade , Síndrome do Desconforto Respiratório/mortalidade , Síndrome do Desconforto Respiratório/patologia , Síndrome do Desconforto Respiratório/prevenção & controle , Alinhamento de Sequência , Taxa de SobrevidaRESUMO
Cytosolic phospholipase A(2)gamma (cPLA(2)gamma) is a calcium-independent, membrane-associated phospholipase A(2) that possesses a C-terminal prenylation motif (-CCLA) whose covalent structure cannot be deduced from the primary sequence alone. Accordingly, we overexpressed human cPLA(2)gamma containing an N-terminal His tag ((His)(6)cPLA(2)gamma) in Sf9 cells and quantitatively solubilized and purified the enzyme by sequential immobilized metal affinity and Mono Q column chromatographies. The final preparation appeared as a single 61 kDa band after SDS-PAGE/silver-staining, possessed high lysophospholipase activity (50 micromol min(-1) mg(-1)), and was inhibited by, but did not hydrolyze, palmitoyl-CoA. Radiolabeling of recombinant human cPLA(2)gamma with [(3)H]-mevalonolactone in the absence of statins and subsequent cleavage of prenyl groups with Raney nickel revealed that the enzyme is only farnesylated and is not geranylgeranylated. Analysis of CNBr-digested cPLA(2)gamma by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI/TOF-TOF) mass spectrometry demonstrated the presence of a farnesyl moiety at Cys-538, cleavage of the Cys(538)-Cys(539) bond, and carboxymethylation of the resultant C-terminal prenylated cysteine. Collectively, these results describe the solubilization and purification of recombinant cPLA(2)gamma to homogeneity and identify cPLA(2)gamma as a farnesylated protein that undergoes at least three sequential posttranslational modifications that likely facilitate its targeting and interactions with its membrane substrates.
Assuntos
Endopeptidases/metabolismo , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Fosfolipases A/isolamento & purificação , Fosfolipases A/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Citosol/enzimologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo IV , Humanos , Hidrólise , Lisofosfolipase/antagonistas & inibidores , Lisofosfolipase/metabolismo , Metilação , Dados de Sequência Molecular , Palmitoil Coenzima A/farmacologia , Fragmentos de Peptídeos/genética , Fosfolipases A/biossíntese , Fosfolipases A/genética , Prenilação de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Spodoptera/enzimologia , Spodoptera/genéticaRESUMO
Charcot-Leyden crystal (CLC) protein, initially reported to possess weak lysophospholipase activity, is still considered to be the eosinophil's lysophospholipase, but it shows no sequence similarities to any known lysophospholipases. In contrast, CLC protein has moderate sequence similarity, conserved genomic organization, and near structural identity to members of the galectin superfamily, and it has been designated galectin-10. To definitively determine whether or not CLC protein is a lysophospholipase, we reassessed its enzymatic activity in peripheral blood eosinophils and an eosinophil myelocyte cell line (AML14.3D10). Antibody affinity chromatography was used to fully deplete CLC protein from eosinophil lysates. The CLC-depleted lysates retained their full lysophospholipase activity, and this activity could be blocked by sulfhydryl group-reactive inhibitors, N-ethylmaleimide and p-chloromercuribenzenesulfonate, previously reported to inhibit the eosinophil enzyme. In contrast, the affinity-purified CLC protein lacked significant lysophospholipase activity. X-ray crystallographic structures of CLC protein in complex with the inhibitors showed that p-chloromercuribenzenesulfonate bound CLC protein via disulfide bonds with Cys(29) and with Cys(57) near the carbohydrate recognition domain (CRD), whereas N-ethylmaleimide bound to the galectin-10 CRD via ring stacking interactions with Trp(72), in a manner highly analogous to mannose binding to this CRD. Antibodies to rat pancreatic lysophospholipase identified a protein in eosinophil and AML14.3D10 cell lysates, comparable in size with human pancreatic lysophospholipase, which co-purifies in small quantities with CLC protein. Ligand blotting of human and murine eosinophil lysates with CLC protein as probe showed that it binds proteins also recognized by antibodies to pancreatic lysophospholipase. Our results definitively show that CLC protein is not one of the eosinophil's lysophospholipases but that it does interact with eosinophil lysophospholipases and known inhibitors of this lipolytic activity.
Assuntos
Inibidores Enzimáticos/metabolismo , Glicoproteínas/fisiologia , Lisofosfolipase/antagonistas & inibidores , Compostos de Sulfidrila/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Cristalografia por Raios X , Primers do DNA , Inibidores Enzimáticos/farmacologia , Eosinófilos/enzimologia , Etilmaleimida/farmacologia , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , RNA Mensageiro/genética , Radioimunoensaio , Ratos , Compostos de Sulfidrila/farmacologiaRESUMO
Two species of giant hornet phospholipase B (PLB), alpha and beta, were purified from the venom of Vespa mandarinia. The purification procedure was simplified by two steps of column chromatographies, Sephadex G-100 and SP-Sepharose. The molecular sizes of PLB alpha and beta were 29.5 and 26.0 kDa, respectively. The isoelectric point of alpha and beta enzymes were pH 10.6 and 10.7, respectively. The temperature optimum for egg yolk lecithin was a broad peak at 40-60 degrees C for both enzymes. Amino acid compositions of both enzymes were high contents of aspartic acid, glycine, leucine, lysine and other aliphatic amino acids. Cystine was similar amounts to other species of phospholipases (PLs). The K(m) values of alpha and beta enzymes were 8.29 and 7.53 mg/ml for egg yolk lecithin, respectively. In the catalytic specificity for L-alpha-phosphatidylcholine-beta-oleoil-gamma-palmitoil, the K(m) values of alpha enzyme for gamma-palmitoil and beta-oleoil residues were 0.528 and 1.392 mM, respectively. While the K(m) values of beta enzyme for gamma-palmitoil and beta-oleoil residues were 7.91 and 2. 68 mM, respectively. Both alpha and beta enzymes were inhibited strongly by cepharanthine. The lecithin hydrolysis of alpha enzyme was competitively inhibited, but beta enzyme was uncompetitive. Cepharanthine also inhibited noncompetitively PLA(2)s of bovine pancreas, bee venom and Naja mossambica mossambica.
Assuntos
Alcaloides/farmacologia , Venenos de Abelha/enzimologia , Inibidores Enzimáticos/farmacologia , Lisofosfolipase/isolamento & purificação , Vespas , Aminoácidos/análise , Animais , Benzilisoquinolinas , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Lisofosfolipase/antagonistas & inibidores , Fosfatidilcolinas/metabolismoRESUMO
Infection caused by the fungus Cryptococcus neoformans is potentially fatal. A highly active extracellular phospholipase, demonstrating phospholipase B (PLB), lysophospholipase (LPL) and lysophospholipase/transacylase (LPTA) activities, was purified to homogeneity from C. neoformans using (NH(4))(2)SO(4) fractionation, and hydrophobic-interaction, anion-exchange and gel-filtration chromatography. All three enzyme activities co-purified as a single protein with an apparent molecular mass of 70-90 kDa by SDS/PAGE and 160-180 kDa by gel filtration. The ratio of the three activities remained constant after each purification step. The amino acid composition, as well as the sequences of the N-terminus and of five internal peptide fragments were novel. The protein was an acidic glycoprotein containing N-linked carbohydrate moieties, with pI values of 5.5 and 3.5. The apparent V(max) values for PLB and LPL activities were 12.3 and 870 micromol/min per mg of protein respectively; the corresponding K(m) values were approx. 185.3 and 92.2 microM. The enzyme was active only at acidic pH (pH optimum of 4.0 for PLB and 4.0-5.0 for LPL and LPTA). Enzyme activity did not require added cations, but was inhibited by Fe(3+). LPL and LPTA activities were decreased by 0.1% (v/v) Triton X-100 to 50% of the control value. Palmitoylcarnitine (0.5 mM) inhibited PLB (97% inhibition) and LPL and LPTA activities (35% inhibition) competitively. All phospholipids except phosphatidic acid were degraded by PLB, but dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylcholine were the preferred substrates. This is the first complete description of the purification and properties of a phospholipase, which may be involved in virulence, from a pathogenic fungus.
Assuntos
Aciltransferases/isolamento & purificação , Aciltransferases/metabolismo , Cryptococcus neoformans/enzimologia , Lisofosfolipase/isolamento & purificação , Lisofosfolipase/metabolismo , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/química , Cátions/farmacologia , Cryptococcus neoformans/patogenicidade , Estabilidade Enzimática , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Glicosilação , Temperatura Alta , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Cinética , Lisofosfolipase/antagonistas & inibidores , Lisofosfolipase/química , Metais/farmacologia , Peso Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , Octoxinol/farmacologia , Palmitoilcarnitina/farmacologia , Análise de Sequência de Proteína , Especificidade por SubstratoRESUMO
Neuronal nuclei isolated from rabbit cerebral cortex were found to be enriched in an NEM-insensitive lysophosphatidic acid (lysoPA) phosphohydrolase activity. LysoPA is an inhibitor of the nuclear lysophosphatidylcholine (lysoPC) lysophospholipase, and by preserving lysoPC levels, lysoPA boosted the nuclear production of the acyl analogue of platelet-activating factor by promoting the acetylation of lysoPC (Baker and Chang, Mol. Cell Biochem., 1999, in press). The nuclear phosphohydrolase converts lysoPA to 1-monoacylglycerol, and thus eliminates this lysoPA inhibition of lysoPC lysophospholipase. The nuclear lysoPA phosphohydrolase specific activity was more than three times that observed for the nuclear lysoPA lysophospholipase (Baker and Chang, Biochim. Biophys. Acta 1438 (1999) 253-263) and represents a more active route for nuclear lysoPA removal. The neuronal nuclear lysoPA phosphohydrolase was inhibited at acidic pH, and also inhibited by calcium ions. The 1-monoacylglycerol product of the phosphohydrolase is rapidly degraded by neuronal monoacylglycerol lipase, an enzyme some sevenfold more active than the phosphohydrolase and sensitive to inhibition by arachidonoyl trifluoromethyl ketone (AACOCF(3)). Both acidic pH and free fatty acid inhibited the lipase. In the absence of AACOCF(3), production of fatty acid from lysoPA substrate could be largely attributed to the sequential actions of the nuclear phosphohydrolase and lipase. This facilitates fatty acid recycling back into phospholipid by lysophospholipid acylation when ATP levels are restored following periods of brain ischemia. At relatively low concentrations, sphingosine-1-phosphate, and alkylglycerophosphate were the most effective phosphohydrolase inhibitors while phosphatidic acid, alkylacetylglycerophosphate and ceramide were without effect. LysoPA is an interesting regulatory molecule that can potentially preserve lysophosphatidylcholine within the nuclear membrane for use in acetylation reactions. Thus conditions relevant to brain ischemia such as falling pH, falling ATP concentrations, rising fatty acid and intracellular calcium levels may, by slowing this metabolic path for lysoPA loss, promote the production of acyl PAF and contribute to the increased levels of the acetylated lipids noted in ischemia.
Assuntos
Córtex Cerebral/metabolismo , Lisofosfolipase/antagonistas & inibidores , Lisofosfolipídeos/metabolismo , Animais , Núcleo Celular/metabolismo , Etilmaleimida/farmacologia , Glicerídeos/metabolismo , Concentração de Íons de Hidrogênio , Monoacilglicerol Lipases/metabolismo , Neurônios/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Coelhos , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismoRESUMO
Neuronal nuclei were isolated from rabbit cerebral cortex, and lipid acetylation reactions were studied because of the high nuclear concentration of acetyltransferases that generate platelet activating factor (PAF) and its acyl analogue AcylPAF. The neuronal nuclear acetylation of 1-palmitoyl lysophosphatidylcholine (lyso PC) was found to be increased more than twofold when low concentrations of lyso PC were incubated in acetylation assays in the presence of 1-palmitoyl lysophosphatidic acid (lyso PA) or 1-hexadecyl glycerophosphate (AGP). This effect was not found for a variety of other acidic and neutral 1-acyl lysoglycerophospholipids. At 4 microM concentrations, AGP was the more effective in increasing rates of lyso PC acetylation, while lyso PA was more effective at 25-35 microM. 1-Stearoyl, 1-alkenyl and 1-decanoyl analogues of lyso PA were all less effective than 1-palmitoyl lyso PA. Phosphatidic acid was considerably less effective than lyso PA, while the acetylated analogue of AGP, AAcGP (alkylacetylglycerophosphate), increased rates of lyso PC acetylation to maxima similar to those seen with lyso PA or AGP. In addition, AAcGP promoted these maxima at considerably lower concentrations (2-4 microM). A mechanism for these effects was suggested when nuclear envelopes (NE), isolated in the presence of PMSF, showed these maximal acetylation rates at low lyso PC concentrations, and these rates were not elevated by the presence of lyso PA. PMSF is a protease inhibitor but can also inhibit lysophospholipase activity. We found a nuclear lysophospholipase that degraded lyso PC at rates more than 13 times those of nuclear lyso PC acetylation. PMSF did inhibit this nuclear lysophospholipase, as did lyso PA, AGP and AAcGP. Kinetic analyses of the effects of lyso PA, AGP and AAcGP on lyso PC lysophospholipase indicated that these three lipids acted as competitive inhibitors for the lyso PC substrate. It is possible that low rates of lyso PC acetylation seen in neuronal nuclei at low lyso PC concentrations, are caused by lyso PC loss mediated by a very strong nuclear lysophospholipase. The effects of lyso PA, AGP and AAcGP in boosting rates of lyso PC acetylation likely come from the inhibition of nuclear lysophospholipase and a preservation of lyso PC concentrations. Competing neuronal nuclear reactions for low endogenous levels of lyso PC may regulate the formation of AcylPAF, and rising lyso PA, AGP or AAcGP concentrations can increase rates of nuclear AcylPAF synthesis.