RESUMEN
Phospholipase A2 (PLA2) is a superfamily of phospholipase enzymes that dock at the water/oil interface of phospholipid assemblies, hydrolyzing the ester bond at the sn-2 position. The enzymatic activity of these enzymes differs based on the nature of the substrate, its supramolecular assemblies (micelle, liposomes), and their composition, reflecting the interfacial nature of the PLA2s and requiring assays able to directly quantify this interaction of the enzyme(s) with these supramolecular assemblies. We developed and optimized a simple, universal assay method employing the pH-sensitive indicator dye bromothymol blue (BTB), in which different POPC (3-palmitoyl-2-oleoyl-sn-glycero-1-phosphocholine) self-assemblies (liposomes or mixed micelles with Triton X-100 at different molar ratios) were used to assess the enzymatic activity. We used this assay to perform a comparative analysis of PLA2 kinetics on these supramolecular assemblies and to determine the kinetic parameters of PLA2 isozymes IB and IIA for each supramolecular POPC assembly. This assay is suitable for assessing the inhibition of PLA2s with great accuracy using UV-VIS spectrophotometry, being thus amenable for screening of PLA2 enzymes and their substrates and inhibitors in conditions very similar to physiologic ones.
Asunto(s)
Fosfatidilcolinas , Fosfolipasas A2 , Fosfolipasas A2/metabolismo , Fosfolipasas A2/química , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Cinética , Micelas , Liposomas/química , Concentración de Iones de Hidrógeno , Pruebas de Enzimas/métodos , Octoxinol/químicaRESUMEN
In this study, two types of microgel particles from egg yolk components were prepared by combining enzymatic hydrolysis with high-pressure homogenization (HPH), and their differences in physicochemical properties, foaming properties, and microstructure were compared. Results showed that the particle size of both types of microgel particles had decreased from 2744.07 ± 408.26 nm (egg yolk, EY) to 144.97 ± 3.19 nm (PLA2 hydrolyzed egg yolk microgel particles, PYM) and 535.07 ± 46.07 nm (egg yolk microgel particles hydrolyzed by PLA2, YMP), from 736.24 ± 34.61 nm (EG) to 182.76 ± 4.12 nm (PLA2 hydrolyzed egg yolk granules microgel particles, PGM) and 443.98 ± 27.09 nm (egg yolk granules microgel particles hydrolyzed by PLA2, GMP). Besides, their interfacial adsorption abilities were significantly improved, reflected in the increase values in overrun, from161.90 % ± 9.84 % (EY) to 269.64 % ± 16.73 % (PMY) and 307.20 % ± 16.09 % (YMP), from 189.21 % ± 5.02 % (EG) to 280.38 % ± 36.05 % (PGM) and 261.91 % ± 34.03 % (GMP). Their structural properties showed higher stabilities after treatments. When the microgel particles are applied to cakes, the specific volume was increased from 2.05 ± 0.1 mL/g (EY) to 2.25 ± 0.13 mL/g (PYM) and 2.45 ± 0.03 mL/g (YPM), and from 2.00 ± 0.09 mL/g (EG) to 2.51 ± 0.13 mL/g (PGM) and 2.75 ± 0.21 mL/g (GMP), respectively. The hardness and chewiness were reduced with both types of microgel particles from egg yolk components, which indicated their potential value as edible foam stabilizers in the baking industry.
Asunto(s)
Proteínas del Huevo , Yema de Huevo , Geles , Tamaño de la Partícula , Fosfolipasas A2 , Presión , Yema de Huevo/química , Fosfolipasas A2/química , Fosfolipasas A2/metabolismo , Geles/química , Proteínas del Huevo/química , Hidrólisis , Fenómenos Químicos , Animales , Pollos , Estabilidad ProteicaRESUMEN
Reducing the allergenicity of edible insects is crucial for the comprehensive utilization of insect resources. Phospholipase A2 (PLA2) exists in various edible insects and mammalian tissues, which can cause serious allergic reactions. Herein, we constructed a magnetic nanocomposite with photo/chemical synergistic capability to mitigate the allergenicity of PLA2. The formation of prepared nanocomposite was systematically confirmed using various techniques. The nanocomposite exhibited uniform diameters, abundant functional groups, excellent magnetic capabilities. An effective photo/chemical method was established to reduce the allergenicity of PLA2 in vitro. The feasibility of the method was demonstrated through circular dichroism, fluorescence spectrum and IgE-binding analysis. The allergenicity and IgE-binding effect of PLA2 were significantly reduced due to conformational changes after nanomaterial treatment. These results demonstrate the sensitivity and effectiveness a strategy for reducing PLA2 allergenicity, providing a basis for development of nanomaterials to reduce the risk of novel food allergies in response to edible insect products.
Asunto(s)
Alérgenos , Fosfolipasas A2 , Fosfolipasas A2/química , Fosfolipasas A2/inmunología , Alérgenos/inmunología , Alérgenos/química , Animales , Humanos , Hipersensibilidad a los Alimentos/inmunología , Hipersensibilidad a los Alimentos/prevención & control , Nanoestructuras/química , Inmunoglobulina E/inmunología , Proteínas de Insectos/inmunología , Proteínas de Insectos/química , Nanocompuestos/química , Insectos Comestibles/química , Insectos Comestibles/inmunologíaRESUMEN
Snake venoms are a complex mixture of proteins and polypeptides that represent a valuable source of potential molecular tools for understanding physiological processes for the development of new drugs. In this study two major PLA2s, named PLA2-I (Asp49) and PLA2-II (Lys49), isolated from the venom of Bothrops diporus from Northeastern Argentina, have shown cytotoxic effects on LM3 murine mammary tumor cells, with PLA2-II-like exhibiting a stronger effect compared to PLA2-I. At sub-cytotoxic levels, both PLA2s inhibited adhesion, migration, and invasion of these adenocarcinoma cells. Moreover, these toxins hindered tubulogenesis in endothelial cells, implicating a potential role in inhibiting tumor angiogenesis. All these inhibitory effects were more pronounced for the catalytically-inactive toxin. Additionally, in silico studies strongly suggest that this PLA2-II-like myotoxin could effectively block fibronectin binding to the integrin receptor, offering a dual advantage over PLA2-I in interacting with the αVß3 integrin. In conclusion, this study reports for the first time, integrating both in vitro and in silico approaches, a comparative analysis of the antimetastatic and antiangiogenic potential effects of two isoforms, an Asp49 PLA2-I and a Lys49 PLA2-II-like, both isolated from Bothrops diporus venom.
Asunto(s)
Bothrops , Venenos de Crotálidos , Fosfolipasas A2 , Animales , Bothrops/metabolismo , Ratones , Fosfolipasas A2/metabolismo , Fosfolipasas A2/química , Fosfolipasas A2/farmacología , Línea Celular Tumoral , Venenos de Crotálidos/química , Movimiento Celular/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Neovascularización Patológica/metabolismo , Adhesión Celular/efectos de los fármacos , Femenino , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/citología , Metástasis de la Neoplasia , Integrina alfaVbeta3/metabolismo , Integrina alfaVbeta3/antagonistas & inhibidores , Fibronectinas/metabolismo , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/química , Humanos , Lisina/química , Lisina/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/patología , Neoplasias Mamarias Animales/metabolismo , AngiogénesisRESUMEN
Snakebite primarily impacts rural communities of Africa, Asia, and Latin America. The sharp-nosed viper (Deinagkistrodon acutus) is among the snakes of highest medical importance in Asia. Despite various studies on its venom using modern venomics techniques, a comprehensive understanding of composition and function of this species' venom remains lacking. We combined proteogenomics with extensive bioactivity profiling to present the first genome-level catalogue of D. acutus venom proteins and their exochemistry. Our analysis identified an unusually simple venom containing 45 components from 20 distinct protein families. Relative toxin abundances indicate that C-type lectin and C-type lectin-related protein (CTL), snake venom metalloproteinase (svMP), snake venom serine protease (svSP), and phospholipase A2 (PLA2) constitute 90 % of the venom. Bioassays targeting key aspects of viperid envenomation showed considerable concentration-dependent cytotoxicity, particularly in kidney and lung cells, and potent protease and PLA2 activity. Factor Xa and thrombin activities were minor, and no plasmin activity was observed. Effects on haemolysis, intracellular calcium (Ca2+) release, and nitric oxide (NO) synthesis were negligible. Our analysis provides the first holistic genome-based overview of the toxin arsenal of D. acutus, predicting the molecular and functional basis of its life-threatening effects, and opens novel avenues for treating envenomation by this highly dangerous snake.
Asunto(s)
Proteogenómica , Animales , Proteogenómica/métodos , Fosfolipasas A2/metabolismo , Humanos , Viperidae/metabolismo , Serina Proteasas/metabolismo , Serina Proteasas/genética , Crotalinae , Serpientes VenenosasRESUMEN
Numerous species of venomous snakes of medical importance exist in Iran. Pseudocerastes persicus (P. persicus), one of the medically important snakes, also called the Persian horned viper, has a geographical spread that extends to the east, southwest, and central areas of Iran and is endemic across the wider region. As a result, this species is responsible for many snakebite occurrences. Venom from P. persicus found in the central province of Semnan contains phospholipase A2 and L-amino acid oxidase activities, and high toxic potency. The venom was fractionated by reverse-phase high-performance liquid chromatography (HPLC) and analyzed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and two-dimensional electrophoresis. Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), a range of components were identified, consistent with the biochemical and toxicological properties of the venom. Proteins identified from 2D electrophoresis and shotgun methods included metallo- and serine proteases, phospholipases, oxidases, and Kunitz trypsin inhibitors, along with many other components at lower qualitative abundance. This study provides a more detailed understanding of the protein profile of Iranian P. persicus venom, which can be effective in the production of an effective antidote against it. The analysis of the resulting data shows that there is a wide range of proteins in the venom of the Persian horned viper. This information can provide a better understanding of how venom is neutralized by polyclonal antivenom. Considering the wide presence of this snake and its related species in Iran and surrounding countries, knowing the venom protein profile of this family can be of great support to antivenom producers such as Razi Vaccine & Serum Research Institute in the preparation of regional antivenoms.
Asunto(s)
Proteómica , Venenos de Víboras , Viperidae , Irán , Animales , Venenos de Víboras/química , Espectrometría de Masas en Tándem , Electroforesis en Gel de Poliacrilamida , Fosfolipasas A2/análisis , Fosfolipasas A2/química , L-Aminoácido Oxidasa/química , L-Aminoácido Oxidasa/análisis , Cromatografía Líquida de Alta Presión , Western Blotting , Electroforesis en Gel BidimensionalRESUMEN
Human parvovirus B19 (B19V), like most parvoviruses, possesses phospholipase A2 (PLA2) activity, which is thought to mediate endosomal escape by membrane disruption. Here, we challenge this model and find evidence for a mechanism of B19V entry mediated by the glycosphingolipid globoside without endosome disruption and retrograde transport to the Golgi. We show that B19V PLA2 activity requires specific calcium levels and pH conditions that are not optimal in endosomes. Accordingly, endosomal membrane integrity was maintained during B19V entry. Furthermore, endosomes remained intact when loaded with MS2 bacteriophage particles pseudotyped with multiple B19V PLA2 subunits, providing superior enzymatic potential compared to native B19V. In globoside knockout cells, incoming viruses are arrested in the endosomal compartment and the infection is blocked. Infection can be rescued by promoting endosomal leakage with polyethyleneimine (PEI), demonstrating the essential role of globoside in facilitating endosomal escape. Incoming virus colocalizes with Golgi markers and interfering with Golgi function blocks infection, suggesting that globoside-mediated entry involves the Golgi compartment, which provides conditions favorable for the lipolytic PLA2. Our study challenges the current model of B19V entry and identifies globoside as an essential intracellular receptor required for endosomal escape.
Asunto(s)
Endosomas , Globósidos , Aparato de Golgi , Parvovirus B19 Humano , Internalización del Virus , Endosomas/metabolismo , Endosomas/virología , Humanos , Aparato de Golgi/metabolismo , Aparato de Golgi/virología , Parvovirus B19 Humano/metabolismo , Parvovirus B19 Humano/fisiología , Parvovirus B19 Humano/genética , Globósidos/metabolismo , Fosfolipasas A2/metabolismo , Calcio/metabolismoRESUMEN
It is well known that C. d. terrificus venom causes pathophysiological effects such as neuropathies, coagulopathies, and even death. Previous studies have reported that ASC16 can interact with monomeric phospholipases A2 from the venom of various snake species (e.g., Vipera russelli and Echis carinatus). As a result, ASC16 has been proposed as an inhibitor of the toxic effects induced by the heterodimeric complex (crotoxin) and other components of the venom of C. d. terrificus. To investigate this further, in silico studies were designed using the crotoxin (CTX) protein complex as a model, and experimental assays were conducted to evaluate the inhibitory effect of ASC16 on CTX, as well as on other venom enzymes such as thrombin-like enzyme (TLE), phosphodiesterase (PDE) and l-aminoxidase (LAAO). For in vitro assays, specific substrates were used, and lethal activity was measured over 48 h using an in vivo murine experimental model (CF01). In silico studies have indicated that the hydrophilic portion of ASC16 adopts a stable conformation while interacting with the catalytic site of crotoxin. At the highest concentrations, ASC16 significantly inhibited the activities of PLA2 (40.89 ± 0.09 %), TLE (11.03 ± 0.69 %), PDE (51.33 ± 2.83 %), and LAAO (56.79 ± 2.91 %). Furthermore, ASC16 neutralized the 2 LD50 lethality of crotalic venom. These findings lay the groundwork for designing promising adjuvants that can facilitate the incorporation of a larger quantity of proteins in immunization schemes. Consequently, this approach aims to achieve higher antibody titers, reduce the number of required immunizations, and minimize local damage in the producer animal.
Asunto(s)
Crotalus , Crotoxina , Serpientes Venenosas , Animales , Masculino , Ratones , Antivenenos/farmacología , Crotoxina/antagonistas & inhibidores , Crotoxina/toxicidad , Simulación del Acoplamiento Molecular , Fosfolipasas A2/toxicidad , Fosfolipasas A2/metabolismo , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/farmacologíaRESUMEN
Spectroscopic studies on domains and peptides of large proteins are complicated because of the tendency of short peptides to form oligomers in aquatic buffers, but conjugation of a peptide with a carrier protein may be helpful. In this study we approved that a fragment of SK30 peptide from phospholipase A2 domain of VP1 Parvovirus B19 capsid protein (residues: 144-159; 164; 171-183; sequence: SAVDSAARIHDFRYSQLAKLGINPYTHWTVADEELLKNIK) turns from random coil to alpha helix in the acidic medium only in case if it had been conjugated with BSA (through additional N-terminal Cys residue, turning it into CSK31 peptide, and SMCC linker) according to CD-spectroscopy results. In contrast, unconjugated SK30 peptide does not undergo such shift because it forms stable oligomers connected by intermolecular antiparallel beta sheet, according to IR-spectroscopy, CD-spectroscopy, blue native gel electrophoresis and centrifugal ultrafiltration, as, probably, the whole isolated phospholipase domain of VP1 protein does. However, being a part of the long VP1 capsid protein, phospholipase domain may change its fold during the acidification of the medium in the endolysosome by the way of the formation of contacts between protonated His153 and Asp175, promoting the shift from random coil to alpha helix in its N-terminal part. This study opens up a perspective of vaccine development, since rabbit polyclonal antibodies against the conjugate of CSK31 peptide with BSA, in which the structure of the second alpha helix from the phospholipase A2 domain should be reproduced, can bind epitopes of the complete recombinant unique part of VP1 Parvovirus B19 capsid (residues: 1-227).
Asunto(s)
Proteínas de la Cápside , Parvovirus B19 Humano , Parvovirus B19 Humano/química , Parvovirus B19 Humano/genética , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Fosfolipasas A2/química , Fosfolipasas A2/metabolismo , Concentración de Iones de Hidrógeno , Péptidos/química , Péptidos/metabolismo , Dominios Proteicos , Albúmina Sérica Bovina/química , AnimalesRESUMEN
OBJECTIVE: To determine whether moxibustion had an anti-inflammatory effect on rheumatoid arthritis (RA) by regulating Annexin 1 expression and interfering with the phospholipaseA2 signaling pathway. METHODS: Thirty male Sprague-Dawley rats were randomly categorized into five groups (six rats per group): blank control (CON) group, RA model (RA) group, moxibustion (MOX) group, Annexin 1 lentiviral intervention (RNAi-Anxa1) group, and Annexin 1 lentiviral intervention + moxibustion (RNAi-Anxa1 + MOX) group. The rats in the RNAi-Anxa1 and the RNAi-Anxa1 + MOX groups were injected with the lentiviral vector-mediated RNAi-Anxa1 into the rat foot pad. An experimental RA rat model was established by injecting Freund's complete adjuvant (FCA) into the RA, MOX, RNAi-Anxa1, and RNAi-Anxa1 + MOX groups. Rats in the MOX and RNAi-Anxa1 + MOX groups received moxibustion treatment. After modeling, using moxibustion "Shenshu (BL23)" and "Zusanli (ST36)", each point is 5 times, bilateral alternating, once a day, 6 times for a course of treatment, between the courses of rest for a one day. A total of three treatment courses were conducted. Both bilateral pad thicknesses were measured using Vernier calipers on experimental days 1, 7, 14, 21, and 28. The expression of cPLA2α signaling in the synovium of diseased joints was observed using Western blot. The pathology of the rat ankle synovium was observed using hematoxylin-eosin (HE) staining. Interleukin (IL)-1ß, IL-10, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) were detected using enzyme-linked immunosorbent assay. RESULTS: Moxibustion increased the levels of Annexin 1 and decreased the inflammatory response in rats with RA. After increasing the expression of Annexin 1, the phosphorylated expression of cPLA2α was inhibited, the serum levels of IL-1ß, PGE2, and LTB4 decreased, and the level of IL-10 increased. In moxibustion treated RA rats after the Annexin 1 lentiviral intervention, the serum levels of IL-1ß, PGE2, LTB4, and IL-10 were almost unchanged. CONCLUSION: Moxibustion enhanced the negative regulation of the cPLA2α signaling pathway, increased the synovial Annexin 1 expression, inhibited the cPLA2α signaling pathway, indirectly inhibited the expression of downstream inflammatory factors, and played a role in reducing inflammation.
Asunto(s)
Anexina A1 , Artritis Reumatoide , Moxibustión , Transducción de Señal , Animales , Humanos , Masculino , Ratas , Anexina A1/genética , Anexina A1/metabolismo , Artritis Reumatoide/genética , Artritis Reumatoide/terapia , Artritis Reumatoide/metabolismo , Artritis Reumatoide/inmunología , Dinoprostona/metabolismo , Inhibidores de Fosfolipasa A2 , Fosfolipasas A2/metabolismo , Fosfolipasas A2/genética , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Epilepsy poses a significant global health challenge, particularly in regions with limited financial resources hindering access to treatment. Recent research highlights neuroinflammation, particularly involving cyclooxygenase-2 (COX-2) pathways, as a promising avenue for epilepsy management. METHODS: This study aimed to develop a Cyclooxygenase-2 inhibitor with potential anticonvulsant properties. A promising drug candidate was identified and chemically linked with phospholipids through docking analyses. The activation of this prodrug was assessed using phospholipase A2 (PLA2)-mediated hydrolysis studies. The conjugate's confirmation and cytotoxicity were evaluated using Fourier Transform Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), and Sulphoramide B (SRB) assays. RESULTS: Docking studies revealed that the Celecoxib-Phospholipid conjugate exhibited a superior affinity for PLA2 compared to other drug-phospholipid conjugates. FT-IR spectroscopy confirmed the successful synthesis of the conjugate, while DSC analysis confirmed its purity and formation. PLA2-mediated hydrolysis experiments demonstrated selective activation of the prodrug depending on PLA2 concentration. SRB experiments indicated dose-dependent cytotoxic effects of Celecoxib, phospholipid non-toxicity, and efficient celecoxib-phospholipid conjugation. CONCLUSION: This study successfully developed a Celecoxib-phospholipid conjugate with potential anticonvulsant properties. The prodrug's specific activation and cytotoxicity profile makes it a promising therapeutic candidate. Further investigation into underlying mechanisms and in vivo studies is necessary to assess its translational potential fully.
Asunto(s)
Anticonvulsivantes , Celecoxib , Simulación del Acoplamiento Molecular , Fosfolipasas A2 , Fosfolípidos , Profármacos , Celecoxib/farmacología , Fosfolípidos/química , Anticonvulsivantes/farmacología , Anticonvulsivantes/síntesis química , Anticonvulsivantes/química , Profármacos/farmacología , Profármacos/química , Profármacos/síntesis química , Fosfolipasas A2/metabolismo , Humanos , Inhibidores de la Ciclooxigenasa 2/farmacología , Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Ciclooxigenasa 2/síntesis química , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Animales , Rastreo Diferencial de Calorimetría , Epilepsia/tratamiento farmacológico , Hidrólisis , Supervivencia Celular/efectos de los fármacosRESUMEN
Currently, the search for new alternatives to conventional antibiotics to combat bacterial resistance is an urgent task, as many microorganisms threaten human health due to increasing bacterial resistance to traditional medicines. Thus, new molecules such as antimicrobial peptides have emerged as promising alternatives because of their low induction of resistance and broad spectrum of action. In this context, in the past few years, our research group has synthesized and characterized a peptide derived from the C-terminal region of the Lys49 PLA2-like BthTX-I, named p-BthTX-I. After several studies, the peptide (p-BthTX-I)2K was proposed as the molecule with the most considerable biotechnological potential. As such, the present work aimed to evaluate whether the modifications made on the peptide (p-BthTX-I)2K can be applied to other molecules originating from the C-terminal region of PLA2-like Lys49 from snake venoms. The peptides were obtained through the solid-phase peptide synthesis technique, and biochemical and functional characterization was carried out using dichroism techniques, mass spectrometry, antimicrobial activity against ESKAPE strains, hemolytic activity, and permeabilization of lipid vesicles. The antimicrobial activity of the peptides was promising, especially for the peptides (p-AppK)2K and (p-ACL)2K, which demonstrated activity against all strains that were tested, surpassing the model molecule (p-BthTX-I)2K in most cases and maintaining low hemolytic activity. The modifications initially proposed for the (p-BthTX-I)2K peptide were shown to apply to other peptides derived from Lys49 PLA2-like from snake venoms, showing promising results for antimicrobial activity. Future assays comparing the activity of the dimers obtained through this strategy with the monomers of these peptides should be carried out.
Asunto(s)
Fosfolipasas A2 , Fosfolipasas A2/farmacología , Fosfolipasas A2/química , Hemólisis/efectos de los fármacos , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/síntesis química , Animales , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Humanos , Antiinfecciosos/farmacología , Antiinfecciosos/química , Antiinfecciosos/síntesis química , Bacterias/efectos de los fármacosRESUMEN
Phospholipase A2 (PLA2) is an enzyme present in appreciable quantity in snake venoms which catalyze the hydrolysis of glycerophospholipids at sn-2 position and promote the release of lysophospholipids and fatty acids. 5-methylcoumarin-4-ß-glucoside (5MC4BG) and lupeol were previously isolated from the leaves of V. glaberrima. The aim of this research was to evaluate effect of these compounds as potential inhibitors of snake venom toxins of Naja nigricollis using an in vitro and in silico studies. Antisnake venom studies was conducted using acidimetry while the molecular docking analysis against PLA2 enzyme from N. nigricollis was performed using Auto Dock Vina and ADME-Tox analysis was evaluated using swissADME and ProTox-II online servers. The two compounds (5MC4BG and Lupeol) were able to inhibit the hydrolytic actions of PLA2 enzyme with percentage inhibition ranging from 23.99 to 72.36 % and 21.97-24.82 % at 0.0625-1.00 mg/mL respectively while the standard ASV had 82.63 % at 1.00 mg/mL after 10 min incubation at 37 °C. Similar effects were observed after 30 min incubation, although there was significant increase in percentage inhibition of 5MC4BG and lupeol ranging from 66.51 to 83.73 % and 54.87-59.60 % at similar concentrations. Furthermore, the compounds were able to bind to the active site of PLA2 enzyme with high affinity (-7.7 to -6.3 kcal/mol); the standard ligand, Varespladib had a docking score of -6.9 kcal/mol and they exhibited favorable drug-likeness and pharmacokinetic properties and according to toxicity predictions, the two compounds are toxic. In conclusion, the leaf of V. glaberrima contains phytoconstituents with antisnake activity and thus, validates the hypothesis that, the phytoconstituents of V. glaberrima leaves has antisnake venom activity against N. nigricollis venom and thus, should be studied further for the development as antisnake venom agents.
Asunto(s)
Simulación del Acoplamiento Molecular , Triterpenos Pentacíclicos , Fosfolipasas A2 , Fitoquímicos , Hojas de la Planta , Vernonia , Fitoquímicos/farmacología , Fitoquímicos/química , Hojas de la Planta/química , Animales , Vernonia/química , Fosfolipasas A2/farmacología , Triterpenos Pentacíclicos/farmacología , Triterpenos Pentacíclicos/química , Venenos Elapídicos/química , Venenos Elapídicos/toxicidad , Naja , Cumarinas/farmacología , Cumarinas/química , Inhibidores de Fosfolipasa A2/farmacología , Extractos Vegetales/farmacología , Extractos Vegetales/química , Simulación por Computador , LupanosRESUMEN
Eicosanoids mediate insect immune responses and synthesized by the catalytic activity of phospholipase A2 (PLA2). A uniquely encoded secretory PLA2 (sPLA2) is associated with immune responses of a lepidopteran insect, Spodoptera exigua. Its deletion mutant was generated using a CRISPR/Cas9 genome editing technology. Both wild and mutant lines were then immune-challenged, and the resulting transcripts were compared with their naïve transcripts by RNASeq using the Illumina-HiSeq platform. In total, 12,878 unigenes were further analyzed by differentially expressed gene tools. Over 69% of the expressed genes in S. exigua larvae are modulated in their expression levels by eicosanoids, recorded from CRISPR/Cas9 mutagenesis against an eicosanoid-synthetic gene, Se-sPLA2. Further, about 36% of the immune-associated genes are controlled by the eicosanoids in S. exigua. Indeed, the deletion mutant suffered significant immunosuppression in both cellular and humoral responses in response to bacterial challenge as well as severely reduced developmental and reproductive potentials.
Asunto(s)
Sistemas CRISPR-Cas , Eicosanoides , Fosfolipasas A2 , Spodoptera , Animales , Eicosanoides/metabolismo , Fosfolipasas A2/genética , Fosfolipasas A2/metabolismo , Transducción de Señal , Larva/genética , Larva/inmunología , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Eliminación de Secuencia , Genes de Insecto , Edición Génica , Eliminación de GenRESUMEN
Phospholipase A2's (PLA2's) constitute a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain on glycerophospholipids. We have previously reported that each PLA2 Type shows a unique substrate specificity for the molecular species it hydrolyzes, especially the acyl chain that is cleaved from the sn-2 position and to some extent the polar group. However, phosphatidylinositol (PI) and PI phosphates (PIPs) have not been as well studied as substrates as other phospholipids because the PIPs require adaptation of the standard analysis methods, but they are important in vivo. We determined the in vitro activity of the three major types of human PLA2's, namely the cytosolic (c), calcium-independent (i), and secreted (s) PLA2's toward PI, PI-4-phosphate (PI(4)P), and PI-4,5-bisphosphate (PI(4,5)P2). The in vitro assay revealed that Group IVA cPLA2 (GIVA cPLA2) showed relatively high activity toward PI and PI(4)P among the tested PLA2's; nevertheless, the highly hydrophilic headgroup disrupted the interaction between the lipid surface and the enzyme. GIVA cPLA2 and GVIA iPLA2 showed detectable activity toward PI(4,5)P2, but it appeared to be a poorer substrate for all of the PLA2's tested. Furthermore, molecular dynamics (MD) simulations demonstrated that Thr416 and Glu418 of GIVA cPLA2 contribute significantly to accommodating the hydrophilic head groups of PI and PI(4)P, which could explain some selectivity for PI and PI(4)P. These results indicated that GIVA cPLA2 can accommodate PI and PI(4)P in its active site and hydrolyze them, suggesting that the GIVA cPLA2 may best account for the PI and PIP hydrolysis in living cells.
Asunto(s)
Fosfatos de Fosfatidilinositol , Fosfatidilinositoles , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Especificidad por Sustrato , Fosfatidilinositoles/metabolismo , Fosfolipasas A2/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , HidrólisisRESUMEN
Central nervous system (CNS) disorders are one of the leading health problems today, accounting for a large proportion of global morbidity and mortality. Most these disorders are characterized by high levels of oxidative stress and intense inflammatory responses in degenerated neuronal tissues. While extensive research has been conducted on CNS diseases, but few breakthroughs have been made in treatment methods. To date, there are no disease-modifying drugs available for CNS treatment, underscoring the urgent need for finding effective medications. Bee venom (BV), which is produced by honeybee workers' stingers, has been a subject of interest and study across various cultures. Over the past few decades, extensive research has focused on BV and its therapeutic potentials. BV consists a variety of substances, mainly proteins and peptides like melittin and phospholipase A2 (PLA2). Research has proven that BV is effective in various medical conditions, including pain, arthritis and inflammation and CNS disorders such as Multiple sclerosis, Alzheimer's disease and Parkinson's disease. This review provides a comprehensive overview of the existing knowledge concerning the therapeutic effects of BV and its primary compounds on various CNS diseases. Additionally, we aim to shed light on the potential cellular and molecular mechanisms underlying these effects.
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Venenos de Abeja , Enfermedades del Sistema Nervioso Central , Venenos de Abeja/uso terapéutico , Venenos de Abeja/farmacología , Humanos , Animales , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Enfermedades del Sistema Nervioso Central/metabolismo , Fosfolipasas A2/metabolismoRESUMEN
The enzyme phospholipase A2 (PLA2) plays a crucial role in acyl remodeling of phospholipids via the Lands' cycle, and consequently alters fatty acid compositions in triacylglycerol (TAG). In this study, a full-length cDNA sequence coding Myrmecia incisa phospholipase A2 (MiPLA2) was cloned using the technique of rapid amplification of cDNA ends. Comparison of the 1082-bp cDNA with its corresponding cloned DNA sequence revealed that MiPLA2 contained 3 introns. Mature MiPLA2 (mMiPLA2) had a conserved Ca2+-binding loop and a catalytic site motif that has been recognized in plant secretory PLA2 (sPLA2) proteins. Correspondingly, phylogenetic analysis illustrated that MiPLA2 was clustered within GroupXIA of plant sPLA2 proteins. To ascertain the function of MiPLA2, the cDNA coding for mMiPLA2 was subcloned into the vector pET-32a to facilitate the production of recombinant mMiPLA2 in Escherichia coli. Recombinant mMiPLA2 was purified and used for the in vitro enzyme reaction. Thin-layer chromatography profiles of the catalytic products generated by recombinant mMiPLA2 indicated a specificity for cleaving sn-2 acyl chains from phospholipids, thereby functionally characterizing MiPLA2. Although recombinant mMiPLA2 displayed a strong preference for phosphatidylethanolamine, it preferentially hydrolyzes arachidonic acid (ArA) at the sn-2 position of phosphatidylcholine. Results from the fused expression of p1300-sp-EGFP-mMiPLA2 illustrated that MiPLA2 was localized in the intercellular space of onion epidermis. Furthermore, the positive correlation between MiPLA2 transcription and free ArA levels were established. Consequently, the role of mMiPLA2 in the biosynthesis of ArA-rich TAG was elucidated. This study helps to understand how M. incisa preferentially uses ArA to synthesize TAG.
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Ácido Araquidónico , Fosfatidilcolinas , Fosfolipasas A2 , Fosfolipasas A2/metabolismo , Fosfolipasas A2/genética , Ácido Araquidónico/metabolismo , Fosfatidilcolinas/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especificidad por Sustrato , Secuencia de Aminoácidos , Microalgas/genética , Microalgas/enzimología , Microalgas/metabolismo , Clonación MolecularRESUMEN
The cockroach allergen Bla g 1 encloses an exceptionally large hydrophobic cavity, which allows it to bind and deliver unsaturated fatty acid ligands. Bla g 1-mediated delivery of naturally occurring (nMix) ligands has been shown to destabilize lipid membranes, contributing to its digestive/antiviral functions within the source organism. However, the consequences of this activity on Bla g 1 allergenicity following human exposure remain unknown. In this work, we show that Bla g 1-mediated membrane disruption can induce a proinflammatory immune response in mammalian cells via two complementary pathways. At high concentrations, the cytotoxic activity of Bla g 1 induces the release of proinflammatory cytosolic contents including damage-associated molecular patterns (DAMPs) such as heat-shock Protein-70 (HSP70) and the cytokine interleukin-1 (IL-1ß). Sublytic concentrations of Bla g 1 enhanced the ability of phospholipase A2 (PLA2) to extract and hydrolyze phospholipid substrates from cellular membranes, stimulating the production of free polyunsaturated fatty acids (PUFAs) and various downstream inflammatory lipid mediators. Both of these effects are dependent on the presence of Bla g 1's natural fatty-acid (nMix) ligands with CC50 values corresponding to the concentrations required for membrane destabilization reported in previous studies. Taken together, these results suggest that mechanisms through which Bla g 1-mediated lipid delivery and membrane destabilization could directly contribute to cockroach allergic sensitization.
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Alérgenos , Membrana Celular , Cucarachas , Animales , Humanos , Membrana Celular/metabolismo , Cucarachas/inmunología , Cucarachas/metabolismo , Alérgenos/metabolismo , Alérgenos/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Fosfolipasas A2/metabolismo , Fosfolipasas A2/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Ácidos Grasos Insaturados/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de Insectos/químicaRESUMEN
Phospholipases A2 (PLA2) comprise a superfamily of enzymes that specifically catalyze hydrolysis of the ester bond at the sn-2 position of glycerophospholipids, generating lysophospholipids and fatty acids. In Rhodnius prolixus, one of the main vectors of the Chagas's disease etiologic agent Trypanosoma cruzi, it was previously shown that lysophosphatidylcholine, a bioactive lipid, found in the insect's saliva, contributes to the inhibition of platelet aggregation, and increases the production of nitric oxide, an important vasodilator. Due to its role in potentially generating LPC, here we studied the PLA2 present in the salivary glands of R. prolixus. PLA2 activity is approximately 100 times greater in the epithelium than in the contents of salivary glands. Our study reveals the role of the RpPLA2XIIA gene in the insect feeding performance and in the fatty acids composition of phospholipids extracted from the salivary glands. Knockdown of RpPLA2XIIA significantly altered the relative amounts of palmitic, palmitoleic, oleic and linoleic acids. A short-term decrease in the expression of RpPLA2III and RpPLA2XIIA in the salivary glands of R. prolixus was evident on the third day after infection by T. cruzi. Taken together, our results contribute to the understanding of the role of PLA2 in the salivary glands of hematophagous insects and show that the parasite is capable of modulating even tissues that are not colonized by it.
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Fosfolipasas A2 , Rhodnius , Glándulas Salivales , Trypanosoma cruzi , Animales , Rhodnius/parasitología , Rhodnius/enzimología , Rhodnius/genética , Glándulas Salivales/parasitología , Glándulas Salivales/enzimología , Glándulas Salivales/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/enzimología , Fosfolipasas A2/metabolismo , Fosfolipasas A2/genética , Ácidos Grasos/metabolismo , Enfermedad de Chagas/parasitología , Insectos Vectores/parasitología , Insectos Vectores/enzimologíaRESUMEN
Bis(monoacylglycerol)phosphate (BMP) is an acidic glycerophospholipid localized to late endosomes and lysosomes. However, the metabolism of BMP is poorly understood. Because many drugs that cause phospholipidosis inhibit lysosomal phospholipase A2 (LPLA2, PLA2G15, LYPLA3) activity, we investigated whether this enzyme has a role in BMPcatabolism. The incubation of recombinant human LPLA2 (hLPLA2) and liposomes containing the naturally occurring BMP (sn-(2-oleoyl-3-hydroxy)-glycerol-1-phospho-sn-1'-(2'-oleoyl-3'-hydroxy)-glycerol (S,S-(2,2',C18:1)-BMP) resulted in the deacylation of this BMP isomer. The deacylation rate was 70 times lower than that of dioleoyl phosphatidylglycerol (DOPG), an isomer and precursor of BMP. The release rates of oleic acid from DOPG and four BMP stereoisomers by LPLA2 differed. The rank order of the rates of hydrolysis were DOPG>S,S-(3,3',C18:1)-BMP>R,S-(3,1',C18:1)-BMP>R,R-(1,1',C18:1)>S,S-(2,2')-BMP. The cationic amphiphilic drug amiodarone (AMD) inhibited the deacylation of DOPG and BMP isomers by hLPLA2 in a concentration-dependent manner. Under these experimental conditions, the IC50s of amiodarone-induced inhibition of the four BMP isomers and DOPG were less than 20 µM and approximately 30 µM, respectively. BMP accumulation was observed in AMD-treated RAW 264.7 cells. The accumulated BMP was significantly reduced by exogenous treatment of cells with active recombinant hLPLA2 but not with diisopropylfluorophosphate-inactivated recombinant hLPLA2. Finally, a series of cationic amphiphilic drugs known to cause phospholipidosis were screened for inhibition of LPLA2 activity as measured by either the transacylation or fatty acid hydrolysis of BMP or phosphatidylcholine as substrates. Fifteen compounds demonstrated significant inhibition with IC50s ranging from 6.8 to 63.3 µM. These results indicate that LPLA2 degrades BMP isomers with different substrate specificities under acidic conditions and may be the key enzyme associated with BMP accumulation in drug-induced phospholipidosis.