Trypanosoma cruzi PLA2: In silico study of enzyme-substrate interaction

The struggle against Chagas disease prompted the study of the parasite Trypanosoma cruzi's metabolism and, in particular, enzyme-substrate interactions to find pharmacological targets necessary to eliminate the parasite without compromising the host. An enzyme that could meet thes...

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Autor principal: Cossy Isasi, S
Formato: Artículo revista
Lenguaje:Español
Publicado: Universidad Nacional Córdoba. Facultad de Ciencias Médicas. Secretaria de Ciencia y Tecnología 2019
Materias:
enzyme-substrate
affinity
trypanosoma cruzi
molecular modeling
computational pharmacology
enzima-sustrato
afinidad
modelado molecular
farmacología computacional
Acceso en línea:https://revistas.unc.edu.ar/index.php/med/article/view/25783
Aporte de:
Revista de la Facultad de Ciencias Médicas de Córdoba de Universidad Nacional de Córdoba
id I10-R327-article-25783
record_format ojs
institution Universidad Nacional de Córdoba
institution_str I-10
repository_str R-327
container_title_str Revista de la Facultad de Ciencias Médicas de Córdoba
language Español
format Artículo revista
topic enzyme-substrate
affinity
trypanosoma cruzi
molecular modeling
computational pharmacology
enzima-sustrato
afinidad
trypanosoma cruzi
modelado molecular
farmacología computacional
spellingShingle enzyme-substrate
affinity
trypanosoma cruzi
molecular modeling
computational pharmacology
enzima-sustrato
afinidad
trypanosoma cruzi
modelado molecular
farmacología computacional
Cossy Isasi, S
Trypanosoma cruzi PLA2: In silico study of enzyme-substrate interaction
topic_facet enzyme-substrate
affinity
trypanosoma cruzi
molecular modeling
computational pharmacology
enzima-sustrato
afinidad
trypanosoma cruzi
modelado molecular
farmacología computacional
author Cossy Isasi, S
author_facet Cossy Isasi, S
author_sort Cossy Isasi, S
title Trypanosoma cruzi PLA2: In silico study of enzyme-substrate interaction
title_short Trypanosoma cruzi PLA2: In silico study of enzyme-substrate interaction
title_full Trypanosoma cruzi PLA2: In silico study of enzyme-substrate interaction
title_fullStr Trypanosoma cruzi PLA2: In silico study of enzyme-substrate interaction
title_full_unstemmed Trypanosoma cruzi PLA2: In silico study of enzyme-substrate interaction
title_sort trypanosoma cruzi pla2: in silico study of enzyme-substrate interaction
description The struggle against Chagas disease prompted the study of the parasite Trypanosoma cruzi's metabolism and, in particular, enzyme-substrate interactions to find pharmacological targets necessary to eliminate the parasite without compromising the host. An enzyme that could meet these requirements is T. cruzi phospholipase A2 (PLA2) whose existence as an individual protein has been suggested experimentally but has not yet been obtained and purified.  In vitro: PLA2 activity was measured by hydrolysis of the bis-pyrenoyl phosphatidylcholine fluorescent substrate (10 mol%) incorporated in small unilamellar liposomes, SUVs, of dipalmitoylphosphatidylcholine (DPPC) in 10 mM HEPES buffer pH 7.2. 50 ml of filtered plasma from mice with different parasitemia were tested. The enzyme was activated with 20 mM calcium. Emission spectra were obtained before and after each kinetic determination. In silico: the Tc00.1047053510743.50 DNA sequence was translated into amino acids and modeled by homology on the phyre2 server. The highest scoring model was selected and resulted in a PLA2-like molecule with similarities to the human platelet activating factor (PAF) acetylhydrolases (PAF-hydrolase). The interaction of the structural model with possible substrates and with GM1 monosialoganglioside (inhibitor of PLA2 from other sources) was analyzed. The gene was identified by the presence of the amino acids HIS, SER, ASP (GLU) conforming the active site, the triad responsible for the nucleophilic attack on the carbon atom of the carbon ester bond, an activity characteristic of PAF-hydrolases. Ligand binding was originally studied by Linear Interaction Energies. The enzyme-substrate complexes (PAF or DPPC as ligands) were obtained with the PatchDock program. The simulations of bound and unbound structures in implicit solvent (GBIS) were developed with NAMD. The results were visualized with VMD and Chimera. The electrostatic contribution increases approximately 20kcal / mol in the bound state, but VdW interactions decrease 28kcal / mol, which makes the term favorable for the bound state. The binding of GM1, a hypothetical inhibitor, was more favorable than for PAF, which would allow us to propose GM1 as a competitive inhibitor.
publisher Universidad Nacional Córdoba. Facultad de Ciencias Médicas. Secretaria de Ciencia y Tecnología
publishDate 2019
url https://revistas.unc.edu.ar/index.php/med/article/view/25783
work_keys_str_mv AT cossyisasis trypanosomacruzipla2insilicostudyofenzymesubstrateinteraction
AT cossyisasis pla2fosfolipasaa2detrypanosomacruzicaracterizacioninsilicodelainteraccionenzimasustrato
first_indexed 2024-09-03T21:01:02Z
last_indexed 2024-09-03T21:01:02Z
_version_ 1809210159737602048
spelling I10-R327-article-257832024-08-27T18:26:16Z Trypanosoma cruzi PLA2: In silico study of enzyme-substrate interaction PLA2 fosfolipasa A2 de Trypanosoma cruzi: caracterización in silico de la interacción enzima-sustrato. Cossy Isasi, S enzyme-substrate affinity trypanosoma cruzi molecular modeling computational pharmacology enzima-sustrato afinidad trypanosoma cruzi modelado molecular farmacología computacional The struggle against Chagas disease prompted the study of the parasite Trypanosoma cruzi's metabolism and, in particular, enzyme-substrate interactions to find pharmacological targets necessary to eliminate the parasite without compromising the host. An enzyme that could meet these requirements is T. cruzi phospholipase A2 (PLA2) whose existence as an individual protein has been suggested experimentally but has not yet been obtained and purified.  In vitro: PLA2 activity was measured by hydrolysis of the bis-pyrenoyl phosphatidylcholine fluorescent substrate (10 mol%) incorporated in small unilamellar liposomes, SUVs, of dipalmitoylphosphatidylcholine (DPPC) in 10 mM HEPES buffer pH 7.2. 50 ml of filtered plasma from mice with different parasitemia were tested. The enzyme was activated with 20 mM calcium. Emission spectra were obtained before and after each kinetic determination. In silico: the Tc00.1047053510743.50 DNA sequence was translated into amino acids and modeled by homology on the phyre2 server. The highest scoring model was selected and resulted in a PLA2-like molecule with similarities to the human platelet activating factor (PAF) acetylhydrolases (PAF-hydrolase). The interaction of the structural model with possible substrates and with GM1 monosialoganglioside (inhibitor of PLA2 from other sources) was analyzed. The gene was identified by the presence of the amino acids HIS, SER, ASP (GLU) conforming the active site, the triad responsible for the nucleophilic attack on the carbon atom of the carbon ester bond, an activity characteristic of PAF-hydrolases. Ligand binding was originally studied by Linear Interaction Energies. The enzyme-substrate complexes (PAF or DPPC as ligands) were obtained with the PatchDock program. The simulations of bound and unbound structures in implicit solvent (GBIS) were developed with NAMD. The results were visualized with VMD and Chimera. The electrostatic contribution increases approximately 20kcal / mol in the bound state, but VdW interactions decrease 28kcal / mol, which makes the term favorable for the bound state. The binding of GM1, a hypothetical inhibitor, was more favorable than for PAF, which would allow us to propose GM1 as a competitive inhibitor. La lucha contra la enfermedad de Chagas ha inspirado el estudio del metabolismo del parásito Trypanosoma cruzi y, en particular, las interacciones enzima-sustrato para encontrar objetivos farmacológicos necesarios para eliminar el parásito sin el compromiso del huésped. Una enzima que podría cumplir estos requisitos es la fosfolipasa A2 (PLA2) de T. cruzi cuya existencia como proteína individual se ha sugerido con base experimental pero no se ha demostrado. In vitro: se midió la actividad PLA2 mediante la hidrólisis del sustrato fluorescente bis-pirenoilfosfatidilcolina (10 mol %) incorporado en liposomas unilamelares pequeños, SUV, de dipalmitoilfosfatidilcolina (DPPC) en buffer HEPES 10 mM pH 7,2. Se ensayaron 50 ml de plasma filtrado  de ratones con diferentes parasitemias. Se activó la enzima llevando el calcio a 20 mM. Se obtuvieron espectros antes y después de cada determinación cinética. In silico: la secuencia de ADN Tc00.1047053510743.50 se tradujo en aminoácidos y se modeló por homología en el servidor phyre2. Se eligió el modelo de puntuación más alta que resultó en una molécula de tipo PLA2 con similitudes con la Factor activador de plaquetas (PAF) acetilhidrolasas (PAF-hidrolasa) humana. Se analizó la interacción del modelo estructural con sustratos posibles y con moniosialogangliósido GM1 que inhibe PLA2 de otras fuentes. El gen se identificó por la presencia de los aminoácidos que constituyen el sitio activo, la triada HIS, SER, ASP (GLU) que son responsables del ataque nuclefílico en el átomo de carbonilo del enlace carbono éster, actividad característica de PAF-hidrolasas. La unión al sustrato fue estudiada originalmente por Energías de Interacción Linear. Los complejos enzima-sustrato (PAF o DPPC como ligandos) se obtuvieron con el programa PatchDock. Las simulaciones de estados unido y disociados en solvente implícito (GBIS) se desarrollaron con NAMD. Los resultados fueron visualizados con VMD y Chimera. La contribución electrostática aumenta aproximadamente 20kcal / mol en el estado enlazado, pero las interacciones VdW disminuyen 28kcal / mol, lo que hace que el término sea favorable para el estado enlazado. La unión de GM1, hipotético inhibidor, fue más favorable que para el PAF, lo que permitiría proponer una actividad inhibitoria de tipo competitivo. Universidad Nacional Córdoba. Facultad de Ciencias Médicas. Secretaria de Ciencia y Tecnología 2019-10-16 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion application/pdf https://revistas.unc.edu.ar/index.php/med/article/view/25783 Revista de la Facultad de Ciencias Médicas de Córdoba.; 2019: Suplemento JIC XX Revista de la Facultad de Ciencias Médicas de Córdoba; 2019: Suplemento JIC XX Revista da Faculdade de Ciências Médicas de Córdoba; 2019: Suplemento JIC XX 1853-0605 0014-6722 10.31053/1853.0605.v76.nSuplemento spa https://revistas.unc.edu.ar/index.php/med/article/view/25783/27533 Derechos de autor 2019 Universidad Nacional de Córdoba https://creativecommons.org/licenses/by-nc/4.0

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