Browsing by Subject "ATP"
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- PublicationOpen AccessAmino acid residues in the P2X7 receptor that mediate differential sensitivity to ATP and BzATP(American Society for Pharmacology and Experimental Therapeutics, 2007) Young, Mark T.; Pelegrin, Pablo; Surprenant, Annmarie; Bioquímica y Biología Molecular B e InmunologíaAgonist properties of the P2X7 receptor (P2X7R) differ strikingly from other P2X receptors in two main ways: high concentrations of ATP (> 100 microM) are required to activate the receptor, and the ATP analog 2',3'-O-(4-benzoyl-benzoyl)ATP (BzATP) is both more potent than ATP and evokes a higher maximum current. However, there are striking species differences in these properties. We sought to exploit the large differences in ATP and BzATP responses between rat and mouse P2X7R to delineate regions or specific residues that may be responsible for the unique actions of these agonists at the P2X7R. We measured membrane currents in response to ATP and BzATP at wild-type rat and mouse P2X7R, at chimeric P2X7Rs, and at mouse P2X7Rs bearing point mutations. Wild-type rat P2X7R was 10 times more sensitive to ATP and 100 times more sensitive to BzATP than wild-type mouse P2X7R. We found that agonist EC50 values were determined solely by the ectodomain of the P2X7R. Two segments (residues 115-136 and 282-288), when transposed together, converted mouse sensitivities to those of rat. Point mutations through these regions revealed a single residue, asparagine284, in the rat P2X7R that fully accounted for the 10-fold difference in ATP sensitivity, whereas the 100-fold difference in BzATP sensitivity required the transfer of both Lys127 and Asn284 from rat to mouse. Thus, single amino acid differences between species can account for large changes in agonist effectiveness and differentiate between the two widely used agonists at P2X7 receptors.
- PublicationOpen AccessCharacterization of acetyl-CoA synthetase kinetics and ATP-binding(Elsevier, 2019) Gallego Jara, Julia; Lozano Terol, Gema; Écija Conesa, Ana; Zambelli, Barbara; Cánovas Díaz, Manuel; De Diego Puente, María Teresa; Bioquímica y Biología Molecular B e InmunologíaBackground The superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo. Methods In this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry. Results The results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although kcat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding. Conclusions The differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated. General Significance The results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.
- PublicationOpen AccessDescripción y análisis del impacto de las estrategias de control de legionelosis en un hospital de tercer nivel(Universidad de Murcia, 2024-07-29) Tomás Borja, Antonio; Torres Cantero, Alberto Manuel; Segovia Hernández, Manuel; Escuela Internacional de DoctoradoIntroducción: Aunque los aspectos relacionados con la Legionella han sido ampliamente estudiados sigue habiendo una tasa creciente de legionelosis en los hospitales. El análisis de temperatura y cloro en instalaciones de agua sanitaria (AS) y de resultados analíticos de muestras ambientales de AS son indicadores de nivel de riesgo de infección. Objetivos: Describir el grado de colonización de Legionella en el periodo 2016-2022 en el sistema de AS del Hospital Clínico Universitario Virgen de la Arrixaca (HCUVA), valorando la efectividad de las medidas de control adoptadas en 2019 en el pabellón Hospital General (HG) y el comportamiento de la instalación, correlacionando los resultados de parámetros fisicoquímicos de AS. Material y método: Estudio longitudinal con los resultados analíticos disponibles de la base de datos de muestras ambientales del periodo 2016 a 2023, incluyendo los parámetros fisicoquímicos y microbiológicos realizados antes y después de las actuaciones de control en 2019 en la instalación de AS del HG. Resultados: Se obtienen valores del comportamiento de la instalación a nivel general, observando la inexistencia de patrón estacional exacto de la bacteria y un riesgo en el sistema de producción central. Los resultados de las actuaciones en hospitalización de HG proporcionan información para la confección del mapa de riesgos a partir de los parámetros fisicoquímicos y los resultados de análisis de Legionella en muestras ambientales por la técnica de PCR y qPCR. Discusión: Los resultados muestran la importancia que tiene el correcto diseño y mantenimiento de las instalaciones de agua sanitaria, evitar condiciones de estancamiento del agua y de mal funcionamiento de los circuitos de retorno de ACS como medidas para evitar la presencia de Legionella en las instalaciones de fontanería, así como alcanzar y mantener homogéneamente niveles de temperatura y cloro en dichas instalaciones. Indicadores como nivel de ATP en AS y técnicas de determinación de Legionella por PCR y qPCR son complementarias a la técnica validada de cultivo y sirven para diseñar el mapa de riesgos de la instalación de AS y priorizar actuaciones de control. Conclusiones: En instalaciones complejas y de muchos años de uso colonizadas no se consigue la erradicación de Legionella aunque se haga correctamente el mantenimiento técnico legal. Es por ello que se hace necesario realizar intervenciones de reforma y limpiezas severas en las instalaciones hídricas como estrategia para poder minimizar la legionelosis en este tipo de edificaciones. Es necesario el exhaustivo mantenimiento y medición de niveles de temperaturas, cloro y ATP en puntos terminales por ser éstas herramientas eficaces para evitar el crecimiento y proliferación de Legionella. Es necesario el control analítico de la bacteria por técnicas más sensibles y rápidas como PCR y PCRq, junto con el método validado de cultivo, herramientas de control para la gestión de la prevención de legionelosis.
- PublicationOpen AccessEndometrial epithelial cell organoids as tools for studying the CD39 family of enzymes and for validating enzyme inhibitors(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2025) Rodríguez Martínez, Aitor; Torrejón Escribano, Benjamín; Eritja, Núria; Dorca Arévalo, Jonatan; Gabaldón, Clara; Sévigny, Jean; Matias Guiu, Xavier; Martín Satué, MireiaExtracellular adenosine triphosphate (ATP) conducts a complex dynamic system of broadly represented cell signaling. Ectonucleotidases are the enzymes with nucleotide hydrolytic ability that regulate ATP levels in physiological and pathological conditions, thus playing a key role in the so-called purinergic signaling. Altered ectonucleotidase expression has been reported in cancer, and the ectonucleoside triphosphate diphosphohydrolase (NTPDase) family of enzymes, with its best-known form NTPDase1 (CD39), is targeted in cancer immunotherapy. The tandem of enzymes CD39-CD73 is responsible for the generation of immuno-suppressive adenosine in the tumor microenvironment, and inhibition strategies are of great interest. Organoids have emerged as very convenient models for the study of tumors since they are three-dimensional cultures that retain many of the features of tissue. The present study aims to contribute to improving the methodology and the molecular tools needed for the study of ecto-nucleotidases in healthy and disease conditions. The study, performed in an endometrial cancer cell model, could be extended to other types of tumors and pathologies in which the purinergic system is involved. We generated organoids from endometrial cancer cells overexpressing NTPDase2 (CD39L1) and NTPDase3 (CD39L3) as fusion proteins with EGFP, and we performed functional assays by adapting in situ cytochemistry protocols. This allowed us to simultaneously detect enzyme activity and protein expression and to demonstrate that organoids can be used to test ectonucleotidase inhibitors—a result that can be used to develop new cancer treatment options
- PublicationOpen AccessExtracellular ATP activates the NLRP3 inflammasome and is an early danger signal of skin allograft rejection(Cell Press, 2017) Amores-Iniesta, Joaquín; Barberá-Cremades, Maria; Martínez, Carlos M.; Pons, José A.; Revilla-Nuín, Beatriz; Martínez-Alarcón, Laura; Di Virgilio, Francesco; Parrilla, Pascual; Baroja-Mazo, Alberto; Pelegrín, Pablo; Bioquímica y Biología Molecular B e InmunologíaImmune cells are equipped with a number of receptors that recognize sterile injury and pathogens. We find that host immune cells release ATP as an inflammatory signal in response to allogeneic transplantation. ATP then acts via a feedback mechanism on the P2X7 channel to activate the NLRP3 inflammasome and subsequently process and release interleukin (IL)-18. This process is a necessary stage in the deleterious Th1 response against allotransplantation via interferon-g production. Lack of IL-18 resulted in a decrease in graft- infiltrated CD8 cells, but an increase in regulatory T cells. In human liver transplant patients subjected to progressive immunosuppressive drug withdrawal, we found that patients suffering acute rejection had higher levels of the P2X7 receptor in circulating inflammatory monocytes compared to tolerant patients. These data suggest that the pharmacological inhibition of the P2X7 receptor or the NLRP3 inflammasome will aid in inducing transplant tolerance without complete immunoparalysis.
- PublicationEmbargoP2X7 receptor activation impairs antitumour activity of natural killer cells(Wiley, 2022) Baroja-Mazo, Alberto; Peñín-Franch, Alejandro; Lucas-Ruiz, Fernando; Torre-Minguela, Carlos de; Alarcón-Vila, Cristina; Hernández-Caselles, Trinidad; Pelegrín, Pablo; Bioquímica y Biología Molecular B e InmunologíaBackground and purpose: A high number of intratumoural infiltrating natural killer (NK) cells is associated with better survival in several types of cancer, constituting an important first line of defence against tumours. Hypoxia in the core of solid tumours induces cellular stress and ATP release into the extracellular space where it triggers purinergic receptor activation on tumour-associated immune cells. The aim of this study was to assess whether activation of the purinergic receptor P2X7 by extracellular ATP plays a role in the NK cells antitumour activity. Experimental approach: We carried out in vitro experiments using purified human NK cells triggered through P2X7 by extracellular ATP. NK cell killing activity against the tumour target cells K562 was studied by means of NK cytotoxicity assays. Likewise, we designed a subcutaneous solid tumour in vivo mouse model. Key results: In this study we found that human NK cells, expressing a functional plasma membrane P2X7, acquired an anergic state after ATP treatment, which impaired their antitumour activity and decreased IFN-γ secretion. This effect was reversed by specific P2X7 antagonists and pretreatment with either IL-2 or IL-15. Furthermore, genetic P2rx7 knockdown resulted in improved control of tumour size by NK cells. In addition, IL-2 therapy restored the ability of NK cells to diminish the size of tumours. Conclusions and implications: Our results show that P2X7 activation represents a new mechanism whereby NK cells may lose antitumour effectiveness, opening the possibility of generating modified NK cells lacking P2X7 but with improved antitumour capacity.
- PublicationOpen AccessProduction and mechanism of secretion of interleukin-1b from the marine fish gilthead seabream(2004) Pablo, Pelegrin; Elena, Chaves-Pozo; José, Meseguer; Victoriano, Mulero; Bioquímica y Biología Molecular B e InmunologíaInterleukin-1b (IL-1b) is a secretory cytokine lacking a signal peptide, and does not follow the classical endoplasmic reticulum to Golgi pathway of secretion. Its post-translational processing by IL-1b-converting enzyme (ICE) and subsequent release from activated macrophages requires ATP acting on P2X7 receptors. No information is available on the production and release of fish IL-1b, but the IL-1b gene sequences reported to date lack a conserved ICE recognition site. We show for the first time that lipopolysaccharide (LPS)/macrophage-activating factor (MAF)/bacterial DNA (VaDNA)-primed immune cells of fish accumulate intracellular IL-1b as a ~30 kDa polypeptide (proIL-1b). The combination of LPS and VaDNA was found to be synergistic, suggesting that each ligand is recognized by a different pattern recognition receptor (PRR). More importantly, addition of extracellular ATP does not promote IL-1b secretion by immune cells and fails to induce phosphatidylserine (PS) flip. In contrast, fish SAF-1 fibroblasts shed microvesicles containing a 22 kDa IL-1b form within 30 min of activation with ATP. Notably, the post-translational processing of IL-1b by SAF-1 cells is abrogated by a specific ICE inhibitor.