Molecular Bases of Catalysis and ADP-Ribose Preference of Human Mn2+-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase and Conversion by Mutagenesis to a Preferential Cyclic ADP-Ribose Phosphohydrolase

Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type.info:eu-repo/semantics/publishedVersio

The characterization of Escherichia coli CpdB as a recombinantpProtein reveals that, besides having the expected 3´-nucleotidase and 2´,3´-cyclic mononucleotide phosphodiesterase activities, it is also active as cyclic dinucleotide phosphodiesterase

Endogenous cyclic diadenylate phosphodiesterase activity was accidentally detected in lysates of Escherichia coli BL21. Since this kind of activity is uncommon in Gram-negative bacteria, its identification was undertaken. After partial purification and analysis by denaturing gel electrophoresis, renatured activity correlated with a protein identified by fingerprinting as CpdB (cpdB gene product), which is annotated as 3´-nucleotidase / 2´,3´- cyclicmononucleotide phosphodiesterase, and it is synthesized as a precursor protein with a signal sequence removable upon export to the periplasm. It has never been studied as a recombinant protein. The coding sequence of mature CpdB was cloned and expressed as a GST fusion protein. The study of the purified recombinant protein, separated from GST, confirmed CpdB annotation. The assay of catalytic efficiencies (kcat/Km) for a large substrate set revealed novel CpdB features, including very high efficiencies for 3´-AMP and 2´,3´- cyclic mononucleotides, and previously unknown activities on cyclic and linear dinucleotides. The catalytic efficiencies of the latter activities, though low in relative terms when compared to the major ones, are far from negligible. Actually, they are perfectly comparable to those of the ‘average’ enzyme and the known, bona fide cyclic dinucleotide phosphodiesterases. On the other hand, CpdB differs from these enzymes in its extracytoplasmic location and in the absence of EAL, HD and DHH domains. Instead, it contains the domains of the 5´-nucleotidase family pertaining to the metallophosphoesterase superfamily, although CpdB lacks 5´-nucleotidase activity. The possibility that the extracytoplasmic activity of CpdB on cyclic dinucleotides could have physiological meaning is discussed.Trabajo financiado por: Junta de Extremadura y Fondos FEDER. Ayudas GR10133 y GR15143 Donación privada para María Jesús Costas Vázquez. Contrato 2015/00481/001peerReviewe

Molecular bases of catalysis and ADP-ribose preference of human Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase and conversion by mutagenesis to a preferential cyclic ADP-ribose phosphohydrolase

Entre las fosfatasas métalo-dependientes, ADP-ribosa/CDP-alcohol diphosphatases forman una familia de proteínas (ADPRibase-Mn-like) principalmente restringida, en eucariotas, vertebrados y plantas, con expresión preferencial, al menos en roedores, en células inmunológicas. La rata y el pez cebra, el único Ribase-Mn ADP bioquímicamente estudiados, son ADP-ribosa phosphohydrolases y algo menos eficiente, de CDP-alcoholes y 2',3'-cAMP. Además, la rata, pero no el pez cebra, muestra una única enzima con actividad ADP-ribosa phosphohydrolytic cíclica. La base molecular de tal especificidad es desconocida. Las ADPRibase-Mn de humanos mostraron actividades similares, incluyendo ADP-ribosa phosphohydrolase cíclica, lo que parece común a los mamíferos Ribase-Mn ADP. Acoplamiento del sustrato en un modelo de homología de ADPRibase-Mn de humanos sugiere posibles interacciones de ADP-ribosa con siete residuos ubicado, con una sola excepción (Cys253), ya sea dentro de la metalo-fosfatasas dependientes de la firma (Gln27, Asn110, Su111), o en regiones estructurales exclusivos de la familia ADPRibase-Mn: s2s3 (PHE37 y Arg43) y h7h8 (Phe210), alrededor de la entrada del sitio activo. Se construyeron mutantes y parámetros cinéticos para ADP-ribosa, CDP-colina, 2',3'-cAMP y cíclica de ADP-ribosa fueron determinadas. Phe37 fue necesaria para ADP-ribosa preferencia sin efecto catalítico, según lo indicado por el aumento de la ADP-ribosa Km y kcat invariable de F37A-ADPRibase-Mn Km, mientras que los valores para los otros sustratos apenas se vieron afectadas. Arg43 era esencial para la catálisis como indicado por la drástica pérdida de eficacia demostrada por R43A-ADPRibase-Mn. Inesperadamente, Cys253 obstaculizaba para cADPR phosphohydrolase, según lo indicado por las diez veces la ganancia de eficiencia de C253A-ADPRibase-Mn con ADP-ribosa cíclica. Esto permitió el diseño de un mutante triple (F37A L196F C253A) para que ADP-ribosa cíclica era el mejor sustrato, con una eficiencia catalítica de 3,5'104 M-1s-1 versus 4'103 M-1s-1 del tipo salvaje.Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose Km and unchanged kcat of F37A-ADPRibase-Mn, while the Km values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type.Trabajo patrocinado por: Ministerio de Ciencia e Innovación. Proyecto BFU2009-07296 (I+D+i) Junta de Extremadura. Proyecto GRU09135 y GR10133 Cofinanciación por el Fondo Europeo de Desarrollo Regional y Fondo Social EuropeopeerReviewe

Microenvironment Eradication of Hepatitis C: A Novel Treatment Paradigm

OBJECTIVES: Prisons are major reservoirs of hepatitis C virus (HCV) in which a therapeutic approach has been particularly difficult so far. Our aim was to create a permanent program of HCV elimination in a prison based on a "test and treat" strategy. METHODS: This open-label clinical trial was conducted in the Spanish prison "El Dueso" between May 2016 and July 2017. Viremic patients were treated with a ledipasvir-sofosbuvir regimen (8-12 weeks) according to the 2015 Spanish Guidelines. A teleconsultation program was established to follow-up patients from the hospital. Non-responders were submitted for a phylogenetic analysis and offered retreatment. An evaluation of new cases of HCV infection was performed every 6 months and upon release in all inmates. RESULTS: 847 (99.5%) inmates accepted to participate. HCV antibodies were present in 110 (13.0%) and 86 (10.2%) had detectable viremia. Most of them were genotype 1 or 3 (82.6%) and had <F2 fibrosis (52.2%). Treatment was started in the 69 inmates whose stay in prison was longer than 30 days. Sustained virological response was achieved in 64 out of 66 patients (96.9%), three of whom were successfully rescued with a salvage regimen after treatment failure. Two patients were lost to follow-up and three are currently on treatment without viremia. As a result, by July 2017 none of the 409 imprisoned was viremic, and neither reinfections nor de novo infections were detected. CONCLUSIONS: A sustained "test-and-treat" strategy against HCV in prisons is feasible and beneficial. Spreading this strategy should entail a public health impact.Supported by Plan Nacional de I+D+i 2013–2016 and Instituto de Salud Carlos III, Ministerio de Economía, Industria y Competitividad, cofinanced by European Development Regional Fund “A way to achieve Europe”,Operative program Intelligent Growth 2014–2020 and grant PIE15/00079. This study received funding assistance from Gilead Sciences, Spain (IN-ES-337-2089), C/Vía de los Poblados, 3, 28033 Madrid, Spain, http://www.gilead. com/about/worldwide-operations/europe/spain; phone number: +34 913789830), who played no part in study design, data analysis, or in the preparation of the manuscript. All study investigators declare to be independent from funders

Ciencia y profesión : el farmacéutico en la historia

474 páginas. Versiones pdf / epubLas IV Jornadas Científicas de la Sociedad de Docentes Universitarios de Historia de la Farmacia (SDUHFE) se celebraron en la Sede de La Rábida en junio de 2016. Esta obra presenta diversas investigaciones y comunicaciones, con varias temáticas que pueden desglosarse en cuatro bloques: 1) En un primer grupo podemos considerar todos los capítulos que abordan la historia de los colegios farmacéuticos así como los avatares de la profesión. Se da cuenta en la provincia de Sevilla de las dificultades del Colegio de Farmacéuticos en el periodo de la Guerra Civil y la Posguerra (1936-1949), del proceso de colegiación obligatoria a partir de 1916, pinceladas históricas sobre los farmacéuticos cántabros del siglo XIX, del Colegio de Farmacéuticos de Filipinas a finales del XIX, de los conflictos de los farmacéuticos en las reuniones sanitarias de mitad del XX, y del papel de los farmacéuticos titulares en la potabilización de las aguas de consumo en Plentzia (Vizcaya). 2) Podemos destacar también todos los trabajos que giran en torno a diferentes medicamentos y productos farmacéuticos, entre ellos estudios históricos sobre piedras preciosas, medicamentos para tratar heridas, quina contra las tercianas, opio, alexifármacos, medicamentos homeopáticos, talidomida o curiosos productos como el Licor del Polo. 3) El papel de los laboratorios farmacéuticos como la Casa Nestlé durante la Guerra Civil española y el franquismo, diferentes laboratorios onubenses durante este mismo periodo, el papel del Instituto de Higiene Militar y la experimentación con insecticidas clorados sintéticos en la posguerra española, aglutinan el tercer cuerpo temático. 4) Finalmente, podemos destacar los trabajos que tienen una componente publicitaria, divulgadora y social entre los que cabe destacar el estudio del NO-DO y los diferentes noticieros y documentales sobre temas farmacéuticos que resultan muy ilustrativos. La propaganda farmacéutica desarrollada en la revista Matronas, el inventario del patrimonio farmacéutico catalán, junto a la percepción social de la farmacia a través de las fallas valencianas conforma este último grupo

Gestión del conocimiento: perspectiva multidisciplinaria. Volumen 12

El libro “Gestión del Conocimiento. Perspectiva Multidisciplinaria”, Volumen 12, de la Colección Unión Global, es resultado de investigaciones. Los capítulos del libro, son resultados de investigaciones desarrolladas por sus autores. El libro cuenta con el apoyo de los grupos de investigación: Universidad Sur del Lago “Jesús María Semprúm” (UNESUR), Zulia – Venezuela; Universidad Politécnica Territorial de Falcón Alonso Gamero (UPTAG), Falcón – Venezuela; Universidad Politécnica Territorial de Mérida Kleber Ramírez (UPTM), Mérida – Venezuela; Universidad Guanajuato (UG) - Campus Celaya - Salvatierra - Cuerpo Académico de Biodesarrollo y Bioeconomía en las Organizaciones y Políticas Públicas (C.A.B.B.O.P.P), Guanajuato – México; Centro de Altos Estudios de Venezuela (CEALEVE), Zulia – Venezuela, Centro Integral de Formación Educativa Especializada del Sur (CIFE - SUR) - Zulia - Venezuela, Centro de Investigaciones Internacionales SAS (CIN), Antioquia - Colombia.y diferentes grupos de investigación del ámbito nacional e internacional que hoy se unen para estrechar vínculos investigativos, para que sus aportes científicos formen parte de los libros que se publiquen en formatos digital e impreso

Cuentos de nunca acabar. Aproximaciones desde la interculturalidad

Cuentos de nunca acabar. Aproximaciones desde la interculturalidad, surge después de la pandemia y su imposibilidad de socializar “en persona” con los compañeros de eventuales encuentros, porque la Comprensión Lectora tenía que reinventarse para su nueva reflexión cognitiva, adaptación contextual y reconstrucción del conocimiento. Este renovado enfoque de la realidad postpandemia, concebido en el marco de la educación intercultural comunitaria, busca potencializar los entornos naturales, sociales y culturales como recursos de aprendizaje multidisciplinario a través del lenguaje animado de los cuentos. En este marco, había que dinamizar la asignatura de Comunicación Oral y Escrita, que se dicta en los Primeros Niveles de los Centros de Apoyo de Otavalo, Cayambe, Latacunga y Riobamba, mediante un eje transversal donde los estudiantes escriban fundamentados en valores de la cosmovisión andina, considerando que provienen de varios lugares de la sierra y amazonía ecuatoriana. Todo surgió del encuentro presencial de un sábado cualquiera donde los estudiantes realizaban ejercicios narrativos, logrando una apreciable respuesta de imaginación, más emotiva que la clásica tarea de las Unidades, tanto así que, pasados unos días, seguían llegando sus escritos a mi correo. Entonces nos pusimos manos a la obra, cada estudiante tendría dos opciones como Actividad Integradora, la primera consistía en escribir un cuento de su propia inspiración, y la segunda analizar un clásico para comentar sus valores y antivalores. La mayor parte de estudiantes decidió escribir su propio cuento, de donde se escogieron algunas participaciones que podrían considerarse originales, para una edición que, respetando la transcripción de la tradición oral que prima en los sectores comunitarios, nos concretamos en revisar la puntuación y ortografía para publicarlos. Con esto buscamos innovar la Actividad Integradora, por algo más práctico y operativo para configurar los Objetos de Aprendizaje que buscamos. Así nació, en medio del camino, este libro de Cuentos de nunca acabar. Aproximaciones desde la interculturalidad, que ponemos en sus manos. Hernán Hermosa Mantilla Quito, junio de 202

Specific cyclic ADP-ribose phosphohydrolase obtained by mutagenic engineering of Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase

Abstract Cyclic ADP-ribose (cADPR) is a messenger for Ca2+ mobilization. Its turnover is believed to occur by glycohydrolysis to ADP-ribose. However, ADP-ribose/CDP-alcohol diphosphatase (ADPRibase-Mn) acts as cADPR phosphohydrolase with much lower efficiency than on its major substrates. Recently, we showed that mutagenesis of human ADPRibase-Mn at Phe37, Leu196 and Cys253 alters its specificity: the best substrate of the mutant F37A + L196F + C253A is cADPR by a short difference, Cys253 mutation being essential for cADPR preference. Its proximity to the ‘northern’ ribose of cADPR in docking models indicates Cys253 is a steric constraint for cADPR positioning. Aiming to obtain a specific cADPR phosphohydrolase, new mutations were tested at Asp250, Val252, Cys253 and Thr279, all near the ‘northern’ ribose. First, the mutant F37A + L196F + C253G, with a smaller residue 253 (Ala > Gly), showed increased cADPR specificity. Then, the mutant F37A + L196F + V252A + C253G, with another residue made smaller (Val > Ala), displayed the desired specificity, with cADPR kcat/KM ≈20–200-fold larger than for any other substrate. When tested in nucleotide mixtures, cADPR was exhausted while others remained unaltered. We suggest that the specific cADPR phosphohydrolase, by cell or organism transgenesis, or the designed mutations, by genome editing, provide opportunities to study the effect of cADPR depletion on the many systems where it intervenes

Molecular Dissection of Escherichia coli CpdB: Roles of the N Domain in Catalysis and Phosphate Inhibition, and of the C Domain in Substrate Specificity and Adenosine Inhibition

CpdB is a 3′-nucleotidase/2′3′-cyclic nucleotide phosphodiesterase, active also with reasonable efficiency on cyclic dinucleotides like c-di-AMP (3′,5′-cyclic diadenosine monophosphate) and c-di-GMP (3′,5′-cyclic diadenosine monophosphate). These are regulators of bacterial physiology, but are also pathogen-associated molecular patterns recognized by STING to induce IFN-β response in infected hosts. The cpdB gene of Gram-negative and its homologs of gram-positive bacteria are virulence factors. Their protein products are extracytoplasmic enzymes (either periplasmic or cell–wall anchored) and can hydrolyze extracellular cyclic dinucleotides, thus reducing the innate immune responses of infected hosts. This makes CpdB(-like) enzymes potential targets for novel therapeutic strategies in infectious diseases, bringing about the necessity to gain insight into the molecular bases of their catalytic behavior. We have dissected the two-domain structure of Escherichia coli CpdB to study the role of its N-terminal and C-terminal domains (CpdB_Ndom and CpdB_Cdom). The specificity, kinetics and inhibitor sensitivity of point mutants of CpdB, and truncated proteins CpdB_Ndom and CpdB_Cdom were investigated. CpdB_Ndom contains the catalytic site, is inhibited by phosphate but not by adenosine, while CpdB_Cdom is inactive but contains a substrate-binding site that determines substrate specificity and adenosine inhibition of CpdB. Among CpdB substrates, 3′-AMP, cyclic dinucleotides and linear dinucleotides are strongly dependent on the CpdB_Cdom binding site for activity, as the isolated CpdB_Ndom showed much-diminished activity on them. In contrast, 2′,3′-cyclic mononucleotides and bis-4-nitrophenylphosphate were actively hydrolyzed by CpdB_Ndom, indicating that they are rather independent of the CpdB_Cdom binding site