Structural and functional studies on cad, the anti-tumoral target protein leading de novo biosynthesis of pyrimidines

CAD is a large multifunctional protein catalyzing the initial three steps in de novo biosynthesis of pyrimidine nucleotides in animals. Since pyrimidines are the building blocks of nucleic acids and the precursors of other key macromolecular substances, the up-regulation of CAD’s activity is essential for cell growth and proliferation, especially in neoplastic cells. Thus, CAD has been considered an attractive target for the development of anti-tumoral compounds. However, despite the central metabolic role and its therapeutic potential, due to the lack of knowledge about its organization and the structure and function of its different enzymatic domains, no robust inhibitor that could be used as anti-proliferative drug has been designed thus far. In this bachelor thesis, a research study on CAD’s DHOase domain was conducted, particularly focused in understanding the role of a flexible loop that appears to play a conserved catalytic role from E.coli to humans. To examine the catalytic mechanism of this loop, a cloning approach was designed to generate a human DHOase construct bearing the equivalent flexible loop of the E.coli enzyme. This chimeric protein was expressed either in mammalian cells or in bacteria cultures and purified using different chromatographic techniques. Activity assays on both the forward and reverse directions of the reaction were then performed in the chimeric huDHOase to estimate the turnover rate of the mutant. With a negligible enzymatic activity (less than 2% of the wild type) the experiments here presented prove that, despite having a conserved functional role, the flexible loop of E.coli and human DHOases are not interchangeable. Overall, the results confirmed the implication of the flexible loop in oligomerization and in the catalytic mechanism of DHOases, highlighting key differences in the functioning of both mammalian and bacterial enzymes that will be exploited in future work for the rational design of specific inhibitors against CAD.Ingeniería Biomédica (Plan 2010

Characterization of the catalytic flexible loop in the dihydroorotase domain of the human multi-enzymatic protein CAD

The dihydroorotase (DHOase) domain of the multifunctional protein carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) catalyzes the third step in the de novo biosynthesis of pyrimidine nucleotides in animals. The crystal structure of the DHOase domain of human CAD (huDHOase) revealed that, despite evolutionary divergence, its active site components are highly conserved with those in bacterial DHOases, encoded as monofunctional enzymes. An important element for catalysis, conserved from Escherichia coli to humans, is a flexible loop that closes as a lid over the active site. Here, we combined mutagenic, structural, biochemical, and molecular dynamics analyses to characterize the function of the flexible loop in the activity of CAD's DHOase domain. A huDHOase chimera bearing the E. coli DHOase flexible loop was inactive, suggesting the presence of distinctive elements in the flexible loop of huDHOase that cannot be replaced by the bacterial sequence. We pinpointed Phe-1563, a residue absolutely conserved at the tip of the flexible loop in CAD's DHOase domain, as a critical element for the conformational equilibrium between the two catalytic states of the protein. Substitutions of Phe-1563 with Ala, Leu, or Thr prevented the closure of the flexible loop and inactivated the protein, whereas substitution with Tyr enhanced the interactions of the loop in the closed position and reduced fluctuations and the reaction rate. Our results confirm the importance of the flexible loop in CAD's DHOase domain and explain the key role of Phe-1563 in configuring the active site and in promoting substrate strain and catalysi

Corporate reputation in the spanish context: An interaction between reporting to stakeholders and industry.

ABSTRACT: The authors describe the intensity and orientation of the corporate social responsibility (CSR) reporting in four Spanish industries and explore the relationship that exists between both concepts and an independent measurement of reputation for CSR (CSRR). The results demonstrate that the CSR reporting is especially relevant and useful in the finance industry. Finance companies report significantly more CSR information than most industries in Spain, and this reporting is more closely linked to their CSRR than the CSR reporting of basic, consumer goods and services industries. Borra

Hypertension, hemorrhagic stroke and smoking cessation

El artículo trata sobre la hipertensión arterial, las enfermedades cerebrovasculares y el hecho de dejar de fumar.Smoking cessation is considered as a good solution for stroke prevention. Unfortunately, first-line therapies to reduce cigarette smoking are not always useful. Some clinical effects of these drugs are able to produce cardiovascular adverse events

The long-term control of nitric oxide on aortic structure and function in uninephrectomized spontaneously hypertensive rats

Ponencia presentada en el 3º Congreso de Nefrología por Internet celebrado del 10 al 25 de noviembre de 2003 , sobre el control a largo plazo del óxido nítrico en la estructura y la función aórticas en ratas hipertensas.Higher blood pressure and organ damage were always observed in chronic hypertension. Here, we have investigated in female uninephrectomized spontaneously hypertensive rats (UNX-SHRs) the role of NO on changes in blood pressure, renal function, thoracic aortic relaxations and structure

Educar en el 2000 : revista de formación del profesorado

El artículo forma parte de una sección de la revista dedicada a difusión de programasEl Proyecto Albádiora ha conseguido financiación por parte de la UE para que 50 alumnos de centros formativos de la Región de Murcia realicen estancias formativas de un mes de duración en empresas europeas, a lo largo de los cursos académicos 2003/04 y 2004/05 que suponen la realización de una parte de la formación en centros de trabajo, fuera de España.MurciaES

Studying nucleoid-associated protein–DNA interactions using polymer microgels as synthetic mimics

9 p.-6 fig.-1 graph. abst.Microfluidically fabricated polymer microgels are used as an experimental platform to analyze protein–DNA interactions regulating bacterial cell division. In particular, we focused on the nucleoid-associated protein SlmA, which forms a nucleoprotein complex with short DNA binding sequences (SBS) that acts as a negative regulator of the division ring stability in Escherichia coli. To mimic the bacterial nucleoid as a dense DNA region of a bacterial cell and investigate the influence of charge and permeability on protein binding and diffusion in there, we have chosen nonionic polyethylene glycol and anionic hyaluronic acid as precursor materials for hydrogel formation, previously functionalized with SBS. SlmA binds specifically to the coupled SBS for both types of microgels while preferentially accumulating at the microgels’ surface. We could control the binding specificity by adjusting the buffer composition of the DNA-functionalized microgels. The microgel charge did not impact protein binding; however, hyaluronic acid-based microgels exhibit a higher permeability, promoting protein diffusion; thus, they were the preferred choice for preparing nucleoid mimics. The approaches described here provide attractive tools for bottom-up reconstitution of essential cellular processes in media that more faithfully reproduce intracellular environments.Financial support by the Volkswagen Foundation (“Change of Course”, Grant Agreement No. 96619) and the Spanish Government (Grant Agreements No. PID2019-104544GB-I00 and PID2022-136951NB-I00) is gratefully acknowledged.Peer reviewe

Protein-based patterning to spatially functionalize biomimetic membranes

10 p.-3 figThe bottom-up reconstitution of proteins for their modular engineering into synthetic cellular systems can reveal hidden protein functions in vitro. This is particularly evident for the bacterial Min proteins, a paradigm for self-organizing reaction-diffusion systems that displays an unexpected functionality of potential interest for bioengineering: the directional active transport of any diffusible cargo molecule on membranes. Here, the MinDE protein system is reported as a versatile surface patterning tool for the rational design of synthetically assembled 3D systems. Employing two-photon lithography, microswimmer-like structures coated with tailored lipid bilayers are fabricated and demonstrate that Min proteins can uniformly pattern bioactive molecules on their surface. Moreover, it is shown that the MinDE system can form stationary patterns inside lipid vesicles, which allow the targeting and distinctive clustering of higher-order protein structures on their inner leaflet. Given their facile use and robust function, Min proteins thus constitute a valuable molecular toolkit for spatially patterned functionalization of artificial biosystems like cell mimics and microcarriers.This work was supported by the Max Planck-Bristol Centre for Minimal Biology (A.M.-S., H.E.) and the Deutsche Forschungsgemeinschaft (P.S.). M.R.-L., S.G. and A.M.-S. are part of IMPRS-LS, and M.R.-L. and S.G. are part of the ONE MUNICH Project supported by the Federal Ministry of Education and Research (BMBF) as well as the Free State of Bavaria under the Excellence Strategy of the Federal Government and the Länder.Peer reviewe

Proyecto IRIS: Educar en la Transparencia y el Buen Gobierno. Memoria del curso 2018-19

Se describen los objetivos, actividades desarrolladas, resultados, propuestas de mejora y conclusiones generales del proyecto IRIS en su segunda edición realizada entre septiembre de 2018 y junio de 2019. Su fin fue diseñar y aplicar un programa de enseñanza del derecho a la información, la transparencia, el buen gobierno y la participación social destinado a estudiantes de primero de bachillerato de la Región de Murcia. Iniciativa de un grupo de docentes y voluntarios con la participación de la Sociedad de Filosofía de la Región de Murcia y la Presidencia del Consejo de Transparencia de la Región de Murcia. Sus objetivos fueron: a) Elaborar e impartir una unidad didáctica relativa a los contenidos a enseñar, de acuerdo con los estándares del currículo de Filosofía de la Región de Murcia b) Identificar y valorar las percepciones iniciales del alumnado de Bachillerato de la Región, c) Diseñar las Bases y desarrollar un Concurso de disertaciones y videos sobre transparencia y buen gobierno, destinado a los y las estudiantes participantes en el proyecto. e) Evaluar los resultados del conjunto de actividades e identificar las propuestas de mejora necesarias para la extensión y consolidación del programa: un plan específico de formación para el profesorado, la inclusión de un estándar sobre transparencia en el currículum autonómico y el desarrollo de materiales, textos y tareas prácticas que faciliten al profesorado su aplicación. En esta edición colaboraron diez Institutos de Enseñanza Secundaria participando en el Programa un total de 736 estudiantesConsejo de Transparencia de la Región de Murci

Memoria del Proyecto IRIS: Educar en la Transparencia y el Buen Gobierno. Proyecto piloto para enseñar y aprender los conceptos de transparencia, la participación y el buen gobierno en Primer Curso de Bachillerato

Síntesis de los objetivos, actividades desarrolladas, resultados, propuestas de mejora y conclusiones generales del proyecto IRIS, cuyo fin fue diseñar y aplicar un programa de enseñanza del derecho a la información, la transparencia, el buen gobierno y la participación social destinado a estudiantes de primero de bachillerato de la Región de Murcia. Iniciativa de un grupo de docentes y voluntarios con la participación de la Sociedad de Filosofía de la Región de Murcia y la Presidencia del Consejo de Transparencia de la Región de Murcia. Sus objetivos fueron: a) Elaborar e impartir una unidad didáctica relativa a los contenidos a enseñar, de acuerdo con los estándares del currículo de Filosofía de la Región de Murcia b) Identificar y valorar las percepciones iniciales del alumnado de Bachillerato de la Región, c) Diseñar las Bases y desarrollar un Concurso de disertaciones y videos sobre transparencia y buen gobierno, destinado a los y las estudiantes participantes en el proyecto. e) Evaluar los resultados del conjunto de actividades e identificar las propuestas de mejora necesarias para la extensión y consolidación del programa: un plan específico de formación para el profesorado, la inclusión de un estándar sobre transparencia en el currículum autonómico y el desarrollo de materiales, textos y tareas prácticas que faciliten al profesorado su aplicaciónSociedad de Filosofía de la Región de Murcia y Consejo de Transparencia de la Región de Murci