Snail2: Estudios estructura-función y análisis in vivo de su papel en el desarrollo del folícullo piloso y la carcinogénesis química de la piel

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 17-10-2014Snail1 y Snail2 son factores de transcripción de dedos de zinc (ZFs) de la superfamilia Snail que se caracterizan por su capacidad para inducir el proceso denominado transición epitelio-mesénquima (TEM), el cual es de gran importancia en desarrollo embrionario y en determinadas situaciones patológicas como la progresión tumoral. Un evento clave durante la TEM es la pérdida de expresión de cadherina-E, que ocurre fundamentalmente por represión transcripcional. El promotor de cadherina-E presenta unas regiones denominadas cajas-E a las que son capaces de unirse diversos factores de transcripción, entre los que se encuentran Snail1 y Snail2. Ambos factores presentan un dominio conservado N-terminal SNAG; una región central divergente que en Snail1 está formada por una caja de destrucción (DB) y secuencias de exportación nuclear (NES), mientras que Snail2 presenta un dominio SLUG y un dominio de interacción con CtBP (CID) degenerado; y una región de unión al DNA (DBD), formada por cuatro y cinco ZFs en Snail1 y Snail2, respectivamente. Análisis previos de expresión génica evidenciaron que Snail1 y Snail2 inducen programas genéticos comunes y específicos a cada factor, pero no se conoce en detalle los motivos de dichas diferencias. Esta tesis ha analizado la contribución de cada uno de los ZFs de Snail1 y Snail2 a su capacidad represora y funcionalidad, la relevancia de los dominios SNAG y SLUG de Snail2, así como el papel en concreto de Snail2 en el ciclo del folículo piloso (FP) y en el desarrollo de las lesiones en el proceso de carcinogénesis química. El análisis de los ZFs de Snail1 y Snail2 se basó en el estudio de su capacidad represora sobre cadherina-E y de inducción de TEM, y demostramos que los ZFs necesarios para su función son diferentes en estos dos factores. En el caso de Snail1, se requiere que esté intacta la estructura del primer (ZF1) y el segundo dedo de zinc (ZF2) conjuntamente, mientras que en Snail2 son el ZF3 o ZF4 los esenciales para su función. Adicionalmente, caracterizamos el dominio SNAG de Snail2 como fundamental para la inducción de TEM, siendo relevante la fosforilación de la serina 4 en dicha capacidad, mientras que la presencia del dominio SLUG modula negativamente la inducción de TEM. El estudio de la relevancia de Snail2 en el ciclo del pelo mediante la utilización del ratón mutante nulo de Snail2 (Snail2-/-) indicó que su ausencia modifica el ciclo del FP a nivel de la telogén refractaria. Por último, datos previos del laboratorio mostraron que los ratones Snail2-/- presentaban una mayor progresión tumoral con un alto componente inflamatorio en respuesta a la carcinogénesis química en piel. En el presente trabajo, mostramos que la dosis génica de Snail2 es importante en la respuesta a la carcinogénesis de piel, y que es, fundamentalmente, la ausencia de Snail2 en la población de células del sistema inmune conocidas como precursores mieloides, la causante de la progresión de las lesiones en ratones Snail2-/-.Snail1 and Snail2 are zinc finger transcription factors that belong to the Snail superfamily, and are characterized by their ability to induce epithelial-to-mesenchymal transition (EMT), linked to embryonic development and pathological conditions like tumoral progression. One key event in EMT is the loss of E-cadherin, that occurs mainly by transcriptional repression through the recognition of specific-sequence regions, called E-boxes, by transcription factors, like Snail1 and Snail2. Both factors present a conserved N-terminal SNAG domain; a central region that differs between them, Snail1 presents nuclear export signals (NES) and a destruction box (DB), whereas Snail2 possesses a SLUG domain and a degenerated CtBP interaction domain (CID); and a DNA binding domain (DBD), formed by four and five zinc fingers (ZFs) in Snail1 and Snail2, respectively. Previous genetic expression profile analyses showed that Snail1 and Snail2 induce common and specific subsets of genes, but little is known about the reasons for such differences. The present thesis describes the contribution of the Snail1 and Snail2 ZFs to their repressor and EMT induction ability, the relevance of Snail2 SNAG and SLUG domains, and the specific role of Snail2 in skin carcinogenesis and hair follicle (HF) cycle. The Snail1 and Snail2 ZFs analyses revealed that the ZFs required for their function are different for each factor; ZF1 and ZF2 of Snail1, and ZF3 or ZF4 of Snail2 are needed for E-cadherin repression and EMT induction. Moreover, we describe that Snail2 SNAG domain is essential for EMT induction, and show that serine 4 phosphorylation, located in this domain, is a key event for this process. On the other hand, SLUG domain impairs the Snail2 ability to induce EMT. The analyses of HF cycle in Snail2 knock out mice (Snail2-/-) showed that Snail2 modifies the response in refractory telogen. Finally, previous data from our lab described that Snail2-/- mice showed an increase in tumoral progression in response to the mice skin chemical carcinogenesis. In the present work, the obtained data point that Snail2 gene dosage is a key factor in the response to skin carcinogenesis, and that the Snail2 absence in hematopoietic precursors, and in particular in myeloid progenitors, triggers the tumoral progression observed in Snail2-/- mice

Characterization of the SNAG and SLUG Domains of Snail2 in the Repression of E-Cadherin and EMT Induction: Modulation by Serine 4 Phosphorylation

Snail1 and Snail2, two highly related members of the Snail superfamily, are direct transcriptional repressors of E-cadherin and EMT inducers. Previous comparative gene profiling analyses have revealed important differences in the gene expression pattern regulated by Snail1 and Snail2, indicating functional differences between both factors. The molecular mechanism of Snail1-mediated repression has been elucidated to some extent, but very little is presently known on the repression mediated by Snail2. In the present work, we report on the characterization of Snail2 repression of E-cadherin and its regulation by phosphorylation. Both the N-terminal SNAG and the central SLUG domains of Snail2 are required for efficient repression of the E-cadherin promoter. The co-repressor NCoR interacts with Snail2 through the SNAG domain, while CtBP1 is recruited through the SLUG domain. Interestingly, the SNAG domain is absolutely required for EMT induction while the SLUG domain plays a negative modulation of Snail2 mediated EMT. Additionally, we identify here novel in vivo phosphorylation sites at serine 4 and serine 88 of Snail2 and demonstrate the functional implication of serine 4 in the regulation of Snail2-mediated repressor activity of E-cadherin and in Snail2 induction of EMT

Autonomous learning of the bioinorganic chemistry and biomaterials laboratory and characterisation techniques used through the use of e-learning tools. Enhancing the understanding of students with disabilities, hearing difficulties or foreigners

Concesión por parte de la Oficina para la Calidad de la prorroga para la entrega de la memoria del proyecto nº 52 hasta el 31 de diciembre de 2022El proyecto tiene como objetivo fundamental la elaboración de material audiovisual para la mejora de las prácticas de la asignatura de Química Bioinorgánica y Biomateriales de cuarto curso del grado en Farmacia para su satisfactorio desarrollo tanto en un escenario totalmente on-line como presencial. Para el buen desarrollo de las prácticas en el laboratorio es necesario que el alumno disponga de una información adecuada que le permita conocer los distintos aspectos del proceso experimental a realizar a través de la elaboración de una página web disponible en el campus virtual. Para ello, la práctica habitual es utilizar un manual de prácticas de laboratorio en el que se aporta información sobre conceptos básicos, material a utilizar, procedimiento experimental, seguridad, etc… de cada práctica. Hay tres aspectos de gran importancia desde el punto de vista formativo: 1) afianzar bien los conceptos básicos en los que se apoya el trabajo experimental posterior, 2) el manejo de los software que permitan el tratamiento de los resultados e interpretación de los datos utilizando herramientas e-learning y 3) facilitar la comprensión de los dos objetivos anteriores a personas con discapacidades auditivas y/o idioma incluyendo subtítulos en español e inglés en los videos elaborados. En dicha elaboración se tendrá muy en cuenta, la inclusión de los alumnos con diversidad presentando dicha información de manera visual, estructurada, secuencial y en pasos ordenados. Estas herramientas de aprendizaje estarán dirigidas inicialmente a los alumnos de cursos superiores del Grado en Farmacia, Química, Ingeniería de Materiales y Máster Universitario en Biomateriales por lo que para comprobar su impacto y eficacia seleccionaemos a estudiantes de los departamentos implicados antes de ponerlos a disposición de los alumnos. Este proyecto se basa en tres Competencias transversales instrumentales del Espacio Europeo de Educación Superior (EEES): Comunicación oral y escrita en la/s lengua/s materna/s, Comunicación en lengua extranjera, y Utilización de las Tecnologías de la información y la Comunicación (TIC) en el ámbito de estudio y contexto profesional. Estas competencias son clave para el desarrollo académico y profesional de los alumnos/as, especialmente para alumnos/as con algún tipo de discapacidad. Creemos que, mediante este proyecto, se realiza un esfuerzo para mejorar la inclusión de estos alumnos/as tanto en un contexto online como presencial en un laboratorio de Química. Por las razones anteriormente expuestas, un grupo de profesores de los Departamentos de Química en Ciencias Farmacéuticas y el de Bioquímica y Biología Molecular se propone elaborar un material didáctico que permita el autoaprendizaje del alumno en la parte experimental de la asignatura optativa de Química Bioinorgánica y Biomateriales del Grado en Farmacia, si bien, este material también será de utilidad para complementar otras asignaturas presentes en los Grados de Biología o de Ingeniería de Materiales e, incluso en el Máster en Biomateriales de la UCM.The main objective of the project is the development of audiovisual material to improve the practices of the Bioinorganic Chemistry and Biomaterials subject in the fourth year of the degree in Pharmacy for its satisfactory development both in a fully online and on-site scenario. For the good development of the laboratory practicals it is necessary that the student has adequate information that allows him/her to know the different aspects of the experimental process to be carried out through the development of a web page available on the virtual campus. To this end, the usual practice is to use a laboratory practice manual which provides information on basic concepts, materials to be used, experimental procedure, safety, etc., for each practice. There are three aspects of great importance from the training point of view: 1) to consolidate the basic concepts on which the subsequent experimental work is based, 2) the use of software that allows the processing of the results and interpretation of the data using e-learning tools and 3) to facilitate the understanding of the two previous objectives for people with hearing and/or language disabilities by including subtitles in Spanish and English in the videos produced. The inclusion of students with diversity will be taken into account in the elaboration of the videos by presenting the information in a visual, structured, sequential and orderly manner. These learning tools will initially be aimed at students in the upper years of the Bachelor's Degree in Pharmacy, Chemistry, Materials Engineering and the Master's Degree in Biomaterials, so in order to check their impact and effectiveness we will select students from the departments involved before making them available to students. This project is based on three transversal instrumental competences of the European Higher Education Area: Oral and written communication in the mother tongue(s), Communication in a foreign language, and use of information and communication technologies (ICT) in the field of study and professional context. These competences are key for the academic and professional development of students, especially for students with disabilities. We believe that, through this project, an effort is being made to improve the inclusion of these students both in an online and face-to-face context in a chemistry laboratory. For the above reasons, a group of lecturers from the Departments of Chemistry in Pharmaceutical Sciences and Biochemistry and Molecular Biology propose to develop a teaching material that allows students to learn by themselves in the experimental part of the optional subject of Bioinorganic Chemistry and Biomaterials of the Degree in Pharmacy, although this material will also be useful to complement other subjects in the Degrees in Biology or Materials Engineering and even in the Master's Degree in Biomaterials of the University of Valencia, where it will also be useful to complement other subjects in the Degrees in Biology or Materials Engineering and even in the Master's Degree in Biomaterials of the Universidad Complutense.Depto. de Química en Ciencias FarmacéuticasFac. de FarmaciaFALSEunpu

Loss of Snail2 favors skin tumor progression by promoting the recruitment of myeloid progenitors

Snail2 is a zinc finger transcription factor involved in driving epithelial to mesenchymal transitions. Snail2 null mice are viable, but display defects in melanogenesis, gametogenesis and hematopoiesis, and are markedly radiosensitive. Here, using mouse genetics, we have studied the contributions of Snail2 to epidermal homeostasis and skin carcinogenesis. Snail2 −/− mice presented a defective epidermal terminal differentiation and, unexpectedly, an increase in number, size and malignancy of tumor lesions when subjected to the two-stage mouse skin chemical carcinogenesis protocol, compared with controls. Additionally, tumor lesions from Snail2 −/− mice presented a high inflammatory component with an elevated percentage of myeloid precursors in tumor lesions that was further increased in the presence of the anti-inflammatory agent dexamethasone. In vitro studies in Snail2 null keratinocytes showed that loss of Snail2 leads to a decrease in proliferation indicating a non-cell autonomous role for Snail2 in the skin carcinogenic response observed in vivo. Bone marrow (BM) cross-reconstitution assays between Snail2 wild-type and null mice showed that Snail2 absence in the hematopoietic system fully reproduces the tumor behavior of the Snail2 null mice and triggers the accumulation of myeloid precursors in the BM, blood and tumor lesions. These results indicate a new role for Snail2 in preventing myeloid precursors recruitment impairing skin chemical carcinogenesis progression

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field