Person:
Rodríguez Peña, José Manuel

First Name

José Manuel

Last Name

Rodríguez Peña

URI

https://hdl.handle.net/20.500.14352/77543

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Farmacia

Department

Microbiología y Parasitología

Area

Microbiología

Identifiers

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Now showing 1 - 10 of 21

Poacic acid, a β‐1,3‐glucan–binding antifungal agent, inhibits cell‐wall remodeling and activates transcriptional responses regulated by the cell‐wall integrity and high‐osmolarity glycerol pathways in yeast
(The FASEB Journal, 2021) García Sánchez, Raúl; Itto‐Nakama, Kaori; Rodríguez Peña, José Manuel; Chen, Xiaolin; Sanz Santamaría, Ana Belén; Lorenzo, Alba de; Pavón Vergés, Mónica; Kubo, Karen; Ohnuki, Shinsuke; Nombela Cano, César; Popolo, Laura; Ohya, Yoshikazu; Arroyo, Javier
As a result of the relatively few available antifungals and the increasing frequency of resistance to them, the development of novel antifungals is increasingly important. The plant natural product poacic acid (PA) inhibits β-1,3-glucan synthesis in Saccharomyces cerevisiae and has antifungal activity against a wide range of plant pathogens. However, the mode of action of PA is unclear. Here, we reveal that PA specifically binds to β-1,3-glucan, its affinity for which is ~30-fold that for chitin. Besides its effect on β-1,3-glucan synthase activity, PA inhibited the yeast glucan-elongating activity of Gas1 and Gas2 and the chitin–glucan transglycosylase activity of Crh1. Regarding the cellular response to PA, transcriptional co-regulation was mediated by parallel activation of the cell-wall integrity (CWI) and high-osmolarity glycerol signaling pathways. Despite targeting β-1,3-glucan remodeling, the transcriptional profiles and regulatory circuits activated by caspofungin, zymolyase, and PA differed, indicating that their effects on CWI have different mechanisms. The effects of PA on the growth of yeast strains indicated that it has a mode of action distinct from that of echinocandins, suggesting it is a unique antifungal agent.
Structural and functional analysis of yeast Crh1 and Crh2 transglycosylases
(FEBS Journal, 2015) Blanco, Noelia; Sanz Santamaría, Ana Belén; Rodríguez Peña, José Manuel; Nombela Cano, César; Vladimir, Farkas; Hurtado-Guerrero, Ramón; Arroyo Nombela, Francisco Javier
Covalent cross-links between chitin and glucan at the yeast cell wall are created by the transglycosylase activity of redundant proteins Crh1 and Crh2, with cleavage of β-1,4 linkages of the chitin backbone and transfer of the generated molecule containing newly created reducing end onto the glucan acceptor. A three-dimensional structure of Crh1 was generated by homology modeling based on the crystal structure of bacterial 1,3-1,4-β-d-glucanase, followed by site-directed mutagenesis to obtain molecular insights into how these enzymes achieve catalysis. The residues of both proteins that are involved in their catalytic and binding activities have been characterized by measuring the ability of yeast cells expressing different versions of these proteins to transglycosylate oligosaccharides derived from β-1,3-glucan, β-1,6-glucan and chitin to the chitin at the cell wall. Within the catalytic site, residues E134 and E138 of Crh1, as well as E166 and E170 of Crh2, corresponding to the nucleophile and general acid/base, and also the auxiliary D136 and D168 of Crh1 and Crh2, respectively, are shown to be essential for catalysis. Mutations of aromatic residues F152, Y160 and W219, located within the carbohydrate-binding cleft of the Crh1 model, also affect the transglycosylase activity. Unlike Crh1, Crh2 contains a putative carbohydrate-binding module (CBM18) of unknown function. Modeling and functional analysis of site-directed mutant residues of this CBM identified essential amino acids for protein folding and stability, as well as residues that tune the catalytic activity of Crh2.
Activation of the yeast cell wall integrity MAPK pathway by zymolyase depends on protease and glucanase activities and requires the mucin-like protein Hkr1 but not Msb2
(FEBS Letters, 2013) Rodríguez Peña, José Manuel; Diez-Muñiz, Sonia; Bermejo, Clara; Nombela, César; Arroyo, Javier
Yeast adaptation to conditions in which cell wall integrity is compromised mainly relies on the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway. Zymolyase, a mixture of cell wall-digesting enzymes, triggers a peculiar signaling mechanism in which activation of the CWI pathway is dependent on the high-osmolarity glycerol MAPK pathway. We have identified inhibitors of the principal enzyme activities present in zymolyase and tested their effect on the activation of the MAPK of the CWI pathway, Slt2/Mpk1. Eventually, only β-1,3-glucanase and protease activities were essential to elicit Slt2 activation and confer lytic power to zymolyase. Moreover, we show that the osmosensor Hkr1 is required for signaling, being the most upstream element identified to date.
Cooperation between SAGA and SWI/SNF complexes is required for efficient transcriptional responses regulated by the yeast MAPK Slt2
(Nucleic Acids Research, 2016) Sanz Santamaría, Ana Belén; García Sánchez, Raúl; Rodríguez Peña, José Manuel; Nombela Cano, César; Arroyo Nombela, Francisco Javier
The transcriptional response of Saccharomyces cerevisiae to cell wall stress is mainly mediated by the cell wall integrity (CWI) pathway through the MAPK Slt2 and the transcription factor Rlm1. Once activated, Rlm1 interacts with the chromatin remodeling SWI/SNF complex which locally alters nucleosome positioning at the target promoters. Here we show that the SAGA complex plays along with the SWI/SNF complex an important role for eliciting both early induction and sustained gene expression upon stress. Gcn5 co-regulates together with Swi3 the majority of the CWI transcriptional program, except for a group of genes which are only dependent on the SWI/SNF complex. SAGA subunits are recruited to the promoter of CWI-responsive genes in a Slt2, Rlm1 and SWI/SNF-dependent manner. However, Gcn5 mediates acetylation and nucleosome eviction only at the promoters of the SAGA-dependent genes. This process is not essential for pre-initiation transcriptional complex assembly but rather increase the extent of the remodeling mediated by SWI/SNF. As a consequence, H3 eviction and Rlm1 recruitment is completely blocked in a swi3Δ gcn5Δ double mutant. Therefore, SAGA complex, through its histone acetylase activity, cooperates with the SWI/SNF complex for the mandatory nucleosome displacement required for full gene expression through the CWI pathway.
Project number: 11
Diseño, desarrollo y elaboración de un soporte informático interactivo para el estudio de las prácticas de la asignatura de Microbiología Clínica mediante el uso de imágenes reales de pruebas microbiológicas obtenidas en el laboratorio
(2020) Rodríguez Peña, José Manuel; Monteoliva Díaz, Lucía; Molero Martín-Portugués, María Gloria; Alonso Monge, Rebeca María Del Mar; García Sánchez, Raúl; Amador García, Ahinara
En las diferentes clases prácticas relacionadas con asignaturas de Microbiología, como sucede en otras áreas de conocimiento, un aspecto que requiere especial atención es el diseño y la elaboración de herramientas que contribuyan a facilitar y mejorar el aprendizaje por parte del alumnado de los múltiples conceptos microbiológicos que se imparten en las mismas, facilitando en lo posible la enseñanza no presencial. En este sentido, pensamos que la existencia de un soporte virtual interactivo de los distintos abordajes prácticos realizados en el laboratorio y sus posibles resultados facilitaría en gran medida el estudio, y por tanto, la adquisición de las competencias correspondientes a la docencia práctica de Microbiología. En los dos cursos académicos anteriores (2017/18 y 2018/19), a través de sendos proyectos UCM-INNOVA (Proyectos 161 y 76, respectivamente), se ha recopilado, con la participación activa de los alumnos, una gran cantidad de información gráfica de toda la labor práctica realizada durante la impartición de las asignaturas incluidas en diversas titulaciones, habiéndose generado de esta manera un amplio banco de imágenes de calidad para diversos usos docentes. El proyecto realizado (UCM-INNOVA 19/20 Proyecto 11), ha consistido en el diseño, generación e implementación de una herramienta informática en entorno PowerPoint, a la que hemos denominado EMC (Evaluación Microbiología Clínica), que pone en valor todo el esfuerzo realizado por parte del profesorado y los alumnos en los proyectos anteriores, en relación a la asignatura de Microbiología Clínica del 4º curso del Grado en Farmacia de la Universidad Complutense de Madrid. En la aplicación EMC se ha incluido y estructurado cada práctica concreta siguiendo la misma organización realizada en el laboratorio, donde se sigue una rutina secuencial y jerárquica en la que el alumno debe interpretar los resultados posibles, fundamentalmente presentados en formato gráfico, de cada una de las pruebas realizadas según el tipo/s de microorganismo/s implicados, siendo redirigido a otro panel de pruebas cada vez más específicas hasta completar cada una de las prácticas. Esta herramienta se ha mostrado útil, no sólo para facilitar el estudio y el aprendizaje de los contenidos temáticos prácticos, sino también para servir de soporte y complemento al estudio del programa teórico de la asignatura. Es importante destacar que, durante las prácticas de Microbiología Clínica se explican los síndromes clínicos relacionados con las enfermedades infecciosas más frecuentes y su diagnóstico microbiológico, incluyendo desde la obtención y procesamiento de las muestras biológicas problema, hasta su posterior análisis microbiológico diferencial con el objeto de identificar a los potenciales microorganismos patógenos responsables de cada cuadro. Tras la realización de cada práctica, las pruebas microbiológicas deben ser adecuadamente eliminadas, no pudiendo guardarse las pruebas de forma indefinida. Por lo tanto, poder revisitar cuantas veces sea necesario por el alumno todas las pruebas y sus correspondientes resultados e interpretación de forma sencilla y visual en un entorno amigable (tipo presentación PowerPoint), supone un apoyo al estudio y al aprendizaje. Finalmente, pensamos que es importante remarcar que incluso en Universidades donde la metodología docente es eminentemente presencial, el desarrollo y utilización de forma complementaria de herramientas que faciliten el aprendizaje on-line es crucial en los tiempos que vivimos, cómo ha dejado patente la pandemia de la COVID-19.
Genomic profiling of fungal cell wall-interfering compounds: identification of a common gene signature.
(BMC genomics, 2015) García Sánchez, Raúl; Botet, Javier; Rodríguez Peña, José Manuel; Bermejo, Clara; Ribas, Juan Carlos; Revuelta, José Luis; Nombela, César; Arroyo, Javier
BACKGROUND The fungal cell wall forms a compact network whose integrity is essential for cell morphology and viability. Thus, fungal cells have evolved mechanisms to elicit adequate adaptive responses when cell wall integrity (CWI) is compromised. Functional genomic approaches provide a unique opportunity to globally characterize these adaptive mechanisms. To provide a global perspective on these CWI regulatory mechanisms, we developed chemical-genomic profiling of haploid mutant budding yeast cells to systematically identify in parallel those genes required to cope with stresses interfering the cell wall by different modes of action: β-1,3 glucanase and chitinase activities (zymolyase), inhibition of β-1,3 glucan synthase (caspofungin) and binding to chitin (Congo red). RESULTS Measurement of the relative fitness of the whole collection of 4786 haploid budding yeast knock-out mutants identified 222 mutants hypersensitive to caspofungin, 154 mutants hypersensitive to zymolyase, and 446 mutants hypersensitive to Congo red. Functional profiling uncovered both common and specific requirements to cope with different cell wall damages. We identified a cluster of 43 genes highly important for the integrity of the cell wall as the common "signature of cell wall maintenance (CWM)". This cluster was enriched in genes related to vesicular trafficking and transport, cell wall remodeling and morphogenesis, transcription and chromatin remodeling, signal transduction and RNA metabolism. Although the CWI pathway is the main MAPK pathway regulating cell wall integrity, the collaboration with other signal transduction pathways like the HOG pathway and the invasive growth pathway is also required to cope with the cell wall damage depending on the nature of the stress. Finally, 25 mutant strains showed enhanced caspofungin resistance, including 13 that had not been previously identified. Only three of them, wsc1Δ, elo2Δ and elo3Δ, showed a significant decrease in β-1,3-glucan synthase activity. CONCLUSIONS This work provides a global perspective about the mechanisms involved in cell wall stress adaptive responses and the cellular functions required for cell wall integrity. The results may be useful to uncover new potential antifungal targets and develop efficient antifungal strategies by combination of two drugs, one targeting the cell wall and the other interfering with the adaptive mechanisms.
Structural and functional analysis of yeast Crh1 and Crh2 transglycosylases.
(The FEBS journal, 2015) Blanco, Noelia; Sanz Santamaría, Ana Belén; Rodríguez Peña, José Manuel; Nombela, César; Farkaš, Vladimír; Hurtado Guerrero, Ramón; Arroyo, Javier
Covalent cross-links between chitin and glucan at the yeast cell wall are created by the transglycosylase activity of redundant proteins Crh1 and Crh2, with cleavage of β-1,4 linkages of the chitin backbone and transfer of the generated molecule containing newly created reducing end onto the glucan acceptor. A three-dimensional structure of Crh1 was generated by homology modeling based on the crystal structure of bacterial 1,3-1,4-β-d-glucanase, followed by site-directed mutagenesis to obtain molecular insights into how these enzymes achieve catalysis. The residues of both proteins that are involved in their catalytic and binding activities have been characterized by measuring the ability of yeast cells expressing different versions of these proteins to transglycosylate oligosaccharides derived from β-1,3-glucan, β-1,6-glucan and chitin to the chitin at the cell wall. Within the catalytic site, residues E134 and E138 of Crh1, as well as E166 and E170 of Crh2, corresponding to the nucleophile and general acid/base, and also the auxiliary D136 and D168 of Crh1 and Crh2, respectively, are shown to be essential for catalysis. Mutations of aromatic residues F152, Y160 and W219, located within the carbohydrate-binding cleft of the Crh1 model, also affect the transglycosylase activity. Unlike Crh1, Crh2 contains a putative carbohydrate-binding module (CBM18) of unknown function. Modeling and functional analysis of site-directed mutant residues of this CBM identified essential amino acids for protein folding and stability, as well as residues that tune the catalytic activity of Crh2.
Mapa génico del plásmido de virulencia de Salmonella enteritides y caracterización de regiones homólogas del cromosoma de Salmonella typhi
(2002) Rodríguez Peña, José Manuel; Rotger Anglada, Rafael
Se ha procedido a la obtención del mapa génico del plásmido de virulencia de Salmonella enteritidis mediante la secuenciación parcial de subciones procedentes de la genoteca plasmídica hindill. Las secuencias obtenidas fueron enviadas a bancos de secuencias internacionales con el fin de detectar secuencias homólogas. De esta forma se han podido situar sobre el plásmido en estudio todos los genes anteriormente descritos en los distintos plásmidos de virulencia de diferentes serotipos de Salmonella; así como, detectar un nuevo orf no descrito hasta el momento cuya secuencia deducida de aminoácidos mostró homología con la endonucleasa nuc del plásmido pkm101. Se ha localizado y caracterizado la región tra del plásmido, la de máxima divergencia frente al plásmido de virulencia de Salmonella typhimurium, encontrándose incompleta con respecto a la del plásmido f. Se ha llevado a cabo la detección, localización y clonación de la región homóloga al plásmido de virulencia presente en el cromosoma de Salmonella typhi. Tras la secuenciación parcial de las regiones cromosómicas homólogas se determinó la presencia de secuencias correspondientes a pefl, orf7, orf8 y orf9 descritas en el plásmido de S. typhimurium. debido a que el producto deducido del orf8 presenta identidad con proteínas tipo dsba se realizo la secuenciación en doble cadena tanto del orf cromosómico de S. typhi como del plasmídico de S. enteritidis homólogos al orf8, denominándose dlt y dle respectivamente. Las secuencias de aminoácidos deducidas de las nucleotídicas obtenidas mostraron características propias de proteínas tipo dsba, pudiendo por tanto presentar esa misma actividad en Salmonella
Slt2 MAPK association with chromatin is required for transcriptional activation of Rlm1 dependent genes upon cell wall stress.
(Biochimica et biophysica acta. Gene regulatory mechanisms, 2018) Sanz Santamaría, Ana Belén; García, Raúl; Rodríguez Peña, José Manuel; Nombela, César; Arroyo, Francisco
The regulation of gene expression through the cell wall integrity (CWI) pathway in yeast is mainly coordinated by the MAPK Slt2 and the transcription factor Rlm1. In this work, we elucidate a new role for Slt2 as a part of the transcriptional activation machinery that regulates CWI gene expression in response to cell wall stress. We show that Slt2 is recruited to promoters and coding regions of CWI Rlm1-dependent genes in response to stress. This phenomenon is dependent both on the activation of the MAPK and its kinase activity. Slt2 binding is also dependent on Rlm1 and SWI/SNF and SAGA complexes. During the initial steps of transcription, the catalytic activity of Slt2 on Rlm1 is critical for the binding of the activator to promoters in response to stress. In addition, Slt2 itself acts as a transactivator, as it is able to induce the transcription of CWI responsive genes when it is bound to promoters through the Rlm1 binding domain independently of its catalytic activity. Slt2 interacts with RNA Pol II in a Rlm1-dependent manner to provide further support to a role of this MAPK as an integral component of the transcriptional complexes under cell wall stress. Selective recruitment and progression of the complex Slt2-RNA Pol II from the promoters to the coding regions of Rlm1-dependent genes does not rely on Paf1, suggesting a different mechanism from that which is exerted by Slt2 on the Swi4/Swi6 (SBF)-regulated genes.
Chromatin remodeling by the SWI/SNF complex is essential for transcription mediated by the yeast cell wall integrity MAPK pathway.
(Molecular biology of the cell, 2012) Sanz Santamaría, Ana Belén; García Sánchez, Raúl; Rodríguez Peña, José Manuel; Díez Muñiz, Sonia; Nombela Cano, César; Peterson, Craig L.; Arroyo Nombela, Francisco Javier
In Saccharomyces cerevisiae, the transcriptional program triggered by cell wall stress is coordinated by Slt2/Mpk1, the mitogen-activated protein kinase (MAPK) of the cell wall integrity (CWI) pathway, and is mostly mediated by the transcription factor Rlm1. Here we show that the SWI/SNF chromatin-remodeling complex plays a critical role in orchestrating the transcriptional response regulated by Rlm1. swi/snf mutants show drastically reduced expression of cell wall stress-responsive genes and hypersensitivity to cell wall-interfering compounds. On stress, binding of RNA Pol II to the promoters of these genes depends on Rlm1, Slt2, and SWI/SNF. Rlm1 physically interacts with SWI/SNF to direct its association to target promoters. Finally, we observe nucleosome displacement at the CWI-responsive gene MLP1/KDX1, which relies on the SWI/SNF complex. Taken together, our results identify the SWI/SNF complex as a key element of the CWI MAPK pathway that mediates the chromatin remodeling necessary for adequate transcriptional response to cell wall stress.