Person: Rodríguez Peña, José Manuel
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First Name
José Manuel
Last Name
Rodríguez Peña
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Farmacia
Department
Microbiología y Parasitología
Area
Microbiología
Identifiers
7 results
Search Results
Now showing 1 - 7 of 7
Item 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, JavierAs 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.Item 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, JavierYeast 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.Item 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, JavierBACKGROUND 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.Item 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, JavierCovalent 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.Item 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, FranciscoThe 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.Item 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 JavierIn 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.Item Signalling through the yeast MAPK Cell Wall Integrity pathway controls P-body assembly upon cell wall stress.(Scientific reports, 2019) García, Raúl; Pulido, Verónica; Orellana Muñoz, Sara; Nombela, César; Vázquez de Aldana, Carlos R.; Rodríguez Peña, José Manuel; Arroyo, JavierPost-transcriptional control of mRNA is a key event in the regulation of gene expression. From yeast to human cells, P-bodies are cytoplasmic RNA-protein aggregates that play an essential role in this process, particularly under stress conditions. In this work, we show that in the model yeast Saccharomyces cerevisiae cell wall stress induces the formation of these structures. This effect is dependent on multiple elements in the Cell Wall Integrity (CWI) MAPK signalling pathway, a signal transduction cascade responsible for the maintenance of cell integrity under adverse environmental conditions. Remarkably, P-body assembly requires the catalytic activity of the MAPK of the pathway, Slt2/Mpk1. In accordance with the control exerted by this signalling pathway, the timing of P-body formation is similar to that of the activation of the CWI pathway. Noticeably, mRNAs whose expression is regulated by this pathway localize in P-bodies after the cell is exposed to stress following a temporal pattern coincident with CWI pathway activation. Moreover, when these mRNAs are overexpressed in a mutant background unable to form visible P-bodies, the cells show hypersensitivity to agents that interfere with cell wall integrity, supporting that they play a role in the mRNA lifecycle under stress conditions.