Person:
Rodríguez Escudero, María Isabel

First Name

María Isabel

Last Name

Rodríguez Escudero

URI

https://hdl.handle.net/20.500.14352/77769

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

Heterologous mammalian Akt disrupts plasma membrane homeostasis by taking over TORC2 signaling in Saccharomyces cerevisiae
(Scientific Reports, 2018) Rodríguez Escudero, María Isabel; Fernández-Acero Bascones, Teresa; Jiménez Cid, Víctor; Molina Martín, María
The Akt protein kinase is the main transducer of phosphatidylinositol-3,4,5-trisphosphate (PtdIns3,4,5P3) signaling in higher eukaryotes, controlling cell growth, motility, proliferation and survival. By co-expression of mammalian class I phosphatidylinositol 3-kinase (PI3K) and Akt in the Saccharomyces cerevisiae heterologous model, we previously described an inhibitory effect on yeast growth that relied on Akt kinase activity. Here we report that PI3K-Akt expression in yeast triggers the formation of large plasma membrane (PM) invaginations that were marked by actin patches, enriched in PtdIns4,5P2 and associated to abnormal intracellular cell wall deposits. These effects of Akt were mimicked by overproduction of the PtdIns4,5P2 effector Slm1, an adaptor of the Ypk1 and Ypk2 kinases in the TORC2 pathway. Although Slm1 was phosphorylated in vivo by Akt, TORC2-dependent Ypk1 activation did not occur. However, PI3K-activated Akt suppressed the lethality derived from inactivation of either TORC2 or Ypk protein kinases. Thus, heterologous co-expression of PI3K and Akt in yeast short-circuits PtdIns4,5P2- and TORC2-signaling at the level of the Slm-Ypk complex, overriding some of its functions. Our results underscore the importance of phosphoinositide-dependent kinases as key actors in the homeostasis and dynamics of the PM.
Functional analysis of PTEN variants of unknown significance from PHTS patients unveils complex patterns of PTEN biological activity in disease
(Eur J Hum Genet., 2023) Torices, Leire; Mingo, Janire; Rodríguez Escudero, María Isabel; Fernández-Acero Bascones, Teresa; Luna, Sandra; Nunes-Xavier, Caroline E.; López, José I.; Mercadillo, Fátima; Currás, María; Urioste, Miguel; Molina Martín, María; Jiménez Cid, Víctor; Pulido, Rafael
Heterozygous germline mutations in PTEN gene predispose to hamartomas and tumors in different tissues, as well as to neurodevelopmental disorders, and define at genetic level the PTEN Hamartoma Tumor Syndrome (PHTS). The major physiologic role of PTEN protein is the dephosphorylation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), counteracting the pro-oncogenic function of phosphatidylinositol 3-kinase (PI3K), and PTEN mutations in PHTS patients frequently abrogate PTEN PIP3 catalytic activity. PTEN also displays non-canonical PIP3-independent functions, but their involvement in PHTS pathogeny is less understood. We have previously identified and described, at clinical and genetic level, novel PTEN variants of unknown functional significance in PHTS patients. Here, we have performed an extensive functional characterization of these PTEN variants (c.77 C > T, p.(Thr26Ile), T26I; c.284 C > G, p.(Pro95Arg), P95R; c.529 T > A, p.(Tyr177Asn), Y177N; c.781 C > G, p.(Gln261Glu), Q261E; c.829 A > G, p.(Thr277Ala), T277A; and c.929 A > G, p.(Asp310Gly), D310G), including cell expression levels and protein stability, PIP3-phosphatase activity, and subcellular localization. In addition, caspase-3 cleavage analysis in cells has been assessed using a C2-domain caspase-3 cleavage-specific anti-PTEN antibody. We have found complex patterns of functional activity on PTEN variants, ranging from loss of PIP3-phosphatase activity, diminished protein expression and stability, and altered nuclear/cytoplasmic localization, to intact functional properties, when compared with PTEN wild type. Furthermore, we have found that PTEN cleavage at the C2-domain by the pro-apoptotic protease caspase-3 is diminished in specific PTEN PHTS variants. Our findings illustrate the multifaceted molecular features of pathogenic PTEN protein variants, which could account for the complexity in the genotype/phenotype manifestations of PHTS patients.
A global analysis of the reconstitution of PTEN function by translational readthrough of PTEN pathogenic premature termination codons
(Human mutation, 2021) Luna, Sandra; Torices, Leire; Mingo, Janire; Amo, Laura; Ruiz‐Ibarlucea, Pablo; Erramuzpe, Asier; Cortés, Jesús M.; Tejada, María I.; Nunes‐Xavier, Caroline E.; López, José I.; Pulido, Rafael; Rodríguez Escudero, María Isabel; Jiménez Cid, Víctor; Molina Martín, María
The PTEN tumor suppressor gene is mutated with high incidence in tumors and in thegermline of patients with cancer predisposition or with macrocephaly associatedwith autism.PTENnonsense mutations generating premature termination codons (PTC)and producing nonfunctional truncated PTEN proteins are frequent in association withhuman disease. However, there are no studies addressing the restoration of full‐lengthPTEN proteins from the PTC‐mutatedPTENgene by translational readthrough. Here,we have performed a global translational and functional readthrough analysis of thecomplete collection ofPTENPTC somatic or hereditary mutations found in tumors or inthegermlineofpatients(disease‐associated PTEN PTCome), and we set standards forthe analysis of the potential of readthrough functional reconstitution in disease‐relevant genes. Our analysis indicates that prevalent pathogenicPTENPTC mutationsare susceptible to PTEN functional restoration in response to readthrough‐inducingcompounds. Comprehensive readthrough analyses of disease‐associated PTComes willbe valuable tools for the implementation of readthrough‐based precision interventionsin specific groups of patients
The TIR-domain containing effectors BtpA and BtpB from Brucella abortus impact NAD metabolism
(Plos Pathogens, 2020) Coronas Serna, Julia María; Louche, Arthur; Roussin, Morgane; Imbert, Paul RC; Rodríguez Escudero, María Isabel; Terradot, laurent; Molina Martín, María; Gorvel, Jean-Pierre; Jiménez Cid, Víctor; Salcedo, Suzana P; Rodríguez-Escudero, Isabel
Brucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.
A trans-kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection
(Nat. Commun., 2023) Sá-Pessoa, Joana; López-Montesino, Sara; Przybyszewska, Kornelia; Marshall, Helina; Ova, Adelia; Schroeder, Gunnar N; Barabas, Peter; Curtis, Tim; Bengoechea, Jose A; Rodríguez Escudero, María Isabel; Jiménez Cid, Víctor; Molina Martín, María
Bacteria can inhibit the growth of other bacteria by injecting effectors using a type VI secretion system (T6SS). T6SS effectors can also be injected into eukaryotic cells to facilitate bacterial survival, often by targeting the cytoskeleton. Here, we show that the trans-kingdom antimicrobial T6SS effector VgrG4 from Klebsiella pneumoniae triggers the fragmentation of the mitochondrial network. VgrG4 colocalizes with the endoplasmic reticulum (ER) protein mitofusin 2. VgrG4 induces the transfer of Ca2+ from the ER to the mitochondria, activating Drp1 (a regulator of mitochondrial fission) thus leading to mitochondrial network fragmentation. Ca2+ elevation also induces the activation of the innate immunity receptor NLRX1 to produce reactive oxygen species (ROS). NLRX1-induced ROS limits NF-κB activation by modulating the degradation of the NF-κB inhibitor IκBα. The degradation of IκBα is triggered by the ubiquitin ligase SCFβ-TrCP, which requires the modification of the cullin-1 subunit by NEDD8. VgrG4 abrogates the NEDDylation of cullin-1 by inactivation of Ubc12, the NEDD8-conjugating enzyme. Our work provides an example of T6SS manipulation of eukaryotic cells via alteration of the mitochondria.
Project number: 21
De la “lección magistral” al Aprendizaje Basado en Proyectos (ABP) "personalizado" a las características del alumnado de Ciencias de la Salud
(2020) Rodríguez Escudero, María Isabel; Díez Orejas, Rosalía María; Navarro García, Federico; Pitarch Velasco, Aída; Pérez Chica, María Almudena
En el presente Proyecto llevamos a cabo la implantación del Aprendizaje Basado en Proyectos (ABP) en el aula universitaria utilizando la asignatura de Microbiología del Grado en Farmacia, en Ciencia y Tecnología de los Alimentos, y en Óptica y Optometría. En cada uno de dichos Grados se comparó esta metodología con la tradicional clase magistral y además se adaptó el ABP en los tres Grados, según la idiosincrasia del alumnado en cada uno de ellos, constituyendo lo que llamamos ABP “personalizado”.
Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent
(Plos pathogens, 2020) Storey, Daniel; McNally, Allan; Astrand, Mia; Sa-Pessoa Graca Santos, Joana; Rodriguez-escudero, Isabel; Elmore, Bronagh; Palacios, leyre; Marshall, Helina; Hobley, Laura; Molina, Maria; Iiménez Cid, Victor; Salminen, Tiina A; Bengoechea, Jose A; Molina Martín, María; Rodríguez Escudero, María Isabel; Molina, Maria; Jiménez Cid, Víctor; Molina Martín, María
Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possesses an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition and identified VgrG4 as a T6SS effector. The DUF2345 domain of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition.
MicroMundo@UCM: Ciencia ciudadana y aprendizaje-servicio frente a la resistencia a antibióticos desde la comunidad educativa
(2023) Jiménez Cid, Víctor; Ayllón Santiago, Tania; Amaro Torres, Francisco; Calvo De Pablo, Pilar; Domenech Lucas, Miriam; De Francisco Martínez, Patricia; Patiño Álvarez, Aurora Belén; Vázquez Estévez, María Covadonga Inmaculada; Barbero Úriz, Óscar; Blesa Esteban, Alba Mercedes; Borrajo López, Ana; Cortés Prieto, Isabel; Fernández-Acero Bascones, Teresa; Fernández-Vega Granado, Alejandro; García Pastor, Lucía; González Rubio, Gema; Lavilla García, Beatriz; López Montesino, Sara; Martínez López, Raquel María; Molina Martín, María; Mascaraque Martín, Victoria; Parra Giraldo, Claudia Marcela; Rodríguez Escudero, María Isabel; Rodríguez Fernández, Carmina; Román González, Elvira; Rubio Lozano, Alba Victoria; Del Val Oriza, Elba; Valentí Sanguino, Marta; Bezos Garrido, Javier; Borrero Del Pino, Juan; Díaz Formoso, Lara; Escudero García-Calderón, José Antonio; García Benzaquén, Nerea; González Zorn, Bruno; Hipólito Carrillo de Albornoz, Alberto; Muñoz Atienza, Estefanía; Pérez Sancho, Marta; Pulido Vadillo, Mario; Romero Martínez, Beatriz; Sánchez Méndez, Irene; Serna Bernaldo, Carlos; Suárez Rodríguez, Mónica; Vergara González, Ester; Arias López, Patricia; González de Figueras, Carolina; Prieto Orzanco, Alicia; Sempere García, Julio; Gil Serna, Jessica; Maestro García-Donas, María Beatriz; García García, Aina; Gil Serna, Jessica
Según ha establecido la OMS y la Asamblea de las Naciones Unidas, la resistencia a los antibióticos es una de las prioridades en Salud Global para el s. XXI, al mismo nivel que la preparación frente a pandemias emergentes y el desarrollo de vacunas y tratamientos frente a tuberculosis, malaria, HIV y otras enfermedades infecciosas. El abordaje de estos problemas sólo será eficaz desde la perspectiva multidisciplinar One Health (Un Mundo: Una Salud), que integra Salud Medioambiental, Animal y Humana. Desde 2016 la UCM ha sido pionera en implicar a las comunidades universitaria y preuniversitaria en la estrategia de aprendizaje activo mediante ApS “MicroMundo” (www.ucm.es/small-world-initiative), un proyecto multidisciplinar basado en ApS en el ámbito de la Biomedicina. Se trata de una adaptación al ApS de la estrategia internacional de studentsourcing Tiny Earth (www.tinyearthnet.org). MicroMundo implica a estudiantes de ESO y Bachillerato a participar como investigadores en un proyecto real para el descubrimiento de nuevos antibióticos a partir de muestras de suelos. El objetivo final del servicio es acercar la cultura científica, la perspectiva One Health y la investigación biomédica a la sociedad. Para ello, colaboramos con una red de colegios e institutos de la Comunidad de Madrid. Un objetivo adicional es fomentar la vocación por Grados STEM y por la I+D. Para lograr estos objetivos, los estudiantes universitarios, objeto del aprendizaje, se responsabilizan de facilitar a los jóvenes estudiantes el entrenamiento y material necesarios para llevar a cabo los experimentos, así como dirigir el trabajo de investigación y diversas estrategias de divulgación hacia la comunidad. El éxito del proyecto en la UCM ha inspirado su implementación en más de 30 universidades en España y Portugal y ha merecido el Premio PRAN 2021 a la comunicación y sensibilización de la población sobre la resistencia a los antibióticos. En esta publicación presentamos la memoria final del proyecto MicroMundo@UCM realizado durante el curso 2022-23 en 33 centros educativos de la Comunidad de Madrid.
Project number: 265
SWI@UCM 2.0: Consolidación de Small World Initiative: descubrimiento y uso racional de antibióticos mediante aprendizaje-servicio en la Comunidad de Madrid
(2018) Jiménez Cid, Víctor; Rodríguez Escudero, María Isabel; Díez Orejas, Rosalía María; Molina Martín, María; Rodríguez Fernández, Carmina; Navarro García, Federico; Arroyo Nombela, Francisco Javier; Román González, Elvira; Martín Brieva, Humberto; Fernández-Acero Bascones, Teresa; Sanz Santamaría, Ana Belén; Díaz Del Toro, Silvia; Calvo De Pablo, Pilar; Patiño Álvarez, Aurora Belén; González Zorn, Bruno; Suárez Rodríguez, Mónica; Goyache Goñi, Joaquín; Escudero García-Calderón, José Antonio; Prieto Prieto, Antonio Daniel; Ugarte Ruiz, María; Gil Serna, Jessica
Durante el curso anterior un equipo de la UCM en el marco de un proyecto previo INNOVA-Docencia UCM instauró en España de manera pionera la iniciativa de aprendizaje activo Small World Initiative, de origen norteamericano. Los objetivos de esta iniciativa son (1) Crear cultura científica y acercar la investigación biomédica a niveles educativos en los que los estudiantes tienen aún capacidad de decisión sobre su futura orientación formativa con el fin de fomentar la vocación en I+D; y (2) Promover la concienciación social sobre el uso racional de los antibióticos y la amenaza de la resistencia bacteriana a estos fármacos. En el entorno español se propuso implementar esta estrategia mediante Aprendizaje-Servicio. En esta segunda edición (SWI@UCM 2.0) se ha trabajado en la consolidación, expansión y mejora del proyecto, con énfasis en la integración de los diversos niveles educativos que integran el proyecto (universitario y preuniversitario) y la divulgación científica.
Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation.
(PloS one, 2016) Cid, Víctor J.; Molina, María; Schulze Luehrmann, Jan; Lührmann, Anja; Rodríguez Escudero, María Isabel
Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors.