Stabilization of the RAS:PDE6D complex is a novel strategy to inhibit RAS signaling

This work was supported by Cancer Research UK core funding number A17196.RAS is a major anticancer drug target which requires membrane localization to activate downstream signal transduction. The direct inhibition of RAS has proven to be challenging. Here, we present a novel strategy for targeting RAS by stabilizing its interaction with the prenyl-binding protein PDE6D and disrupting its localization. Using rationally designed RAS point mutations, we were able to stabilize the RAS:PDE6D complex by increasing the affinity of RAS for PDE6D, which resulted in the redirection of RAS to the cytoplasm and the primary cilium and inhibition of oncogenic RAS/ERK signaling. We developed an SPR fragment screening and identified fragments that bind at the KRAS:PDE6D interface, as shown through cocrystal structures. Finally, we show that the stoichiometric ratios of KRAS:PDE6D vary in different cell lines, suggesting that the impact of this strategy might be cell-type-dependent. This study forms the foundation from which a potential anticancer small-molecule RAS:PDE6D complex stabilizer could be developed.Publisher PDFPeer reviewe

Structure-based design, synthesis and biological evaluation of a novel series of isoquinolone and pyrazolo[4,3-c]pyridine inhibitors of fascin 1 as potential anti-metastatic agents

Fascin is an actin binding and bundling protein that is not expressed in normal epithelial tissues but overexpressed in a variety of invasive epithelial tumors. It has a critical role in cancer cell metastasis by promoting cell migration and invasion. Here we report the crystal structures of fascin in complex with a series of novel and potent inhibitors. Structure-based elaboration of these compounds enabled the development of a series with nanomolar affinities for fascin, good physicochemical properties and the ability to inhibit fascin-mediated bundling of filamentous actin. These compounds provide promising starting points for fascin-targeted anti-metastatic therapies

The Level of the Transcription Factor Pax6 Is Essential for Controlling the Balance between Neural Stem Cell Self-Renewal and Neurogenesis

Neural stem cell self-renewal, neurogenesis, and cell fate determination are processes that control the generation of specific classes of neurons at the correct place and time. The transcription factor Pax6 is essential for neural stem cell proliferation, multipotency, and neurogenesis in many regions of the central nervous system, including the cerebral cortex. We used Pax6 as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in stem cells of the developing mouse cerebral cortex. We identified the genomic binding locations of Pax6 in neocortical stem cells during normal development and ascertained the functional significance of genes that we found to be regulated by Pax6, finding that Pax6 positively and directly regulates cohorts of genes that promote neural stem cell self-renewal, basal progenitor cell genesis, and neurogenesis. Notably, we defined a core network regulating neocortical stem cell decision-making in which Pax6 interacts with three other regulators of neurogenesis, Neurog2, Ascl1, and Hes1. Analyses of the biological function of Pax6 in neural stem cells through phenotypic analyses of Pax6 gain- and loss-of-function mutant cortices demonstrated that the Pax6-regulated networks operating in neural stem cells are highly dosage sensitive. Increasing Pax6 levels drives the system towards neurogenesis and basal progenitor cell genesis by increasing expression of a cohort of basal progenitor cell determinants, including the key transcription factor Eomes/Tbr2, and thus towards neurogenesis at the expense of self-renewal. Removing Pax6 reduces cortical stem cell self-renewal by decreasing expression of key cell cycle regulators, resulting in excess early neurogenesis. We find that the relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-renewal

Genome-Wide Scan on Total Serum IgE Levels Identifies FCER1A as Novel Susceptibility Locus

High levels of serum IgE are considered markers of parasite and helminth exposure. In addition, they are associated with allergic disorders, play a key role in anti-tumoral defence, and are crucial mediators of autoimmune diseases. Total IgE is a strongly heritable trait. In a genome-wide association study (GWAS), we tested 353,569 SNPs for association with serum IgE levels in 1,530 individuals from the population-based KORA S3/F3 study. Replication was performed in four independent population-based study samples (total n = 9,769 individuals). Functional variants in the gene encoding the alpha chain of the high affinity receptor for IgE (FCER1A) on chromosome 1q23 (rs2251746 and rs2427837) were strongly associated with total IgE levels in all cohorts with P values of 1.85×10−20 and 7.08×10−19 in a combined analysis, and in a post-hoc analysis showed additional associations with allergic sensitization (P = 7.78×10−4 and P = 1.95×10−3). The “top” SNP significantly influenced the cell surface expression of FCER1A on basophils, and genome-wide expression profiles indicated an interesting novel regulatory mechanism of FCER1A expression via GATA-2. Polymorphisms within the RAD50 gene on chromosome 5q31 were consistently associated with IgE levels (P values 6.28×10−7−4.46×10−8) and increased the risk for atopic eczema and asthma. Furthermore, STAT6 was confirmed as susceptibility locus modulating IgE levels. In this first GWAS on total IgE FCER1A was identified and replicated as new susceptibility locus at which common genetic variation influences serum IgE levels. In addition, variants within the RAD50 gene might represent additional factors within cytokine gene cluster on chromosome 5q31, emphasizing the need for further investigations in this intriguing region. Our data furthermore confirm association of STAT6 variation with serum IgE levels

I Jornada de Aulas Abiertas: Encuentro de Docentes de la Facultad de Ciencias Económicas

La Jornada de Aulas Abiertas quiere ser una oportunidad para que los docentes de la Facultad de Ciencias Económicas nos encontremos en un espacio de reflexión y revisión de nuestras prácticas, distendido, cálido y respetuoso, que nos permita compartir nuestras experiencias cotidianas en las aulas, tanto presenciales como virtuales. Es la posibilidad de conocernos, intercambiar, aprender y contagiarnos de las inquietudes y el entusiasmo que muchos docentes ponen en juego cotidianamente. En el marco de propuestas de enseñanza, se analizaron recursos multimediales, materiales de estudio, aulas virtuales, redes sociales, aplicaciones web, juegos y actividades de evaluación y coevaluación originales; también se abordaron problemáticas y propuestas para favorecer vinculaciones con la práctica profesional. Estas fueron algunas de las cuestiones abordadas y compartidas en las presentaciones de nuestros colegas. Distintas propuestas, pero siempre con el propósito de favorecer las oportunidades de aprendizaje de nuestros estudiantes. Esta publicación pretende ampliar el alcance de esta actividad. Es una invitación para que los y las docentes que participaron puedan revisar nuevamente aquellas actividades que les parecieron valiosas, o las que no pudieron presenciar. Y para aquellos/as que no tuvieron la posibilidad de estar presentes, puedan descubrir cuánto podemos hacer para que nuestros estudiantes aprendan más y mejor, y se animen a iniciar sus propios recorridos. Esperamos repetir este evento para seguir aprendiendo de las iniciativas de los/las docentes de nuestra Facultad, poder hablar de lo que nos preocupa y nos enorgullece, en particular de las propuestas que desarrollamos en el aula para favorecer la comprensión, promover el entusiasmo, abordar temas complejos y errores frecuentes de nuestros estudiantes. Desde el Área de Formación Docente y Producción Educativa queremos agradecer a las autoridades de nuestra Facultad por acompañarnos en este desafío y a los/las docentes que estuvieron presentes compartiendo sus experiencias.Fil: Sabulsky, Gabriela. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Margaría, Oscar A. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Iturralde, Ivan. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Domenech, Roberto. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Torrico, Julieta. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Estigarribia, Lucrecia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Gohlke, Guillermo. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Rosenfeld, Valeria. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Montenjano, Franco. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Atienza, Bárbara. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Becerra, Natalia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Alonso, Micaela. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Tomatis, Karina. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Saunders, Shirley. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: David, María Laura. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Flores, Verónica Andrea. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Heckmann, Gerardo. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Vega, Juan José. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Trucchi, Carlos. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Ferro, Flavia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Díaz, Cecilia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Peretto, Claudia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Racagni, Josefina. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Guardiola, Mariana. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: López, Sonia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Beltrán, Natacha. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Russo, Paulo. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Sánchez, Pablo. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Rocha Vargas, Marcelo. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Flores, Norma. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Arévalo, Eliana. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Pacheco, Verónica. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Delmonte, Laura. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Stanecka, Nancy. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Caminos, Ana Belén. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Ahumada, María Inés. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Caro, Norma Patricia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Bravino, Laura. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Giménez, Siria Miriam. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Perona, Eugenia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Cuttica, Mariela. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: García, Gladys Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Cohen, Natalia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Tapia, Sebastián. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Erazu, Damián. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Torres, César. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Casini, Rosanna Beatriz. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Rosales, Julio. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Infante, Roberto Adrián. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Ricci, María Beatriz. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Römer, Gabriela. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Goyeneche, Noel. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Marzo, Emanuel. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Olmos, Mariano. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Bottino, Cecilia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Cacciagiú, Victor. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Scidá, María Florencia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Guajardo Molina, Vanesa. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Batistella, Silvana del V. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Huanchicay, Silvia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Jones, Carola. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Cassutti, Marcela Beatriz. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Sánchez, Juan Nicolás. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Arónica, Sandra. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Ortega, Fernando. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Peretti, Florencia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Tagle, María Mercedes. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Asís, Gloria Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Ortiz Figueroa, Ana María. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Giménez, Miriam Mónica. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Magnano, Cecilia. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina.Fil: Arias, Verónica. Universidad Nacional de Córdoba. Facultad de Ciencias Económicas; Argentina

Hydrophobic alkyl chains substituted to the 8-position of cyclic nucleotides enhance activation of CNG and HCN channels by an intricate enthalpy - entropy compensation

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are tetrameric non-specific cation channels in the plasma membrane that are activated by either cAMP or cGMP binding to specific binding domains incorporated in each subunit. Typical apparent affinities of these channels for these cyclic nucleotides range from several hundred nanomolar to tens of micromolar. Here we synthesized and characterized novel cAMP and cGMP derivatives by substituting either hydrophobic alkyl chains or similar-sized more hydrophilic heteroalkyl chains to the 8-position of the purine ring with the aim to obtain full agonists of higher potency. The compounds were tested in homotetrameric CNGA2, heterotetrameric CNGA2:CNGA4:CNGB1b and homotetrameric HCN2 channels. We show that nearly all compounds are full agonists and that longer alkyl chains systematically increase the apparent affinity, at the best more than 30 times. The effects are stronger in CNG than HCN2 channels which, however, are constitutively more sensitive to cAMP. Kinetic analyses reveal that the off-rate is significantly slowed by the hydrophobic alkyl chains. Molecular dynamics simulations and free energy calculations suggest that an intricate enthalpy - entropy compensation underlies the higher apparent affinity of the derivatives with the longer alkyl chains, which is shown to result from a reduced loss of configurational entropy upon binding

High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis

International audienceIn vivo membrane fusion primarily occurs between highly curved vesicles and planar membranes. A better understanding of fusion entails an accurate in vitro reproduction of the process. To date, supported bilayers have been commonly used to mimic the planar membranes. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that induce membrane fusion usually have limited fluidity when embedded in supported bilayers. This alters the kinetics and prevents correct reconstitution of the overall fusion process. Also, observing content release across the membrane is hindered by the lack of a second aqueous compartment. Recently, a step toward resolving these issues was achieved by using membranes spread on holey substrates. The mobility of proteins was preserved but vesicles were prone to bind to the substrate when reaching the edge of the hole, preventing the observation of many fusion events over the suspended membrane. Building on this recent advance, we designed a method for the formation of pore-spanning lipid bilayers containing t-SNARE proteins on Si/SiO2 holey chips, allowing the observation of many individual vesicle fusion events by both lipid mixing and content release. With this setup, proteins embedded in the suspended membrane bounced back when they reached the edge of the hole which ensured vesicles did not bind to the substrate. We observed SNARE-dependent membrane fusion with the freestanding bilayer of about 500 vesicles. The time between vesicle docking and fusion is ∼1 s. We also present a new multimodal open-source software, Fusion Analyzer Software, which is required for fast data analysis

Calmodulin extracts the Ras family protein RalA from lipid bilayers by engagement with two membrane-targeting motifs.

RalA is a small GTPase and a member of the Ras family. This molecular switch is activated downstream of Ras and is widely implicated in tumor formation and growth. Previous work has shown that the ubiquitous Ca2+-sensor calmodulin (CaM) binds to small GTPases such as RalA and K-Ras4B, but a lack of structural information has obscured the functional consequences of these interactions. Here, we have investigated the binding of CaM to RalA and found that CaM interacts exclusively with the C terminus of RalA, which is lipidated with a prenyl group in vivo to aid membrane attachment. Biophysical and structural analyses show that the two RalA membrane-targeting motifs (the prenyl anchor and the polybasic motif) are engaged by distinct lobes of CaM and that CaM binding leads to removal of RalA from its membrane environment. The structure of this complex, along with a biophysical investigation into membrane removal, provides a framework with which to understand how CaM regulates the function of RalA and sheds light on the interaction of CaM with other small GTPases, including K-Ras4B