CENTRO DE BIOLOGÍA MOLECULAR SEVERO OCHOACaptura de pantalla 2022 09 14 a las 10.27.10    

Representative publications

Rosario Francisco-Velilla, Azman Embarc-Buh, Salvador Abellan, Francisco del Caño-Ochoa, Santiago Ramón-Maiques, Encarnacion Martinez-Salas

Gemin5 is a multifunctional RNA binding protein (RBP) organized in domains with a distinctive structural organization. The protein is a hub for several protein networks performing diverse RNA-dependent func-tions including regulation of translation, and recognition of small nuclear RNAs (snRNAs). Here we sought to identify the presence of phosphoresidues on the C-terminal half of Gemin5, a region of the protein that harbors a tetratricopeptide repeat (TPR)-like dimerization domain and a non-canonical RNA binding site (RBS1). We identified two phosphoresidues in the purified protein: P-T897 in the dimerization domain and P-T1355 in RBS1. Replacing T897 and T1355 with alanine led to decreased translation, and mass spectrometry analysis revealed that mutation T897A strongly abrogates the association with cellular pro-teins related to the regulation of translation. In contrast, the phosphomimetic substitutions to glutamate partially rescued the translation regulatory activity. The structural analysis of the TPR dimerization domain indicates that local rearrangements caused by phosphorylation of T897 affect the conformation of the flexible loop 2–3, and propagate across the dimerization interface, impacting the position of the C-terminal helices and the loop 12–13 shown to be mutated in patients with neurological disorders. Computational analysis of the potential relationship between post-translation modifications and cur-rently known pathogenic variants indicates a lack of overlapping of the affected residues within the func-tional domains of the protein and provides molecular insights for the implication of the phosphorylated residues in translation regulation.
 2022 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY-NC-ND license (http://creative-commons.org/licenses/by-nc-nd/4.0/).

Karolina Jodkowska, Vera Pancaldi, Maria Rigau, Ricardo Almeida,
José M. Fernández-Justel, Osvaldo Graña-Castro, Sara Rodríguez-Acebes,
Miriam Rubio-Camarillo, Enrique Carrillo-de Santa Pau, David Pisano,
Fátima Al-Shahrour, Alfonso Valencia, María Gómez, and Juan Méndez

In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are as-sembled. Replicative stress triggers additional initi-ation events from ‘dormant’ origins whose genomic distribution and regulation are not well understood. In this study, we have analyzed origin activity in mouse embryonic stem cells in the absence or pres-ence of mild replicative stress induced by aphidi-colin, a DNA polymerase inhibitor, or by deregula-tion of origin licensing factor CDC6. In both cases, we observe that the majority of stress-responsive origins are also active in a small fraction of the cell population in a normal S phase, and stress in-creases their frequency of activation. In a search for the molecular determinants of origin efficiency, we compared the genetic and epigenetic features of ori-gins displaying different levels of activation, and integrated their genomic positions in three-dimensional chromatin interaction networks derived from high-depth Hi-C and promoter-capture Hi-C data. We re-port that origin efficiency is directly proportional to the proximity to transcriptional start sites and to the number of contacts established between origin-containing chromatin fragments, supporting the or-ganization of origins in higher-level DNA replication factories.

Roberto Del Amparo, Luis Daniel González‑Vázquez, Laura Rodríguez‑Moure, Ugo Bastolla, Miguel Arenas

Genetic recombination is a common evolutionary mechanism that produces molecular diversity. However, its consequences on protein folding stability have not attracted the same attention as in the case of point mutations. Here, we studied the effects of homologous recombination on the computationally predicted protein folding stability for several protein families, finding less detrimental effects than we previously expected. Although recombination can affect multiple protein sites, we found that the fraction of recombined proteins that are eliminated by negative selection because of insufficient stability is not significantly larger than the corresponding fraction of proteins produced by mutation events. Indeed, although recombi-nation disrupts epistatic interactions, the mean stability of recombinant proteins is not lower than that of their parents. On the other hand, the difference of stability between recombined proteins is amplified with respect to the parents, promoting phenotypic diversity. As a result, at least one third of recombined proteins present stability between those of their parents, and a substantial fraction have higher or lower stability than those of both parents. As expected, we found that parents with similar sequences tend to produce recombined proteins with stability close to that of the parents. Finally, the simulation of protein evolution along the ancestral recombination graph with empirical substitution models commonly used in phylogenet-ics, which ignore constraints on protein folding stability, showed that recombination favors the decrease of folding stability, supporting the convenience of adopting structurally constrained models when possible for inferences of protein evolutionary histories with recombination.

David G. Mı́guez, Antonella Iannini, Diana García-Morales, and Fernando Casares

Morphogens of the Hh family trigger gene expression changes in receiving cells in a concentration-dependent manner to regulate their identity, proliferation, death or metabolism, depending on the tissue or organ. This variety of responses relies on a conserved signaling pathway. Its logic includes a negative-feedback loop involving the Hh receptor Ptc. Here, using experiments and computational models we study and compare the different spatial signaling profiles downstream of Hh in several developing Drosophila organs. We show that the spatial distributions of Ptc and the activator transcription factor CiA in wing, antenna and ocellus show similar features, but are markedly different from that in the compound eye. We propose that these two profile types represent two time points along the signaling dynamics, and that the interplay between the spatial displacement of the Hh source in the compound eye and the negative-feedback loop maintains the receiving cells effectively in an earlier stage of signaling. These results show how the interaction between spatial and temporal dynamics of signaling and differentiation processes may contribute to the informational versatility of the conserved Hh signaling pathway.

Marı́a Hernández-Bejarano, Gaia Gestri, Clinton Monfries, Lisa Tucker, Elena I. Dragomir, Isaac H. Bianco, Paola Bovolenta, Stephen W. Wilson and Florencia Cavodeassi

Appropriate patterning of the retina during embryonic development is assumed to underlie the establishment of spatially localised specialisations that mediate the perception of specific visual features. For example, in zebrafish, an area involved in high acuity vision (HAA) is thought to be present in the ventro-temporal retina. Here, we show that the interplay of the transcription factor Rx3 with Fibroblast Growth Factor and Hedgehog signals initiates and restricts foxd1 expression to the prospective temporal retina, initiating naso-temporal regionalisation of the retina. Abrogation of Foxd1 results in the loss of temporal and expansion of nasal retinal character, and consequent absence of the HAA. These structural defects correlate with severe visual defects, as assessed in optokinetic and optomotor response assays. In contrast, optokinetic responses are unaffected in the opposite condition, in which nasal retinal character is lost at the expense of expanded temporal character. Our study indicates that the establishment of temporal retinal character during early retinal development is required for the specification of the HAA, and suggests a prominent role of the temporal retina in controlling specific visual functions.

Irene Bravo-Alonso, Matías Morin, Laura Arribas-Carreira,
Mar Álvarez, Consuelo Pedrón-Giner, Lucia Soletto, Carlos Santolaria, Santiago Ramón-Maiques, Magdalena Ugarte, Pilar Rodríguez-Pombo, Joaquín Ariño, Miguel Ángel Moreno-Pelayo, Belén Pérez

Coenzyme A (CoA) is an essential cofactor involved in a range of metabolic pathways including the activation of long-chain fatty acids for catabolism. Cells synthesize CoA de novo from vitamin B5 (pantothenate) via a pathway strongly conserved across prokaryotes and eukaryotes. In humans, it involves five enzymatic steps catalyzed by four enzymes: pantothenate kinase (PANK [isoforms 1–4]), 40-phosphopantothenoylcysteine synthetase (PPCS), phospho-pantothenoylcysteine decarboxylase (PPCDC), and CoA synthase (COASY). To date, inborn errors of metabolism associated with all of these genes, except PPCDC, have been described, two related to neurodegeneration with brain iron accumulation (NBIA), and one associated with a cardiac phenotype. This paper reports another defect in this pathway (detected in two sisters), associ-ated with a fatal cardiac phenotype, caused by biallelic variants (p.Thr53Pro and p.Ala95Val) of PPCDC. PPCDC enzyme (EC catalyzes the decar-boxylation of 40-phosphopantothenoylcysteine to 40-phosphopantetheine in CoA biosynthesis. The variants p.Thr53Pro and p.Ala95Val affect residues highly conserved across different species; p.Thr53Pro is involved in the binding of flavin mononucleotide, and p.Ala95Val is likely a destabilizing mutation. Patient-derived fibroblasts showed an absence of PPCDC protein, and nearly 50% reductions in CoA levels. The cells showed clear energy deficiency prob-lems, with defects in mitochondrial respiration, and mostly glycolytic ATP synthesis. Functional studies performed in yeast suggest these mutations to be functionally relevant. In summary, this work describes a new, ultra-rare, severe inborn error of metabolism due to pathogenic variants of PPCDC.

Ángel Gaudioso, Teresa P. Silva, María Dolores Ledesma

The lack of available treatments and fatal outcome in most lysosomal storage disorders (LSDs) have spurred research on pathological mechanisms and novel therapies in recent years. In this effort, experi-mental methodology in cellular and animal models have been developed, with aims to address major challenges in many LSDs such as patient-to-patient variability and brain condition. These techniques and models have advanced knowledge not only of LSDs but also for other lysosomal disorders and have provided fundamental insights into the biological roles of lysosomes. They can also serve to assess the efficacy of classical therapies and modern drug delivery systems. Here, we summarize the techniques and models used in LSD research, which include both established and recently developed in vitro meth-ods, with general utility or specifically addressing lysosomal features. We also review animal models of LSDs together with cutting-edge technology that may reduce the need for animals in the study of these devastating diseases.

Carla Sánchez-Castillo, María I. Cuartero, Alba Fernández-Rodrigo, Víctor Briz, Sergio López-García, Raquel Jiménez-Sánchez, Juan A. López, Mariona Graupera,
José A. Esteban

Neuronal connectivity and activity-dependent synaptic plasticity are fundamental properties that support brain function and cognitive performance. Phosphatidylinositol 3-kinase (PI3K) intracellular signaling controls multi-ple mechanisms mediating neuronal growth, synaptic structure, and plasticity. However, it is still unclear how these pleiotropic functions are integrated at molecular and cellular levels. To address this issue, we used neuron-
specific virally delivered Cre expression to delete either p110α or p110β (the two major catalytic isoforms of type I PI3K) from the hippocampus of adult mice. We found that dendritic and postsynaptic structures are almost
exclusively supported by p110α activity, whereas p110β controls neurotransmitter release and metabotropic
glutamate receptor–dependent long-term depression at the presynaptic terminal. In addition to these separate functions, p110α and p110β jointly contribute to N-methyl-D-aspartate receptor–dependent postsynaptic long-
term potentiation. This molecular and functional specialization is reflected in different proteomes controlled by each isoform and in distinct behavioral alterations for learning/memory and sociability in mice lacking p110α or p110β.

Carlos Verdú, Patricia Pérez-Arnaiz, Ana Peropadre, José Berenguer and Mario Mencía

DNA primase-polymerases (Ppol) have been shown to play active roles in DNA repair and damage tolerance, both in prokaryotes and eukaryotes. The ancestral thermophilic bacterium Thermus thermophilus strain HB27 encodes a Ppol protein among the genes present in mobile element ICETh2, absent in other T. thermophilus strains. Using different strategies we ablated the function of Ppol in HB27 cells, either by knocking out the gene through insertional mutagenesis, markerless deletion or through abolition of its catalytic activity. Whole genome sequencing of this diverse collection of Ppol mutants showed spontaneous loss of function mutation in the helicase-nuclease AddAB in every ppol mutant isolated. Given that AddAB is a major player in recombinational repair in many prokaryotes, with similar activity to the proteobacterial RecBCD complex, we have performed a detailed characterization of the ppol mutants in combination with addAB mutants. The results show that knockout addAB mutants are more sensitive to DNA damage agents than the wild type, and present a dramatic three orders of magnitude increase in natural transformation efficiencies with both plasmid and lineal DNA, whereas ppol mutants show defects in plasmid stability. Interestingly, DNA-integrity comet assays showed that the genome of all the ppol and/or addAB mutants was severely affected by widespread fragmentation, however, this did not translate in neat loss of viability of the strains.

Cristina Cacheiro-Llaguno, Elena Hernández-Subirá, Manuel D. Díaz-Muñoz, Manuel Fresno, Juan M. Serrador and Miguel A. Íñiguez

Cyclooxygenase (COX) is the key enzyme in prostanoid synthesis from arachidonic acid (AA). Two isoforms, named COX-1 and COX-2, are expressed in mammalian tissues. The expression of COX-2 isoform is induced by several stimuli including cytokines and mitogens, and this induction is inhibited by glucocorticoids (GCs). We have previously shown that the transcriptional induction of COX-2 occurs early after T cell receptor (TCR) triggering, suggesting functional implications of this enzyme in T cell activation. Here, we show that dexamethasone (Dex) inhibits nuclear factor of activated T cells (NFAT)-mediated COX-2 transcriptional induction upon T cell activation. This effect is dependent on the presence of the GC receptor (GR), but independent of a functional DNA binding domain, as the activation-deficient GRLS7 mutant was as effective as the wild-type GR in the repression of NFAT-dependent transcription. Dex treatment did not disturb NFAT dephosphorylation, but interfered with activation mediated by the N-terminal transactivation domain (TAD) of NFAT, thus pointing to a negative cross-talk between GR and NFAT at the nuclear level. These results unveil the ability of GCs to interfere with NFAT activation and the induction of pro-inflammatory genes such as COX-2, and explain some of their immunomodulatory properties in activated human T cells.

Daniel Pérez-Núñez, Sun-Young Sunwoo, Raquel García-Belmonte, Chansong Kim,
Gonzalo Vigara-Astillero, Elena Riera, Dae-min Kim, Jiyun Jeong, Dongseob Tark, Young-Seung Ko, Young-Kook You and Yolanda Revilla

African swine fever (ASF) is an obligated declaration swine disease, provoking farm isola-tion measures and the closing of affected country boarders. ASF virus (ASFV) is currently the cause of a pandemic across China and Eurasia. By the end of 2019, ASF was detected in nine EU Member States: Bulgaria, Romania, Slovakia, Estonia, Hungary, Latvia, Lithuania, Poland and Belgium. The affected area of the EU extended progressively, moving mostly in a southwestern direction (EFSA). Inactivated and/or subunit vaccines have proven to fail since certain virus replication is needed for protection. LAVs are thus the most realistic option, which must be safe, effective and industrially scalable. We here generated a vaccine prototype from the Arm/07/CBM/c2 genotype II strain, in which we have deleted the EP402R (CD2v) and A238L genes by CRISPR/Cas9 in COS-1 cells, without detectable further genetic changes. The successful immunization of pigs has proven this vaccine to be safe and fully protective against the circulating Korean Paju genotype II strain, opening the possibility of a new vaccine on the market in the near future.

Luis Valiente, Silvia López-Argüello, Alicia Rodríguez-Huete, Alejandro Valbuena, Mauricio G. Mateu

Human rhinovirus (HRV), one of the most frequent human pathogens, is the major causative agent of common colds. HRVs also cause or exacerbate severe respi-ratory diseases, such as asthma or chronic obstructive pulmonary disease. Despite the bio-medical and socioeconomic importance of this virus, no anti-HRV vaccines or drugs are available yet. Protein-protein interfaces in virus capsids have increasingly been recognized as promising virus-specific targets for the development of antiviral drugs. However, the specific structural elements and residues responsible for the biological functions of these extended capsid regions are largely unknown. In this study, we performed a thorough mutational analysis to determine which particular residues along the capsid interpentamer interfaces are relevant to HRV infection as well as the stage(s) in the viral cycle in which they are involved. The effect on the virion infectivity of the individual mutation to alanine of 32 interfacial residues that, together, removed most of the interpentamer interactions was analyzed. Then, a representative sample that included many of those 32 single mutants were tested for capsid and virion assembly as well as virion conformational sta-bility. The results indicate that most of the interfacial residues, and the interactions they establish, are biologically relevant, largely because of their important roles in virion assem-bly and/or stability. The HRV interpentamer interface is revealed as an atypical protein-protein interface, in which infectivity-determining residues are distributed at a high den-sity along the entire interface. Implications for a better understanding of the relationship between the molecular structure and function of HRV and the development of novel cap-sid interface-binding anti-HRV agents are discussed.

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