Artículos (Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER))

URI permanente para esta colecciónhttps://hdl.handle.net/11441/11090

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Mostrando 1 - 20 de 198

Lack of sugar discrimination by human pol requires a single glycine residue
(2003) Blanco Dávila, Luis; Ruiz Pérez, José Francisco; García Díaz, Miguel; Juárez, Raquel; Terrados, Gloria; Picher, Ángel J.; González Barrera, Sergio; Fernández de Henestrosa, Antonio R.; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER)
An improved system for estradiol-dependent regulation of gene expression in yeast
(2007) Chávez de Diego, Sebastián; Cebolla Ramírez, Ángel; Arévalo Rodríguez, Miguel; Quintero Carrasco, María José; Maya Miles, Douglas; Universidad de Sevilla. Departamento de Genética; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER)
CDK targets Sae2 to control DNA-end resection and homologous recombination
(Nature Publishing Group, 2008) Huertas Sánchez, Pablo; Cortés Ledesma, Felipe; Sartori, Alessandro A.; Aguilera López, Andrés; Jackson, Stephen P.; Universidad de Sevilla. Departamento de Genética
DNA double-strand breaks (DSBs) are repaired by two principal mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR)1. HR is the most accurate DSB repair mechanism but is generally restricted to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as a repair template2-5. By contrast, NHEJ operates throughout the cell cycle but assumes most importance in G1 (refs 4, 6). The choice between repair pathways is governed by cyclin-dependent protein kinases (CDKs)2,3,5,7, with a major site of control being at the level of DSB resection, an event that is necessary for HR but not NHEJ, and which takes place most effectively in S and G2 (refs 2, 5). Here we establish that cell-cycle control of DSB resection in Saccharomyces cerevisiae results from the phosphorylation by CDK of an evolutionarily conserved motif in the Sae2 protein. We show that mutating Ser 267 of Sae2 to a non-phosphorylatable residue causes phenotypes comparable to those of a sae2Δ null mutant, including hypersensitivity to camptothecin, defective sporulation, reduced hairpin-induced recombination, severely impaired DNA-end processing and faulty assembly and disassembly of HR factors. Furthermore, a Sae2 mutation that mimics constitutive Ser 267 phosphorylation complements these phenotypes and overcomes the necessity of CDK activity for DSB resection. The Sae2 mutations also cause cell-cycle-stage specific hypersensitivity to DNA damage and affect the balance between HR and NHEJ. These findings therefore provide a mechanistic basis for cell-cycle control of DSB repair and highlight the importance of regulating DSB resection.
Dysfunction of the unfolded protein response increases neurodegeneration in aged rat hippocampus following proteasome inhibition
(Wiley-Blackwell, 2009) Gavilán Dorronzoro, María de la Paz; Pintado Losa, Cristina; Gavilán Dorronzoro, Elena; Jiménez Muñoz, Sebastián; Ríos Sánchez, Rosa María; Vitorica Ferrández, Francisco Javier; Castaño Navarro, Angélica; Ruano Caballero, Diego; Universidad de Sevilla. Departamento de Bioquímica y Biología Molecular; Universidad de Sevilla. Departamento de Citología e Histología Normal y Patológica
Dysfunctions of the ubiquitin proteasome system (UPS) have been proposed to be involved in the aetiology and/or progression of several age-related neurodegenerative disorders. However, the mechanisms linking proteasome dysfunction to cell degeneration are poorly understood. We examined in young and aged rat hippocampus the activation of the unfolded protein response (UPR) under cellular stress induced by proteasome inhibition. Lactacystin injection blocked proteasome activity in young and aged animals in a similar extent and increased the amount of ubiquitinated proteins. Young animals activated the three UPR arms, IRE1α, ATF6α and PERK, whereas aged rats failed to induce the IRE1α and ATF6α pathways. In consequence, aged animals did not induce the expression of pro-survival factors (chaperones, Bcl-XL and Bcl-2), displayed a more sustained expression of proapoptotic markers (CHOP, Bax, Bak and JKN), an increased caspase-3 processing. At the cellular level, proteasome inhibition induced neuronal damage in young and aged animals as assayed using Fluorojade-B staining. However, degenerating neurons were evident as soon as 24 h postinjection in aged rats, but it was delayed up to 3 days in young animals. Our findings show evidence supporting age-related dysfunctions in the UPR activation as a potential mechanism linking protein accumulation to cell degeneration. An imbalance between pro-survival and pro-apoptotic proteins, because of noncanonical activation of the UPR in aged rats, would increase the susceptibility to cell degeneration. These findings add a new molecular vision that might be relevant in the aetiology of several age-related neurodegenerative disorders.
Transcription at the proximity of the nuclear pore: A role for the THP1-SAC3-SUS1-CDC31 (THSC) complex
(Landes Bioscience, 2009) Luna Varo, Rosa María; González Aguilera, Cristina; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética
A key aspect of eukaryotic gene expression is the coupling of transcription with RNA processing, polyadenylation and export. The use of new techniques based on tandem affinity purification (TAP) and chromatin immunoprecipitation (ChIP), and of genetic and cell biology approaches has contributed to the beginning of deciphering the network of protein-mRNA interactions accompanying this coupling. Although an extensive amount of work has shed light on this matter, the order of participation and precise role of the different proteins remain to be deciphered. It seems that different and sequential protein interactions must converge to finally promote the anchoring of genes to the nuclear periphery. Here we discuss the new data on the coupling of gene expression and RNA export, with emphasis on the THP1-SAC3-SUS1-CDC31 complex and the possible implications of these results on transcription at the nuclear pore.
R-loops do not accumulate in transcription-defective hpr1-101 mutants: implications for the functional role of THO/TREX
(2009) Aguilera López, Andrés; Gómez González, Belén; Universidad de Sevilla. Departamento de Genética; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER)
Golgi localisation of GMAP210 requires two distinct cis-membrane binding mechanisms
(BMC, 2009) Cárdenas, Jesús; Rivero Canalejo, Sabrina; Goud, Bruno; Bornens, Michel; Ríos, Rosa M.; Universidad de Sevilla. Departamento de Citología e Histología Normal y Patológica
Background The Golgi apparatus in mammals appears as a ribbon made up of interconnected stacks of flattened cisternae that is positioned close to the centrosome in a microtubule-dependent manner. How this organisation is achieved and retained is not well understood. GMAP210 is a long coiled-coil cis-Golgi associated protein that plays a role in maintaining Golgi ribbon integrity and position and contributes to the formation of the primary cilium. An amphipathic alpha-helix able to bind liposomes in vitro has been recently identified at the first 38 amino acids of the protein (amphipathic lipid-packing sensor motif), and an ARF1-binding domain (Grip-related Arf-binding domain) was found at the C-terminus. To which type of membranes these two GMAP210 regions bind in vivo and how this contributes to GMAP210 localisation and function remains to be investigated. Results By using truncated as well as chimeric mutants and videomicroscopy we found that both the N-terminus and the C-terminus of GMAP210 are targeted to the cis-Golgi in vivo. The ALPS motif was identified as the N-terminal binding motif and appeared concentrated in the periphery of Golgi elements and between Golgi stacks. On the contrary, the C-terminal domain appeared uniformly distributed in the cis-cisternae of the Golgi apparatus. Strikingly, the two ends of the protein also behave differently in response to the drug Brefeldin A. The N-terminal domain redistributed to the endoplasmic reticulum (ER) exit sites, as does the full-length protein, whereas the C-terminal domain rapidly dissociated from the Golgi apparatus to the cytosol. Mutants comprising the full-length protein but lacking one of the terminal motifs also associated with the cis-Golgi with distribution patterns similar to those of the corresponding terminal end whereas a mutant consisting in fused N- and C-terminal ends exhibits identical localisation as the endogenous protein. Conclusion We conclude that the Golgi localisation of GMAP210 is the result of the combined action of the two N- and C-terminal domains that recognise different sub-regions of the cis-GA. Based on present and previous data, we propose a model in which GMAP210 would participate in homotypic fusion of cis-cisternae by anchoring the surface of cisternae via its C-terminus and projecting its distal N-terminus to bind the rims or to stabilise tubular structures connecting neighbouring cis-cisternae.
The SWR1 histone replacement complex causes genetic instability and genome-wide transcription misregulation in the absence of H2A.Z
(2010) Andújar, Eloísa; Morillo Huesca, Macarena; Prado Velasco, José Félix; Clemente Ruiz, Marta; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER)
Low concentrations of nitric oxide delay the differentiation of embryonic stem cells and promote their survival
(2010) Martín, F.; Tejedo Huamán, Juan Rigoberto; Tapia Limonchi, R.; Mora Castilla, S.; Hmadcha, Abdelkrim; Soria Escoms, Bernat; Bedoya Bergua, Francisco Javier; Cahuana Macedo, Gladys Margot; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER)
Application of flow focusing to the break-up of a magnetite suspension jet for the production of paramagnetic microparticles
(2011) Martín Banderas, Lucía; González Prieto, Román; Rodríguez Gil, Alfonso; Fernández Arévalo, María Mercedes; Flores Mosquera, María; Chávez de Diego, Sebastián; Gañán-Calvo, Alfonso M.; Universidad de Sevilla. Departamento de Farmacia y Tecnología Farmacéutica; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Ingeniería Aeroespacial y Mecánica de Fluidos; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER)
Paramagnetic particles offer an extensive improvement in the magnetic separation or purification of a wide variety of protein molecules. Most commercial paramagnetic particles are synthesized by laborious and costly procedures. A straightforward production of paramagnetic microparticles with homogeneous and selectable sizes using flow focusing (FF) technology is described in this work. The development of an initial formulation of a stable iron oxide suspension compatible with the FF requirements is also reported. The obtained particles, below 10 microns in diameter and presenting smooth and reactive surface, were codified with an organic fluorophore and showed excellent properties for covalent attachment of biomolecules such as proteins and its subsequent recognition by flow cytometry. Furthermore, particles with suitable magnetite content resulted as well-suited for commercial magnet separators for these purposes.
Immunohistochemical assessment of Pax8 expression during pancreatic islet development and in human neuroendocrine tumors
(Springer Verlag, 2011) Lorenzo, Petra I.; Jiménez Moreno, Carmen María; Delgado, Irene; Cobo Vuilleumier, Nadia; Meier, Rafael; Gómez Izquierdo, Lourdes; Berney, Thierry; García Carbonero, Rocío; Rojas, Anabel; Gauthier, Benoit R.; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía
The paired box transcription factor Pax8 is critical for development of the eye, thyroid gland as well as the urinary and reproductive organs. In adult, Pax8 overexpression is associated with kidney, ovarian and thyroid tumors and has emerged as a specific marker for these cancers. Recently, Pax8 expression was also reported in human pancreatic islets and in neuroendocrine tumors, identifying Pax8 as a novel member of the Pax family expressed in the pancreas. Herein, we sought to provide a comprehensive analysis of Pax8 expression during pancreogenesis and in adult islets. Immunohistochemical analysis using the most employed Pax8 polyclonal antibody revealed strong nuclear staining in the developing mouse pancreas and in mature human and mouse islets. Astonishingly, Pax8 mRNA in mouse islets was undetectable while human islets exhibited low levels. These discrepancies raised the possibility of antibody cross-reactivity. This premise was confirmed by demonstrating that the polyclonal Pax8 antibody also recognized the islet-enriched Pax6 protein both by Western blotting and immunohistochemistry. Thus, in islets polyclonal Pax8 staining corresponds mainly to Pax6. In order to circumvent this caveat, a novel Pax8 monoclonal antibody was used to re-evaluate whether Pax8 was indeed expressed in islets. Surprisingly, Pax8 was not detected in neither the developing pancreas or in mature islets. Reappraisal of pancreatic neuroendocrine tumors using this Pax8 monoclonal antibody exhibited no immunostaining as compared to the Pax8 polyclonal antibody. In conclusion, Pax8 is not expressed in the pancreas and cast doubts on the value of Pax8 as a pancreatic neuroendocrine tumor marker.
The Rad50 coiled-coil domain is indispensable for Mre11 complex functions
(Nature Publishing Group, 2011) Hohl, Marcel; Muñoz Galván, Sandra; Tous Rivera, Cristina; Aguilera López, Andrés; Petrini, John H. J.; Universidad de Sevilla. Departamento de Genética; Swiss National Science Foundation (SNFS); Ministerio de Ciencia e Innovación (MICIN). España
The Mre11 complex (Mre11, Rad50 and Xrs2 in Saccharomyces cerevisiae) influences diverse functions in the DNA damage response. The complex comprises the globular DNA-binding domain and the Rad50 hook domain, which are linked by a long and extended Rad50 coiled-coil domain. In this study, we constructed rad50 alleles encoding truncations of the coiled-coil domain to determine which Mre11 complex functions required the full length of the coils. These mutations abolished telomere maintenance and meiotic double-strand break (DSB) formation, and severely impaired homologous recombination, indicating a requirement for long-range action. Nonhomologous end joining, which is probably mediated by the globular domain of the Mre11 complex, was also severely impaired by alteration of the coiled-coil and hook domains, providing the first evidence of their influence on this process. These data show that functions of Mre11 complex are integrated by the coiled coils of Rad50.
TDP2/TTRAP Is the Major 5′-Tyrosyl DNA Phosphodiesterase Activity in Vertebrate Cells and Is Critical for Cellular Resistance to Topoisomerase II-induced DNA Damage
(American Society for Biochemistry and Molecular Biology, 2011) Zeng, Zhihong; Cortés Ledesma, Felipe; El Khamisy, Sherif F.; Caldecott, Keith W.
Topoisomerase II (Top2) activity involves an intermediate in which the topoisomerase is covalently bound to a DNA double-strand break via a 5′-phosphotyrosyl bond. Although these intermediates are normally transient, they can be stabilized by antitumor agents that act as Top2 “poisons,” resulting in the induction of cytotoxic double-strand breaks, and they are implicated in the formation of site-specific translocations that are commonly associated with cancer. Recently, we revealed that TRAF and TNF receptor-associated protein (TTRAP) is a 5′-tyrosyl DNA phosphodiesterase (5′-TDP) that can cleave 5′-phosphotyrosyl bonds, and we denoted this protein tyrosyl DNA phosphodiesterase-2 (TDP2). Here, we have generated TDP2-deleted DT40 cells, and we show that TDP2 is the major if not the only 5′-TDP activity present in vertebrate cells. We also show that TDP2-deleted DT40 cells are highly sensitive to the anticancer Top2 poison, etoposide, but are not hypersensitive to the Top1 poison camptothecin or the DNA-alkyating agent methyl methanesulfonate. These data identify an important mechanism for resistance to Top2-induced chromosome breakage and raise the possibility that TDP2 is a significant factor in cancer development and treatment.
Zim17/Tim15 links mitochondrial iron–sulfur cluster biosynthesis to nuclear genome stability
(Oxford University Press, 2011) Díaz de la Loza, María del Carmen; Gallardo Ortega, Mercedes; García Rubio, María Luisa; Izquierdo, Alicia; Herrero, Enrique; Aguilera López, Andrés; Wellinger, Ralf Erik; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía
Genomic instability is related to a wide-range of human diseases. Here, we show that mitochondrial iron–sulfur cluster biosynthesis is important for the maintenance of nuclear genome stability in Saccharomyces cerevisiae. Cells lacking the mitochondrial chaperone Zim17 (Tim15/Hep1), a component of the iron–sulfur biosynthesis machinery, have limited respiration activity, mimic the metabolic response to iron starvation and suffer a dramatic increase in nuclear genome recombination. Increased oxidative damage or deficient DNA repair do not account for the observed genomic hyperrecombination. Impaired cell-cycle progression and genetic interactions of ZIM17 with components of the RFC-like complex involved in mitotic checkpoints indicate that replicative stress causes hyperrecombination in zim17Δ mutants. Furthermore, nuclear accumulation of pre-ribosomal particles in zim17Δ mutants reinforces the importance of iron–sulfur clusters in normal ribosome biosynthesis. We propose that compromised ribosome biosynthesis and cell-cycle progression are interconnected, together contributing to replicative stress and nuclear genome instability in zim17Δ mutants.
Brahma is required for proper expression of the floral repressor FLC in Arabidopsis
(2011) Amasino, Richard M.; Farrona Cortés, Sara; Hurtado Álvarez, Lidia; Florencio Bellido, Francisco Javier; Reyes Rosa, José Carlos; March Díaz, Rosana Rocío; Schmitz, Robert J.; Turck, Franziska; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER); Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular
PS Integrins and Laminins: Key Regulators of Cell Migration during Drosophila Embryogenesis
(Public Library of Science, 2011) Urbano, José M.; Domínguez Jiménez, Paloma; Estrada Martín, Beatriz; Martín Bermudo, María D.; Ministerio de Ciencia y Tecnología (MCYT). España; Junta de Andalucía
During embryonic development, there are numerous cases where organ or tissue formation depends upon the migration of primordial cells. In the Drosophila embryo, the visceral mesoderm (vm) acts as a substrate for the migration of several cell populations of epithelial origin, including the endoderm, the trachea and the salivary glands. These migratory processes require both integrins and laminins. The current model is that αPS1βPS (PS1) and/or αPS3βPS (PS3) integrins are required in migrating cells, whereas αPS2βPS (PS2) integrin is required in the vm, where it performs an as yet unidentified function. Here, we show that PS1 integrins are also required for the migration over the vm of cells of mesodermal origin, the caudal visceral mesoderm (CVM). These results support a model in which PS1 might have evolved to acquire the migratory function of integrins, irrespective of the origin of the tissue. This integrin function is highly specific and its specificity resides mainly in the extracellular domain. In addition, we have identified the Laminin α1,2 trimer, as the key extracellular matrix (ECM) component regulating CVM migration. Furthermore, we show that, as it is the case in vertebrates, integrins, and specifically PS2, contributes to CVM movement by participating in the correct assembly of the ECM that serves as tracks for migration.
The replication checkpoint protects fork stability by releasing transcribed genes from nuclear pores
(Elsevier, 2011) Bermejo, Rodrigo; Gómez González, Belén; Aguilera López, Andrés; Foiani, Marco; Capra, Thelma; Cocito, Andrea; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia e Innovación (MICIN). España; National Institutes of Health. United States
Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.
Topological constraints impair RNA polymerase II transcription and causes instability of plasmid-borne convergent genes
(2011) García Rubio, María Luisa; Aguilera López, Andrés; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER); Universidad de Sevilla. Departamento de Genética
Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers
(2011) Luna Varo, Rosa María; Japón Rodríguez, Miguel Ángel; Sáez, Carmen; Domínguez Sánchez, María Salud; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER)
One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples.
Inhibition of RhoA GTPase and the subsequent activation of PTP1B protects cultured hippocampal neurons against amyloid b toxicity
(BioMed Central, 2011) Chacón Fernández, Pedro José; García Mejías, Rosa; Rodríguez Tebar, Alfredo; Ministerio de Educación y Ciencia (MEC). España
Background: Amyloid beta (Ab) is the main agent responsible for the advent and progression of Alzheimer’s disease. This peptide can at least partially antagonize nerve growth factor (NGF) signalling in neurons, which may be responsible for some of the effects produced by Ab. Accordingly, better understanding the NGF signalling pathway may provide clues as to how to protect neurons from the toxic effects of Ab. Results: We show here that Ab activates the RhoA GTPase by binding to p75NTR, thereby preventing the NGFinduced activation of protein tyrosine phosphatase 1B (PTP1B) that is required for neuron survival. We also show that the inactivation of RhoA GTPase and the activation of PTP1B protect cultured hippocampal neurons against the noxious effects of Ab. Indeed, either pharmacological inhibition of RhoA with C3 ADP ribosyl transferase or the transfection of cultured neurons with a dominant negative form of RhoA protects cultured hippocampal neurons from the effects of Ab. In addition, over-expression of PTP1B also prevents the deleterious effects of Ab on cultured hippocampal neurons. Conclusion: Our findings indicate that potentiating the activity of NGF at the level of RhoA inactivation and PTP1B activation may represent a new means to combat the noxious effects of Ab in Alzheimer’s disease.

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