Efecto de la infección de Newcastle Disease Virus en distintas fases del ciclo celular

Trabajo Fin de Máster presentado por la licenciada en Biotecnología Elena Martín Doncel para optar al título de Máster en Biología y Clínica del Cáncer por la Universidad de Salamanca, que ha sido realizado en el Instituto de Biología Molecular y Celular del Cáncer (Curso 2013/2014).Peer Reviewe

Oncogenic Sox2 regulates and cooperates with VRK1 in cell cycle progression and differentiation.

Sox2 is a pluripotency transcription factor that as an oncogene can also regulate cell proliferation. Therefore, genes implicated in several different aspects of cell proliferation, such as the VRK1 chromatin-kinase, are candidates to be targets of Sox2. Sox 2 and VRK1 colocalize in nuclei of proliferating cells forming a stable complex. Sox2 knockdown abrogates VRK1 gene expression. Depletion of either Sox2 or VRK1 caused a reduction of cell proliferation. Sox2 up-regulates VRK1 expression and both proteins cooperate in the activation of CCND1. The accumulation of VRK1 protein downregulates SOX2 expression and both proteins are lost in terminally differentiated cells. Induction of neural differentiation with retinoic acid resulted in downregulation of Sox2 and VRK1 that inversely correlated with the expression of differentiation markers such as N-cadherin, Pax6, mH2A1.2 and mH2A2. Differentiation-associated macro histones mH2A1.2and mH2A2 inhibit CCND1 and VRK1 expression and also block the activation of the VRK1 promoter by Sox2. VRK1 is a downstream target of Sox2 and both form an autoregulatory loop in epithelial cell differentiation.Ministerio de Economía y Competitividad [SAF2013-44810R, SAF2014-57791-REDC].Consejería de Educación de la Junta de Castilla y León [CSI002U14 and UIC-017] to P.A.L

VRK1 (Y213H) homozygous mutant impairs Cajal bodies in a hereditary case of distal motor neuropathy.

Background: Distal motor neuropathies with a genetic origin have a heterogeneous clinical presentation with overlapping features affecting distal nerves and including spinal muscular atrophies and amyotrophic lateral sclerosis. This indicates that their genetic background is heterogeneous. Patient and methods: In this work, we have identified and characterized the genetic and molecular base of a patient with a distal sensorimotor neuropathy of unknown origin. For this study, we performed whole-exome sequencing, molecular modelling, cloning and expression of mutant gene, and biochemical and cell biology analysis of the mutant protein. Results: A novel homozygous recessive mutation in the human VRK1 gene, coding for a chromatin kinase, causing a substitution (c.637T > C; p.Tyr213His) in exon 8, was detected in a patient presenting since childhood a progressive distal sensorimotor neuropathy and spinal muscular atrophy syndrome, with normal intellectual development. Molecular modelling predicted this mutant VRK1 has altered the kinase activation loop by disrupting its interaction with the C-terminal regulatory region. The p.Y213H mutant protein has a reduced kinase activity with different substrates, including histones H3 and H2AX, proteins involved in DNA damage responses, such as p53 and 53BP1, and coilin, the scaffold for Cajal bodies. The mutant VRK1(Y213H) protein is unable to rescue the formation of Cajal bodies assembled on coilin, in the absence of wild-type VRK1. Conclusion: The VRK1(Y213H) mutant protein alters the activation loop, impairs the kinase activity of VRK1 causing a functional insufficiency that impairs the formation of Cajal bodies assembled on coilin, a protein that regulates SMN1 and Cajal body formation.post-print2120 K

Viral RNA load in plasma is associated with critical illness and a dysregulated host response in COVID‑19

Background. COVID-19 can course with respiratory and extrapulmonary disease. SARS-CoV-2 RNA is detected in respiratory samples but also in blood, stool and urine. Severe COVID-19 is characterized by a dysregulated host response to this virus. We studied whether viral RNAemia or viral RNA load in plasma is associated with severe COVID-19 and also to this dysregulated response. Methods. A total of 250 patients with COVID-19 were recruited (50 outpatients, 100 hospitalized ward patients and 100 critically ill). Viral RNA detection and quantification in plasma was performed using droplet digital PCR, targeting the N1 and N2 regions of the SARS-CoV-2 nucleoprotein gene. The association between SARS-CoV-2 RNAemia and viral RNA load in plasma with severity was evaluated by multivariate logistic regression. Correlations between viral RNA load and biomarkers evidencing dysregulation of host response were evaluated by calculating the Spearman correlation coefficients. Results. The frequency of viral RNAemia was higher in the critically ill patients (78%) compared to ward patients (27%) and outpatients (2%) (p < 0.001). Critical patients had higher viral RNA loads in plasma than non-critically ill patients, with non-survivors showing the highest values. When outpatients and ward patients were compared, viral RNAemia did not show significant associations in the multivariate analysis. In contrast, when ward patients were compared with ICU patients, both viral RNAemia and viral RNA load in plasma were associated with critical illness (OR [CI 95%], p): RNAemia (3.92 [1.183–12.968], 0.025), viral RNA load (N1) (1.962 [1.244–3.096], 0.004); viral RNA load (N2) (2.229 [1.382–3.595], 0.001). Viral RNA load in plasma correlated with higher levels of chemokines (CXCL10, CCL2), biomarkers indicative of a systemic inflammatory response (IL-6, CRP, ferritin), activation of NK cells (IL-15), endothelial dysfunction (VCAM-1, angiopoietin-2, ICAM-1), coagulation activation (D-Dimer and INR), tissue damage (LDH, GPT), neutrophil response (neutrophils counts, myeloperoxidase, GM-CSF) and immunodepression (PD-L1, IL-10, lymphopenia and monocytopenia). Conclusions. SARS-CoV-2 RNAemia and viral RNA load in plasma are associated with critical illness in COVID-19. Viral RNA load in plasma correlates with key signatures of dysregulated host responses, suggesting a major role of uncontrolled viral replication in the pathogenesis of this disease.This work was supported by awards from the Canadian Institutes of Health Research, the Canadian 2019 Novel Coronavirus (COVID-19) Rapid Research Funding initiative (CIHR OV2 – 170357), Research Nova Scotia (DJK), Atlantic Genome/Genome Canada (DJK), Li-Ka Shing Foundation (DJK), Dalhousie Medical Research Foundation (DJK), the “Subvenciones de concesión directa para proyectos y programas de investigación del virus SARS‐CoV2, causante del COVID‐19”, FONDO–COVID19, Instituto de Salud Carlos III (COV20/00110, CIBERES, 06/06/0028), (AT) and fnally by the “Convocatoria extraordinaria y urgente de la Gerencia Regional de Salud de Castilla y León, para la fnanciación de proyectos de investigación en enfermedad COVID-19” (GRS COVID 53/A/20) (CA). DJK is a recipient of the Canada Research Chair in Translational Vaccinology and Infammation. APT was funded by the Sara Borrell Research Grant CD018/0123 funded by Instituto de Salud Carlos III and co-fnanced by the European Development Regional Fund (A Way to Achieve Europe programme). The funding sources did not play any role neither in the design of the study and collection, not in the analysis, in the interpretation of data or in writing the manuscript

Gestión del conocimiento: perspectiva multidisciplinaria. Volumen 13

El libro “Gestión del Conocimiento. Perspectiva Multidisciplinaria”, Volumen 13 de la Colección Unión Global, es resultado de investigaciones. Los capítulos del libro, son resultados de investigaciones desarrolladas por sus autores. El libro es una publicación internacional, seriada, continua, arbitrada, de acceso abierto a todas las áreas del conocimiento, orientada a contribuir con procesos de gestión del conocimiento científico, tecnológico y humanístico. Con esta colección, se aspira contribuir con el cultivo, la comprensión, la recopilación y la apropiación social del conocimiento en cuanto a patrimonio intangible de la humanidad, con el propósito de hacer aportes con la transformación de las relaciones socioculturales que sustentan la construcción social de los saberes y su reconocimiento como bien público. El libro “Gestión del Conocimiento. Perspectiva Multidisciplinaria”, Volumen 13, de la Colección Unión Global, es resultado de investigaciones. Los capítulos del libro, son resultados de investigaciones desarrolladas por sus autores. El libro cuenta con el apoyo de los grupos de investigación: Universidad Sur del Lago “Jesús María Semprúm” (UNESUR) - Zulia – Venezuela; Universidad Politécnica Territorial de Falcón Alonso Gamero (UPTFAG) - Falcón – Venezuela; Universidad Politécnica Territorial de Mérida Kléber Ramírez (UPTM) - Mérida - Venezuela; Universidad Guanajuato (UG) - Campus Celaya - Salvatierra - Cuerpo Académico de Biodesarrollo y Bioeconomía en las Organizaciones y Políticas Públicas (CABBOPP) - Guanajuato – México; Centro de Altos Estudios de Venezuela (CEALEVE) - Zulia – Venezuela, Centro Integral de Formación Educativa Especializada del Sur (CIFE - SUR) - Zulia – Venezuela; Centro de Investigaciones Internacionales SAS (CEDINTER) - Antioquia – Colombia y diferentes grupos de investigación del ámbito nacional e internacional que hoy se unen para estrechar vínculos investigativos, para que sus aportes científicos formen parte de los libros que se publiquen en formatos digital e impreso

Implicación de la quinasa humana VRK1 en enfermedades neurodegenerativas

[ES]VRK1 es una serina-treonina quinasa implicada en múltiples procesos biológicos, entre los más destacados se encuentra la regulación del ciclo celular a través de la fosforilación de ciertos factores de transcripción, su implicación en respuesta en el daño al ADN, en la rotura de la envuelta nuclear durante la división celular, en la regulación de la dinámica de ensamblaje y desensamblaje de los cuerpos de Cajal, en la condensación de la cromatina, en la fragmentación del aparato de Golgi, y también se encuentra implicada en procesos de desarrollo y mantenimiento del sistema nervioso. En los últimos años se han reportado casos de pacientes que presentan ciertas variantes de VRK1 muy poco frecuentes en la población, que generan distintas neuropatías severas. En este trabajo de tesis doctoral caracterizamos las variantes patogénicas de VRK1 a distintos niveles, para comprender el papel de la quinasa en el desarrollo del sistema nervioso, y también estudiamos la implicación de VRK1 en la enfermedad Ataxia espinocerebelosa tipo I o SCA1, una enfermedad rara, a través del estudio de la relación entre VRK1 y la proteína responsable de SCA1, Ataxina1. En la primera parte de este trabajo, estudiamos la estabilidad y la actividad catalítica de las proteínas generadas a partir de las variantes patogénicas de VRK1, y el efecto sobre el fenotipo de líneas celulares estables, y en sus capacidades de motilidad, migración, proliferación, respuesta al daño generado en el ADN, y capacidad para ensamblar cuerpos de Cajal. Observamos que hay dos grupos de variantes, unas que generan proteínas estables (R89Q, H119R y V236M), y otras que generan proteínas inestables (R133C, G135R, L195V, R321C, R358X). En el caso de la actividad catalíticas, solo las variantes R89Q y L195V resultaron activas, tanto en cuanto a autofosforilación como a transfosforilación. En cuanto a la capacidad de progresión en el ciclo celular, todas las variantes presentaron alteraciones. Del mismo modo, ninguna de las variantes fue capaz de inducir una respuesta al daño generado en el ADN con el agente quimioterapéutico doxorrubicina. Tres de las variantes (H119R. R133C y G135R) presentaron defectos en la capacidad de migración celular y en motilidad, y salvo L195V, ninguna de las variantes permitió el correcto ensamblaje de los cuerpos de Cajal. En la segunda parte, comprobamos que Ataxina1 y VRK1 interaccionan por la región de contacto entre el extremo carboxilo terminal y el dominio catalítico, siendo la interacción más fuerte con la Ataxina1 expandida, y en el núcleo de las células. Además, determinamos que VRK1 ejerce un papel protector sobre la degradación de Ataxina1, puesto que al silenciar a la quinasa disminuyen los niveles proteicos de Ataxina1. Por último, verificamos que VRK1 fosforila a Ataxina1 normal frente a la expandida, y proponemos el residuo serina en posición 239 como el candidato a ser diana de fosforilación de VRK1

Characterization of VRKl and Atxnl interaction and its functional implications

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.Atxn1 is the protein involved in the inherited neurodegenerative disease SCA1, an autosomal dominant disorder caused by the expansion of a triplet CAG, encoding a polyglutarnine tract (polyQ) which confers toxic properties to the protein. Nevertheless, polyQ is necessary but not sufficient to cause pathology. Indeed, is also important the nuclear localization signal, the AXH domain and a serine 776 residue. VRK1 is a Ser-Thr kinase associated with multiple processes, including the regulation ofcell cycle progression, chromatin remodeling or Cajal Bodies dynamics. Also VRK1 is mutated in neurodegenerative diseases. Previously we identified Atxnl like a potential target for VRK1, through the use of linear peptide chips. Our aim is to characterize the interaction VRKl-Atxnl and its potential functional implication. Through immunoprecipitation and nuclei isolation assays, we detected an interaction between both proteins. Besides, by HPLC fractionation is proved that Atxn1 and VRK1 elute in the same high molecular size fractions. By immunofluorescence, we did not detect any colocalization, which may indicate a transient interaction between both proteins. In a kinase assay, we observed distinct phosphorylation levels between Arxn1 with a physiological polyQ and the protein with an expanded polyQ. Only wild-type Atxn1 can be phosphorylated by VRK1. Atxn1 is degraded via ubiquitin proteasome system. Here, we showed that different mutants of Atxn1 have a similar protein accumulation in the nuclear inclusions when proteasome is inhibited with MG-132. Yet, we have evidence that VRK1 downregulation enhances the degradation of Atxn1. We believe other proteins, Iike VCP, which colocalized with Atxn1 nuclear inclusions with expanded polyQ, might be involved in this process.Peer Reviewe

Characterization of VRK1 and Atxn1 interaction and its functional implication

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.Atxn1 is the protein involved in the inherited neurodegenerative disease SCAI, an autosomal dominant disorder caused by the expansion of a triplet CAG, encoding a polyglutamine tract (polyQ) which confers toxic properties to the protein. Nevertheless, polyQ is necessary but not sufficient to cause pathology. Indeed, is also important the nuclear localization signal, the AXH domain and a serine 776 residue. VRK1 is a Ser-Thr kinase associated with multiple processes, including the regulation of cell cycle progression, chromatin remodeling or Cajal Bodies dynamics. Also VRK1 is mutated in neurodegenerative diseases. Previously we identified Atxn1 like a potential target for VRK1, through the use oflinear peptide chips. Our aim is to characterize üe interaction VRKl-Atxnl and its potential functional implication. Through immunoprecipitation and nuclei isolation assays, we detected an interaction between both proteins. Besides, by HPLC lractionation is proved that Atxn1 and VRK1 elute in the same high molecular size fractions. By immunofluorescence, we did not detect any colocalization, which may indicate a transient interaction between both proteins. In a kinase assay, we observed distinct phosphorylation levels between Arxn1 with a physiological polyQ and the protein with an expanded polyQ. Only wild-type Atxnl can be phosphorylated by VRK1. Atxnl is degraded via ubiquitin proteasome system. Here, we showed that different mutants of Atxn1 have a similar protein accumulation in the nuclear inclusions when proteasome is inhibited with MG-132. Yet, we have eyidence that VRK1 downregulation enhances the degradation of Atxn1. We believe other proteins, Iike VCP, which colocalized with Atxn1 nuclear inclusions with expanded polyQ, might be involved in this process.Peer Reviewe

Vaccinia related kinase 1 (VRK1) regulates the stability of Ataxin 1 (Atxn1)

Resumen del póster presentado al 1st Joint Meeting of the French-Portuguese-Spanish Biochemical and Molecular Biology Societies y al XL Spanish Society of Biochemistry and Molecular Biology (SEBBM) Congress, celebrado en Barcelona (España) del 23 al 26 de octubre de 2017.VRK1 is a Ser-Thr kinase that participates in multiple processes, among which we highlight the chromatin remodelling, regulation of cell cycle progression or Cajal Bodies dynamics. VRK1 is mutated in several neurodegenerative diseases, like SMA or ALS, and forms complexes with SMN, Coilin and Atxn1. Atxn1 is a protein that causes the neurodegenerative disease SCA1. SCA1 is one of the nine-polyglutamine diseases where there is an expansion of a triplet CAG. This expansion is necessary but no suffi cient to cause the disorder, there are also important the pos translational modifi cations of Atxn1. Knowing the interaction between both proteins, our aim is to study variations in phosphorylation linked to the length of polyQ tract and identify the functional consequences of this phosphorylation in the normal stability of the protein. By immunoprecipitation, we detected a diff erential interaction between VRK1 and mutants of Atxn1 with distinct length of polyQ. We also observed that VRK1 can phosphorylate wild type Atxn1 and not polyQ expanded Atxn1. We generated mutants of the Ser239 to Ala or Asp,a candidate to be phosphorylated by the CK1 family, to which VRK1 belongs. Phosphorylation of this residue is essential to the stability of Atxn1. Furthermore, we showed a signifi cant increase in the VRK1 interaction with SCA1 mutants that cannot be phosphorylated. Depletion of VRK1 promotes a quick degradation of Atxn1. Also, we know that expanded Atxn1 interacts and colocalizes with VCP, an important protein involved in the removal of misfolded proteins. All this suggests that the absence of VRK1 promotes a quick degradation of Atxn1 by proteasome 26s through VCP activity. Our conclusion is that the phosphorylation of Atxn1 by VRK1 regulates its degradation after ubiquitination.Peer Reviewe

VRK1 as a novel regulator of Cajal Body dynamics and stability in cell cycle, and its pathological implications

Resumen del trabajo presentado al 15th ASEICA International Congress, celebrado en Sevilla (España) del 21 al 23 de octubre de 2015.[Introduction]: Spinal muscular atrophies (SMA) and ataxias are neurodegenerative diseases that often cause death at an early age. Numerous genes involved in these diseases have been identified, but their molecular mechanisms of pathogenesis are still unknown. The most well-known SMAs are associated with SMN1 (Survival Motor Neuron) gene disruption. SMN localizes in the cytosol and accumulates in Cajal Bodies (CBs) taking part of the SMN complex with the Gemins or interacting with Coilin. CBs are nuclear organelles associated with ribonucleoprotein functions and RNA maturation. CBs are assembled on coilin, its main scaffold protein, in a cell cycle dependent manner. Coilin is regulated by phosphorylation and methylation. Recently it have been identified mutations in the human kinase VRK1 (vaccinia related kinase 1), that cause a child pontocerebellar atrophy, manifested as an early ataxia, spinal muscular atrophy and microcephaly with death at an early age. Furthermore, it has been described that VRK1 interacts and phosphorylates Coilin at Ser184 residue. [Objectives]: We hypothesize that VRK1 plays an essential role in the formation of CBs through the phosphorylation of coilin in a cell cycle dependent manner. [Methods]: The interaction between VRK1 and coilin was assessed by reciprocal immunoprecipitations in HEK-293T and HeLa cells. We evaluated the effect of inactive VRK1 in CBs formation by rescue experiments, and we analyzed the effect of VRK1 knockdown in coilin oligomerization by HLPC experiment. We also studied the phosphorylation in Ser184 residue by kinase assay and cell cycle experiments arresting HeLa cells in G1/S, after thymidine block or in G2/M, after thymidine/nocodazole block. We have evaluated he degradation of coilin with ubiquitination assays, and we also studied the export of coilin to the cytosol treating the cells with leptomycin B.[Results]: In this work, we show that VRK1, whose activity is cell cycle regulated, interacts with and phosphorylates coilin regulating the assembly of CBs. Coilin phosphorylation is not necessary for its interaction with VRK1, but it occurs in mitosis and regulates coilin stability. Knockdown of VRK1 or VRK1 inactivation by serum deprivation causes a loss of coilin phosphorylation in Ser184 and CBs formation, which are rescued with an active VRK1. The phosphorylation of coilin in Ser184 occurs during mitosis before assembly of CBs. Loss of coilin phosphorylation results in disintegration of CBs, and of coilin degradation that is prevented by proteasome inhibitors. After depletion of VRK1, coilin is ubiquitinated in nuclei, which is partly mediated by mdm2, but its proteasomal degradation occurs in cytosol and is prevented by blocking its nuclear export. Also, VRK1 gene silencing and subsequent disassembly of CBs produces a massive exit of SMN protein to the cytosol. [Conclusions]: We conclude that VRK1 is a novel regulator of CBs dynamics and stability, in cell cycle, by protecting coilin from ubiquitination and degradation in the proteasome, and propose a model of CB dynamics. Previous studies describe VRK1 as an important protein in cancer, but now it is presented as a new player in neurodegenerative diseases and may be a functional link with other proteins regulating their interactions and their biological and pathological effects.Peer Reviewe