Mecanismos moleculares implicados en la patogénesis de las variantes de la quinasa humana VRK1 en síndromes neuromotores

[ES] Las proteínas quinasas son enzimas encargadas de fosforilar proteínas específicas, es decir, catalizan la unión covalente de un grupo fosfato a residuos de treonina, serina o tirosina de las proteínas diana. El dominio quinasa o dominio catalítico de estas proteínas, formado por una secuencia de unos 250‐300 aminoácidos, está altamente conservado en la evolución [1]. La fosforilación es una modificación postraduccional que regula distintos procesos celulares, como la proliferación, diferenciación o el metabolismo, mediante cambios en la localización subcelular de las proteínas, en su interacción con otras moléculas, actividad o en su estabilidad [2–3]. Esta modificación está muy regulada en la célula, dada la importancia de los procesos en los que participa. Las fosfatasas son otro tipo de proteínas capaces de eliminar el grupo fosfato añadido por las quinasas [4]. En 1995 se describió el quinoma humano, en el que se encuentran clasificadas filogenéticamente las quinasas humanas. Esta clasificación fue ampliada en 2002 por Manning y colaboradores. Hasta la fecha, se han descrito 518 genes codificantes y 106 pseudogenes con secuencias de dominio quinasa. El quinoma se divide en 8 grupos y estos a su vez, en familias. La familia de las serina‐treonina quinasas VRK (del inglés Vacciniarelated kinase) se encuentra dentro del grupo caseína quinasa de tipo 1 (CK1) [1–5–6] y son en las que nos centraremos en este trabajo

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

El sexismo en las y los profesionales de salud mental: un factor de riesgo psicosocial para la detección de la desigualdad de género

Diversos organismos recomiendan a las y los profesionales de salud superar las propias creencias sexistas y apuntan a la importancia de abordar la desigualdad de género. En este sentido, este trabajo se centra en: (1) conocer los niveles de sexismo ambivalente en profesionales de salud mental, (2) analizar la asociación entre sexismo y características sociodemográficas y de perspectiva de género y (3) estudiar la asociación entre sexismo y detección de desigualdad de género en consulta. 449 profesionales de salud mental completaron la Escala de Sexismo Ambivalente, así como otro cuestionario desarrollado ad-hoc. Se realizaron análisis no paramétricos. Los datos mostraron, entre otros aspectos, un nivel bajo de sexismo en la muestra, así como que las y los profesionales que manifestaron niveles significativamente más bajos de sexismo, detectaron más problemas de desigualdad en consulta. Los resultados subrayan la importancia de reducir el sexismo de las y los profesionales para identificar los problemas de desigualdad de género.Some organizations recommend that healthcare professionals overcome their own sexist beliefs and emphasize the importance of dealing with gender inequality. Thus, this study focused on: (1) find out the levels of ambivalent sexism in a network of mental health professionals in Spain, (2) analyze the association between sexism, sociodemographic cha-racteristics and gender perspective, and (3) study the association between sexism and gender inequality detection during clinical attention. The sample was comprised of 449 mental health professionals who filled in the Ambivalent Sexism Inventory and another ad hoc questionnaire developed. Variables were compared by nonparametric analysis. The data showed low levels of sexism in the sample, and that the professionals who obtained lower levels of sexism found more problems related to gender inequality during clinical attention. The results underline the importance of intervention for reducing sexism of professionals in order to contribute to a more unbiased detection of problems of gender inequality

Effectiveness of a universal personalized intervention for the prevention of anxiety disorders: Protocol of a randomized controlled trial (the prevANS project)

Background: To date, all preventive anxiety disorders interventions are one-fit-all and none of them are based on individual level and risk profile. The aim of this project is to design, develop and evaluate an online personalized intervention based on a risk algorithm for the universal prevention of anxiety disorders in the general population. Methods: A randomized controlled trial (RCT) with two parallel arms (prevANS vs usual care) and 1-year follow- up including 2000 participants without anxiety disorders from Spain and Portugal will be conducted. The prevANS intervention will be self-guided and can be implemented from the prevANS web or from the participants' Smartphone (through an App). The prevANS intervention will have different intensities depending on the risk level of the population, evaluated from the risk algorithm for anxiety: predictA. Both low and moderate-high risk participants will receive information on their level and profile (risk factors) of anxiety disorders, will have access to stress management tools and psychoeducational information periodically. In addition, participants with a moderate-high risk of anxiety disorders will also have access to cognitive-behavioral training (problem-solving, decision-making, communication skills, and working with thoughts). The control group will not receive any intervention, but they will fill out the same questionnaires as the intervention group. Assessments will be completed at baseline, 6 and 12-month follow-up. The primary outcome is the cumulative incidence of anxiety disorders. Secondary outcomes include depressive and anxiety symptoms, risk probability of anxiety disorders (predictA algorithm) and depression (predictD algorithm), improvement in physical and mental quality of life, and acceptability and satisfaction with the intervention. In addition, cost-effectiveness and cost-utility analyses will also be carried out from two perspectives, societal and health system, and analyses of mediators and moderators will also be performedSpanish Ministry of Health, the Institute of Health Carlos III, co-funded by the European Social Fund “Investing in your future” (grant references: CP19/00056), and the Chronicity, Primary Care and Health Promotion Research Network ‘RICAPPS’ (RD21/0016/0012); and Spanish Ministry of Science and Innovation, the State Investigation Agency (PID2020-119652RA-l00). These funding sources had no role in the design of this study and will not have any role during its execution, analyses, interpretation of the data, or decision to submit resultsS

A personalized intervention to prevent depression in primary care: cost-effectiveness study nested into a clustered randomized trial

Abstract Background: Depression is viewed as a major and increasing public health issue, as it causes high distress in the people experiencing it and considerable financial costs to society. Efforts are being made to reduce this burden by preventing depression. A critical component of this strategy is the ability to assess the individual level and profile of risk for the development of major depression. This paper presents the cost-effectiveness of a personalized intervention based on the risk of developing depression carried out in primary care, compared with usual care. Methods: Cost-effectiveness analyses are nested within a multicentre, clustered, randomized controlled trial of a personalized intervention to prevent depression. The study was carried out in 70 primary care centres from seven cities in Spain. Two general practitioners (GPs) were randomly sampled from those prepared to participate in each centre (i.e. 140 GPs), and 3326 participants consented and were eligible to participate. The intervention included the GP communicating to the patient his/her individual risk for depression and personal risk factors and the construction by both GPs and patients of a psychosocial programme tailored to prevent depression. In addition, GPs carried out measures to activate and empower the patients, who also received a leaflet about preventing depression. GPs were trained in a 10- to 15-h workshop. Costs were measured from a societal and National Health care perspective. Qualityadjustedlife years were assessed using the EuroQOL five dimensions questionnaire. The time horizon was 18 months.This work was supported by grants from the Spanish Ministry of Health, the Institute of Health Carlos III (ISCIII) and the European Regional Development Fund (ERDF) ’A way to build Europe’(grant references PS09/02272, PS09/02147, PS09/01095, PS09/00849 and PS09/00461); the Andalusian Council of Health (grant reference PI-0569-2010); the Spanish Network of Primary Care Research ’redIAPP’ (RD06/0018, RD12/0005/0001); the ’Aragón group’ (RD06/0018/0020, RD12/0005/0006); the ’Bizkaya group’ (RD06/0018/0018, RD12/0005/0010); the Castilla-León Group (RD06/0018/0027); the Mental Health (SJD) Barcelona Group (RD06/0018/0017, RD12/0005/0008); and the Mental-Health, Services and Primary Care (SAMSERAP) MálagaGroup (RD06/0018/0039, RD12/0005/0005)

Caracterización del complejo proteico WRK1-SMN-coilina-VCP implicado en procesos neurodegenerativos

Memoria presentada por la graduada en Biología Patricia Morejón García, que ha sido realizada en el Instituto de Biología Molecular y Celular del Cáncer de Salamanca para optar al título de Máster en Biología y Clínica del Cáncer por la Universidad de Salamanca.[ES]: Las atrofias musculares espinales (SMA) son un conjunto de enfermedades neurodegenerativas para las que no existe actualmente tratamiento, que representan la principal causa genética de muerte infantil. Se deben a defectos en proteínas que llevan a cabo diferentes funciones en la célula, pero aún no está claro el mecanismo molecular exacto por el que las neuronas degeneran. Se han descrito mutaciones en los genes VRK1, SMN y VCP asociadas tanto a esta patología neuromuscular como a ataxia, degeneración pontocerebelar y esclerosis lateral amiotrófica. En este trabajo nos hemos propuesto caracterizar las relaciones entre las proteínas SMN, VCP y VRK1 en líneas celulares tumorales, para entender la base molecular común de estas patologías neurodegenerativas. Hemos visto mediante ensayos de inmunoprecipitación, que estas tres proteínas interaccionan entre sí en alguna fase del ciclo celular formando un complejo proteico. Lo siguiente a evaluar será en qué compartimento celular se producen dichas interacciones y cómo la alteración de cada uno de estos componentes afecta al conjunto y causa estas patologías neurodegenerativas.[EN]: Spinal muscular atrophy (SMA), a hereditary neurodegenerative disease, is the leading genetic cause of infant mortality. It is caused because of the loss of important proteins which carry different functions in the cells. However, the molecular mechanism by neurons degenerate is not yet clear. Mutations in the VRK1, SMN and VCP genes have been observed in SMA, ataxia, pontocerebellar hipoplasia, amyotrophic lateral sclerosis. In this work, we study the relationship between SMN, VCP and VRK1, using tumor cell lines, in order to understand the common molecular basis of these neurodegenerative diseases. We have seen that these three proteins interact forming a complex in the cell. The next to investigate will be in which cellular compartment such interactions occur and how the alterations of these components affect the whole and cause neurodegenerative diseases.Peer Reviewe

VRK1 phosphorylates Tip60/KAT5 and is required for H4K16 acetylation in response to DNA damage

Dynamic remodeling of chromatin requires acetylation and methylation of histones, frequently affecting the same lysine residue. These alternative epigenetic modifications require the coordination of enzymes, writers and erasers, mediating them such as acetylases and deacetylases. In cells in G0/G1, DNA damage induced by doxorubicin causes an increase in histone H4K16ac, a marker of chromatin relaxation. In this context, we studied the role that VRK1, a chromatin kinase activated by DNA damage, plays in this early step. VRK1 depletion or MG149, a Tip60/KAT5 inhibitor, cause a loss of H4K16ac. DNA damage induces the phosphorylation of Tip60 mediated by VRK1 in the chromatin fraction. VRK1 directly interacts with and phosphorylates Tip60. Furthermore, the phosphorylation of Tip60 induced by doxorubicin is lost by depletion of VRK1 in both ATM +/+ and ATM−/− cells. Kinase-active VRK1, but not kinase-dead VRK1, rescues Tip60 phosphorylation induced by DNA damage independently of ATM. The Tip60 phosphorylation by VRK1 is necessary for the activating acetylation of ATM, and subsequent ATM autophosphorylation, and both are lost by VRK1 depletion. These results support that the VRK1 chromatin kinase is an upstream regulator of the initial acetylation of histones, and an early step in DNA damage responses (DDR).This work was supported by grants from Agencia Estatal de Investigación-Ministerio de Economía y Competitividad-FEDER [SAF2016-75744-R; RED2018-102801-T, PID2019-105610RB-I00] Consejería de Educación de la Junta de Castilla y León-ERDF [CLC-2017-01, and UIC-258] to P.A.L., R.G.-G, P.M.-G. and I.C.-M. were supported by Consejería de Educación-Junta de Castilla y León-Fondo Social Europeo (ESF), Ministerio de Educación y Universidades [FPU 16-01883] and MINECO-FPI [BES-2014-067729] predoctoral fellowships respectively

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

Characterization of VRK1 mutants implicated in human neurodegenerative diseases

Resumen del trabajo presentado en el 41 Congreso de la SEBBM (Sociedad Española de Bioquímica y Biología Molecular), celebrado en Santander (España) del 10 al 13 de septiembre de 2018.VRK1 is a nuclear and chromatin Ser-Thr kinase that appeared late in evolution. VRK1 gene expression is ubiquitous, and it is highly expressed in proliferating cells, and in many tumours associated to a poorer prognosis. VRK1 participates in several processes that include regulation of cell cycle progression and proliferation, its implication on chromatin remodeling during transcription, replication and DNA repair, the regulation of transcription factors like p53, its implication on DNA-Damage Response, and the regulation of the Cajal Bodies dynamics and nuclear envelope assembly. Furthermore, VRK1 has a role both in the development and in the maintenance of the nervous system. Mutations in the human VRK1 gene are associated to multiple neurodegenerative diseases, like Spinal Muscular Atrophy or Aminotrophic Lateral Sclerosis. In the present work, we have generated and characterized all known VRK1 mutants that have been identified in patients with neurodegenerative diseases ([R89Q], [H119R], [R133C], [G135R], [L195V], [V236M], [R321C], [R358X]). We determined the effect of the mutations on VRK1 stability and its autophosphorylation and transphosphorylation of its known substrates, such as p53, histone H3, 53BP1, NBS1 and coilin. VRK1 is a very stable protein. Some mutants have a significantly reduced half-life. Regarding to the kinase activity, some mutants have a reduced autophosphorylation but they maintain the phosphorylation of its targets. Others have altered the auto-phosphorylation and the substrate-specific phosphorylation activity. We conclude that the functional alteration of the VRK1 mutations and the impairment of its function contribute to the pathogenesis of the neurodegenerative phenotypes observed in patient

The VRK1 chromatin kinase regulates the acetyltransferase activity of Tip60/KAT5 by sequential phosphorylations in response to DNA damage

The regulation of histone epigenetic modifications mediates the adaptation of chromatin to different biological processes. DNA damage causes a local relaxation of chromatin associated to histone H4 acetylation in K16, mediated by Tip60/KAT5. In this work, we have studied the role that the VRK1 chromatin kinase plays on the activation of Tip60 during this process. In the DNA damage response induced by doxorubicin, VRK1 directly phosphorylates Tip60. However, the phosphorylated Tip60 residues and their functional roles are unknown. In DDR, we have identified these two Tip60 phosphorylated residues and the cooperation of the participating kinases. The T158 phosphorylation, mediated by VRK1, is early and transient, preceding that of S199, which is more sustained in time, and mediated by DNA-PK. The role of each phosphorylated residues was determined by using phosphomimetic and phosphonull mutants and their combination. T158 phosphorylation protects Tip60 from ubiquitin-mediated degradation, promotes its recruitment to chromatin from the nucleoplasm, and is necessary for its full trans-acetylase activity. The phosphorylation in S199 by DNA-PK directly facilitates Tip60 autoacetylation, but it is not enough for trans-acetylation of two of its targets, histone H4 and ATM, which requires a double phosphorylation of Tip60 in T158 and S199. DNA-PK inhibitors block the phosphorylation of S199. We propose a model in which the cooperation between VRK1 and DNA-PK mediates the sequential phosphorylation of Tip60/KAT5, and contributes to the recruitment of this protein to initiate the sequential remodeling of chromatin in DDR. Both proteins are candidates for novel synthetic lethality strategies in cancer treatment.This work was supported by grants from Agencia Estatal de Investigación (doi: 10.13039/501100011033) - Ministerio de Ciencia e Innovación-FEDER (RED2018-102801-T, PID2019-105610RB-I00), Consejería de Educación de la Junta de Castilla y León-ERDF (CSI264P20, CLC-2017-01) to P.A.L. P. M.-G. was supported by Ministerio de Educación predoctoral fellowship (FPU16/01883). R. G-G and E. M-S. were supported (Consejería de Educación-Junta de Castilla y León-Fondo Social Europeo (CSI004-18), predoctoral fellowships respectively. E. N-C was supported by Agencia Estatal de Investigación-MINECO predoctoral fellowship (BES-2017-080543)