RESUMEN
BACKGROUND: Cells deploy quality control mechanisms to remove damaged or misfolded proteins. Recently, we have reported that a mutation (R43W) in the Frank-ter Haar syndrome protein Tks4 resulted in aberrant intracellular localization. RESULTS: Here we demonstrate that the accumulation of Tks4(R43W) depends on the intact microtubule network. Detergent-insoluble Tks4 mutant colocalizes with the centrosome and its aggregate is encaged by the intermediate filament protein vimentin. Both the microtubule inhibitor nocodazole and the histone deacetylase inhibitor Trichostatin A inhibit markedly the aggresome formation in cells expressing Tks4(R43W). Finally, pretreatment of cells with the proteasome inhibitor MG132 markedly increases the level of aggresomes formed by Tks4(R43W). Furthermore, two additional mutant Tks4 proteins (Tks4(1-48) or Tks4(1-341)) have been investigated. Whereas the shorter Tks4 mutant, Tks4(1-48), shows no expression at all, the longer Tks4 truncation mutant accumulates in the nuclei of the cells. CONCLUSIONS: Our results suggest that misfolded Frank-ter Haar syndrome protein Tks4(R43W) is transported via the microtubule system to the aggresomes. Lack of expression of Tks4(1-48) or aberrant intracellular expressions of Tks4(R43W) and Tks4(1-341) strongly suggest that these mutations result in dysfunctional proteins which are not capable of operating properly, leading to the development of FTHS.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Anomalías Craneofaciales/genética , Cardiopatías Congénitas/genética , Microtúbulos/patología , Osteocondrodisplasias/congénito , Mutación Puntual , Agregación Patológica de Proteínas/genética , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células COS , Chlorocebus aethiops , Anomalías Craneofaciales/metabolismo , Anomalías Craneofaciales/patología , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Cardiopatías Congénitas/metabolismo , Cardiopatías Congénitas/patología , Humanos , Microtúbulos/genética , Microtúbulos/metabolismo , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patología , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Pliegue de Proteína , Estructura Terciaria de Proteína , Transporte de ProteínasRESUMEN
Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular , Anomalías Craneofaciales/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Cardiopatías Congénitas/metabolismo , Osteocondrodisplasias/congénito , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células COS , Chlorocebus aethiops , Cromonas/farmacología , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/mortalidad , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/mortalidad , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Silenciador del Gen , Células HeLa , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/mortalidad , Humanos , Morfolinas/farmacología , Mutación , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/mortalidad , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Estructura Terciaria de Proteína , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
People's knowledge about the arts shapes how they experience and engage with art. Since its introduction, the 10-item Aesthetic Fluency Scale has been widely used to measure self-reported art knowledge. Drawing from findings and researchers' experience since then, the present work develops and evaluates a Revised Aesthetic Fluency Scale using item response theory to broaden its scope (36 items) and refine its response scale. In a large sample (n = 2,089 English-speaking adults), Study 1 found strong evidence for unidimensionality, good item fit, and a difficulty level suitable for its targeted population; Study 2 (n = 392) provided initial evidence for score validity via relationships with art engagement, Openness to Experience, and aesthetic responsiveness; and Study 3 derived a brief, 10-item form for time-constrained projects. Taken together, the revised scales build upon lessons learned from the original and appear promising for the next generation of research.
Asunto(s)
Estética , Adulto , Humanos , Psicometría , Encuestas y Cuestionarios , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Scaffold proteins have an important role in the regulation of signal propagation. These proteins do not possess any enzymatic activity but can contribute to the formation of multiprotein complexes. Although scaffold proteins are present in all cell types, the nervous system contains them in the largest amount. Caskin proteins are typically present in neuronal cells, particularly, in the synapses. However, the signaling mechanisms by which Caskin proteins are regulated are largely unknown. RESULTS: Here we demonstrate that EphB1 receptor tyrosine kinase can recruit Caskin1 through the adaptor protein Nck. Upon activation of the receptor kinase, the SH2 domain of Nck binds to one of its tyrosine residues, while Nck SH3 domains interact with the proline-rich domain of Caskin1. Complex formation of the receptor, adaptor and scaffold proteins results in the tyrosine phosphorylation of Caskin1 on its SH3 domain. The phosphorylation sites were identified by mass-spectrometry as tyrosines 296 and 336. To reveal the structural consequence of this phosphorylation, CD spectroscopy was performed. This measurement suggests that upon tyrosine phosphorylation the structure of the Caskin1 SH3 domain changes significantly. CONCLUSION: Taken together, we propose that the scaffold protein Caskin1 can form a complex with the EphB1 tyrosine kinase via the Nck protein as a linker. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain. Although we were not able to identify any physiological partner of the SH3 domain so far, we could demonstrate that phosphorylation on conserved tyrosine residues results in marked changes in the structure of the SH3 domain.
RESUMEN
The pain- and stress-reducing effects of music are well-known, but the effects of visual art, and the combination of these two, are much less investigated. We aim to (1) investigate the pain- and (2) stress-reducing effects of multimodal (music + visual art) aesthetic experience as we expect this to have stronger effects than a single modal aesthetic experience (music/ visual art), and in an exploratory manner, (3) investigate the underlying mechanisms of aesthetic experience, and the (4) individual differences. In a repeated-measures design (music, visual art, multimodal aesthetic experience, control) participants bring self-selected "movingly beautiful" visual artworks and pieces of music to the lab, where pain and stress are induced by the cold pressor test. Activity of the pain and stress responsive systems are measured by subjective reports, autonomic (electrocardiography, electrodermal activity, salivary alpha-amylase) and endocrine markers (salivary cortisol).
Asunto(s)
Música , alfa-Amilasas Salivales , Estética , Humanos , Dolor , Manejo del DolorRESUMEN
Background: Tyrosinaemia type 1 is a rare inherited metabolic disease caused by an enzyme defect in the tyrosine degradation pathway. It is treated using nitisinone and a low-protein diet. In a workshop in 2013, a group of nutritional specialists from Germany, Switzerland and Austria agreed to advocate a simplified low-protein diet and to allow more natural protein intake in patients with tyrosinaemia type 1. This retrospective study evaluates the recommendations made at different treatment centers and their impact on clinical symptoms and metabolic control. Methods: For this multicenter study, questionnaires were sent to nine participating treatment centers to collect data on the general therapeutic approach and data of 47 individual patients treated by those centers. Results: Dietary simplification allocating food to 3 categories led to increased tyrosine and phenylalanine blood concentrations without weighing food. Phenylalanine levels were significantly higher in comparison to a strict dietary regimen whereas tyrosine levels in plasma did not change. Non-inferiority was shown for the simplification and liberalization of the diet. Compliance with dietary recommendations was higher using the simplified diet in comparison to the stricter approach. Age correlates negatively with compliance. Conclusions: Simplification of the diet with increased natural protein intake based on three categories of food may be implemented in the diet of patients with tyrosinaemia type 1 without significantly altering metabolic control. Patient compliance is strongly influencing tyrosine blood concentrations. A subsequent prospective study with a larger sample size is necessary to get a better insight into the effect of dietary recommendations on metabolic control.
Asunto(s)
Ciclohexanonas/administración & dosificación , Dieta con Restricción de Proteínas/métodos , Proteínas en la Dieta/administración & dosificación , Inhibidores Enzimáticos/administración & dosificación , Nitrobenzoatos/administración & dosificación , Tirosinemias/terapia , Adolescente , Austria , Niño , Preescolar , Terapia Combinada/métodos , Terapia Combinada/normas , Dieta con Restricción de Proteínas/normas , Femenino , Alemania , Humanos , Masculino , Cooperación del Paciente/estadística & datos numéricos , Fenilalanina/sangre , Guías de Práctica Clínica como Asunto , Estudios Prospectivos , Estudios Retrospectivos , Encuestas y Cuestionarios/estadística & datos numéricos , Suiza , Resultado del Tratamiento , Tirosina/sangre , Tirosinemias/sangre , Tirosinemias/diagnóstico , Tirosinemias/metabolismo , Adulto JovenRESUMEN
Background The dietary management of methylmalonic acidaemia (MMA) is a low-protein diet providing sufficient energy to avoid catabolism and to limit production of methylmalonic acid. The goal is to achieve normal growth, good nutritional status and the maintenance of metabolic stability. Aim To describe the dietary management of patients with MMA across Europe. Methods A cross-sectional questionnaire was sent to European colleagues managing inherited metabolic disorders (IMDs) (n=53) with 27 questions about the nutritional management of organic acidaemias. Data were analysed by different age ranges (0-6 months; 7-12 months; 1-10 years; 11-16 years; >16 years). Results Questionnaires were returned from 53 centres. Twenty-five centres cared for 80 patients with MMA vitamin B12 responsive (MMAB12r) and 43 centres managed 215 patients with MMA vitamin B12 non-responsive (MMAB12nr). For MMAB12r patients, 44% of centres (n=11/25) prescribed natural protein below the World Health Organization/Food and Agriculture Organization/United Nations University (WHO/FAO/UNU) 2007 safe levels of protein intake in at least one age range. Precursor-free amino acids (PFAA) were prescribed by 40% of centres (10/25) caring for 36% (29/80) of all the patients. For MMAB12nr patients, 72% of centres (n=31/43) prescribed natural protein below the safe levels of protein intake (WHO/FAO/UNU 2007) in at least one age range. PFAA were prescribed by 77% of centres (n=33/43) managing 81% (n=174/215) of patients. In MMAB12nr patients, 90 (42%) required tube feeding: 25 via a nasogastric tube and 65 via a gastrostomy. Conclusions A high percentage of centres used PFAA in MMA patients together with a protein prescription that provided less than the safe levels of natural protein intake. However, there was inconsistent practices across Europe. Long-term efficacy studies are needed to study patient outcome when using PFAA with different severities of natural protein restrictions in patients with MMA to guide future practice.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/dietoterapia , Proteínas en la Dieta/administración & dosificación , Encuestas y Cuestionarios/normas , Adolescente , Errores Innatos del Metabolismo de los Aminoácidos/epidemiología , Niño , Preescolar , Estudios Transversales , Europa (Continente)/epidemiología , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Apoyo NutricionalRESUMEN
CASK-interactive proteins, Caskin1 and Caskin2, are multidomain neuronal scaffold proteins. Recent data from Caskin1 knockout animals indicated only a mild role of Caskin1 in anxiety and pain perception. In this work, we show that deletion of both Caskins leads to severe deficits in novelty recognition and spatial memory. Ultrastructural analyses revealed a reduction in synaptic profiles and dendritic spine areas of CA1 hippocampal pyramidal neurons of double knockout mice. Loss of Caskin proteins impaired LTP induction in hippocampal slices, while miniature EPSCs in dissociated hippocampal cultures appeared to be unaffected. In cultured Caskin knockout hippocampal neurons, overexpressed Caskin1 was enriched in dendritic spine heads and increased the amount of mushroom-shaped dendritic spines. Chemically induced LTP (cLTP) mediated enlargement of spine heads was augmented in the knockout mice and was not influenced by Caskin1. Immunocytochemistry and immunoprecipitation confirmed that Shank2, a master scaffold of the postsynaptic density, and Caskin1 co-localized within the same complex. Phosphorylation of AMPA receptors was specifically altered by Caskin deficiency and was not elevated by cLTP treatment further. Taken together, our results prove a previously unnoticed postsynaptic role of Caskin scaffold proteins and indicate that Caskins influence learning abilities via regulating spine morphology and AMPA receptor localisation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Hipocampo/patología , Proteínas del Tejido Nervioso/genética , Aprendizaje Espacial/fisiología , Memoria Espacial/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Cultivadas , Espinas Dendríticas/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Cultivo Primario de Células , Receptores AMPA/metabolismoRESUMEN
The commitment steps of mesenchymal stromal cells (MSCs) to adipogenic and other lineages have been widely studied but not fully understood. Therefore, it is critical to understand which molecules contribute to the conversion of stem cells into differentiated cells. The scaffold protein Tks4 plays a role in podosome formation, EGFR signaling and ROS production. Dysfunction of Tks4 causes a hereditary disease called Frank-ter Haar syndrome with a variety of defects concerning certain mesenchymal tissues (bone, fat and cartilage) throughout embryogenic and postnatal development. In this study, we aimed to analyze how the mutation of Tks4 affects the differentiation potential of multipotent bone marrow MSCs (BM-MSCs). We generated a Tks4 knock-out mouse strain on C57Bl/6 background, and characterized BM-MSCs isolated from wild type and Tks4-/- mice to evaluate their differentiation. Tks4-/- BM-MSCs had reduced ability to differentiate into osteogenic and adipogenic lineages compared to wild type. Studying the expression profile of a panel of lipid-regulated genes during adipogenic induction revealed that the expression of adipogenic transcription factors, genes responsible for lipid droplet formation, sterol and fatty acid metabolism was delayed or reduced in Tks4-/- BM-MSCs. Taken together, these results establish a novel function for Tks4 in the regulation of MSC differentiation.
Asunto(s)
Adipogénesis , Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Fosfoproteínas/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/metabolismo , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/metabolismo , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Ratones , Ratones Noqueados , Osteocondrodisplasias/congénito , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Fosfoproteínas/genéticaRESUMEN
BACKGROUND: Tks5/FISH is a scaffold protein comprising of five SH3 domains and one PX domain. Tks5 is a substrate of the tyrosine kinase Src and is required for the organization of podosomes/invadopodia implicated in invasion of tumor cells. Recent data have suggested that a close homologue of Tks5, Tks4, is implicated in the EGF signaling. RESULTS: Here, we report that Tks5 is a component of the EGF signaling pathway. In EGF-treated cells, Tks5 is tyrosine phosphorylated within minutes and the level of phosphorylation is sustained for at least 2 hours. Using specific kinase inhibitors, we demonstrate that tyrosine phosphorylation of Tks5 is catalyzed by Src tyrosine kinase. We show that treatment of cells with EGF results in plasma membrane translocation of Tks5. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutation of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks5. CONCLUSIONS: Our results identify Tks5 as a novel component of the EGF signaling pathway.
RESUMEN
The important regulatory role of the guanine-quadruplex (GQ) structure, present in the nuclease hypersensitive element (NHE) III(1) region of the human c-myc (h c-myc) gene's promoter, in the regulation of the transcription of that gene has been documented. Here we present evidences, that the human nuclear poly(ADP-ribose)polymerase-1 (h PARP-1) protein participates in the regulation of the h c-myc gene expression through its interaction with this GQ structure, characterized by binding assays, fluorescence energy transfer (FRET) experiments and by affinity pull-down experiments in vitro, and by chromatin immunoprecipitation (ChIP)-qPCR analysis and h c-myc-promoter-luciferase reporter determinations in vivo. We surmise that h PARP-1 binds to the GQ structure and participates in the conversion of that structure into the transcriptionally more active B-DNA form. The first Zn-finger structure present in h PARP-1 participates in this interaction. PARP-1 might be a new member of the group of proteins participating in the regulation of transcription through their interactions with GQ structures present in the promoters of different genes.
Asunto(s)
ADN Forma B/metabolismo , G-Cuádruplex , Poli(ADP-Ribosa) Polimerasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Inmunoprecipitación de Cromatina , ADN Forma B/química , Fibroblastos/enzimología , Transferencia Resonante de Energía de Fluorescencia , Genes Reporteros , Células HL-60 , Células HeLa , Humanos , Cinética , Luciferasas/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Reacción en Cadena de la Polimerasa , Unión Proteica , Temperatura , TransfecciónRESUMEN
The recognition of poly ADP-ribose transferase-1 (PARP-1) as an ATP sensor receiving this energy source by way of a specific adenylate kinase ATP wire (AK) from mitochondrial ATP synthase (F0F1), and directly regulating cellular mRNA and DNA synthesis, was the first step towards the identification of an effect by PARP-1 that is of fundamental significance. The molecular target of AK-ATP is Arg 34 of the Zn finger I of PARP-1, which is also a site for cation-π interactions as a target of π-electron donors. We now identify this π-electron receptor site as the second active center of PARP-1 which by interaction with a π-electron donor-inducible MgATPase reversibly controls a malignant vs. non-malignant phenotype through energizing the NADHâNADP+ transhydrogenase, a reaction which is the metabolic connection of PARP-1 to cell function. The specific enzyme-inducing action of the π-electrons is executed by the PARP-1 -topoisomerase I - DNA complex of the nuclei regulating both the nature and the quantity of cellular enzymes that constitute cell-specific physiology.