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1.
Mol Neurobiol ; 61(2): 693-706, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-37656313

RESUMEN

Members of the Shank family of postsynaptic scaffold proteins (Shank1-3) link neurotransmitter receptors to the actin cytoskeleton in dendritic spines through establishing numerous interactions within the postsynaptic density (PSD) of excitatory synapses. Large Shank isoforms carry at their N-termini a highly conserved domain termed the Shank/ProSAP N-terminal (SPN) domain, followed by a set of Ankyrin repeats. Both domains are involved in an intramolecular interaction which is believed to regulate accessibility for additional interaction partners, such as Ras family G-proteins, αCaMKII, and cytoskeletal proteins. Here, we analyze the functional relevance of the SPN-Ank module; we show that binding of active Ras or Rap1a to the SPN domain can differentially regulate the localization of Shank3 in dendrites. In Shank1 and Shank3, the linker between the SPN and Ank domains binds to inactive αCaMKII. Due to this interaction, both Shank1 and Shank3 exert a negative effect on αCaMKII activity at postsynaptic sites in mice in vivo. The relevance of the SPN-Ank intramolecular interaction was further analyzed in primary cultured neurons; here, we observed that in the context of full-length Shank3, a closed conformation of the SPN-Ank tandem is necessary for proper clustering of Shank3 on the head of dendritic spines. Shank3 variants carrying Ank repeats which are not associated with the SPN domain lead to the atypical formation of postsynaptic clusters on dendritic shafts, at the expense of clusters in spine-like protrusions. Our data show that the SPN-Ank tandem motif contributes to the regulation of postsynaptic signaling and is also necessary for proper targeting of Shank3 to postsynaptic sites. Our data also suggest how missense variants found in autistic patients which alter SPN and Ank domains affect the synaptic function of Shank3.


Asunto(s)
Proteínas del Tejido Nervioso , Transducción de Señal , Ratones , Humanos , Animales , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Sinapsis/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Microfilamentos/metabolismo
2.
Mol Autism ; 11(1): 85, 2020 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-33115499

RESUMEN

BACKGROUND: Neurodevelopmental disorders such as autism spectrum disorder (ASD) may be caused by alterations in genes encoding proteins that are involved in synapse formation and function. This includes scaffold proteins such as Shank3, and synaptic adhesion proteins such as Neurexins or Neuroligins. An important question is whether the products of individual risk genes cooperate functionally (exemplified in the interaction of Neurexin with Neuroligin isoforms). This might suggest a common pathway in pathogenesis. For the SHANK3 gene, heterozygous loss of function, as well as missense mutations have been observed in ASD cases. Several missense mutations affect the N-terminal part of Shank3 which contains the highly conserved Shank/ProSAP N-terminal (SPN) and Ankyrin repeat (Ank) domains. The role of these domains and the relevance of these mutations for synaptic function of Shank3 are widely unknown. METHODS: We used purification from a synaptic protein fraction, as well as a variety of biochemical and cell biological approaches to identify proteins which associate with the Shank3 N-terminus at postsynaptic sites. RESULTS: We report here that δ-catenin, which is encoded by CTNND2, an autism candidate gene, directly interacts with the Ank domain of Shank3 at postsynaptic sites through its Armadillo-repeat domain. The interaction is not affected by well-known posttranslational modifications of δ-catenin, i.e. by phosphorylation or palmitoylation. However, an ASD-associated mutation in the SPN domain of Shank3, L68P, significantly increases the interaction of Shank3 with δ-catenin. By analysis of postsynaptic fractions from mice, we show that the lack of SPN-Ank containing, large isoforms of Shank3 results in the loss of postsynaptic δ-catenin. Further, expression of Shank3 variants containing the N-terminal domains in primary cultured neurons significantly increased the presence of coexpressed δ-catenin at postsynaptic sites. LIMITATIONS: Work in model organisms such as mice, and in primary cultured neurons may not reproduce faithfully the situation in human brain neurons. Work in primary cultured neurons was also hampered by lack of a specific antibody for endogenous δ-catenin. CONCLUSIONS: Our data show that the interaction between Shank3 N-terminus and δ-catenin is required for the postsynaptic targeting of δ-catenin. Failure of proper targeting of δ-catenin to postsynaptic sites may contribute to the pathogenesis of autism spectrum disorder.


Asunto(s)
Cateninas/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sinapsis/metabolismo , Animales , Células HEK293 , Humanos , Ratones Noqueados , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Mutación Missense/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Ratas , Catenina delta
3.
Nat Commun ; 11(1): 5797, 2020 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-33199684

RESUMEN

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development.


Asunto(s)
Proteínas Argonautas/genética , Células Germinativas/metabolismo , Mutación/genética , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Interferencia de ARN , Adolescente , Animales , Proteínas Argonautas/química , Niño , Preescolar , Análisis por Conglomerados , Dendritas/metabolismo , Fibroblastos/metabolismo , Silenciador del Gen , Células HEK293 , Hipocampo/patología , Humanos , Ratones , Simulación de Dinámica Molecular , Neuronas/metabolismo , Fosforilación , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , Ratas , Transcriptoma/genética
4.
Blood Adv ; 2(19): 2554-2567, 2018 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-30301811

RESUMEN

Receptor tyrosine kinase (RTK)-dependent signaling has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL) of childhood. However, the RTK-dependent signaling state and its interpretation with regard to biological behavior are often elusive. To decipher signaling circuits that link RTK activity with biological output in vivo, we established patient-derived xenograft ALL (PDX-ALL) models with dependencies on fms-like tyrosine kinase 3 (FLT3) and platelet-derived growth factor receptor ß (PDGFRB), which were interrogated by phosphoproteomics using iTRAQ mass spectrometry. Signaling circuits were determined by receptor type and cellular context with few generic features, among which we identified group I p21-activated kinases (PAKs) as potential therapeutic targets. Growth factor stimulation markedly increased catalytic activities of PAK1 and PAK2. RNA interference (RNAi)-mediated or pharmacological inhibition of PAKs using allosteric or adenosine triphosphate (ATP)-competitive compounds attenuated cell growth and increased apoptosis in vitro. Notably, PAK1- or PAK2-directed RNAi enhanced the antiproliferative effects of the type III RTK and protein kinase C inhibitor midostaurin. Treatment of FLT3- or PDGFRB-dependent ALLs with ATP-competitive PAK inhibitors markedly decreased catalytic activities of both PAK isoforms. In FLT3-driven ALL, this effect was augmented by coadministration of midostaurin resulting in synergistic effects on growth inhibition and apoptosis. Finally, combined treatment of FLT3 D835H PDX-ALL with the ATP-competitive group I PAK inhibitor FRAX486 and midostaurin in vivo significantly prolonged leukemia progression-free survival compared with midostaurin monotherapy or control. Our study establishes PAKs as potential downstream targets in RTK-dependent ALL of childhood, the inhibition of which might help prevent the selection or acquisition of resistance mutations toward tyrosine kinase inhibitors.


Asunto(s)
Antineoplásicos/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinasas p21 Activadas/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Niño , Modelos Animales de Enfermedad , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Linfopoyesis/genética , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/etiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteoma , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismo
5.
Nat Cell Biol ; 19(4): 292-305, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28263956

RESUMEN

SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1-RIAM-talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes.


Asunto(s)
Integrina beta1/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Celular , Línea Celular , Movimiento Celular , Extensiones de la Superficie Celular/metabolismo , Femenino , Citometría de Flujo , Ratones Endogámicos C57BL , Modelos Biológicos , Mutación/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Reacción en Cadena de la Polimerasa , Unión Proteica , Dominios Proteicos , Ratas Wistar , Alineación de Secuencia , Talina/metabolismo , Ubiquitinas/genética
6.
PLoS One ; 7(2): e31998, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22384124

RESUMEN

In multiple myeloma, circulating "clonotypic" B cells, that express the immunoglobulin rearrangement of the malignant plasma cell clone, can be indirectly detected by PCR. Their role as potential "feeder" cells for the malignant plasma cell pool remains controversial. Here we established for the first time an approach that allows direct tracking of such clonotypic cells by labeling with patient-specific immunoglobulin ligands in 15 patients with myeloma. Fifty percent of patients showed evidence of clonotypic B cells in blood or bone marrow by PCR. Epitope-mimicking peptides from random libraries were selected on each patient's individual immunoglobulin and used as ligands to trace cells expressing the idiotypic immunoglobulin on their surface. We established a flow cytometry and immunofluorescence protocol to track clonotypic B cells and validated it in two independent monoclonal B cell systems. Using this method, we found clonotypic B cells in only one out of 15 myeloma patients. In view of the assay's validated sensitivity level of 10(-3), this surprising data suggests that the abundance of such cells has been vastly overestimated in the past and that they apparently represent a very rare population in myeloma. Our novel tracing approach may open perspectives to isolate and analyze clonotypic B cells and determine their role in myeloma pathobiology.


Asunto(s)
Linfocitos B/inmunología , Inmunoglobulinas/química , Mieloma Múltiple/inmunología , Estudios de Cohortes , Epítopos/química , Glutatión Transferasa/metabolismo , Humanos , Ligandos , Mieloma Múltiple/sangre , Mieloma Múltiple/patología , Biblioteca de Péptidos , Péptidos/química , Fenotipo , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/química , Inducción de Remisión , Reproducibilidad de los Resultados , Trasplante de Células Madre
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