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1.
Mol Cell Proteomics ; 8(4): 681-95, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19054758

RESUMO

Mitogen-activated protein kinases (MAPKs) control neuronal synaptic function; however, little is known about the synaptic substrates regulated by MAPKs. A phosphopeptide library incorporating the MAPK consensus motif (PX(pS/pT)P where pS is phosphoserine and pT is phosphothreonine) was used to raise a phosphospecific antibody that detected MAPK-mediated phosphorylation. The antibody (termed "5557") recognized a variety of phosphoproteins in the brain, many of which were enriched in postsynaptic density fractions. The immunoblot pattern changed rapidly in response to altered synaptic activity and with the inhibition of specific MAPKs and protein phosphatases. By immunoaffinity purification with 5557 antibody followed by mass spectrometry, we identified 449 putative MAPK substrates of which many appeared dynamically regulated in neuron cultures. Several of the novel candidate MAPK substrates were validated by in vitro phosphorylation assays. Additionally 82 specific phosphorylation sites were identified in 34 proteins, including Ser-447 in delta-catenin, a component of the cadherin adhesion complex. We further raised another phosphospecific antibody to confirm that delta-catenin Ser-447 is modified in neurons by the MAPK JNK in a synaptic activity-dependent manner. Ser-447 phosphorylation by JNK appears to be correlated with delta-catenin degradation, and a delta-catenin mutant defective in Ser-447 phosphorylation showed enhanced ability to promote dendrite branching in cultured neurons. Thus, phosphomotif-based affinity purification is a powerful approach to identify novel substrates of MAPKs in vivo and to reveal functionally significant phosphorylation events.


Assuntos
Anticorpos Fosfo-Específicos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/enzimologia , Fosfoproteínas/análise , Fosfoproteínas/química , Motivos de Aminoácidos , Animais , Cateninas , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Cromatografia de Afinidade , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Imunoprecipitação , Marcação por Isótopo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Reprodutibilidade dos Testes , Especificidade por Substrato/efeitos dos fármacos , delta Catenina
2.
Curr Biol ; 17(20): 1752-8, 2007 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-17935993

RESUMO

Septins are GTP-binding proteins that polymerize into heteromeric filaments and form microscopic bundles or ring structures in vitro and in vivo. Because of these properties and their ability to associate with membrane, F-actin, and microtubules, septins have been generally regarded as cytoskeletal components [1, 2]. Septins are known to play roles in cytokinesis, in membrane trafficking, and as structural scaffolds; however, their function in neurons is poorly understood. Many members of the septin family, including Septin 7 (Sept7), were found by mass-spectrometry analysis of postsynaptic density (PSD) fractions of the brain [3, 4], suggesting a possible postsynaptic function of septins in neurons. We report that Sept7 is localized at the base of dendritic protrusions and at dendritic branch points in cultured hippocampal neurons--a distribution reminiscent of septin localization in the bud neck of budding yeast. Overexpression of Sept7 increased dendrite branching and the density of dendritic protrusions, whereas RNA interference (RNAi)-mediated knockdown of Sept7 led to reduced dendrite arborization and a greater proportion of immature protrusions. These data suggest that Sept7 is critical for spine morphogenesis and dendrite development during neuronal maturation.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Dendritos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Dendritos/ultraestrutura , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Hipocampo/citologia , Hipocampo/metabolismo , Interferência de RNA , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
3.
Curr Biol ; 25(5): 613-20, 2015 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-25660540

RESUMO

Gall-forming arthropods are highly specialized herbivores that, in combination with their hosts, produce extended phenotypes with unique morphologies [1]. Many are economically important, and others have improved our understanding of ecology and adaptive radiation [2]. However, the mechanisms that these arthropods use to induce plant galls are poorly understood. We sequenced the genome of the Hessian fly (Mayetiola destructor; Diptera: Cecidomyiidae), a plant parasitic gall midge and a pest of wheat (Triticum spp.), with the aim of identifying genic modifications that contribute to its plant-parasitic lifestyle. Among several adaptive modifications, we discovered an expansive reservoir of potential effector proteins. Nearly 5% of the 20,163 predicted gene models matched putative effector gene transcripts present in the M. destructor larval salivary gland. Another 466 putative effectors were discovered among the genes that have no sequence similarities in other organisms. The largest known arthropod gene family (family SSGP-71) was also discovered within the effector reservoir. SSGP-71 proteins lack sequence homologies to other proteins, but their structures resemble both ubiquitin E3 ligases in plants and E3-ligase-mimicking effectors in plant pathogenic bacteria. SSGP-71 proteins and wheat Skp proteins interact in vivo. Mutations in different SSGP-71 genes avoid the effector-triggered immunity that is directed by the wheat resistance genes H6 and H9. Results point to effectors as the agents responsible for arthropod-induced plant gall formation.


Assuntos
Cromossomos/genética , Dípteros/genética , Família Multigênica/genética , Filogenia , Tumores de Planta/genética , Triticum/parasitologia , Adaptação Biológica/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Dípteros/metabolismo , Larva/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência , Comportamento Sexual Animal/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genética
4.
G3 (Bethesda) ; 4(12): 2433-49, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-25326424

RESUMO

There has been a renewed interest in investigating the role of stabilizing selection acting on genome-wide traits such as codon usage bias. Codon bias, when synonymous codons are used at unequal frequencies, occurs in a wide variety of taxa. Standard evolutionary models explain the maintenance of codon bias through a balance of genetic drift, mutation and weak purifying selection. The efficacy of selection is expected to be reduced in regions of suppressed recombination. Contrary to observations in Drosophila melanogaster, some recent studies have failed to detect a relationship between the recombination rate, intensity of selection acting at synonymous sites, and the magnitude of codon bias as predicted under these standard models. Here, we examined codon bias in 2798 protein coding loci on the third chromosome of D. pseudoobscura using whole-genome sequences of 47 individuals, representing five common third chromosome gene arrangements. Fine-scale recombination maps were constructed using more than 1 million segregating sites. As expected, recombination was demonstrated to be significantly suppressed between chromosome arrangements, allowing for a direct examination of the relationship between recombination, selection, and codon bias. As with other Drosophila species, we observe a strong mutational bias away from the most frequently used codons. We find the rate of synonymous and nonsynonymous polymorphism is variable between different amino acids. However, we do not observe a reduction in codon bias or the strength of selection in regions of suppressed recombination as expected. Instead, we find that the interaction between weak stabilizing selection and mutational bias likely plays a role in shaping the composition of synonymous codons across the third chromosome in D. pseudoobscura.


Assuntos
Cromossomos/genética , Drosophila/genética , Animais , Códon , Sequenciamento de Nucleotídeos em Larga Escala , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Seleção Genética , Análise de Sequência de DNA
5.
Neuron ; 65(3): 373-84, 2010 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-20159450

RESUMO

MicroRNAs (miRNAs) are noncoding RNAs that suppress translation of specific mRNAs. The miRNA machinery interacts with fragile X mental retardation protein (FMRP), which functions as translational repressor. We show that miR-125b and miR-132, as well as several other miRNAs, are associated with FMRP in mouse brain. miR-125b and miR-132 had largely opposing effects on dendritic spine morphology and synaptic physiology in hippocampal neurons. FMRP knockdown ameliorates the effect of miRNA overexpression on spine morphology. We identified NMDA receptor subunit NR2A as a target of miR-125b and show that NR2A mRNA is specifically associated with FMRP in brain. In hippocampal neurons, NR2A expression is negatively regulated through its 3' UTR by FMRP, miR-125b, and Argonaute 1. Regulation of NR2A 3'UTR by FMRP depends in part on miR-125b. Because NMDA receptor subunit composition profoundly affects synaptic plasticity, these observations have implications for the pathophysiology of fragile X syndrome, in which plasticity is altered.


Assuntos
Proteína do X Frágil da Deficiência Intelectual/fisiologia , MicroRNAs/metabolismo , Neurônios/fisiologia , Sinapses/fisiologia , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Células Cultivadas , Espinhas Dendríticas/metabolismo , Embrião de Mamíferos , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Imunoprecipitação/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Neurônios/citologia , RNA Mensageiro/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transfecção/métodos
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