RESUMO
Neurofibromatosis type 1 (NF1) is a dominant genetic disorder manifesting, in part, as cognitive defects. Previous study indicated that neurofibromin (NF1 protein) interacts with valosin-containing protein (VCP)/P97 to control dendritic spine formation, but the mechanism is unknown. Here, using Nf1+/- mice and transgenic mice overexpressing wild-type Vcp/p97, we demonstrate that neurofibromin acts with VCP to control endoplasmic reticulum (ER) formation and consequent protein synthesis and regulates dendritic spine formation, thereby modulating contextual fear memory and social interaction. To validate the role of protein synthesis, we perform leucine supplementation in vitro and in vivo. Our results suggest that leucine can effectively enter the brain and increase protein synthesis and dendritic spine density of Nf1+/- neurons. Contextual memory and social behavior of Nf1+/- mice are also restored by leucine supplementation. Our study suggests that the "ER-protein synthesis" pathway downstream of neurofibromin and VCP is a critical regulator of dendritic spinogenesis and brain function.
Assuntos
Medo/fisiologia , Leucina/administração & dosagem , Memória/fisiologia , Neurofibromina 1/metabolismo , Biossíntese de Proteínas , Comportamento Social , Proteína com Valosina/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Células Cultivadas , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Suplementos Nutricionais , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Camundongos Mutantes , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteoma/metabolismo , Sirolimo/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismoRESUMO
Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gαi1, which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1·RGS4 mechanism. This movement induces a charge shift toward ß-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/química , Arginina/química , Domínio Catalítico , Estabilidade Enzimática , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Neurofibromina 1/química , Neurofibromina 1/metabolismo , Proteínas RGS/química , Proteínas RGS/genética , Proteínas RGS/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
In the present study, we isolated and screened an antitumor polysaccharide (PGP2a) from the roots of Panax ginseng. Chemical composition analysis indicated PGP2a was an acidic protein-polysaccharide. The average molecular weight was estimated to be 3.2 × 10(4)Da. According to gas chromatography (GC) result, PGP2a consisted of galactose, arabinose, glucose and galacturonic acid in the molar ratio of 3.7:1.6:0.5:5.4, respectively. MTT assay showed that PGP2a had a potent inhibitory effect on the growth of HGC-27 cells in a dose-dependent fashion. Furthermore, the number of HGC-27 cells arrested in G2/M phase, and the percentage of apoptotic cells were increased in response to PGP2a treatment along with concentration increasing. Moreover, western blotting analysis showed that protein expressions of Twist and AKR1C2 were suppressed by PGP2a, whereas an increase of NF1 was observed at protein level. Taken together, these findings suggested that PGP2a could be developed as a novel antitumor agent acting on Twist related gene for human gastric cancer therapy.
Assuntos
Apoptose/efeitos dos fármacos , Hidroxiesteroide Desidrogenases/metabolismo , Neurofibromina 1/metabolismo , Proteínas Nucleares/metabolismo , Panax/química , Polissacarídeos/farmacologia , Neoplasias Gástricas/patologia , Proteína 1 Relacionada a Twist/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Cromatografia em Gel , Ativação Enzimática , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Neoplasias Gástricas/metabolismoRESUMO
Neurofibromatosis type 1 (NF1) is the most common genetic disease affecting the nervous system. Patients typically develop many tumors over their lifetime, leading to increased morbidity and mortality. The NF1 gene, mutated in NF1, is also commonly mutated in sporadic glioblastoma multiforme (GBM). Because both NF1 and GBM are currently incurable, new therapeutic approaches are clearly needed. Natural products represent an opportunity to develop new therapies, as they have been evolutionarily selected to play targeted roles in organisms. Schweinfurthin A is a prenylated stilbene natural product that has previously shown specific inhibitory activity against brain and hematopoietic tumor lines. We show that patient-derived GBM and NF1 malignant peripheral nerve sheath tumor (MPNST) lines, as well as tumor lines derived from the Nf1-/+;Trp53-/+ (NPcis) mouse model of astrocytoma and MPNST are highly sensitive to inhibition by schweinfurthin A and its synthetic analogs. In contrast, primary mouse astrocytes are resistant to the growth inhibitory effects of schweinfurthin A, suggesting that schweinfurthin A may act specifically on tumor cells. Stable transfection of the GTPase-activating protein related domain of Nf1 into Nf1-/-;Trp53-/- astrocytoma cells confers resistance to schweinfurthin A. In addition, the profound effect of schweinfurthin A on dynamic reorganization of the actin cytoskeleton led us to discover that schweinfurthin A inhibits growth factor-stimulated Rho signaling. In summary, we have identified a class of small molecules that specifically inhibit growth of cells from both central and peripheral nervous system tumors and seem to act on NF1-deficient cells through cytoskeletal reorganization correlating to changes in Rho signaling.
Assuntos
Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Genes da Neurofibromatose 1 , Glioma/patologia , Neurofibromatose 1/patologia , Estilbenos/farmacologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Animais Recém-Nascidos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Genes da Neurofibromatose 1/fisiologia , Glioma/genética , Glioma/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Neurofibromatose 1/metabolismo , Neurofibromina 1/química , Neurofibromina 1/metabolismo , Neurofibromina 1/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/fisiologiaRESUMO
To study the role of the neurofibromatosis-1 (NF1) gene in mammalian brain development, we recently generated mice in which Nf1 gene inactivation occurs in neuroglial progenitor cells using the brain lipid binding protein (BLBP) promoter. We found that Nf1(BLBP)CKO mice exhibit significantly reduced body weights and anterior pituitary gland sizes. We further demonstrate that the small anterior pituitary size reflects loss of neurofibromin expression in the hypothalamus, leading to reduced growth hormone releasing hormone, pituitary growth hormone (GH) and liver insulin-like growth factor-1 (IGF1) production. Since neurofibromin both negatively regulates Ras activity and positively modulates cAMP levels, we examined the signaling pathway responsible for these abnormalities. While BLBP-mediated expression of an activated Ras molecule did not recapitulate the body weight and hypothalamic/pituitary defects, treatment of Nf1(BLBP)CKO mice with rolipram to increase cAMP levels resulted in a partial restoration of the body weight phenotype. Furthermore, conditional expression of the Ras regulatory GAP domain of neurofibromin also did not rescue the body weight or Igf1 mRNA defects in Nf1(BLBP)CKO mice. Collectively, these data demonstrate a critical role for neurofibromin in hypothalamic-pituitary axis function and provide further insights into the short stature and GH deficits seen in children with NF1.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hipotálamo/crescimento & desenvolvimento , Neurofibromatose 1/metabolismo , Neurofibromina 1/metabolismo , Hipófise/crescimento & desenvolvimento , Animais , Peso Corporal , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Hormônio do Crescimento/metabolismo , Humanos , Hipopituitarismo/genética , Hipopituitarismo/metabolismo , Hipotálamo/química , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurofibromatose 1/genética , Neurofibromina 1/química , Neurofibromina 1/genética , Tamanho do Órgão , Hipófise/química , Hipófise/metabolismo , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
The rodent barrel cortex is a useful system to study the role of genes and neuronal activity in the patterning of the nervous system. Several genes encoding either intracellular signaling molecules or neurotransmitter receptors are required for barrel formation. Neurofibromin is a tumor suppressor protein that has Ras GTPase activity, thus attenuating the MAPK (mitogen-activated protein kinase) and and PI-3 kinase (phosphatidylinositol 3-kinase) pathways, and is mutated in humans with the condition neurofibromatosis type 1 (NF1). Neurofibromin is widely expressed in the developing and adult nervous system, and a common feature of NF1 is deficits in intellectual development. In addition, NF1 is an uncommonly high disorder among individuals with autism. Thus, NF1 may have important roles in normal CNS development and function. To explore roles for neurofibromin in the development of the CNS, we took advantage of a mouse conditional allele. We show that mice that lack neurofibromin in the majority of cortical neurons and astrocytes fail to form cortical barrels in the somatosensory cortex, whereas segregation of thalamic axons within the somatosensory cortex appears unaffected.
Assuntos
Astrócitos/metabolismo , Neurofibromina 1/metabolismo , Neurônios/metabolismo , Córtex Somatossensorial/citologia , Córtex Somatossensorial/metabolismo , Animais , Axônios/fisiologia , Mapeamento Encefálico , Linhagem Celular , Genes da Neurofibromatose 1/fisiologia , Camundongos , Córtex Somatossensorial/crescimento & desenvolvimento , Tálamo/crescimento & desenvolvimentoRESUMO
Bone formation is controlled by a network of transcription factors and signaling molecules. In this issue, , studying the role of the transcription factor ATF4 in a new mouse model of neurofibromatosis type I skeletal defects, demonstrate striking effects of changing dietary protein on bone formation abnormalities.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Doenças do Desenvolvimento Ósseo/dietoterapia , Doenças do Desenvolvimento Ósseo/metabolismo , Proteínas Alimentares/uso terapêutico , Neurofibromina 1/metabolismo , Osteoblastos/metabolismo , Aminoácidos/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/genética , Doenças do Desenvolvimento Ósseo/congênito , Doenças do Desenvolvimento Ósseo/patologia , Reabsorção Óssea/dietoterapia , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Síndrome de Coffin-Lowry/genética , Síndrome de Coffin-Lowry/metabolismo , Síndrome de Coffin-Lowry/patologia , Colágeno/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Camundongos , Camundongos Knockout , Neurofibromina 1/deficiência , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/genética , Ligante RANK/biossíntese , Ligante RANK/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/deficiência , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
The transcription factor ATF4 enhances bone formation by favoring amino acid import and collagen synthesis in osteoblasts, a function requiring its phosphorylation by RSK2, the kinase inactivated in Coffin-Lowry Syndrome. Here, we show that in contrast, RSK2 activity, ATF4-dependent collagen synthesis, and bone formation are increased in mice lacking neurofibromin in osteoblasts (Nf1(ob)(-/-) mice). Independently of RSK2, ATF4 phosphorylation by PKA is enhanced in Nf1(ob)(-/-) mice, thereby increasing Rankl expression, osteoclast differentiation, and bone resorption. In agreement with ATF4 function in amino acid transport, a low-protein diet decreased bone protein synthesis and normalized bone formation and bone mass in Nf1(ob)(-/-) mice without affecting other organ weight, while a high-protein diet overcame Atf4(-/-) and Rsk2(-/-) mice developmental defects, perinatal lethality, and low bone mass. By showing that ATF4-dependent skeletal dysplasiae are treatable by dietary manipulations, this study reveals a molecular connection between nutrition and skeletal development.
Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Doenças do Desenvolvimento Ósseo/dietoterapia , Doenças do Desenvolvimento Ósseo/metabolismo , Proteínas Alimentares/uso terapêutico , Neurofibromina 1/metabolismo , Osteoblastos/metabolismo , Aminoácidos/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/genética , Doenças do Desenvolvimento Ósseo/congênito , Doenças do Desenvolvimento Ósseo/patologia , Reabsorção Óssea/dietoterapia , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Síndrome de Coffin-Lowry/genética , Síndrome de Coffin-Lowry/metabolismo , Síndrome de Coffin-Lowry/patologia , Colágeno/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Camundongos , Camundongos Knockout , Neurofibromina 1/deficiência , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/genética , Ligante RANK/biossíntese , Ligante RANK/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/deficiência , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismoRESUMO
In mammalian intestine, adenosine deaminase (ADA) is expressed at high levels only along the villi of the duodenal epithelium. A duodenum-specific enhancer identified in the second intron of the human ADA gene controls this pattern of expression. This enhancer faithfully recapitulates this expression pattern in transgenic mice, when included in CAT reporter gene constructions. Multiple binding sites for PDX-1 and GATA factors were previously identified within the approximately 300-bp region that encompasses the enhancer. Mutation analyses demonstrated that binding of PDX-1 and of GATA-4 was absolutely essential for enhancer function. In the present study, we have identified additional enhancer binding sites for Cdx factors, for YY1, and for NFI family members. Detailed EMSA studies were used to confirm binding at these sites. This brings the number of confirmed binding sites within the enhancer to thirteen, with five different factors or family of factors contributing to the putative enhanceosome complex. Mutation analysis was utilized to examine the specific roles of the newly identified sites. Two sites were identified that bound both Cdx1 and Cdx2. Mutations were identified in these two sites that completely and specifically eliminated Cdx binding. In transgenic mice, these enhancer mutations dramatically changed the developmental timing of enhancer activation (delaying it by 2-3 weeks) without affecting other aspects of enhancer function. In the chromatin context of certain transgenic insertion sites, mutation of the two YY1 sites to specifically ablate binding caused a delay in enhancer activation similar to that observed with the Cdx mutations. No overt changes were observed from mutation of the NFI site.
Assuntos
Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Duodeno/crescimento & desenvolvimento , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Sequência de Bases , Sítios de Ligação , Fator de Transcrição CDX2 , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA4 , Genes Reporter , Humanos , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Neurofibromina 1/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência do Ácido Nucleico , Fatores de Tempo , Transativadores/metabolismo , Transcrição Gênica , Transgenes , Fator de Transcrição YY1RESUMO
The helix-loop-helix protein Id-1 is a dominant negative regulator of basic helix-loop-helix transcription factors, and plays a key role in the control of breast epithelial cell growth, invasion and differentiation. Previous investigations in our laboratory have shown that Id-1 mRNA was constitutively expressed in highly aggressive and invasive human breast cancer cells in comparison to non-transformed or non-aggressive cancerous cells, and that this loss of regulation is mediated by a 2.2-kb region of the human Id-1 promoter. Here we show that a 31 bp sequence within this 2.2-kb promoter, located 200 bp upstream of the initiation of transcription, is responsible for the constitutive expression of Id-1 in metastatic human breast cancer cells. Using gel shift experiments, we identified a high molecular weight complex present only in non-aggressive breast cancer cells cultured in serum-free medium and which appear to be necessary for proper Id-1 repression. In contrast, nuclear extracts from highly aggressive and metastatic cell lines do not contain this large molecular weight complex. Using DNA affinity precipitation assays (DAPA), we show that this complex contains SP-1, NF-1, Rb and HDAC-1 proteins. On the basis of these findings, we propose a mechanism for the loss of regulation of Id-1 promoter in invasive and metastatic human breast cancer cells.