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1.
Front Cell Neurosci ; 9: 66, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25883547

RESUMO

Megalencephalic leukoencephalopathy with subcortical cysts (MLCs) disease is a rare inherited, autosomal recessive form of childhood-onset spongiform leukodystrophy characterized by macrocephaly, deterioration of motor functions, epileptic seizures and mental decline. Brain edema, subcortical fluid cysts, myelin and astrocyte vacuolation are the histopathological hallmarks of MLC. Mutations in either the MLC1 gene (>75% of patients) or the GlialCAM gene (<20% of patients) are responsible for the disease. Recently, the GlialCAM adhesion protein was found essential for the membrane expression and function of the chloride channel ClC-2 indicating MLC disease caused by mutation in GlialCAM as the first channelopathy among leukodystrophies. On the contrary, the function of MLC1 protein, which binds GlialCAM, its functional relationship with ClC-2 and the molecular mechanisms underlying MLC1 mutation-induced functional defects are not fully understood yet. The human MLC1 gene encodes a 377-amino acid membrane protein with eight predicted transmembrane domains which shows very low homology with voltage-dependent potassium (K(+)) channel subunits. The high expression of MLC1 in brain astrocytes contacting blood vessels and meninges and brain alterations observed in MLC patients have led to hypothesize a role for MLC1 in the regulation of ion and water homeostasis. Recent studies have shown that MLC1 establishes structural and/or functional interactions with several ion/water channels and transporters and ion channel accessory proteins, and that these interactions are affected by MLC1 mutations causing MLC. Here, we review data on MLC1 functional properties obtained in in vitro and in vivo models and discuss evidence linking the effects of MLC1 mutations to brain channelopathies.

2.
Neurobiol Dis ; 66: 1-18, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24561067

RESUMO

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in the gene encoding MLC1, a membrane protein mainly expressed in astrocytes in the central nervous system. Although MLC1 function is unknown, evidence is emerging that it may regulate ion fluxes. Using biochemical and proteomic approaches to identify MLC1 interactors and elucidate MLC1 function we found that MLC1 interacts with the vacuolar ATPase (V-ATPase), the proton pump that regulates endosomal acidity. Because we previously showed that in intracellular organelles MLC1 directly binds Na, K-ATPase, which controls endosomal pH, we studied MLC1 endosomal localization and trafficking and MLC1 effects on endosomal acidity and function using human astrocytoma cells overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. We found that WT MLC1 is abundantly expressed in early (EEA1(+), Rab5(+)) and recycling (Rab11(+)) endosomes and uses the latter compartment to traffic to the plasma membrane during hyposmotic stress. We also showed that WT MLC1 limits early endosomal acidification and influences protein trafficking in astrocytoma cells by stimulating protein recycling, as revealed by FITC-dextran measurement of endosomal pH and transferrin protein recycling assay, respectively. WT MLC1 also favors recycling to the plasma-membrane of the TRPV4 cation channel which cooperates with MLC1 to activate calcium influx in astrocytes during hyposmotic stress. Although MLC disease-causing mutations differentially affect MLC1 localization and trafficking, all the mutated proteins fail to influence endosomal pH and protein recycling. This study demonstrates that MLC1 modulates endosomal pH and protein trafficking suggesting that alteration of these processes contributes to MLC pathogenesis.


Assuntos
Astrócitos/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico , Animais , Encéfalo/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endossomos/efeitos dos fármacos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana/genética , Estresse Oxidativo , Transporte Proteico/efeitos dos fármacos , Ratos , Canais de Cátion TRPV/metabolismo , Transferrina/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
3.
Transl Neurosci ; 4(2)2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24340223

RESUMO

Astrocytes are the predominant glial cell population in the central nervous system (CNS). Once considered only passive scaffolding elements, astrocytes are now recognised as cells playing essential roles in CNS development and function. They control extracellular water and ion homeostasis, provide substrates for energy metabolism, and regulate neurogenesis, myelination and synaptic transmission. Due to these multiple activities astrocytes have been implicated in almost all brain pathologies, contributing to various aspects of disease initiation, progression and resolution. Evidence is emerging that astrocyte dysfunction can be the direct cause of neurodegeneration, as shown in Alexander's disease where myelin degeneration is caused by mutations in the gene encoding the astrocyte-specific cytoskeleton protein glial fibrillary acidic protein. Recent studies point to a primary role for astrocytes in the pathogenesis of other genetic leukodystrophies such as megalencephalic leukoencephalopathy with subcortical cysts and vanishing white matter disease. The aim of this review is to summarize current knowledge of the pathophysiological role of astrocytes focusing on their contribution to the development of the above mentioned leukodystrophies and on new perspectives for the treatment of neurological disorders.

4.
Exp Eye Res ; 116: 1-8, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23954924

RESUMO

Dysbindin, the product of the DTNBP1 gene, was identified by yeast two hybrid assay as a binding partner of dystrobrevin, a cytosolic component of the dystrophin protein complex. Although its functional role has not yet been completely elucidated, the finding that dysbindin assembles into the biogenesis of lysosome related organelles complex 1 (BLOC-1) suggests that it participates in intracellular trafficking and biogenesis of organelles and vesicles. Dysbindin is ubiquitous and in brain is expressed primarily in neurons. Variations at the dysbindin gene have been associated with increased risk for schizophrenia. As anomalies in retinal function have been reported in patients suffering from neuropsychiatric disorders, we investigated the expression of dysbindin in the retina. Our results show that differentially regulated dysbindin isoforms are expressed in rat retina during postnatal maturation. Interestingly, we found that dysbindin is mainly localized in Müller cells. The identification of dysbindin in glial cells may open new perspectives for a better understanding of the functional involvement of this protein in visual alterations associated to neuropsychiatric disorders.


Assuntos
Proteínas de Transporte/genética , Células Ependimogliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , RNA/genética , Retina/crescimento & desenvolvimento , Animais , Western Blotting , Proteínas de Transporte/biossíntese , Disbindina , Proteínas Associadas à Distrofina , Eletroforese , Células Ependimogliais/citologia , Masculino , Proteínas do Tecido Nervoso/biossíntese , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Retina/citologia , Retina/metabolismo
5.
FEBS J ; 279(22): 4131-44, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22978324

RESUMO

Dystrobrevin family members (α and ß) are cytoplasmic components of the dystrophin-associated glycoprotein complex, a multimeric protein complex first isolated from skeletal muscle, which links the extracellular matrix to the actin cytoskeleton. Dystrobrevin shares high homology with the cysteine-rich and C-terminal domains of dystrophin and a common domain organization. The ß-dystrobrevin isoform is restricted to nonmuscle tissues, serves as a scaffold for signaling complexes, and may participate in intracellular transport through its interaction with kinesin heavy chain. We have previously characterized the molecular determinants affecting the ß-dystrobrevin-kinesin heavy chain interaction, among which is cAMP-dependent protein kinase [protein kinase A (PKA)] phosphorylation of ß-dystrobrevin. In this study, we have identified ß-dystrobrevin residues phosphorylated in vitro by PKA with pull-down assays, surface plasmon resonance measurements, and MS analysis. Among the identified phosphorylated residues, we demonstrated, by site-directed mutagenesis, that Thr11 is the regulatory site for the ß-dystrobrevin-kinesin interaction. As dystrobrevin may function as a signaling scaffold for kinases/phosphatases, we also investigated whether ß-dystrobrevin is phosphorylated in vitro by kinases other than PKA. Thr11 was phosphorylated by protein kinase C, suggesting that this represents a key residue modified by the activation of different signaling pathways.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Associadas à Distrofina/metabolismo , Cinesinas/metabolismo , Neuropeptídeos/metabolismo , Proteína Quinase C/metabolismo , Treonina/metabolismo , Sequência de Aminoácidos , Western Blotting , Proteínas Associadas à Distrofina/genética , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Neuropeptídeos/genética , Fosforilação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de Superfície , Treonina/genética
6.
Exp Cell Res ; 318(19): 2460-9, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22814252

RESUMO

Dystroglycan (DG) is an extracellular receptor composed of two subunits, α-DG and ß-DG, connected through the α-DG C-terminal domain and the ß-DG N-terminal domain. We report an alanine scanning of all DG cysteine residues performed on DG-GFP constructs overexpressed in 293-Ebna cells, demonstrating that Cys-669 and Cys-713, both located within the ß-DG N-terminal domain, are key residues for the DG precursor cleavage and trafficking, but not for the interaction between the two DG subunits. In addition, we have used immunprecipitation and confocal microscopy showing that ERp57, a member of the disulfide isomerase family involved in glycoprotein folding, is associated and colocalizes immunohistochemically with ß-DG in the ER and at the plasma membrane of 293-Ebna cells. The ß-DG-ERp57 complex also included α-DG. DG mutants, unable to undergo the precursor cleavage, were still associated to ERp57. ß-DG and ERp57 were also co-immunoprecipitated in rat heart and kidney tissues. In vitro, a mutant ERp57, mimicking the reduced form of the wild-type protein, interacts directly with the recombinant N-terminal domain of both α-DG and ß-DG with apparent dissociation constant values in the micromolar range. ERp57 is likely to be involved in the DG processing/maturation pathway, but its association to the mature DG complex might also suggest some further functional role that needs to be investigated.


Assuntos
Distroglicanas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Distroglicanas/genética , Glicosilação , Células HEK293 , Coração/fisiologia , Humanos , Rim/metabolismo , Rim/fisiologia , Mutação , Ligação Proteica/genética , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Subunidades Proteicas , Transporte Proteico/fisiologia , Ratos
7.
Hum Mol Genet ; 21(10): 2166-80, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22328087

RESUMO

Megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare leukodystrophy characterized by macrocephaly, subcortical fluid cysts and myelin vacuolation, has been linked to mutations in the MLC1 gene. This gene encodes a membrane protein that is highly expressed in astrocytes. Based on MLC pathological features, it was proposed that astrocyte-mediated defects in ion and fluid homeostasis could account for the alterations observed in MLC-affected brains. However, the role of MLC1 and the effects of pathological mutations on astrocyte osmoregulatory functions have still to be demonstrated. Using human astrocytoma cells stably overexpressing wild-type MLC1 or three known MLC-associated pathological mutations, we investigated MLC1 involvement in astrocyte reaction to osmotic changes using biochemical, dynamic video imaging and immunofluorescence techniques. We have found that MLC1 overexpressed in astrocytoma cells is mainly localized in the plasma membrane, is part of the Na,K-ATPase-associated molecular complex that includes the potassium channel Kir4.1, syntrophin and aquaporin-4 and functionally interacts with the calcium permeable channel TRPV4 (transient receptor potential vanilloid-4 cation channel) which mediates swelling-induced cytosolic calcium increase and volume recovery in response to hyposmosis. Pathological MLC mutations cause changes in MLC1 expression and intracellular localization as well as in the astrocyte response to osmotic changes by altering MLC1 molecular interactions with the Na,K-ATPase molecular complex and abolishing the increase in calcium influx induced by hyposmosis and treatment with the TRPV4 agonist 4αPDD. These data demonstrate, for the first time, that MLC1 plays a role in astrocyte osmo-homeostasis and that defects in intracellular calcium dynamics may contribute to MLC pathogenesis.


Assuntos
Astrócitos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Canais de Cátion TRPV/metabolismo , Cálcio/metabolismo , Cátions Bivalentes , Cistos/genética , Cistos/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Mutação , Osmose , Transfecção
8.
BMC Cancer ; 11: 17, 2011 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-21241471

RESUMO

BACKGROUND: "High risk" human papillomavirus strains are the causative agents of the vast majority of carcinomas of the uterine cervix. In these tumors, the physical integration of the HPV genome is a frequent, though not invariable occurrence, but the constitutive expression of the E6 and E7 viral genes is always observed, suggesting key roles for the E6 and E7 oncoproteins in the process of malignant transformation. The "intracellular antibody" technology using recombinant antibodies in single-chain format offers the possibility of targeting a protein in its intracellular environment even at the level of definite domains thus representing a valuable strategy to "knock out" the function of specific proteins. METHODS: In this study, we investigate the in vitro activity of two single-chain antibody fragments directed against the "high-risk" HPV 16 E7 oncoprotein, scFv 43M2 and scFv 51. These scFvs were expressed by retroviral system in different cell compartments of the HPV16-positive SiHa cells, and cell proliferation was analyzed by Colony Formation Assay and EZ4U assay. The binding of these scFvs to E7, and their possible interference with the interaction between E7 and its main target, the tumor suppressor pRb protein, were then investigated by immunoassays, PepSet™ technology and Surface Plasmon Resonance. RESULTS: The expression of the two scFvs in the nucleus and the endoplasmic reticulum of SiHa cells resulted in the selective growth inhibition of these cells. Analysis of binding showed that both scFvs bind E7 via distinct but overlapping epitopes not corresponding to the pRb binding site. Nevertheless, the binding of scFv 43M2 to E7 was inhibited by pRb in a non-competitive manner. CONCLUSIONS: Based on the overall results, the observed inhibition of HPV-positive SiHa cells proliferation could be ascribed to an interaction between scFv and E7, involving non-pRb targets. The study paves the way for the employment of specific scFvs in immunotherapeutic approaches against the HPV-associated lesions.


Assuntos
Proliferação de Células , Papillomavirus Humano 16/imunologia , Proteínas E7 de Papillomavirus/imunologia , Anticorpos de Cadeia Única/imunologia , Ligação Competitiva , Linhagem Celular Tumoral , Mapeamento de Epitopos , Feminino , Imunofluorescência , Células HEK293 , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/fisiologia , Humanos , Proteínas E7 de Papillomavirus/metabolismo , Ligação Proteica , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Ressonância de Plasmônio de Superfície , Transfecção , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
9.
Hum Mol Genet ; 20(1): 90-103, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-20926452

RESUMO

Megalencephalic leucoencephalopathy with subcortical cysts (MLC) is a rare congenital leucodystrophy caused by mutations in MLC1, a membrane protein of unknown function. MLC1 expression in astrocyte end-feet contacting blood vessels and meninges, along with brain swelling, fluid cysts and myelin vacuolation observed in MLC patients, suggests a possible role for MLC1 in the regulation of fluid and ion homeostasis and cellular volume changes. To identify MLC1 direct interactors and dissect the molecular pathways in which MLC1 is involved, we used NH2-MLC1 domain as a bait to screen a human brain library in a yeast two-hybrid assay. We identified the ß1 subunit of the Na,K-ATPase pump as one of the interacting clones and confirmed it by pull-downs, co-fractionation assays and immunofluorescence stainings in human and rat astrocytes in vitro and in brain tissue. By performing ouabain-affinity chromatography on astrocyte and brain extracts, we isolated MLC1 and the whole Na,K-ATPase enzyme in a multiprotein complex that included Kir4.1, syntrophin and dystrobrevin. Because Na,K-ATPase is involved in intracellular osmotic control and volume regulation, we investigated the effect of hypo-osmotic stress on MLC1/Na,K-ATPase relationship in astrocytes. We found that hypo-osmotic conditions increased MLC1 membrane expression and favoured MLC1/Na,K-ATPase-ß1 association. Moreover, hypo-osmosis induced astrocyte swelling and the reversible formation of endosome-derived vacuoles, where the two proteins co-localized. These data suggest that through its interaction with Na,K-ATPase, MLC1 is involved in the control of intracellular osmotic conditions and volume regulation in astrocytes, opening new perspectives for understanding the pathological mechanisms of MLC disease.


Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Proteínas de Membrana/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células Cultivadas , Cistos/genética , Cistos/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Proteínas de Membrana/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Complexos Multiproteicos/metabolismo , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/genética
10.
J Biol Chem ; 285(32): 24740-50, 2010 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-20530487

RESUMO

alpha and beta dystrobrevins are cytoplasmic components of the dystrophin-associated protein complex that are thought to play a role as scaffold proteins in signal transduction and intracellular transport. In the search of new insights into the functions of beta-dystrobrevin, the isoform restricted to non-muscle tissues, we performed a two-hybrid screen of a mouse cDNA library to look for interacting proteins. Among the positive clones, one encodes iBRAF/HMG20a, a high mobility group (HMG)-domain protein that activates REST (RE-1 silencing transcription factor)-responsive genes, playing a key role in the initiation of neuronal differentiation. We characterized the beta-dystrobrevin-iBRAF interaction by in vitro and in vivo association assays, localized the binding region of one protein to the other, and assessed the kinetics of the interaction as one of high affinity. We also found that beta-dystrobrevin directly binds to BRAF35/HMG20b, a close homologue of iBRAF and a member of a co-repressor complex required for the repression of neural specific genes in neuronal progenitors. In vitro assays indicated that beta-dystrobrevin binds to RE-1 and represses the promoter activity of synapsin I, a REST-responsive gene that is a marker for neuronal differentiation. Altogether, our data demonstrate a direct interaction of beta-dystrobrevin with the HMG20 proteins iBRAF and BRAF35 and suggest that beta-dystrobrevin may be involved in regulating chromatin dynamics, possibly playing a role in neuronal differentiation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Associadas à Distrofina/fisiologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Neurônios/citologia , Animais , Células COS , Proteínas de Ciclo Celular , Diferenciação Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Cromatina/química , Humanos , Cinética , Camundongos , Distrofias Musculares/metabolismo , Ratos , Ressonância de Plasmônio de Superfície
11.
DNA Repair (Amst) ; 9(6): 700-7, 2010 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-20418187

RESUMO

The MUTYH DNA glycosylase specifically removes adenine misincorporated by replicative polymerases opposite the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoG). A defective protein activity results in the accumulation of G>T transversions because of unrepaired 8-oxoG:A mismatches. In humans, MUTYH germline mutations are associated with a recessive form of familial adenomatous polyposis and colorectal cancer predisposition (MUTYH-associated polyposis, MAP). Here we studied the repair capacity of the MUTYH variants R171W, E466del, 137insIW, Y165C and G382D, identified in MAP patients. Following expression and purification of human proteins from a bacterial system, we investigated MUTYH incision capacity on an 8-oxoG:A substrate by standard glycosylase assays. For the first time, we employed the surface plasmon resonance (SPR) technology for real-time recording of the association/dissociation of wild-type and MUTYH variants from an 8-oxoG:A DNA substrate. When compared to the wild-type protein, R171W, E466del and Y165C variants showed a severe reduction in the binding affinity towards the substrate, while 137insIW and G382D mutants manifested only a slight decrease mainly due to a slower rate of association. This reduced binding was always associated with impairment of glycosylase activity, with adenine removal being totally abrogated in R171W, E466del and Y165C and only partially reduced in 137insIW and G382D. Our findings demonstrate that SPR analysis is suitable to identify defective enzymatic behaviour even when mutant proteins display minor alterations in substrate recognition.


Assuntos
Polipose Adenomatosa do Colo/genética , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Sequência de Bases , Domínio Catalítico , DNA/genética , DNA/metabolismo , DNA Glicosilases/química , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Cinética , Proteínas Ligantes de Maltose , Proteínas Mutantes/química , Proteínas Periplásmicas de Ligação/metabolismo , Ressonância de Plasmônio de Superfície
12.
Neurobiol Dis ; 35(2): 278-85, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19481149

RESUMO

The early effects of the diabetic milieu on retinal tissue and their relation to the Renin-Angiotensin system (RAS) activation are poorly known. Here we investigated RAS signaling in retinas explanted from adult rats exposed for 48 h to high glucose (HG), with or without the Angiotensin Converting Enzyme inhibitor enalaprilat, which blocks RAS. HG was observed to i) initiate a phosphotyrosine-dependent signaling cascade; ii) up-regulate Angiotensin(1) Receptor (AT(1)R); iii) activate src tyrosine kinase and increase phosphorylation of Pyk2, PLCgamma1 and ERK1/2; and iv) activate Akt and the transcription factor CREB. In the presence of enalaprilat, tyrosine phosphorylation signal and AT(1)R upregulation decreased and activation of PLCgamma1 and CREB reverted, showing their relation to RAS signaling. In line with Akt activation, no apoptosis or synapse degeneration was found. Müller glia was activated, but in a RAS-independent manner. Our results suggest that, in early phases of HG exposure, a pro-survival cell program may be induced in the retina.


Assuntos
Glucose/metabolismo , Sistema Renina-Angiotensina/fisiologia , Retina/fisiologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Enalaprilato/farmacologia , Quinase 2 de Adesão Focal/metabolismo , Técnicas In Vitro , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfolipase C gama/metabolismo , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Retina/efeitos dos fármacos , Neurônios Retinianos/efeitos dos fármacos , Neurônios Retinianos/fisiologia , Fatores de Tempo , Quinases da Família src/metabolismo
13.
Eur J Cell Biol ; 88(6): 343-56, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19304341

RESUMO

In human red blood cells, protein 4.1 (4.1R) is an 80-kDa polypeptide that stabilizes the spectrin-actin network and anchors it to the plasma membrane. In non-erythroid cells there is a great variety of 4.1R isoforms, mainly generated by alternative pre-mRNA splicing, which localize at various intracellular sites, including the nucleus. We studied protein 4.1R distribution in relation to beta-spectrin, actin and cytokeratin during Xenopus oogenesis. Immunoprecipitation experiments indicate that at least two isoforms of protein 4.1R are present in Xenopus laevis oocytes: a 56-kDa form in the cytoplasm and a 37-kDa form in the germinal vesicle (GV). Antibodies to beta-spectrin reveal two bands of 239 and 100 kDa in the cytoplasm. Coimmunoprecipitation experiments indicate that both the 37- and 56-kDa isoforms of protein 4.1R associate with the 100-kDa isoform of beta-spectrin. Moreover, the 56-kDa form coimmunoprecipitates with a cytokeratin of the same molecular weight. Confocal immunolocalization shows that protein 4.1R distribution is in the peripheral cytoplasm, in the mitochondrial cloud (MC) and in the GV of previtellogenic oocytes. In the cytoplasm of vitellogenic oocytes, a loose network of fibers stained by the anti-protein 4.1R antibody spreads across the cytoplasm. beta-Spectrin has a similar distribution. Protein 4.1R was found to colocalize with actin in the cortex of oocytes in the form of fluorescent dots. Double immunolocalization of protein 4.1R and cytokeratin depicts two separate networks that overlap throughout the whole cytoplasm. Protein 4.1R filaments partially colocalize with cytokeratin in both the animal and vegetal hemispheres. We hypothesize that protein 4.1R could function as a linker protein between cytokeratin and the actin-based cytoskeleton.


Assuntos
Citoesqueleto/metabolismo , Oogênese/fisiologia , Proteínas de Xenopus/metabolismo , Actinas/metabolismo , Animais , Western Blotting , Citoplasma/metabolismo , Imunoprecipitação , Queratinas/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Ligação Proteica , Isoformas de Proteínas/metabolismo , Espectrina/metabolismo , Xenopus laevis
14.
Hum Mol Genet ; 17(13): 2018-29, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18372317

RESUMO

Missense PTPN11 mutations cause Noonan and LEOPARD syndromes (NS and LS), two developmental disorders with pleiomorphic phenotypes. PTPN11 encodes SHP2, an SH2 domain-containing protein tyrosine phosphatase functioning as a signal transducer. Generally, different substitutions of a particular amino acid residue are observed in these diseases, indicating that the crucial factor is the residue being replaced. For a few codons, only one substitution is observed, suggesting the possibility of specific roles for the residue introduced. We analyzed the biochemical behavior and ligand-binding properties of all possible substitutions arising from single-base changes affecting codons 42, 139, 279, 282 and 468 to investigate the mechanisms underlying the invariant occurrence of the T42A, E139D and I282V substitutions in NS and the Y279C and T468M changes in LS. Our data demonstrate that the isoleucine-to-valine change at codon 282 is the only substitution at that position perturbing the stability of SHP2's closed conformation without impairing catalysis, while the threonine-to-alanine change at codon 42, but not other substitutions of that residue, promotes increased phosphopeptide-binding affinity. The recognition specificity of the C-SH2 domain bearing the E139D substitution differed substantially from its wild-type counterpart acquiring binding properties similar to those observed for the N-SH2 domain, revealing a novel mechanism of SHP2's functional dysregulation. Finally, while functional selection does not seem to occur for the substitutions at codons 279 and 468, we point to deamination of the methylated cytosine at nucleotide 1403 as the driving factor leading to the high prevalence of the T468M change in LS.


Assuntos
Substituição de Aminoácidos , Síndrome LEOPARD/genética , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Simulação por Computador , Análise Mutacional de DNA , Células HeLa , Humanos , Síndrome LEOPARD/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Síndrome de Noonan/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo
15.
Matrix Biol ; 27(4): 360-70, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18249103

RESUMO

Keratoepithelin (KE) is an extracellular matrix protein that binds collagens, fibronectin, decorin, biglycan and integrins, interconnecting extracellular matrix components with resident cells in several tissues. KE has a molecular mass of 68 kDa and harbours four FAS1 domains named after those identified in the insect cell adhesion molecule fasciclin I. In humans, KE is preferentially expressed by the corneal epithelial layer and liberated towards the corneal stroma but it was also detected in the lung and in the bladder smooth muscle. No detailed information is available on the distribution of this protein in other human tissues. In this work, we have raised a polyclonal antibody against the recombinantly expressed human fourth FAS1 domain which is able to specifically detect KE in human skeletal muscle tissue extracts. Immunofluorescence experiments indicate that KE is localized around the perimysium and endomysium of each skeletal muscle fiber. The same kind of analysis shows that in muscle sections from patients affected by different forms of muscular dystrophy KE is upregulated and widely distributed in fibrotic tissues. The muscle specific expression of KE was also demonstrated by RT-PCR. In human skeletal muscle, KE may help to build up a bridge between collagen VI and yet unidentified muscle receptor(s), adding to the complexity of the adhesive molecular network established between muscle fibers and the surrounding basement membrane.


Assuntos
Proteínas da Matriz Extracelular/imunologia , Proteínas da Matriz Extracelular/metabolismo , Músculo Esquelético/imunologia , Músculo Esquelético/metabolismo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Córnea/metabolismo , Reações Cruzadas , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peso Molecular , RNA Mensageiro/genética , Ratos , Alinhamento de Sequência , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/genética
16.
Mol Cell Neurosci ; 36(2): 174-84, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17698368

RESUMO

Axotomy of superior cervical ganglion (SCG) neurons is characterized by peripheral regeneration of injured axons and temporary disassembly of the intraganglionic synapses, necessary for synaptic silencing. Both events require remodeling of the extracellular matrix achieved through controlled proteolysis of its components by different enzymatic systems. In this study, we investigate the involvement of the plasminogen enzymatic cascade in the response to axotomy of rat SCG neurons. All components of this proteolytic pathway, tissue plasminogen activator (tPA), plasminogen, membrane receptor annexin II and tPA inhibitor (PAI-1), are constitutively expressed in uninjured SCG and increase significantly after SCG neuron axotomy. Immunolocalization of plasminogen, the key protein converted into the enzymatically active plasmin by tPA, in both neurons and non-neuronal cells indicates that all cell types are involved in the response to axotomy. The time course of activation of tPA/plasmin enzymatic pathway suggests its involvement in both intraganglionic synapse remodeling and axonal regeneration.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Neurônios/enzimologia , Plasminogênio/metabolismo , Gânglio Cervical Superior/citologia , Simpatectomia , Animais , Imunoprecipitação/métodos , Masculino , Microscopia Eletrônica de Transmissão/métodos , Neurônios/ultraestrutura , Inibidores de Proteases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Peptídeos/metabolismo , Transdução de Sinais , Fatores de Tempo , Ativador de Plasminogênio Tecidual/metabolismo
17.
J Mol Biol ; 371(5): 1174-87, 2007 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-17610895

RESUMO

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Associadas à Distrofina/química , Proteínas Associadas à Distrofina/fisiologia , Animais , Sítios de Ligação , Encéfalo/metabolismo , Células COS , Chlorocebus aethiops , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico , Matriz Extracelular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido
18.
J Neurosci Res ; 85(12): 2631-9, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17265465

RESUMO

The dystrobrevins (alpha and beta) are components of the dystrophin-associated protein complex (DPC), which links the cytoskeleton to the extracellular matrix and serves as a scaffold for signaling proteins. The precise functions of the beta-dystrobrevin isoform, which is expressed in nonmuscle tissues, have not yet been determined. To gain further insights into the role of beta-dystrobrevin in brain, we performed a yeast two-hybrid screen and identified pancortin-2 as a novel beta-dystrobrevin-binding partner. Pancortins-1-4 are neuron-specific olfactomedin-related glycoproteins, highly expressed during brain development and widely distributed in the mature cerebral cortex of the mouse. Pancortins are important constituents of the extracellular matrix and are thought to play an essential role in neuronal differentiation. We characterized the interaction between pancortin-2 and beta-dystrobrevin by in vitro and in vivo association assays and mapped the binding site of pancortin-2 on beta-dystrobrevin to amino acids 202-236 of the beta-dystrobrevin molecule. We also found that the domain of interaction for beta-dystrobrevin is contained in the B part of pancortin-2, a central region that is common to all four pancortins. Our results indicate that beta-dystrobrevin could interact with all members of the pancortin family, implying that beta-dystrobrevin may be involved in brain development. We suggest that dystrobrevin, a motor protein receptor that binds kinesin heavy chain, might play a role in intracellular transport of pancortin to specific sites in the cell.


Assuntos
Proteínas Associadas à Distrofina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células COS , Chlorocebus aethiops , Clonagem Molecular/métodos , Proteínas da Matriz Extracelular/farmacocinética , Mutação , Ligação Proteica , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Ratos , Isótopos de Enxofre/farmacocinética , Transfecção , Técnicas do Sistema de Duplo-Híbrido
19.
FEBS J ; 273(21): 4929-43, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17018058

RESUMO

The dystroglycan adhesion complex consists of two noncovalently interacting proteins: alpha-dystroglycan, a peripheral extracellular subunit that is extensively glycosylated, and the transmembrane beta-dystroglycan, whose cytosolic tail interacts with dystrophin, thus linking the F-actin cytoskeleton to the extracellular matrix. Dystroglycan is thought to play a crucial role in the stability of the plasmalemma, and forms strong contacts between the extracellular matrix and the cytoskeleton in a wide variety of tissues. Abnormal membrane targeting of dystroglycan subunits and/or their aberrant post-translational modification are often associated with several pathologic conditions, ranging from neuromuscular disorders to carcinomas. A putative functional hotspot of dystroglycan is represented by its intersubunit surface, which is contributed by two amino acid stretches: approximately 30 amino acids of beta-dystroglycan (691-719), and approximately 15 amino acids of alpha-dystroglycan (550-565). Exploiting alanine scanning, we have produced a panel of site-directed mutants of our two consolidated recombinant peptides beta-dystroglycan (654-750), corresponding to the ectodomain of beta-dystroglycan, and alpha-dystroglycan (485-630), spanning the C-terminal domain of alpha-dystroglycan. By solid-phase binding assays and surface plasmon resonance, we have determined the binding affinities of mutated peptides in comparison to those of wild-type alpha-dystroglycan and beta-dystroglycan, and shown the crucial role of two beta-dystroglycan phenylalanines, namely Phe692 and Phe718, for the alpha-beta interaction. Substitution of the alpha-dystroglycan residues Trp551, Phe554 and Asn555 by Ala does not affect the interaction between dystroglycan subunits in vitro. As a preliminary analysis of the possible effects of the aforementioned mutations in vivo, detection through immunofluorescence and western blot of the two dystroglycan subunits was pursued in dystroglycan-transfected 293-Ebna cells.


Assuntos
Distroglicanas/química , Linhagem Celular , Distroglicanas/genética , Distroglicanas/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fenilalanina/genética , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
Cancer Biol Ther ; 5(4): 441-8, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16575202

RESUMO

Dystroglycan (DG) is an integral membrane receptor of extracellular matrix proteins, composed of two subunits alpha and beta derived from a common precursor. In brain DG is expressed in neurons, glia limitans, astrocytic endfeet around vessels and endothelial cells. We investigate whether DG may play a role in brain tumors. Western blot and immunofluorescence analysis showed that, while beta-DG subunit was present, the highly glycosylated alpha-DG subunit was strongly reduced in surgically derived human glioblastoma biopsies, in low passage patient-derived cultures and in glioma cell lines, U87MG and A172MG, but not in all glioma cell lines tested. Immunohistochemistry of tumor frozen sections revealed that the loss of alpha-DG was confined in the tumor area but not around blood vessels. Overexpression of DG decreased the growth rate of the glioma cell lines lacking the highly glycosylated alpha-DG subunit and the colony-forming efficiency. Clonogenic assay in presence of temozolomide showed an additive effect between DG overexpression and drug treatment. Our data suggest that DG may be involved in the progression of primary brain tumors.


Assuntos
Distroglicanas/fisiologia , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Animais , Western Blotting , Encéfalo/embriologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Distroglicanas/química , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência , Invasividade Neoplásica , Ratos , Transfecção
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