RESUMO
Our previous work has correlated permanent alterations in the rat neurosecretory machinery with epileptogenesis. Such findings highlighted the need for a greater understanding of the molecular mechanisms underlying epilepsy so that novel therapeutic regimens can be designed. To this end, we examined kindling in transgenic mice with a defined reduction of a key element of the neurosecretory machinery: the v-SNARE (vesicle-bound SNAP [soluble NSF attachment protein] receptor), synaptobrevin/vesicle-associated membrane protein 2 (VAMP2). Initial analysis of biochemical markers, which previously displayed kindling-dependent alterations in rat hippocampal synaptosomes, showed similar trends in both wild-type and VAMP2(+/-) mice, demonstrating that kindled rat and mouse models are comparable. This report focuses on the effects that a ~50% reduction of synaptosomal VAMP2 has on the progression of electrical kindling and on glutamate release in hippocampal subregions. Our studies show that epileptogenesis is dramatically attenuated in VAMP2(+/-) mice, requiring both higher current and more stimulations to reach a fully kindled state (two successive Racine stage 5 seizures). Progression through the five identifiable Racine stages was slower and more variable in the VAMP2(+/-) animals compared with the almost linear progression seen in wild-type littermates. Consistent with the expected effects of reducing a major neuronal v-SNARE, glutamate-selective, microelectrode array (MEA) measurements in specific hippocampal subregions of VAMP2(+/-) mice showed significant reductions in potassium-evoked glutamate release. Taken together these studies demonstrate that manipulating the levels of the neurosecretory machinery not only affects neurotransmitter release but also mitigates kindling-induced epileptogenesis.
Assuntos
Epilepsia/fisiopatologia , Excitação Neurológica/genética , Excitação Neurológica/fisiologia , Proteína 2 Associada à Membrana da Vesícula/biossíntese , Proteína 2 Associada à Membrana da Vesícula/fisiologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/metabolismo , Região CA2 Hipocampal/efeitos dos fármacos , Região CA2 Hipocampal/metabolismo , Interpretação Estatística de Dados , Giro Denteado/efeitos dos fármacos , Giro Denteado/metabolismo , Estimulação Elétrica , Ácido Glutâmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microeletrodos , Proteínas SNARE/fisiologia , Proteína 2 Associada à Membrana da Vesícula/genéticaRESUMO
BACKGROUND: Variation in platelet reactivity contributes to disorders of hemostasis and thrombosis, but the molecular mechanisms are not well understood. OBJECTIVES: To discover associations between interindividual platelet variability and the responsible platelet genes, and to begin to define the molecular mechanisms altering platelet gene expression. SUBJECTS/METHODS: Two hundred and eighty-eight healthy subjects were phenotyped for platelet responsiveness. Platelet RNA from subjects demonstrating hyperreactivity (n=18) and hyporeactivity (n=11) was used to screen the human transcriptome. RESULTS: Distinctly different mRNA profiles were observed between subjects with differing platelet reactivity. Increased levels of mRNA for VAMP8/endobrevin, a critical v-SNARE involved in platelet granule secretion, were associated with platelet hyperreactivity (Q=0.0275). Validation studies of microarray results showed 4.8-fold higher mean VAMP8 mRNA levels in hyperreactive than hyporeactive platelets (P=0.0023). VAMP8 protein levels varied 13-fold among platelets from these normal subjects, and were 2.5-fold higher in hyperreactive platelets (P=0.05). Among our cohort of 288 subjects, a VAMP8 single-nucleotide polymorphism (rs1010) was associated with platelet reactivity in an age-dependent manner (P<0.003). MicroRNA-96 was predicted to bind to the 3'-untranslated regionof VAMP8 mRNA and was detected in platelets. Overexpression of microRNA-96 in VAMP8-expressing cell lines caused a dose-dependent decrease in VAMP8 protein and mRNA, suggesting a role in VAMP8 mRNA degradation. CONCLUSIONS: These findings support a role for VAMP8/endobrevin in the heterogeneity of platelet reactivity, and suggest a role for microRNA-96 in the regulation of VAMP8 expression.
Assuntos
Plaquetas/metabolismo , MicroRNAs/sangue , Agregação Plaquetária/genética , Polimorfismo de Nucleotídeo Único , Proteínas R-SNARE/genética , Regiões 3' não Traduzidas , Adulto , Fatores Etários , Sítios de Ligação , Epinefrina , Feminino , Perfilação da Expressão Gênica/métodos , Genótipo , Células HCT116 , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Proteínas R-SNARE/sangue , RNA Mensageiro/sangue , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Regulação para Cima , Adulto JovemRESUMO
Kindling is a model of complex partial epilepsy wherein periodic application of an initially subconvulsive stimulus leads to first limbic and then generalized tonic-clonic seizures. Several laboratories have reported that augmented neurotransmitter release of l-glutamate is associated with the chronically kindled state. Neurotransmitter release requires membrane proteins called SNAREs, which form transmembrane complexes that participate in vesicle docking and are required for membrane fusion. We show here that kindling by entorhinal stimulation is associated with an accumulation of 7S SNARE complexes in the ipsilateral hippocampus. This increase of 7S SNARE complexes appears to begin early in the kindling process, achieves a peak with full kindling, and remains at this level for at least a month following cessation of further kindling stimuli. The increase is focal and permanently limited to the ipsilateral hippocampus despite progression to generalized electrographic and behavioral seizures. It is not seen in animals that receive electroconvulsive seizures, suggesting it is related to the kindling process itself. The duration and focality of increased 7S SNARE complexes with entorhinal kindling suggest that this is an altered molecular process associated with epileptogenesis.
Assuntos
Hipocampo/metabolismo , Excitação Neurológica/metabolismo , Proteínas de Membrana/metabolismo , Sinaptossomos/metabolismo , Proteínas de Transporte Vesicular , Animais , Eletroforese em Gel de Poliacrilamida , Hipocampo/química , Substâncias Macromoleculares , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/química , Proteínas Qa-SNARE , Proteínas R-SNARE , Ratos , Ratos Sprague-Dawley , Proteínas SNARE , Sinaptossomos/químicaRESUMO
The role of calpain in platelet function is generally associated with aggregation and clot retraction. In this report, data are presented to show that one component of the platelet secretory machinery, SNAP-23, is specifically cleaved by calpain in activated cells. Other proteins of the membrane fusion machinery, e.g. syntaxins 2 and 4 and alpha-SNAP, are not affected. In vitro studies, using permeabilized platelets, demonstrate that cleavage is time- and calcium-dependent. Analysis of SNAP-23 cleavage products suggests that the calpain cleavage site(s) is in the C-terminal third of the molecule potentially between the cysteine-rich acyl attachment sites and the C-terminal coiled-coil domain. The time course of cleavage is most consistent with late calpain-mediated events such as pp60(c-src) cleavage, but not early events such as protein-tyrosine phosphatase-1B activation. SNAP-23 cleavage is inhibited by calpeptin, calpastatin, calpain inhibitor IV, and E-64d, but not by caspase 3 inhibitor III or cathepsin inhibitor I. When tested for their effect on secretion, none of the calpain-specific inhibitors significantly affected release of soluble components from any of the three platelet granule storage pools. These results indicate that SNAP-23 cleavage occurs after granule release and therefore may play a role in affecting granule membrane exteriorization. This is consistent with the ultrastructural morphology of calpeptin-treated platelets after activation.
Assuntos
Plaquetas/fisiologia , Calpaína/sangue , Proteínas de Transporte/sangue , Membrana Celular/fisiologia , Ativação Plaquetária/fisiologia , Sequência de Aminoácidos , Proteínas de Transporte/química , Membrana Celular/ultraestrutura , Grânulos Citoplasmáticos/fisiologia , Dipeptídeos/química , Exocitose/fisiologia , Humanos , Técnicas In Vitro , Cinética , Fusão de Membrana/fisiologia , Fragmentos de Peptídeos/química , Proteínas Tirosina Fosfatases/sangue , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Our understanding of the molecular mechanisms of membrane trafficking advanced at a rapid rate during the 1990s. As one of the initial protein components of the trafficking machinery to be identified, N-ethylmaleimide sensitive factor (NSF) has served as a reference point in many of these recent studies. This hexameric ATPase is essential for most of the membrane-trafficking events in a cell. Initially, due to its ATPase activity, NSF was thought to be the motor that drove membrane fusion. Subsequent studies have shown that NSF actually plays the role of a chaperone by activating SNAP receptor proteins (SNAREs) so that they can participate in membrane fusion. In this review we will examine the initial characterization of NSF, its role in membrane fusion events, and what new structural information can tell us about NSF's mechanism of action.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Fusão de Membrana/fisiologia , Estrutura Terciária de Proteína , Proteínas de Transporte Vesicular , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Humanos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Sensíveis a N-Etilmaleimida , Proteínas SNARE , Alinhamento de Sequência , Proteínas de Ligação a Fator Solúvel Sensível a N-EtilmaleimidaAssuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Etilmaleimida/farmacologia , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular , Proteínas de Transporte/química , Cristalografia , Proteínas Sensíveis a N-Etilmaleimida , Conformação Proteica , Proteínas SNARERESUMO
Critical to SNARE protein function in neurotransmission are the accessory proteins, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP), and NSF, that play a role in activation of the SNAREs for membrane fusion. In this report, we demonstrate the depolarization-induced, calcium-dependent phosphorylation of NSF in rat synaptosomes. Phosphorylation of NSF is coincident with neurotransmitter release and requires an influx of external calcium. Phosphoamino acid analysis of the radiolabeled NSF indicates a role for a serine/threonine-specific kinase. Synaptosomal phosphorylation of NSF is stimulated by phorbol esters and is inhibited by staurosporine, chelerythrine, bisindolylmaleimide I, calphostin C, and Ro31-8220 but not the calmodulin kinase II inhibitor, Kn-93, suggesting a role for protein kinase C (PKC). Indeed, NSF is phosphorylated by PKC in vitro at Ser-237 of the catalytic D1 domain. Mutation of this residue to glutamic acid or to alanine eliminates in vitro phosphorylation. Molecular modeling studies suggest that Ser-237 is adjacent to an inter-subunit interface at a position where its phosphorylation could affect NSF activity. Consistently, mutation of Ser-237 to Glu, to mimic phosphorylation, results in a hexameric form of NSF that does not bind to SNAP-SNARE complexes, whereas the S237A mutant does form complex. These data suggest a negative regulatory role for PKC phosphorylation of NSF.
Assuntos
Proteínas de Transporte/metabolismo , Neurotransmissores/metabolismo , Sinaptossomos/metabolismo , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Potenciais da Membrana , Dados de Sequência Molecular , Proteínas Sensíveis a N-Etilmaleimida , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Serina/metabolismo , Sinaptossomos/fisiologiaRESUMO
The role of alpha/beta-SNAP (Soluble NSF Attachment Protein) in vesicular trafficking is well established; however, the function of the ubiquitously expressed gamma-SNAP remains unclear. To further characterize the cellular role of this enigmatic protein, a two-hybrid screen was used to identify new, gamma-SNAP-binding proteins and to uncover potentially novel functions for gamma-SNAP. One such SNAP-binding protein, termed Gaf-1 (gamma-SNAP associate factor-1) specifically binds gamma- but not alpha-SNAP. The full-length Gaf-1 (75 kDa) is ubiquitously expressed and is found stoichiometrically associated with gamma-SNAP in cellular extracts. This binding is distinct from other SNAP interactions since no alpha-SNAP or NSF coprecipitated with Gaf-1. Subcellular fractionation and immunofluorescence analysis show that Gaf-1 is peripherally associated with the outer mitochondrial membrane. Only a fraction of gamma-SNAP was mitochondrial with the balance being either cytosolic or associated with other membrane fractions. GFP-gamma-SNAP and the C-terminal domain of Gaf-1 both show a reticular distribution in HEK-293 cells. This reticular structure colocalizes with Gaf-1 and mitochondria as well as with microtubules but not with other cytoskeletal elements. These data identify a class of gamma-SNAP interactions that is distinct from other members of the SNAP family and point to a potential role for gamma-SNAP in mitochondrial dynamics.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Proteínas de Transporte Vesicular , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/análise , Linhagem Celular , Citosol/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Cinética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Especificidade por Substrato , Transfecção , Tubulina (Proteína)/metabolismoRESUMO
On stimulation by strong agonists, platelets release the contents of 3 storage compartments in 2 apparent waves of exocytosis. The first wave is the release of alpha- and dense core granule contents and the second is the release of lysosomal contents. Using a streptolysin O-permeabilized platelet exocytosis assay, we show that hexosaminidase release is stimulated by either Ca(++) or by GTP-gamma-S. This release step retains the same temporal separation from serotonin release as seen in intact platelets. This assay system was also used to dissect the molecular mechanisms of lysosome exocytosis. Lysosome release requires adenosine triphosphate and the general membrane fusion protein, N-ethylmaleimide sensitive factor. Uniquely, 2 syntaxin t-SNAREs, syntaxin 2 and 4, which localize to granules and open canalicular membranes, together with the general target membrane SNAP receptor (t-SNARE) protein SNAP-23 appear to make up the heterodimeric t-SNAREs required for lysosome exocytosis. These studies further show that regardless of stimuli (Ca(++) or GTP-gamma-S) serotonin and hexosaminidase release requires the same membrane fusion machinery. (Blood. 2000;96:1782-1788)
Assuntos
Plaquetas/metabolismo , Exocitose/fisiologia , Proteínas de Transporte Vesicular , Antígenos de Superfície/fisiologia , Plaquetas/efeitos dos fármacos , Plaquetas/ultraestrutura , Cálcio/farmacologia , Proteínas de Transporte/fisiologia , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/ultraestrutura , Exocitose/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas de Membrana/fisiologia , Microscopia Imunoeletrônica , Proteínas do Tecido Nervoso/fisiologia , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas SNARE , Serotonina/metabolismo , Sintaxina 1 , Trítio , beta-N-Acetil-Hexosaminidases/efeitos dos fármacos , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
The mechanisms by which synaptic vesicles are transported and primed to fuse with the presynaptic membrane are important to all chemical synapses. Processes of signal transduction that affect vesicular dynamics, such as the second-messenger cascades induced by neuromodulators, are more readily addressed in assessable synaptic preparations of neuromuscular junctions in the crayfish. We assessed the effects of serotonin (5-HT) through the analysis of the latency jitter and the quantal parameters: n and p in the opener muscle of the walking leg in crayfish. There is an increase in the size of the postsynaptic currents due to more vesicles being released. Quantal analysis reveals a presynaptic mechanism by an increase in the number of vesicles being released. Latency measures show more events occur with a short latency in the presence of 5-HT. No effect on the frequency or size of spontaneous release was detected. Thus, the influence of 5-HT is presynaptic, leading to a release of more vesicles at a faster rate.
Assuntos
Potenciais Evocados/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Músculo Esquelético/inervação , Junção Neuromuscular/fisiologia , Serotonina/farmacologia , Animais , Astacoidea , Potenciais Evocados/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Técnicas In Vitro , Fibras Musculares Esqueléticas , Junção Neuromuscular/efeitos dos fármacos , Teoria Quântica , Tempo de Reação , Sistemas do Segundo Mensageiro , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/fisiologiaRESUMO
Platelets function by secreting components necessary for primary clot formation. This report describes an in vitro assay that measures alpha-granule secretion. Using permeabilized platelets, it is possible to recreate Ca(2+)-stimulated release of platelet factor 4 (PF4) that is ATP- and temperature-dependent. Though other divalent cations can replace Ca(2+) (i.e., Sr(2+), Mn(2+), Zn(2+)), there is no effect of Ba(2+). Analysis by electron microscopy indicates that the in vitro assay also mimics the cytoskeletal rearrangements and granule centralization that occurs upon platelet activation in vivo. Antibody inhibition studies show that PF4 release requires the general membrane fusion protein N-ethylmaleimide-sensitive factor (NSF) and well as the target membrane SNAP receptors (t-SNAREs), syntaxin 2, 4, and SNAP-23. As shown by electron microscopy, the anti-t-SNARE antibodies block granule to target membrane fusion. This finding is unique in that it is the first report of a role for two syntaxins in the same exocytosis event.
Assuntos
Plaquetas/fisiologia , Cálcio/farmacologia , Grânulos Citoplasmáticos/metabolismo , Exocitose/fisiologia , Proteínas de Transporte Vesicular , Trifosfato de Adenosina/sangue , Antígenos de Superfície/sangue , Plaquetas/efeitos dos fármacos , Plaquetas/ultraestrutura , Proteínas de Transporte/sangue , Cátions Bivalentes/farmacologia , Permeabilidade da Membrana Celular , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/ultraestrutura , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Humanos , Técnicas In Vitro , Cinética , Proteínas de Membrana/sangue , Proteínas Sensíveis a N-Etilmaleimida , Proteínas do Tecido Nervoso/sangue , Fator Plaquetário 4/metabolismo , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas SNARE , Sintaxina 1 , TemperaturaRESUMO
To characterize the molecular mechanisms of platelet secretion, we focused on the calcium-induced exocytosis of dense core granules. Platelets contain several known t-SNAREs (soluble N-ethylmaleimide sensitive factor [NSF] attachment protein receptors) such as syntaxins 2, 4, and 7 and SNAP-23 (synaptosomal associated protein 23). By using an in vitro exocytosis assay, we have been able to assign roles for some of these t-SNAREs in dense core granule release. This calcium-induced secretion relies on the SNARE proteins because it is stimulated by the addition of recombinant alpha-SNAP and inhibited by a dominant negative alpha-SNAP-L294A mutant or by anti-alpha-SNAP and anti-NSF antibodies. SNAP-23 antibodies and an inhibitory C-terminal SNAP-23 peptide both blocked dense core granule release, demonstrating a role for SNAP-23. Unlike other cell types, platelets contain a significant pool of soluble SNAP-23, which does not partition into Triton X-114. Of the anti-syntaxin antibodies tested, only anti-syntaxin 2 antibody inhibited dense core granule release. Immunoprecipitation studies showed that the 2 t-SNAREs syntaxin 2 and SNAP-23 do form a complex in vivo. These data clearly show that SNAPs, NSF, and specific t-SNAREs are used for dense core granule release; these data provide a greater understanding of regulated exocytosis in platelets.
Assuntos
Antígenos de Superfície/fisiologia , Plaquetas/metabolismo , Cálcio/farmacologia , Proteínas de Transporte/fisiologia , Grânulos Citoplasmáticos/metabolismo , Exocitose/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Transporte Vesicular , Antígenos de Superfície/imunologia , Antígenos de Superfície/farmacologia , Plaquetas/efeitos dos fármacos , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Detergentes/farmacologia , Exocitose/efeitos dos fármacos , Genes Dominantes , Humanos , L-Lactato Desidrogenase/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/farmacologia , Octoxinol , Fator de Crescimento Derivado de Plaquetas/metabolismo , Polietilenoglicóis/farmacologia , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas Recombinantes/farmacologia , Serotonina/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Sintaxina 1 , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
In this manuscript, we address the role of the soluble N-ethylmaleimide sensitive factor attachment protein (alpha-SNAP) in synaptic transmission at the neuromuscular junction of the crayfish opener muscle. Immunochemical methods confirm the presence of alpha-SNAP in these preparations and show that it is concentrated in the synaptic areas. Microinjection and electrophysiological studies show that alpha-SNAP causes an increase in neurotransmitter release yet does not significantly affect the kinetics. More specific quantal analysis, using focal, macropatch, synaptic current recordings, shows that alpha-SNAP increases transmitter release by increasing the probability of exocytosis but not the number of potential release sites. These data demonstrate that the role of alpha-SNAP is to increase the efficiency of neurotransmission by increasing the probability that a stimulus will result in neurotransmitter release. What this suggests is that alpha-SNAP is critical for the formation and maintenance of a "ready release" pool of synaptic vesicles.
Assuntos
Astacoidea/fisiologia , Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Junção Neuromuscular/fisiologia , Transmissão Sináptica/fisiologia , Proteínas de Transporte Vesicular , Animais , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Imunofluorescência , Técnicas In Vitro , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Microinjeções , Músculos/citologia , Músculos/efeitos dos fármacos , Músculos/fisiologia , Mutação/fisiologia , Junção Neuromuscular/efeitos dos fármacos , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Transmissão Sináptica/efeitos dos fármacosRESUMO
The cytosolic ATPase N-ethylmaleimide-sensitive fusion protein (NSF) disassembles complexes of membrane-bound proteins known as SNAREs, an activity essential for vesicular trafficking. The amino-terminal domain of NSF (NSF-N) is required for the interaction of NSF with the SNARE complex through the adaptor protein alpha-SNAP. The crystal structure of NSF-N reveals two subdomains linked by a single stretch of polypeptide. A polar interface between the two subdomains indicates that they can move with respect to one another during the catalytic cycle of NSF. Structure-based sequence alignments indicate that in addition to NSF orthologues, the p97 family of ATPases contain an amino-terminal domain of similar structure.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Etilmaleimida/farmacologia , Proteínas de Transporte Vesicular , Adenosina Trifosfatases/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Proteínas de Transporte/efeitos dos fármacos , Clonagem Molecular , Cricetinae , Cricetulus , Cristalografia por Raios X/métodos , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Sensíveis a N-Etilmaleimida , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Proteínas SNARE , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Hermansky Pudlak syndrome (HPS) is an autosomal recessive inherited disorder characterized by defects in synthesis and/or secretion of three related subcellular organelles: melanosomes, platelet-dense granules, and lysosomes. In the mouse, mutant forms of any of 14 separate genes result in an HPS-like phenotype. The mouse pearl and mocha genes encode subunits of the AP3 adaptor protein complex, confirming that HPS mutations involve proteins regulating intracellular vesicular trafficking. Therefore, expression of several additional proteins involved in vesicular transport was examined by immunoblotting of platelet extracts from HPS mutant and control mice. Platelet levels of SCAMPS (secretory carrier membrane proteins), Rab11, Rab31, NSF (N-ethylmaleimide-sensitive fusion protein), syntaxin 2, syntaxin 4, munc18c, and p115/TAP (p115/transcytosis-associated protein) were not significantly altered in several different HPS mutants. However, gunmetal (gm/gm) platelets contained decreased amounts of SNAP-23. The Snap23 gene was mapped to mouse chromosome 5, demonstrating it cannot encode the gm gene, which maps to chromosome 14. It is likely therefore that the gm gene functions upstream of SNAP-23 in vesicular trafficking.
Assuntos
Albinismo Oculocutâneo/metabolismo , Plaquetas/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas/metabolismo , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP , Albinismo Oculocutâneo/genética , Animais , Antígenos de Superfície/metabolismo , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Mapeamento Cromossômico , Modelos Animais de Doenças , Proteínas de Ligação ao GTP/metabolismo , Proteínas da Matriz do Complexo de Golgi , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Mutantes , Proteínas Munc18 , Muridae , Proteínas Sensíveis a N-Etilmaleimida , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas/análise , Proteínas/genética , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Sintaxina 1RESUMO
Vesicular transport events appear to be facilitated by the VAMP/synaptobrevin family of membrane proteins in the vesicle (v-SNAREs) and a heterodimeric complex of syntaxin and SNAP-23/25 family members in the target membrane (t-SNAREs). In this manuscript we examine the tissue distribution and composition of the heterodimeric t-SNARE complexes in adult rodent brain. Analysis of protein extracts from brain regions shows that SNAP-25, syntaxin 1, and 4 are broadly distributed, while SNAP-23, syntaxin 3, and 7 show distinct patterns of expression. Further immunohistochemistry and fractionation studies show that while SNAP-25 is enriched in axons and nerve terminals, SNAP-23 is concentrated in cell bodies. Both SNAP-23 and SNAP-25 associate with the plasma membrane and can be metabolically labeled with [(3)H] palmitate in AtT-20 cells. Anti-SNAP-25 antibodies co-immunoprecipitate t-SNARE heterodimers from brain extracts that predominantly contain syntaxin 1 and 2. Contrary to results from in vitro binding assays, SNAP-23 was found predominantly associated with syntaxin 3. These observations suggest that t-SNARE, heterodimer composition is governed more by SNARE expression and localization than by simple protein-protein affinity.
Assuntos
Encéfalo/metabolismo , Proteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Sistemas Neurossecretores/fisiologia , Acilação , Animais , Axônios/química , Linhagem Celular , Imuno-Histoquímica , Neurônios/química , Proteínas R-SNARE , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/química , Proteína 25 Associada a Sinaptossoma , Sinaptossomos/químicaRESUMO
We have reexamined the intracellular localization of the ubiquitously expressed target membrane SNAP receptor (t-SNARE), SNAP-23. While SNAP-23 appears on the plasma membrane, in the cell types examined there is a significant pool associated with endosomal compartments. Immuno-staining and expression of green fluorescent protein-tagged SNAP-23, show that it has a punctate, perinuclear localization in HepG2 and HT4 cells. This distribution overlaps significantly with transferrin receptor and slightly with the late endosome/lysosomal protein LAMP-1. The localization of SNAP-23 changes as HepG2 cells polarize. Initially it is concentrated at sites of cell-cell contact and then almost exclusively to the apical (or bile canalicular) domain of the cell. These data are consistent with a role for SNAP-23 in both endosome-plasma membrane trafficking as well as endosome-endosome transport.
Assuntos
Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Líquido Intracelular/metabolismo , Animais , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/imunologia , Compartimento Celular , Linhagem Celular , Polaridade Celular , Endossomos/química , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Soros Imunes/análise , Líquido Intracelular/química , Camundongos , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Ratos , Coloração e Rotulagem , Células Tumorais CultivadasRESUMO
Several studies suggest membrane trafficking events are mediated by integral, membrane proteins from both transport-vesicle and target membranes, called v- and t-SNAREs (SNAp REceptors), respectively. Previous experiments using antibodies to synaptobrevin/vesicle associated membrane protein (VAMP) 1, 2, or rat cellubrevin failed to detect these v-SNAREs in human platelets, although membrane proteins from these cells could support 20S complex formation. To identify v-SNAREs in platelets, we used a polymerase chain reaction (PCR) approach with degenerate primers to amplify potential VAMP-like v-SNAREs. A cDNA encoding a novel v-SNARE was isolated from a human megakaryocyte cDNA library. Termed human cellubrevin (Hceb), this protein has greater than 93% identity with human VAMP 1, 2, and rat cellubrevin over the conserved core region, but has a unique N-terminal domain. Northern blot analysis showed that the 2. 5-kB mRNA encoding Hceb is expressed in every human tissue tested. Hceb from detergent-solubilized platelet membranes, participated in alpha-SNAP-dependent 20S complex formation and adenosine triphosphate (ATP)-dependent disassembly, showing that Hceb can act as a v-SNARE in platelets. Immunofluorescence microscopy, using an anti-Hceb antibody showed a punctate, intracellular staining pattern in platelets, megakaryocytes, and HEK-293 cells. This same pattern was observed in surface-activated platelets even though all dense core and most alpha-granule contents had been released. These data suggest that Hceb may reside on a platelet organelle that is not primarily involved in the exocytic pathway.
Assuntos
Plaquetas/química , Proteínas de Membrana/análise , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , DNA Complementar/análise , DNA Complementar/química , Imunofluorescência , Humanos , Megacariócitos/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Homologia de Sequência , Distribuição Tecidual , Proteína 3 Associada à Membrana da VesículaRESUMO
Excitatory amino acids may promote microtubular proteolysis observed in ischemic neuronal degeneration by calcium-mediated activation of calpain, a neutral protease. We tested this hypothesis in an animal model of focal cerebral ischemia without reperfusion. Spontaneously hypertensive rats were treated with 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)quinoxaline (NBQX), a competitive antagonist of the neuronal receptor for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or cis-4-[phosphono-methyl]-2-piperidine carboxylic acid (CGS 19755), a competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor. After treatment, all animals were subjected to permanent occlusion of the middle cerebral artery for 6 or 24 h. Infarct volumes measured in animals pretreated with CGS 19755 after 24 h of ischemia were significantly smaller than those quantified in ischemic controls. Rats pretreated with NBQX showed partial amelioration of cytoskeletal injury with preserved immunolabeling of microtubule-associated protein 2 (MAP 2) at 6 and 24 h and reduced accumulation of calpain-cleaved spectrin byproducts only at 6 h. Prevention of cytoskeletal damage was more effective after pretreatment with CGS 19755, as shown by retention of MAP 2 immunolabeling and significant restriction of calpain activity at both 6 and 24 h. Preserved immunolabeling of tau protein was observed at 6 and 24 h only in animals pretreated with CGS 19755. Western analysis performed on ischemic cortex taken from controls or rats pretreated with either NBQX or CGS 19755 suggested that loss of tau protein immunoreactivity was caused by dephosphorylation, rather than proteolysis. These results demonstrate a crucial link between excitotoxic neurotransmission, microtubular proteolysis, and neuronal degeneration in focal cerebral ischemia.
Assuntos
Calpaína/antagonistas & inibidores , Citoesqueleto/enzimologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ataque Isquêmico Transitório/enzimologia , Receptores de Glutamato/efeitos dos fármacos , Animais , Western Blotting , Infarto Cerebral/fisiopatologia , Citoesqueleto/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Ácidos Pipecólicos/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Endogâmicos SHR , Espectrina/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Proteínas tau/metabolismoRESUMO
The ATPase of the N-ethylmaleimide sensitive factor (NSF) appears to be central to the events that culminate in vesicle-target membrane fusion. Complexes containing different combinations of NSF, alpha-SNAP, Vamp-2 (synaptobrevin 2), syntaxin 1, and SNAP-25 were reconstituted and then tested for their effect on the ATPase of NSF. While NSF interacts with all alpha-SNAP-containing complexes, only the alpha-SNAP/t-SNARE complex significantly stimulated ATPase activity. This stimulation was dependent on increasing SNAP/t-SNARE complex and was saturable. The apparent stimulation of ATPase activity is due to a 10-fold increase in initial hydrolysis rate. Complex containing both v- and t-SNAREs bound significantly more alpha-SNAP but did not stimulate the ATPase of NSF.