RESUMO
During the initial phase of their development, sensory neurons of the dorsal root ganglion (DRG) require target-derived trophic support for their survival, but as they mature they lose this requirement. Because many of these neurons express BDNF (brain-derived neurotrophic factor) messenger RNA, we hypothesized that BDNF might act as an autocrine survival factor in adult DRG neurons, thus explaining their lack of dependence on exogenous growth factors. When cultured adult DRG cells were treated with antisense oligonucleotides to BDNF, expression of BDNF protein was reduced by 80%, and neuronal survival was reduced by 35%. These neurons could be rescued by exogenous BDNF or neurotrophin-3, but not by other growth factors. Similar results were obtained with single-neuron microcultures, whereas microcultures derived from mutant mice lacking BDNF were unaffected by antisense oligonucleotides. Our results strongly support an autocrine role for BDNF in mediating the survival of a subpopulation of adult DRG neurons.
Assuntos
Morte Celular , Proteínas do Tecido Nervoso/fisiologia , Neurônios Aferentes/fisiologia , Envelhecimento , Animais , Fator Neurotrófico Derivado do Encéfalo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Células Cultivadas , Gânglios Espinais/citologia , Camundongos , Proteínas do Tecido Nervoso/genética , Oligonucleotídeos Antissenso/farmacologia , Polissacarídeos/farmacologia , RNA Mensageiro/metabolismoRESUMO
Neurotrophin-4 (NT-4) is the most recently discovered member of the neurotrophin family. We have expressed, refolded, and purified recombinant human NT-4 from Escherichia coli and compared it with recombinant human NT-4 secreted into the culture medium of baculovirus-infected insect cells. Both preparations were characterized and determined to be indistinguishable according to several biochemical criteria. Recombinant NT-4 from E. coli was crystallized in a form suitable for x-ray analysis, and characterization of these crystals indicated that NT-4 was present as a dimer within the asymmetric unit. NT-4 was active in promoting the survival of rat TrkB receptor-expressing fibroblasts, but was inactive on embryonic chicken sensory neurons, unlike the other members of the neurotrophin family and in contrast to the reported activities of partially purified NT-4.
Assuntos
Fatores de Crescimento Neural/química , Animais , Baculoviridae/genética , Células Cultivadas , Dicroísmo Circular , Cristalização , Humanos , Mariposas , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Ratos , Receptor do Fator Neutrófico Ciliar , Receptores de Fatores de Crescimento/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Difração de Raios XRESUMO
We have exploited a battery of approaches to address several controversies that have accompanied the expansion of the nerve growth factor (NGF) family of neurotrophic factors and the identification of the Trk tyrosine kinases as receptors for these factors. For example, we find that a recently cloned mammalian neurotrophin, known as either neurotrophin-4 or neurotrophin-5 and assigned widely differing receptor specificities, represents the functional counterpart of Xenopus neurotrophin-4 and is a "preferred" ligand for TrkB. However, its interactions with TrkB can be distinguished from those of brain-derived neurotrophic factor (BDNF) with TrkB. We also find that all of the Trks display similar dose responses to their "preferred" ligands in neuronal as compared with nonneuronal cells (i.e., NGF for TrkA, BDNF and NT-4/5 for TrkB, and NT-3 for TrkC), providing evidence against a role for accessory molecules expressed in neurons in generating receptors that would allow for responses to lower concentrations of the neurotrophins. However, we find that a neuronal environment does restrict the Trks in their ability to respond to their "nonpreferred" neurotrophin ligands.
Assuntos
Proteínas de Membrana/metabolismo , Fatores de Crescimento Neural/farmacologia , Neurônios/metabolismo , Proteínas Tirosina Quinases/metabolismo , Células 3T3 , Animais , Fator Neurotrófico Derivado do Encéfalo , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Células PC12 , Fosforilação , Fosfotirosina , Ratos , Receptor do Fator Neutrófico Ciliar , Tirosina/análogos & derivados , Tirosina/metabolismoAssuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Bactérias/ultraestrutura , Transporte Biológico/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Sinais Direcionadores de Proteínas/metabolismoRESUMO
We determined the nucleotide sequence of the regulatory region of the cya gene of Salmonella typhimurium. A set of nested BAL-31 deletions originating upstream of the promoter/regulatory region and extending into the cya structural gene was constructed in M13mp::cya phages and was tested for complementation of a chromosomal cya deletion mutation. BAL-31 deletion mutants unable to complement cya localized the major cya promoter. The synthetic tac promoter was inserted upstream of the BAL-31 deletions so that expression of cya was dependent on transcription from tac. Those tac derivative phages unable to complement cya localized the translation initiation region. The cya DNA sequence revealed at least three potential promoters capable of transcribing cya, with a CRP binding site straddling the-10 hexamer of the promoter proximal to the structural gene. The leader RNA sequence initiated at the latter promoter is approximately 140 bases long and includes a region that may form a stable secondary structure (delta G = -23.8 kcal). There exist two possible in-frame translation start points, one of which is TTG and the other of which is ATG. The sequence of the S. typhimurium regulatory region was compared with that reported for Escherichia coli.
Assuntos
Adenilil Ciclases/genética , Regulação Bacteriana da Expressão Gênica , Sequências Reguladoras de Ácido Nucleico , Salmonella typhimurium/genética , Adenilil Ciclases/metabolismo , Sequência de Bases , Deleção Cromossômica , Cromossomos Bacterianos , Genes Bacterianos , Teste de Complementação Genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Mapeamento por Restrição , Salmonella typhimurium/enzimologia , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
We studied the expression of the cya promoter(s) in cya-lac fusion strains of Salmonella typhimurium and demonstrated cAMP receptor protein (CRP)-dependent repression by cAMP. Expression of cya was reduced about fourfold in cultures grown in acetate minimal medium as compared to cultures grown in glucose-6-phosphate minimal medium. Expression of cya was also reduced about fourfold by addition of 5 mM cAMP to cultures grown in glucose minimal medium. We constructed in vitro deletion and insertion mutations altering a major cya promoter (P2) and a putative CRP binding site overlapping P2. These mutations were recombined into the chromosome by allele replacement with M13mp::cya recombinant phages and the regulation of the mutant promoters was analyzed. A 4-bp deletion of the CRP binding site and a 4-bp insertion in this site nearly eliminated repression by cAMP. A mutant with the P2 promoter and the CRP binding site both deleted exhibited an 80% reduction in cya expression; the 20% residual expression was insensitive to cAMP repression. This mutant retained a Cya+ phenotype. Taken together, the results establish that the cya gene is transcribed from multiple promoters one of which, P2, is negatively regulated by the cAMP-CRP complex. Correction for the contribution to transcription by the cAMP-CRP nonregulated cya promoters indicates that the P2 promoter is repressed at least eightfold by cAMP-CRP.
Assuntos
Adenilil Ciclases/genética , Regulação Bacteriana da Expressão Gênica , Mutação , Receptores de AMP Cíclico/metabolismo , Salmonella typhimurium/genética , Transcrição Gênica , Acetatos/metabolismo , Adenilil Ciclases/metabolismo , Alelos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Meios de Cultura , AMP Cíclico/metabolismo , Glucofosfatos/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Salmonella typhimurium/enzimologia , Salmonella typhimurium/crescimento & desenvolvimentoRESUMO
Recent years have seen the convergence of both genetic and biochemical approaches in the study of protein translocation in E. coli. The powerful combination of these approaches is exemplified in the use of an in vitro protein synthesis-protein translocation system to analyze the role of genetically defined components of the protein translocation machinery. We describe in this review recent results focusing on the function of the secA, secB, and secY gene products and the demonstration of their requirement for in vitro protein translocation. The SecA protein was recently shown to possess ATPase activity and was proposed to be a component of the translocation ATPase. We present a speculative working model whereby the translocator complex is composed of the integral membrane proteins SecY, SecD, SecE, and SecF, forming an aqueous channel in the cytoplasmic membrane, and the tightly associated peripheral membrane protein SecA functioning as the catalytic subunit of the translocator or "protein-ATPase."
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Transporte Biológico Ativo , Conformação Molecular , Canais de Translocação SECRESUMO
Mutations in the Escherichia coli secB gene lead to protein export defects in vivo (Kumamoto, C.A., and Beckwith, J. (1985) J. Bacteriol. 163, 267-274). To demonstrate directly the participation of the secB gene product (SecB) in protein export, SecB was purified, and its effects on in vitro protein translocation were analyzed. SecB was purified from soluble extracts of a strain that overproduced it, by ammonium sulfate precipitation, DEAE-cellulose chromatography, and differential precipitation at acid pH. The chromatographic behavior on gel filtration columns indicated apparent molecular masses of approximately 90 kDa for both purified SecB and SecB in cytosolic extracts of wild type cells. When added to a translocation mixture, purified SecB stimulated pro-OmpA translocation into membrane vesicles. SecB also suppressed the thermoinduced defect in translocating activity of membranes derived from a secY24 mutant. The results of these in vitro studies and of previous in vivo studies demonstrate that SecB plays a direct role in normal protein export in E. coli.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Genes Bacterianos , Genes , Processamento de Proteína Pós-Traducional , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/isolamento & purificação , Cromatografia DEAE-Celulose , Escherichia coli/metabolismo , Genótipo , Peso Molecular , PlasmídeosRESUMO
Genetic analysis of protein secretion in Escherichia coli has identified secY/prlA and secA as components of the secretory apparatus. We have examined the roles of the secY(prlA) gene product (an integral membrane protein) and the soluble secA gene product in translocation of OmpA and alkaline phosphatase precursors in an in vitro system. The protein translocation defect of the secY24 mutation was recently demonstrated in vitro as was its suppression by an S300 extract. We show here that the extract was essentially inactive in SecY24 suppression when SecA protein was removed from it by immunoaffinity chromatography. Furthermore, purified SecA protein suppressed the SecY24 defect. Preincubation of the inactivated SecY24 membrane vesicles either with S300 containing SecA or with purified SecA protein reconstituted the membranes and restored the translocation activity when assayed in the absence of additional soluble proteins. These results suggest that the SecY24 translocation defect is suppressed by SecA interacting, directly or indirectly, with SecY24 on the cytoplasmic membrane.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Proteínas de Membrana/genética , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Genes Bacterianos , Cinética , Mutação , Plasmídeos , Processamento de Proteína Pós-Traducional , Supressão Genética , TemperaturaRESUMO
The secY (prlA) gene product is an integral membrane protein that has been identified genetically as one of the central components of the Escherichia coli protein translocation machinery. We have examined the effect of the secY24 (temperature-sensitive) mutation on the protein translocation activity of E. coli inverted membrane vesicles. Vesicles isolated from cells carrying this allele and grown at the nonpermissive temperature (42 degrees C) were less than 1% as active in translocation as vesicles isolated from an isogenic secY+ strain under the same conditions. Vesicles from the mutant strain grown at the permissive temperature (32 degrees C) were partially active, but those vesicles preincubated at 40 degrees C lost 90% of their activity. Moreover, the secY24 translocation defect on in vivo- or in vitro-inactivated vesicles was suppressed, or compensated, by an S300 soluble fraction from wild-type cells or from secY24 cells grown at nonpermissive temperature. The suppressing factor(s) was heat-labile and sensitive to proteinase K. These results provide biochemical evidence for the essential role of SecY in the translocation process and indicate that the translocation defect of SecY24 membranes can be compensated for by supplementing with additional soluble cytoplasmic proteins.