Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 16 de 16
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Br J Dermatol ; 156(3): 420-7, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17300228

RESUMO

BACKGROUND: During normal stratification of the epidermis, keratinocytes undergo a complex programme of terminal differentiation. This programmed cell death results in corneocyte accumulation to form the stratum corneum (SC). Terminal differentiation and apoptosis share numerous features such as elimination of nuclei and organelles, changes in cell shape, and activation of transglutaminases and proteases. Caspases are cysteine proteases that play a central role in apoptosis. Therefore they may also be involved in the terminal differentiation of keratinocytes. OBJECTIVES: To identify the caspases expressed in normal human epidermis and to define their pattern of expression and activation. METHODS: We analysed mRNAs from human epidermis by reverse transcription-polymerase chain reaction (RT-PCR), skin cryosections by immunohistological methods, and epidermal protein extracts by Western blotting. RESULTS: The mRNAs encoding caspase-1, -2, -3, -4, -6, -7, -8, -9, -10 and -14 were detected by RT-PCR. Accordingly, the immunohistological analyses showed clear expression in the epidermis of the corresponding proteins except caspase-2 and caspase-8, with only a weak expression of caspase-9. Moreover, procaspase-1, -2, -3, -4, -6, -7, -9, -10 and -14, and the fully processed caspase-14, were immunodetected in total epidermis extracts. However, only procaspase-1 and the processed caspase-14 were detected in extracts of superficial SC. In addition to these two proteins, procaspase-4 was detected in extracts of superficial SC obtained from lesional psoriatic skin. CONCLUSIONS: This study, the first exhaustive description of caspase expression and processing in normal human epidermis, indicates that in vivo granular keratinocytes express nine procaspases, and in addition the activated form of caspase-14. This confirms that only caspase-14 is involved in keratinocyte differentiation, and suggests that keratinocytes are ready to induce apoptosis in response to cutaneous damage.


Assuntos
Caspases/metabolismo , Epiderme/enzimologia , Caspase 14/metabolismo , Caspases/genética , Diferenciação Celular/fisiologia , Células Epidérmicas , Expressão Gênica , Humanos , Queratinócitos/citologia , Queratinócitos/enzimologia , Psoríase/enzimologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
2.
Int J Cosmet Sci ; 29(3): 147-68, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18489346

RESUMO

Deimination (or citrullination) is a recently described post-translational modification, but its consequences are not yet well understood. It is catalysed by peptidylarginine deiminases (PADs). These enzymes transform arginyl residues involved in a peptidyl link into citrullyl residues in a calcium-dependent manner. Several PAD substrates have already been identified like filaggrin and keratins K1 and K10 in the epidermis, trichohyalin in hair follicles, but also ubiquitous proteins like histones. PADs act in a large panel of physiological functions as cellular differentiation or gene regulation. It has been suggested that deimination plays a role in many major diseases such as rheumatoid arthritis, multiple sclerosis, Alzheimer's disease and psoriasis. Five human genes (PADIs), encoding five highly conserved paralogous enzymes (PAD1-4 and 6), have been characterized. These genes are clustered in a single locus, at 1p35-36 in man. Only PAD1-3 are expressed in human epidermis. PADs seem to be controlled at transcriptional, translational and activity levels and they present particular substrate specificities. In this review, we shall discuss these main biochemical, genetic and functional aspects of PADs together with their pathophysiological implications.

3.
Cell Mol Life Sci ; 62(17): 1984-95, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16091842

RESUMO

Deimination, a post-translational modification catalyzed by peptidylarginine deiminases (PADs), appears as a crucial Ca(2+)-dependent event in the last steps of epidermal differentiation. In normal human epidermis, where the deiminated proteins are filaggrin and keratins, PAD1, 2 and 3 are expressed but their relative role is unknown. The three PADs, produced as active recombinant forms, showed distinct synthetic-substrate specificities, various efficiencies to deiminate filaggrin and particular calcium and pH sensitivities. Immunoelectron microscopy demonstrated that PAD1 and PAD3 are co-located with filaggrin within the filamentous matrix of the deeper corneocytes where the protein is deiminated. This result strongly suggests that both isoforms are involved in the deimination of filaggrin, an essential step leading to free amino acid production necessary for epidermal barrier function. Moreover, PAD1 was shown to persist up to the upper corneocytes where it deiminates keratin K1, a modification supposed to be related to ultrastructural changes of the matrix.


Assuntos
Epiderme/enzimologia , Hidrolases/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Anticorpos Monoclonais/imunologia , Cálcio/farmacologia , Epiderme/química , Proteínas Filagrinas , Humanos , Concentração de Íons de Hidrogênio , Hidrolases/análise , Hidrolases/isolamento & purificação , Proteínas de Filamentos Intermediários/análise , Proteínas de Filamentos Intermediários/imunologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína-Arginina Desiminase do Tipo 1 , Proteína-Arginina Desiminase do Tipo 3 , Proteínas Recombinantes/análise , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
4.
Horm Metab Res ; 34(6): 288-92, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12173068

RESUMO

We previously showed that a phosphate-deficient diet resulting in hypophosphatemia upregulated the catalytic subunit p36 of rat liver glucose-6-phosphatase, which is responsible for hepatic glucose production. A possible association between phosphate and glucose homeostasis was now further evaluated in the Hyp mouse, a murine homologue of human X-linked hypophosphatemia. We found that in the Hyp mouse as in the dietary Pi deficiency model, serum insulin was reduced while glycemia was increased, and that liver glucose-6-phosphatase activity was enhanced as a consequence of increased mRNA and protein levels of p36. In contrast, the Hyp model had decreased mRNA and protein levels of the putative glucose-6-phosphate translocase p46 and liver cyclic AMP was not increased as in the phosphate-deficient diet rats. It is concluded that in genetic as in dietary hypophosphatemia, elevated glucose-6-phosphatase activity could be partially responsible for the impaired glucose metabolism albeit through distinct mechanisms.


Assuntos
5'-Nucleotidase , Regulação da Expressão Gênica , Glucose-6-Fosfatase/genética , Hipofosfatemia Familiar/enzimologia , Fígado/enzimologia , Animais , Antiporters , Glicemia/análise , Northern Blotting , Western Blotting , AMP Cíclico/análise , Ligação Genética , Glucose-6-Fosfatase/análise , Glucose-6-Fosfatase/metabolismo , Glicoproteínas/análise , Glicoproteínas/genética , Hipofosfatemia Familiar/genética , Insulina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/química , Proteínas de Transporte de Monossacarídeos , Fosfotransferases/análise , Fosfotransferases/genética , RNA Mensageiro/análise , Cromossomo X
5.
Metabolism ; 49(9): 1200-3, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11016904

RESUMO

The catalytic subunit (p36) and putative glucose-6-phosphate (G6P) transporter (p46) protein levels of the rat glucose-6-phosphatase (G6Pase) system were studied in relation to G6Pase hydrolytic activity and G6P uptake in liver microsomes during the fetal to neonatal period. The mean G6P hydrolytic activity in liver microsomes increased significantly from the 20th to 21st day of gestation (from 6 to 22 mU/mg protein) and was further enhanced by 3-fold 6 hours after birth, with a maximal activity at 1 day of age (112 mU/mg protein). In contrast, G6P uptake into the vesicles was undetectable before birth, appeared after day 1 (656 pmol/mg protein), and decreased after day 2 (about 330 pmol/mg protein). Immunoblot analysis showed that the mean p36 protein level was low (< 1.6 arbitrary units [AU]) during gestation, increased sharply (to about 4.0 AU) during the first day, and remained stable afterward. Unlike p36, p46 protein was present before birth at values comparable to those postpartum. P46 increased from 3.2 AU at 20 days to 4.6 AU at 21 days of gestation, and decreased transiently after birth. These results show that (1) G6Pase hydrolytic activity before birth can occur without detectable G6P uptake function; (2) the presence of the putative G6P transporter protein is not sufficient to elicit G6P uptake; and (3) full G6Pase activity requires optimal expression of both p36 and p46 proteins. These data are discussed in relation to the function of G6Pase.


Assuntos
Glucose-6-Fosfatase/metabolismo , Fígado/crescimento & desenvolvimento , Microssomos Hepáticos/enzimologia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Transporte Biológico , Western Blotting , Catálise , Idade Gestacional , Glucose-6-Fosfatase/química , Glucose-6-Fosfato/metabolismo , Hidrólise , Fígado/embriologia , Dados de Sequência Molecular , Ratos , Ratos Wistar
6.
Proteins ; 41(2): 164-72, 2000 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-10966570

RESUMO

The cDNA encoding the protein (P46) that is mutated in glycogen storage disease type-1b (GSD-1b) has been previously cloned by homology with bacterial sequences of the uhp (upper hexose phosphate) system. Hydropathic profiles, transmembrane-prediction analysis, and a multiple alignment of 14 sequences related to P46 (with percentage of identity around 30%) allowed to identify two large domains in the proteins linked by a large variable loop. Highly conserved transmembrane (TM) segments, TM1 and TM4 in the first domain and TM5 in the second one, were identified almost in all the integral proteins related to P46. The multiple alignment allowed definition of a consensus involving the 14 sequences related to P46. The detailed comparison of the consensus with the UhpT (the bacterial G6P transporter) and with UhpC (the bacterial G6P receptor) sequences reveals that the P46 protein could carry both G6P receptor and transporter functions.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteínas de Escherichia coli , Glucose-6-Fosfatase/química , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras , Proteínas de Transporte de Monossacarídeos , Fosfotransferases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Antiporters , Sequência Consenso , Glucose-6-Fosfato/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Dados de Sequência Molecular , Mutação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
7.
Eur J Biochem ; 267(6): 1533-49, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10712583

RESUMO

The operation of glucose 6-phosphatase (EC 3.1.3.9) (Glc6Pase) stems from the interaction of at least two highly hydrophobic proteins embedded in the ER membrane, a heavily glycosylated catalytic subunit of m 36 kDa (P36) and a 46-kDa putative glucose 6-phosphate (Glc6P) translocase (P46). Topology studies of P36 and P46 predict, respectively, nine and ten transmembrane domains with the N-terminal end of P36 oriented towards the lumen of the ER and both termini of P46 oriented towards the cytoplasm. P36 gene expression is increased by glucose, fructose 2,6-bisphosphate (Fru-2,6-P2) and free fatty acids, as well as by glucocorticoids and cyclic AMP; the latter are counteracted by insulin. P46 gene expression is affected by glucose, insulin and cyclic AMP in a manner similar to P36. Accordingly, several response elements for glucocorticoids, cyclic AMP and insulin regulated by hepatocyte nuclear factors were found in the Glc6Pase promoter. Mutations in P36 and P46 lead to glycogen storage disease (GSD) type-1a and type-1 non a (formerly 1b and 1c), respectively. Adenovirus-mediated overexpression of P36 in hepatocytes and in vivo impairs glycogen metabolism and glycolysis and increases glucose production; P36 overexpression in INS-1 cells results in decreased glycolysis and glucose-induced insulin secretion. The nature of the interaction between P36 and P46 in controling Glc6Pase activity remains to be defined. The latter might also have functions other than Glc6P transport that are related to Glc6P metabolism.


Assuntos
Proteínas de Escherichia coli , Glucose-6-Fosfatase/fisiologia , Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Antiporters , Proteínas de Bactérias/fisiologia , Transporte Biológico , Proteínas de Transporte/fisiologia , AMP Cíclico/farmacologia , AMP Cíclico/fisiologia , Retículo Endoplasmático/enzimologia , Indução Enzimática/efeitos dos fármacos , Ácidos Graxos não Esterificados/metabolismo , Ácidos Graxos não Esterificados/farmacologia , Frutosedifosfatos/metabolismo , Frutosedifosfatos/farmacologia , Regulação da Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Glucocorticoides/farmacologia , Glucocorticoides/fisiologia , Glucose/farmacologia , Glucose/fisiologia , Glucose-6-Fosfatase/química , Glucose-6-Fosfatase/genética , Doença de Depósito de Glicogênio Tipo I/classificação , Doença de Depósito de Glicogênio Tipo I/enzimologia , Doença de Depósito de Glicogênio Tipo I/genética , Doença de Depósito de Glicogênio Tipo I/metabolismo , Glicólise , Humanos , Hidrólise , Insulina/farmacologia , Insulina/fisiologia , Fígado/metabolismo , Substâncias Macromoleculares , Microssomos/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Fosfotransferases/química , Fosfotransferases/genética , Fosfotransferases/fisiologia , Sistemas do Segundo Mensageiro , Relação Estrutura-Atividade
8.
J Biol Chem ; 274(48): 33866-8, 1999 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-10567346

RESUMO

The effect of streptozocin diabetes on the expression of the catalytic subunit (p36) and the putative glucose-6-phosphate translocase (p46) of the glucose-6-phosphatase system (G6Pase) was investigated in rats. In addition to the documented effect of diabetes to increase p36 mRNA and protein in the liver and kidney, a approximately 2-fold increase in the mRNA abundance of p46 was found in liver, kidney, and intestine, and a similar increase was found in the p46 protein level in liver. In HepG2 cells, glucose caused a dose-dependent (1-25 mM) increase (up to 5-fold) in p36 and p46 mRNA and a lesser increase in p46 protein, whereas insulin (1 microM) suppressed p36 mRNA, reduced p46 mRNA level by half, and decreased p46 protein by about 33%. Cyclic AMP (100 microM) increased p36 and p46 mRNA by >2- and 1.5-fold, respectively, but not p46 protein. These data suggest that insulin deficiency and hyperglycemia might each be responsible for up-regulation of G6Pase in diabetes. It is concluded that enhanced hepatic glucose output in insulin-dependent diabetes probably involves dysregulation of both the catalytic subunit and the putative glucose-6-phosphate translocase of the liver G6Pase system.


Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Glucose-6-Fosfatase/genética , Fosfotransferases/genética , Animais , Antiporters , Domínio Catalítico/genética , AMP Cíclico/farmacologia , Diabetes Mellitus Experimental/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Glucose-6-Fosfatase/efeitos dos fármacos , Glucose-6-Fosfatase/metabolismo , Humanos , Insulina/farmacologia , Intestinos/efeitos dos fármacos , Intestinos/enzimologia , Rim/efeitos dos fármacos , Rim/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Proteínas de Transporte de Monossacarídeos , Músculos/efeitos dos fármacos , Músculos/enzimologia , Fosfotransferases/efeitos dos fármacos , Fosfotransferases/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Células Tumorais Cultivadas
9.
Biochem J ; 343 Pt 2: 393-6, 1999 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-10510305

RESUMO

Because P(i) deprivation markedly affects the Na/P(i) co-transporter in kidney and has been related to insulin resistance and glucose intolerance, the effect of a P(i)-deficient diet on the liver microsomal glucose-6-phosphatase (G6Pase) system was investigated. Rats were fed with a control diet (+P(i)) or a diet deficient in phosphate (-P(i)) for 2 days and killed on the morning of the third day, after an overnight fast (fasted) or not (fed). Kinetic parameters of P(i) transport (t((1/2)) and equilibration) into liver microsomes were not changed by the different nutritional conditions. In contrast, it was found that G6Pase activity was significantly increased in the (-P(i)) groups. This was due to an increase in the V(max) of the enzyme, without change in the K(m) for G6P. There was no correlation between liver microsomal glycogen content and G6Pase activity, but both protein abundance and mRNA of liver 36 kDa catalytic subunit of G6Pase (p36) were increased. The mRNA of the putative G6P translocase protein (p46) was changed in parallel with that of the catalytic subunit, but the p46 immunoreactive protein was unchanged. These findings indicate that dietary P(i) deficiency causes increased G6Pase activity by up-regulation of the expression of the 36 kDa-catalytic-subunit gene.


Assuntos
Glucose-6-Fosfatase/metabolismo , Fígado/enzimologia , Fosfatos/deficiência , Animais , Antiporters , Transporte Biológico , Western Blotting , Domínio Catalítico/genética , Indução Enzimática , Jejum , Glucose-6-Fosfatase/biossíntese , Glucose-6-Fosfatase/química , Glucose-6-Fosfatase/genética , Glicogênio/análise , Cinética , Fígado/citologia , Fígado/metabolismo , Masculino , Microssomos Hepáticos/química , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Peso Molecular , Proteínas de Transporte de Monossacarídeos , Fosfatos/administração & dosagem , Fosfatos/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley
10.
Gene ; 179(2): 211-8, 1996 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-8972902

RESUMO

Previously, two B-cell epitopes from the entero-pathogenic transmissible gastroenteritis virus (TGEV), namely the C epitope (TGEV-C) amino acids (aa) 363-371 and the A epitope (TGEV-A) aa 522-531 of the spike S protein (TGEV-S), have been separately expressed on the CS31A fibrillae at the surface of Escherichia coli following insertion into a same region of ClpG. However, the resulting chimeras induced a marginal TGEV-neutralizing antibody (Ab) response in mice. Here, with the view to improving this response, we introduced TGEV-C alone or in different tandem association with TGEV-A (A::C or C::A) in twelve putatively exposed regions of ClpG. Among the 28 resulting engineered proteins only 15, carrying up to 51 extra aa, had not essentially disturbed the correct CS31A fibrillae formation process. Six partially permissive sites accepting only TGEV-C and three highly permissive sites tolerating A::C or C::A tandem peptide, were identified throughout ClpG. Intact bacteria or extracted CS31A hybrid fibrillae expressing TGEV epitopes at any of the permissive sites, were recognized by Ab directed against the foreign parent protein, providing a direct argument for exposure of the corresponding CIpG region at the cell surface and for antigenicity of the epitopes in the polymeric CS31A fibrillae context. The potential of CS31A fibrillae as carriers of the TGEV peptides indicates that there may be three positions (N terminus, aa 202-204 and 202-218) in ClpG which may turn out to be important fusion sites and therefore be relevant for the eventual design of TGEV vaccines. Unexpectedly, TGEV-A, whatever its position in ClpG, mediated the partial proteolytic degradation of the hybrid proteins, suggesting that it functions as a substrate for a cellular protease, and thereby that its suitability as a vaccine antigen candidate is doubtful.


Assuntos
Antígenos de Bactérias , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Vetores Genéticos , Vírus da Gastroenterite Transmissível/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Recombinante , Epitopos/genética , Escherichia coli , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Vírus da Gastroenterite Transmissível/imunologia
11.
J Biol Chem ; 271(37): 22289-92, 1996 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-8798384

RESUMO

Incubation of isolated rat cardiomyocytes with insulin increased 2-deoxyglucose uptake, glycogen synthesis, and fructose 2, 6-bisphosphate content. Half-maximal effects were obtained with 1-2 nM insulin. The insulin-induced increase in fructose 2,6-bisphosphate content was preceded by a 2-3-fold activation of 6-phosphofructo-2-kinase, which was independent of glucose transport. Insulin activated phosphatidylinositol 3-kinase and p70 ribosomal S6 kinase (p70 S6 kinase), but had no significant effect on mitogen-activated protein kinase, although phorbol 12-myristate 13-acetate activated the latter. The effect of insulin on fructose 2, 6-bisphosphate, 6-phosphofructo-2-kinase, and phosphatidylinositol 3-kinase was blocked by wortmannin. However, rapamycin, which inhibited p70 S6 kinase activation, and PD 98059, an inhibitor of the mitogen-activated protein kinase pathway, had no effect on the insulin-induced activation of 6-phosphofructo-2-kinase. Heart 6-phosphofructo-2-kinase can therefore be regarded as a glycolytic target of insulin. Its activation by insulin might be mediated by phosphatidylinositol 3-kinase.


Assuntos
Insulina/farmacologia , Miocárdio/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais , Androstadienos/farmacologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Frutosedifosfatos/metabolismo , Glicólise/efeitos dos fármacos , Técnicas In Vitro , Antagonistas da Insulina/farmacologia , Proteína Quinase 1 Ativada por Mitógeno , Modelos Biológicos , Fosfatidilinositol 3-Quinases , Fosfofrutoquinase-2 , Polienos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Wistar , Proteínas Quinases S6 Ribossômicas , Sirolimo , Wortmanina
12.
Infect Immun ; 64(9): 3555-64, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8751899

RESUMO

As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins. Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A antigen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG. With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located. Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG. In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein. The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A. To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies. Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit. Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A. These data indicate that an immunodominant surface-exposed linear epitope was present in the region from positions 179 to 184 of ClpG in the native CS31A antigen on intact bacterial cells and suggest that the four surface-exposed epitopes constitute potential sites for insertions or substitutions with heterologous peptides.


Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias , Antígenos de Superfície/imunologia , Linfócitos B/imunologia , Aderência Bacteriana , Proteínas de Bactérias/imunologia , Proteínas de Escherichia coli , Escherichia coli/imunologia , Proteínas de Fímbrias , Sequência de Aminoácidos , Animais , Antígenos de Superfície/química , Mapeamento de Epitopos , Escherichia coli/química , Dados de Sequência Molecular , Peptídeos/imunologia , Coelhos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
13.
FEBS Lett ; 364(3): 319-24, 1995 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-7758589

RESUMO

The structural relatedness of K88-related major and minor subunits was deduced from their sequences by hydrophobic cluster analysis (HCA) and secondary structure predictions produced by the profile neural network prediction program (PHD) on multiple sequence alignments. Although the weak residue identity between major and minor subunits is evidence of a high evolutionary distance, an overall structural similarity was observed In addition, clear amphipathic conformations were conserved in predicted secondary structure. On the basis of this predicted structural similarity, a schematic 2D model of ClpG subunit was developed.


Assuntos
Antígenos de Superfície/química , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas de Fímbrias , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Antígenos de Bactérias/química , Fenômenos Químicos , Físico-Química , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína
15.
Gene ; 148(1): 23-32, 1994 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-7523252

RESUMO

The clpG gene, expressing the Escherichia coli major CS31A fimbrial subunit ClpG, was subjected to random mutagenesis by insertion of an EcoRI linker and a kanamycin-resistance (KmR) cassette into the multiple newly generated EcoRI sites. The KmR gene was then excised by PstI, which left a 48-bp linker representing the heterologous sequence. The same procedure was followed to introduce a synthetic oligodeoxyribonucleotide (oligo) corresponding to epitope C from the spike protein S from the porcine transmissible gastroenteritis coronavirus (TGEV). Nine insertion/deletion mutants (indels) that contained long foreign peptides variously located around the ClpG signal peptide (SP) processing site were characterized. A striking feature of this study is the variety of amino acid (aa) insertions in the ClpG prepilin that have little or no effect on CS31A fimbria biogenesis. These 'permissive' sites tolerate inserts of 18 or 19 aa and accept sequences of different natures in view of their aa composition, charge and hydrophobicity. The results obtained here are also interesting in light of the high level of aa sequence conservation seen in the SP and N-terminal domains of the ClpG-related subunits. The structure-function relationship of the ClpG SP is discussed. The TGEV-C epitope fused to the N-terminal end of the mature ClpG protein was cell-surface exposed, as observed on immuno-electron microscopy. Therefore, the CS31A fimbria seems to be a potent tool for the presentation of foreign antigenic determinants or the production of heterologous polypeptides in E. coli.


Assuntos
Antígenos de Bactérias , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Coronavirus/genética , Coronavirus/imunologia , Análise Mutacional de DNA , Epitopos/análise , Fímbrias Bacterianas/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Mutagênese Insercional , Plasmídeos/genética , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/metabolismo , Glicoproteína da Espícula de Coronavírus , Suínos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
16.
Infect Immun ; 62(6): 2553-61, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7514578

RESUMO

CS31A is a multimeric surface protein surrounding certain enterotoxigenic and septicemic bovine Escherichia coli strains. The possibility of using CS31A as a carrier of foreign antigenic determinants was investigated. Introduction of heterologous viral epitopes into the CS31A major subunit, ClpG, was successfully performed in the V3 region of the molecule which encodes for an immunodominant linear epitope. E. coli K-12 strains producing the hybrid CS31A molecules or the purified chimeric proteins were used for mice immunization. By using the C epitope derived from the S protein of the porcine transmissible gastroenteritis virus, significant antiviral antibody titers were elicited and seroneutralization of the virus was demonstrated, confirming that the molecular environment in V3 is favorable for antigen presentation. These results indicate that synthesis of CS31A hybrid proteins by the wild-type strain 31A could become a powerful tool for the development of oral vaccines directed against mucosal pathogens.


Assuntos
Antígenos de Bactérias/genética , Epitopos , Escherichia coli/genética , Vetores Genéticos , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Antígenos de Superfície/genética , Cápsulas Bacterianas , Sequência de Bases , Ensaio de Imunoadsorção Enzimática , Escherichia coli/imunologia , Imunização , Camundongos , Dados de Sequência Molecular , Mutagênese , Vírus da Gastroenterite Transmissível/imunologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...