RESUMEN
BACKGROUND: Epidermolysis bullosa simplex generalized severe (EBS-gen sev) is a genetic disorder caused by mutation in the KRT5 or KRT14 genes. Although it is usually considered a mechanical disease, recent data argue for additional inflammatory mechanisms. OBJECTIVES: To assess the inflammation in the skin of patients with EBS-gen sev. METHODS: A first immunohistochemical retrospective study was performed on frozen skin samples from 17 patients with EBS-gen sev. A second multicentre prospective study was conducted on 10 patients with severe EBS-gen sev. Blister fluid and epidermis were processed for immunochemical analysis and quantitative real-time polymerase chain reaction. Cytokine expression was analysed in blister fluid and compared with that in controls. RESULTS: Histological analysis showed a constant dermal perivascular CD4+ lymphocyte infiltrate in skin biopsies of both blister (n = 17) and rubbed skin (n = 5), an epidermal infiltration of neutrophils and eosinophils in 70% of cases, and increased immunostaining for CXCL9 and CXCL10 in blistering skin. High levels of T helper 17 cytokines were detected in lesional skin. Three adult patients with EBS-gen sev were treated with apremilast, with a dramatic improvement of skin blistering and good tolerance. CONCLUSIONS: Our study demonstrates the importance of inflammation in patients with EBS-gen sev and underlines the key role for T helper 17 cells in its pathogenesis. In addition, this study provides promising new therapeutic approaches for this disabling disorder.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Epidermólisis Ampollosa Simple/inmunología , Piel/efectos de los fármacos , Células Th17/inmunología , Talidomida/análogos & derivados , Adulto , Antiinflamatorios no Esteroideos/uso terapéutico , Niño , Preescolar , Epidermólisis Ampollosa Simple/tratamiento farmacológico , Epidermólisis Ampollosa Simple/genética , Femenino , Humanos , Lactante , Recién Nacido , Queratina-14/genética , Queratina-5/genética , Masculino , Persona de Mediana Edad , Mutación , Proyectos Piloto , Estudios Retrospectivos , Piel/citología , Piel/inmunología , Células Th17/efectos de los fármacos , Talidomida/farmacología , Talidomida/uso terapéutico , Resultado del Tratamiento , Adulto JovenRESUMEN
Extending our previous analyses to the most recently described monoclonal broadly neutralizing antibodies (bNAbs), we confirmed a drift of HIV-1 clade B variants over 2 decades toward higher resistance to bNAbs targeting almost all the identified gp120-neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 membrane-proximal external region remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable of neutralizing efficiently most of the circulating variants.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Flujo Genético , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Animales , Epidemias , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/inmunología , Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Masculino , Ratones , Pruebas de NeutralizaciónRESUMEN
BACKGROUND: Acral peeling skin syndrome (APSS) is a rare skin fragility disorder usually caused by mutations in the transglutaminase 5 gene (TGM5). METHODS: We investigated the mutation spectrum of APSS in the U.K., Germany and Poland. RESULTS: We identified 59 children with APSS from 52 families. The phenotype was readily recognizable, with some variation in severity both within and between families. Most cases had been misdiagnosed as the localized form of epidermolysis bullosa simplex (EBS-loc). Eighteen different TGM5 mutations were identified, 15 of which were novel. Eight mutations were unique to a single family, nine each occurred in two families, while the common p.Gly113Cys mutation linked to a second missense variant p.Thr109Met occurred in 47 of the 52 families and was homozygous in 28. Most patients were of nonconsanguineous white European origin. CONCLUSIONS: We propose that APSS is under-reported and widely misdiagnosed as EBS-loc, with significant counselling implications as APSS is autosomal recessive while EBS-loc is dominant. We recommend screening for TGM5 mutations when EBS-loc is suspected but not confirmed by mutations in KRT5 or KRT14. Our report trebles the number of known TGM5 mutations. It provides further evidence that p.Gly113Cys is a founder mutation in the European population. This is consistent with the striking ethnic distribution of APSS in U.K., where the majority of patients are of nonconsanguineous white European origin, in contrast to the pattern of other recessive skin disorders.
Asunto(s)
Dermatitis Exfoliativa/genética , Mutación/genética , Trastornos de la Pigmentación/genética , Transglutaminasas/genética , Niño , Dermatitis Exfoliativa/diagnóstico , Dermatitis Exfoliativa/etnología , Diagnóstico Diferencial , Epidermólisis Ampollosa Simple/diagnóstico , Efecto Fundador , Pruebas Genéticas , Alemania/etnología , Heterocigoto , Homocigoto , Humanos , Queratina-14/genética , Queratina-5/genética , Trastornos de la Pigmentación/diagnóstico , Trastornos de la Pigmentación/etnología , Polonia/etnología , Enfermedades de la Piel/congénito , Reino Unido/etnologíaRESUMEN
BACKGROUND: Basal epidermolysis bullosa simplex (EBS) is a group of blistering genodermatoses mostly caused by mutations in the keratin genes, KRT5 and KRT14. Recessive mutations represent about 5% of all EBS mutations, being common and specific in populations with high consanguinity, where affected patients show severe phenotypes. OBJECTIVES: To accomplish the first mutational analysis in patients of Spanish origin with EBS and to delineate a comprehensive genotype-phenotype correlation. METHODS: Twenty-one EBS families were analysed. Immunofluorescence mapping at the dermoepidermal junction level was performed on skin biopsies from patients. Mutation screening of the entire coding sequences of KRT5 and KRT14 in genomic DNA was assessed by polymerase chain reaction and direct sequencing. RESULTS: KRT5 or KRT14 causative mutations were identified in 18 of the 21 EBS families. A total of 14 different mutations were disclosed, of which 12 were dominant missense mutations and two truncating recessive mutations. Five of the 14 mutations were novel including three dominant in KRT5 (p.V186E, p.T321P and p.A428T) and two recessive in KRT14 (p.K116X and p.K250RfsX8). The two patients with EBS carrying homozygous recessive mutations were affected by severe phenotypes and belonged to consanguineous families. All five families with the EBS Dowling-Meara subtype carried recurrent mutations affecting the highly conserved ends of the α-helical rod domain of K5 and K14. The seven mutations associated with the localized EBS subtype were widely distributed along the KRT5 and KRT14 genes. Two families with mottled pigmentation carried the P25L mutation in KRT5, commonly associated with this subtype. CONCLUSIONS: This study further confirms the genotype-phenotype correlation established for EBS in other ethnic groups, and is the first in a Mediterranean country (excluding Israel). This study adds two novel recessive mutations to the worldwide record to date, which includes a total of 14 mutations. As in previous reports, the recessive mutations resulted in a lack of keratin K14, giving rise to a generalized and severe presentation.
Asunto(s)
Epidermólisis Ampollosa Simple/genética , Queratina-14/genética , Mutación Missense/genética , Adolescente , Adulto , Preescolar , Estudios de Cohortes , Consanguinidad , Análisis Mutacional de ADN , Femenino , Homocigoto , Humanos , Lactante , Queratina-5/genética , Masculino , Linaje , España , Adulto JovenAsunto(s)
Hiperqueratosis Epidermolítica/genética , Hiperostosis Esquelética Difusa Idiopática/inducido químicamente , Queratina-10/genética , Osteonecrosis/inducido químicamente , Eliminación de Secuencia/genética , Adulto , Fármacos Dermatológicos/efectos adversos , Femenino , Articulación de la Cadera , Humanos , Hiperqueratosis Epidermolítica/tratamiento farmacológico , Hiperostosis Esquelética Difusa Idiopática/genética , Isotretinoína/efectos adversos , Osteonecrosis/genéticaRESUMEN
INTRODUCTION: There is a scarcity of data on height as well as bone densitometry in humans with NOGGIN mutations. METHODS: In 2 families with symphalangism, anthropometry, bone densitometry and genetic analysis of the NOGGIN gene were performed. RESULTS: In family A, the height standard deviation scores of the affected father and son were -0.4 and 3.5, respectively. In family B, the height standard deviation scores of the affected father, twin daughters and another daughter were 1.7, 1.8, 2.4 and 1.8, respectively. In the children, percentage predicted bone mineral content (BMC) for height at the appendicular sites (total femur, femoral neck) was lower than at an axial site lumbar spine. In the 2 fathers, median bone mineral density at total femur and femoral neck was -0.3 standard deviation scores (-0.7, 0.2) and at lumbar spine the scores were -0.4 and 0.9. The children had median tibial and radial speed of sound velocities of -2.1 (-0.9 to -6.4) and -1.4 (-0.2 to -4.9), respectively. DNA analysis revealed a novel missense mutation in family A and family B, resulting in a Met190Val substitution and a Pro42Arg substitution, respectively. CONCLUSION: Heterozygous gene mutations in NOGGIN are associated with tall stature in children but not necessarily in adults. The appendicular BMC and speed of sound may be low in affected children but normalises by adulthood. However, axial BMC seems normal in childhood and is high in adulthood.
Asunto(s)
Estatura/genética , Desarrollo Óseo/genética , Proteínas Portadoras/genética , Mutación Missense/genética , Adolescente , Adulto , Densidad Ósea/genética , Niño , Femenino , Humanos , Masculino , Linaje , Fenotipo , Sinostosis/genéticaRESUMEN
The study of colicin release from producing cells has revealed a novel mechanism of secretion. Instead of a built-in 'tag', such as a signal peptide containing information for secretion, the mechanism employs coordinate expression of a small protein which causes an increase in the envelope permeability, resulting in the release of the colicin as well as other proteins. On the other hand, the mechanism of entry of colicins into sensitive cells involves the same three stages of protein translocation that have been demonstrated for various cellular organelles. They first interact with receptors located at the surface of the outer membrane and are then transferred across the cell envelope in a process that requires energy and depends upon accessory proteins (TolA, TolB, TolC, TolQ, TolR) which might play a role similar to that of the secretory apparatus of eukaryotic and prokaryotic cells. At this point, the type of colicin described in this review interacts specifically with the inner membrane to form an ion channel. The pore-forming colicins are isolated as soluble proteins and yet insert spontaneously into lipid bilayers. The three-dimensional structures of some of these colicins should soon become available and site-directed mutagenesis studies have now provided a large number of modified polypeptides. Their use in model systems, particularly those in which the role of transmembrane potential can be tested for polypeptide insertion and ionic channel gating, constitutes a powerful handle with which to improve our understanding of the dynamics of protein insertion into and across membranes and the molecular basis of membrane excitability. In addition, their immunity proteins, which exist only in one state (membrane-inserted) will also contribute to such an understanding.
Asunto(s)
Colicinas , Proteínas de Escherichia coli , Receptores de Superficie Celular , Secuencia de Aminoácidos , Secuencia de Bases , Colicinas/biosíntesis , Colicinas/genética , Colicinas/inmunología , Colicinas/metabolismo , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Mitomicina , Mitomicinas/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Plásmidos , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Receptores Inmunológicos/metabolismo , Relación Estructura-ActividadRESUMEN
The ion-channel-forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of insertion of a soluble protein into a lipid bilayer. The soluble structure is known from X-ray crystallography and consists of a ten-helix bundle containing a hydrophobic helical hairpin. In this work fluorescence spectroscopy was used to study the membrane-bound structure. An extrinsic probe, N'-(iodoacetyl)-N'-(5-sulfol-naphthyl)ethylenediamine (IAEDANS) was attached to mutant proteins each of which bears a unique cysteine residue. Three mutants K39C (helix 2), T127C (between helices 6 and 7) and S16Crpt (helix 1, which bears a decapeptide repeat before the mutation) gave useful derivatives. In the soluble protein they showed emission wavelengths decreasing in the order K39C greater than T127C greater than S16Crpt and although all showed blue shifts on addition of dimyristoylphosphatidylglycerol (DMPG) this order was maintained in the membrane-bound state. These shifts were not indicative of deep membrane insertion. Polarization of IAEDANS revealed differences in mobility between mutants. The three tryptophan residues were used as a compound donor to IAEDANS in resonance energy transfer distance determinations. The values obtained for the soluble form were 1.2 A to 3.2 A longer than in the crystal structure. On addition of lipids the indicated distances increased: S16Crpt-I(AEDANS) 6.45 A (22%), K39C-I 5.45 A (18%) and T127C-I 2.4 A (14%). N-bromosuccinimide (NBS) completely abolishes the tryptophan emission from the thermolytic fragment. When lipids were added to a mixture containing ten NBS-treated channel-forming fragments to one IAEDANS labelled fragment the indicated distances increased rather more: S16Crpt-I 9.7 A (38%), K39C-I 8.1 A (36%) and T127C-I 2.5 A (16%). This showed that intermolecular transfer reduces the distance estimated in samples containing only labelled protein. The ensemble of results shows that the amphipathic helices of the C-terminal fragment open out on the surface of the lipid bilayer during the initial phase of membrane insertion.
Asunto(s)
Colicinas/química , Cisteína/química , Colicinas/metabolismo , Cisteína/genética , Transferencia de Energía , Colorantes Fluorescentes , Membrana Dobles de Lípidos/metabolismo , Modelos Químicos , Mutagénesis Sitio-Dirigida , Naftalenosulfonatos , Fosfatidilgliceroles/química , Solubilidad , Espectrometría de Fluorescencia , Difracción de Rayos XRESUMEN
The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) inserted into the inner membrane of Escherichia coli and apparently formed a functional channel, when generated in vivo. We investigated pfColA functional activity in vivo by the PhoA gene fusion approach, combined with cell fractionation and protease susceptibility experiments. Alkaline phosphatase was fused to the carboxy-terminal end of each of the ten alpha-helices of sp-pfColA to form a series of differently sized fusion proteins. We suggest that the alpha-helices anchoring pfColA in the membrane are first translocated into the periplasm. We identify two domains that anchor pfColA to the membrane in vivo: domain 1, extending from helix 1 to helix 8, which contains the voltage-responsive segment and domain 2 consisting of the hydrophobic helices 8 and 9. These two domains function independently. Fusion proteins with a mutation inactivating the voltage-responsive segment or with a domain 1 lacking helix 8 were peripherally associated with the outside of the inner membrane, and were therefore digested by proteases added to spheroplasts. In contrast, fusion proteins with a functional domain 1 were protected from proteases, suggesting as expected that most of domain 1 is inserted into the membrane or is indeed translocated to the cytoplasm during pfColA channel opening.
Asunto(s)
Colicinas/química , Colicinas/metabolismo , Citoplasma/metabolismo , Escherichia coli/metabolismo , Membranas Intracelulares/metabolismo , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Precipitación Química , Colicinas/genética , Colicinas/inmunología , Citoplasma/química , Endopeptidasa K/química , Endopeptidasa K/metabolismo , Epítopos , Membranas Intracelulares/química , Datos de Secuencia Molecular , Periplasma/química , Periplasma/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares , Tripsina/química , Tripsina/metabolismoRESUMEN
The structure of complexes of RecA with double-stranded and single-stranded DNA was studied by linear dichroism spectroscopy, fluorescence quenching and fluorescence anisotropy measurements. One of the two tryptophan residues (Trp291) of RecA was replaced by genetic engineering for an ultraviolet light-transparent threonine. This modified RecA protein shows, within experimental errors, the same DNA-binding kinetics and stoichiometry as the wild-type protein and no significant variation with respect to in vivo repair function was observed between cells with the two protein forms. By comparing the dichroic and fluorescence properties of the wild-type versus the modified protein, when bound to DNA, information about orientation and environment of the Trp291 chromophore in the complex could be obtained. The indole chromophore of Trp291Z was found to be oriented with its pseudo-long axis tilted 61 degrees and the aromatic plane is tilted 27 degrees relative to the fibre axis. Trp291 shows low mobility within the protein and therefore the deduced orientation may be used as a "handle" on the protein at the construction of three-dimensional models of RecA-DNA complexes. Comparison with the orientation for this residue in the crystal structure of the RecA homopolymer fibre indicates no measurable reorientation of the C-terminal subdomain of RecA upon DNA binding. Whereas the accuracy of the orientation determination of tryptophan, in absolute terms, is rather poor, changes of its orientation can be detected with high precision. Thus, similar Trp291 orientations are obtained in the complexes with single-stranded and double-stranded DNA, indicating similar structures of the protein fibres. The fluorescence quenching results indicate that the protein region of Trp291 is not involved in the binding of DNA.
Asunto(s)
Reparación del ADN/fisiología , ADN/química , Conformación de Ácido Nucleico , Rec A Recombinasas/química , ADN/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Polarización de Fluorescencia , Poli A/química , Conformación Proteica , Ingeniería de Proteínas , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Escatol/química , Espectrometría de Fluorescencia , Triptófano/químicaRESUMEN
Colicins A and E1 are two pore-forming colicins sharing homology in their C-terminal domains but not in their N-terminal or central domains. Using site-directed mutagenesis, restriction sites were inserted at the proper locations to allow recombination of these domains. Six different constructs were obtained. All these proteins were expressed in Escherichia coli and properly recognized by monoclonal antibodies directed against epitopes located in different domains of colicin A. Out of the six hybrids, only two were released to the extracellular medium. Immunocytolocalization indicated that some of the hybrids aggregated within the cytoplasm. With some hybrids, the defect in release was related to a defect in synthesis of the lysis protein that normally promotes release.
Asunto(s)
Proteínas Bacterianas/genética , Colicinas , Anticuerpos Monoclonales/inmunología , Proteínas Bacterianas/química , Western Blotting , Compartimento Celular , Clonación Molecular , Colicinas/química , Análisis Mutacional de ADN , Escherichia coli/genética , Espacio Extracelular/metabolismo , Inmunohistoquímica , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-ActividadRESUMEN
The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membrane of Escherichia coli from the periplasmic side and forms a functional channel. The soluble structure of pfColA consists of a ten-helix bundle containing a hydrophobic helical hairpin. Here, we generated a series of mutants in which an increasing number of sp-pfColA alpha-helices was deleted. These peptides were tested for their ability to form ion channels in vivo and in vitro. We found that the shortest sp-pfColA mutant protein that killed Escherichia coli was composed of the five last alpha-helices of sp-pfColA, whereas the shortest peptide that formed a channel in planar lipid bilayer membranes similar to that of intact pfColA was the protein composed of the last six alpha-helices. The peptide composed of the last five alpha-helices of pfColA generated a voltage-independent conductance in planar lipid bilayer with properties very different from that of intact pfColA. Thus, helices 1 to 4 are unnecessary for channel formation, while helix 5, or some part of it, is important but not absolutely necessary. Voltage-dependence of colicin is evidently controlled by the first four alpha-helices of pfColA.
Asunto(s)
Colicinas/química , Colicinas/metabolismo , Secuencia de Aminoácidos , Western Blotting , División Celular/efectos de los fármacos , Cloruros/metabolismo , Colicinas/genética , Colicinas/farmacología , Conductividad Eléctrica , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Activación del Canal Iónico , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Potasio/metabolismo , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Especificidad por SustratoRESUMEN
Six different hybrid colicins were constructed by recombining various domains of the two pore-forming colicins A and E1. These hybrid colicins were purified and their properties were studied. All of them were active against sensitive cells, although to varying degrees. From the results, one can conclude that: (1) the binding site of OmpF is located in the N-terminal domain of colicin A; (2) the OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) the TolC dependence for colicin E1 is located in the N-terminal domain of colicin E1; (4) the 183 N-terminal amino acid residues of colicin E1 are sufficient to promote E1AA uptake and thus probably colicin E1 uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that domain interactions can be reconstituted in hybrids that are fully active, whereas in others that are much less active, non-proper domain interactions may interfere with translocation; (7) there is a specific recognition of the C-terminal domains of colicin A and colicin E1 by their respective immunity proteins.
Asunto(s)
Colicinas/metabolismo , Proteínas de Escherichia coli , Canales Iónicos/fisiología , Receptores de Péptidos , Proteínas de la Membrana Bacteriana Externa , Transporte Biológico , Colicinas/química , Conductividad Eléctrica , Escherichia coli/fisiología , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos , Potenciales de la Membrana , Proteínas de Transporte de Membrana , Receptores de Superficie Celular/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Equilibrio HidroelectrolíticoRESUMEN
We have constructed, by recombinant DNA techniques, one hybrid protein, colicin A-beta-lactamase (P24), and two modified colicin As, one (P44) lacking a large central domain and the other (PX-345) with a different C-terminal region. The regulation of synthesis, the release into the medium and the properties of these proteins were studied. Only P44 was released into the medium. This suggests that both ends of the colicin A polypeptide chain might be required for colicin release. None of the three proteins was active on sensitive cells in an assay in vivo. However, P44 was able to form voltage-dependent channels in phospholipid planar bilayers. Its lack of activity in vivo is therefore probably caused by the inability to bind to the receptor in the outer membrane. PX-345 is a colicin in which the last 43 amino acids of colicin A have been replaced by 27 amino acids encoded by another reading frame in the same region of the colicin A structural gene; it was totally unable to form pores in planar bilayers at neutral pH but showed a very slight activity at acidic pH. These results confirm that the C-terminal domain of colicin A is involved in pore formation and indicate that at least the 43 C-terminal amino acid residues of this domain play a significant role in pore formation or pore function. Fifteen monoclonal antibodies directed against colicin A have been isolated by using conventional techniques. Five out of the 15 monoclonal antibodies could preferentially recognize wild-type colicin A. In addition, the altered forms of the colicin A polypeptide were used to map the epitopes of ten monoclonal antibodies reacting specifically with colicin A. Some of the antibodies did not bind to colicin A when it was pre-incubated at acidic pH suggesting that colicin A undergoes conformational change at about pH 4. The effects of monoclonal antibodies on activity in vivo of colicin A were investigated. The degree of inhibition observed was related to the location of the epitopes, with monoclonal antibodies reacting with the N terminus giving greater inhibition. The monoclonal antibodies directed against the C-terminal region promoted an apparent activation of colicin activity in vivo.
Asunto(s)
Colicinas/metabolismo , ADN Recombinante/biosíntesis , Proteínas Recombinantes/metabolismo , Anticuerpos Monoclonales , Colicinas/genética , Colicinas/inmunología , Elementos Transponibles de ADN , Epítopos/inmunología , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , beta-Lactamasas/biosíntesisRESUMEN
The ion-channel-forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a ten-helix bundle containing a hydrophobic helical hairpin. In this study fluorescence resonance energy transfer spectroscopy was used to determine the position of this helical hairpin in the membrane bound state. An extrinsic probe, N'-(iodoacetyl)-N'-(5-sulpho-1-naphthyl)ethylenediamine (I-AEDANS) was attached to mutant proteins each of which bears a unique cysteine residue. Five mutants I26C (helix 1), F105C (between helices 4 and 5), G166CJ (helix 8), A169C (helix 8-9), G176C (helix 9) were used. All mutants show wild-type binding activity to phosphatidylglycerol vesicles as judged by fluorescence polarization anisotropy, emission wavelength changes and brominated lipid quenching. The three tryptophan residues were used as a compound donor to AEDANS in resonance energy transfer distance determinations. The distances obtained for the soluble form were equal to those found in the crystal structure. On adding vesicles under conditions where intermolecular transfer was avoided the indicated distances increased; I26(10.9 A) F105(3.4 A), G166(3.3 A), A169(1.9 A) and G176(2.9 A). This confirms that, in the absence of a membrane potential, helices 1 and 2 open out onto the membrane surface whilst the helical hairpin remains closely packed against the rest of the structure. The insertion of this hairpin is thus not the driving force behind colicin membrane binding.
Asunto(s)
Colicinas/química , Proteínas de la Membrana/química , Colicinas/genética , Polarización de Fluorescencia , Canales Iónicos/química , Cinética , Mutagénesis Sitio-Dirigida , Naftalenosulfonatos , Fosfatidilgliceroles , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Solubilidad , Agua/químicaRESUMEN
Two monoclonal antibodies (mAbs), 5A4 and 6D6, directed against cortisol, have been obtained; 6D6 is used in an assay kit for cortisol. The antibodies also recognize other, structurally related steroids present in the sample assayed. To improve the specificity of the assay, we aimed to minimize the recognition of non-cortisol steroids by the two anti-cortisol mAbs. Our strategy consisted in constructing an efficient expression vector in E. coli which produced the single-chain variable fragment (scFv) of the mAbs in the periplasmic space. We demonstrated that temperature and inducer concentration of the bacterial culture influenced dramatically the yield of active scFv. From the nucleotide sequence we constructed a three-dimensional model of the two variable fragments in order to understand why related steroids are, or are not recognized by the antibody. For both antibodies, we have identified chemical groups which are probably involved in the binding of the steroid haptens and the antibodies. The hydrophobic pocket formed by the antibody comprises two or three tryptophan residues which can interact with the steroid nucleus by stacking. The serine at position 35 of the heavy chain is buried in the back of the pocket and can form a hydrogen bond with the 20-keto group of the cortisol. The stacking interactions and the hydrogen bond orient the steroid in the pocket. This reactivity of the binding site is sustained by the analysis of the cross-reactions of related steroids with the mAbs.
Asunto(s)
Anticuerpos Monoclonales/química , Sitios de Unión de Anticuerpos/inmunología , Hidrocortisona/inmunología , Región Variable de Inmunoglobulina/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Escherichia coli , Vectores Genéticos , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/inmunología , Modelos Inmunológicos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/químicaRESUMEN
To obtain an expression vector that will optimize secretion of proteins with disulfide bridges in Escherichia coli, we fused the phoA gene, encoding the bacterial alkaline phosphatase (PhoA), to the sequence encoding the pectate lyase B signal sequence (PelBSS). We used an extensively degenerate pelBSS with silent mutations to study their effects on the production level and activity of PhoA. 11 representative clones differed by a factor of five between the lowest and the highest level of activity, and by a factor greater than seven for the production levels. The efficiency of translocation seems to be the result of an equilibrium between production and secretion levels that favours the secretion of active PhoA according to the competence of the fusion protein being translocated. Free energy calculations and the predicted mRNA secondary structures of the translation initiation regions showed that the high stability of the secondary structure decreased production and secretion levels of PhoA and vice versa. A stem-loop encompassing the degenerate positions downstream from the AUG start codon appears to be responsible for the differences in the production levels.
Asunto(s)
Fosfatasa Alcalina/biosíntesis , Proteínas Bacterianas/biosíntesis , Cistina/metabolismo , Escherichia coli/metabolismo , Vectores Genéticos/genética , Polisacárido Liasas/genética , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/biosíntesis , Fosfatasa Alcalina/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Transporte Biológico , Inducción Enzimática , Genes Bacterianos , Genes Sintéticos , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Procesamiento Proteico-Postraduccional , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
Ovalbumin is secreted by the tubular gland cells without cleavage of a signal sequence at the N-terminus. In Escherichia coli strains which produce a chicken ovalbumin-like protein (OLP) from a plasmid-cloned gene, the OLP is synthesized on membrane-bound polysomes and secreted without cleavage into the periplasmic space. In contrast, a deleted protein, which lacks 126 amino acids in the N-terminal half, is not secreted and is synthesized from free polysomes. Our results are compatible with the presence, in the N-terminal half of the molecule, of a signal sequence necessary for the transport across the membrane.
Asunto(s)
Ovalbúmina/genética , Animales , Transporte Biológico , Membrana Celular/metabolismo , Pollos/genética , Clonación Molecular , Escherichia coli/genética , Escherichia coli/ultraestructura , Ovalbúmina/metabolismo , Plásmidos , Polirribosomas/metabolismoRESUMEN
DNA constructs coding for human growth hormone (hGH)-releasing factor (hGRF) preceded by the specific recognition sequence for the activated blood coagulation factor X (FXa), fused in frame to the N-terminal 172-amino acid residues of colicin A, have been expressed in Escherichia coli. The construct was placed under the control of the inducible caa promoter in an operon containing a downstream gene coding for the cell lysis protein, Cal. Induction resulted in excretion of only the processed colicin A fragment. Replacement of Cal by the terminator from phage fd resulted in high expression of the hybrid protein, which was recovered as cytoplasmic aggregates. Enzymatic cleavage of the purified and renatured hybrid protein using FXa allowed the recovery of authentic hGRF.