RESUMEN
BACKGROUND: Recombinant hypoallergens that display reduced allergenicity but retain T-cell reactivity represent promising candidates to improve the safety and efficacy of allergen-specific vaccines or immunotherapy. OBJECTIVE: The current study reports the immunologic characterization of a hypoallergenic variant of the major mite allergen Der p 1. METHODS: The recombinant proform of Der p 1 (ProDer p 1) was expressed in Escherichia coli (ProDer p 1 coli), purified and characterized at the level of its secondary structure, and IgE and T-cell reactivities. Moreover, the prophylactic potential of ProDer p 1 coli vaccinations was evaluated in a murine Der p 1 sensitization model. RESULTS: After purification and refolding, ProDer p 1 coli remained aggregated with a higher beta-sheet content and altered Der p 1 conformational epitopes compared with the correctly folded monomeric ProDer p 1 produced in Chinese hamster ovary cells. Both ProDer p 1 forms were able to retain the Der p 1-specific T-cell reactivity but direct ELISA, competitive inhibition, and rat basophil leukemia assays clearly showed that ProDer p 1 coli displays a very weak IgE reactivity. Mice vaccinations with aggregated ProDer p 1 adjuvanted with alum induced a T(H)1-biased immune response that prevented the subsequent allergic response after Der p 1 sensitization and airway challenge with aerosolized mite extracts. Furthermore, ProDer p 1 coli treatment inhibited the development of airway eosinophilia and airway hyperresponsiveness to inhaled methacholine. CONCLUSION: Aggregated forms of Der p 1 could represent hypoallergens suitable for the prevention of mite allergy.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad/prevención & control , Pyroglyphidae/inmunología , Vacunas/inmunología , Animales , Antígenos Dermatofagoides/genética , Antígenos Dermatofagoides/aislamiento & purificación , Antígenos Dermatofagoides/farmacología , Proteínas de Artrópodos , Basófilos/efectos de los fármacos , Basófilos/inmunología , Basófilos/metabolismo , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/prevención & control , Broncoconstrictores/farmacología , Línea Celular Tumoral , Clonación Molecular , Cisteína Endopeptidasas , Modelos Animales de Enfermedad , Eosinofilia/inmunología , Eosinofilia/prevención & control , Femenino , Humanos , Hipersensibilidad/inmunología , Inmunoglobulina E/sangre , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-5/biosíntesis , Interleucina-5/inmunología , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos BALB C , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Saccharomyces cerevisiae cells possess a plasma membrane sensor able to detect the presence of extracellular amino acids and then to activate a signaling pathway leading to transcriptional induction of multiple genes, e.g., AGP1, encoding an amino acid permease. This sensing function requires the permease-like Ssy1 and associated Ptr3 and Ssy5 proteins, all essential to activation, by endoproteolytic processing, of the membrane-bound Stp1 transcription factor. The SCF(Grr1) ubiquitin-ligase complex is also essential to AGP1 induction, but its exact role in the amino acid signaling pathway remains unclear. Here we show that Stp1 undergoes casein kinase I-dependent phosphorylation. In the yck mutant lacking this kinase, Stp1 is not cleaved and AGP1 is not induced in response to amino acids. Furthermore, we provide evidence that Ssy5 is the endoprotease responsible for Stp1 processing. Ssy5 is significantly similar to serine proteases, its self-processing is a prerequisite for Stp1 cleavage, and its overexpression causes inducer-independent Stp1 cleavage and high-level AGP1 transcription. We further show that Stp1 processing also requires the SCF(Grr1) complex but is insensitive to proteasome inhibition. However, Stp1 processing does not require SCF(Grr1), Ssy1, or Ptr3 when Ssy5 is overproduced. Finally, we describe the properties of a particular ptr3 mutant that suggest that Ptr3 acts with Ssy1 in amino acid detection and signal initiation. We propose that Ssy1 and Ptr3 form the core components of the amino acid sensor. Upon detection of external amino acids, Ssy1-Ptr3 likely allows-in a manner dependent on SCF(Grr1)-the Ssy5 endoprotease to gain access to and to cleave Stp1, this requiring prior phosphorylation of Stp1 by casein kinase I.
Asunto(s)
Aminoácidos/metabolismo , Quinasa de la Caseína I/metabolismo , Proteínas Nucleares/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , ADN de Hongos/genética , Genes Fúngicos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Proteínas Nucleares/genética , Péptido Hidrolasas/genética , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
Organization of proteins into complexes is crucial for many cellular functions. However, most proteomic approaches primarily detect protein interactions for soluble proteins but are less suitable for membrane-associated complexes. Here we describe a mating-based split ubiquitin system (mbSUS) for systematic identification of interactions between membrane proteins as well as between membrane and soluble proteins. mbSUS allows in vivo cloning of PCR products into a vector set, detection of interactions via mating, regulated expression of baits, and improved selection of interacting proteins. Cloning is simplified by introduction of lambda attachment sites for GATEWAY. Homo- and heteromeric interactions between Arabidopsis K(+) channels KAT1, AKT1, and AKT2 were identified. Tests with deletion mutants demonstrate that the C terminus of KAT1 and AKT1 is necessary for physical assembly of complexes. Screening of a sorted collection of 84 plant proteins with K(+) channels as bait revealed differences in oligomerization between KAT1, AKT1, and AtKC1, and allowed detection of putative interacting partners of KAT1 and AtKC1. These results show that mbSUS is suited for systematic analysis of membrane protein interactions.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/genética , Canales de Potasio/metabolismo , Proteómica/métodos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clonación Molecular , Proteínas de Plantas , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismoRESUMEN
In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh). A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts. Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization. In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system. LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a approximately 35-kDa polypeptide by denaturation. To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH4+ uniporter. Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes. Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake.
Asunto(s)
Proteínas Portadoras/química , Proteínas de Transporte de Catión , Proteínas de Plantas/química , Secuencia de Aminoácidos , Animales , Western Blotting , Membrana Celular/metabolismo , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Electrofisiología , Femenino , Genes Dominantes , Proteínas Fluorescentes Verdes , Concentración de Iones de Hidrógeno , Cinética , Proteínas Luminiscentes/metabolismo , Solanum lycopersicum , Metilaminas/química , Datos de Secuencia Molecular , Mutación , Oocitos/metabolismo , Péptidos/química , Plásmidos/metabolismo , Mutación Puntual , Estructura Terciaria de Proteína , Compuestos de Amonio Cuaternario , ARN Complementario/metabolismo , Técnicas del Sistema de Dos Híbridos , XenopusRESUMEN
Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.