RESUMO
A comprehensive search system for the bioscience databases is in progress. We constructed a search service, Life science database cross search system (https://biosciencedbc.jp/dbsearch/index. php?lang=en) by integrating numerous biomedical databases using database crawling algorithms. The described system integrates 600 databases containing over 90 million entries indexed for biomedical research and development.
RESUMO
In the life sciences, researchers increasingly want to access multiple databases in an integrated way. However, different databases currently use different formats and vocabularies, hindering the proper integration of heterogeneous life science data. Adopting the Resource Description Framework (RDF) has the potential to address such issues by improving database interoperability, leading to advances in automatic data processing. Based on this idea, we have advised many Japanese database development groups to expose their databases in RDF. To further promote such activities, we have developed an RDF-based life science dataset repository called the National Bioscience Database Center (NBDC) RDF portal. All the datasets in this repository have been reviewed by the NBDC to ensure interoperability and queryability. As of July 2018, the service includes 21 RDF datasets, comprising over 45.5 billion triples. It provides SPARQL endpoints for all datasets, useful metadata and the ability to download RDF files. The NBDC RDF portal can be accessed at https://integbio.jp/rdf/.
Assuntos
Disciplinas das Ciências Biológicas , Sistemas de Gerenciamento de Base de Dados , Bases de Dados Factuais , Semântica , Internet , Interface Usuário-ComputadorRESUMO
We have fully integrated public chromatin chromatin immunoprecipitation sequencing (ChIP-seq) and DNase-seq data (n > 70,000) derived from six representative model organisms (human, mouse, rat, fruit fly, nematode, and budding yeast), and have devised a data-mining platform-designated ChIP-Atlas (http://chip-atlas.org). ChIP-Atlas is able to show alignment and peak-call results for all public ChIP-seq and DNase-seq data archived in the NCBI Sequence Read Archive (SRA), which encompasses data derived from GEO, ArrayExpress, DDBJ, ENCODE, Roadmap Epigenomics, and the scientific literature. All peak-call data are integrated to visualize multiple histone modifications and binding sites of transcriptional regulators (TRs) at given genomic loci. The integrated data can be further analyzed to show TR-gene and TR-TR interactions, as well as to examine enrichment of protein binding for given multiple genomic coordinates or gene names. ChIP-Atlas is superior to other platforms in terms of data number and functionality for data mining across thousands of ChIP-seq experiments, and it provides insight into gene regulatory networks and epigenetic mechanisms.
Assuntos
Imunoprecipitação da Cromatina , Mineração de Dados , Análise de Sequência de DNA , Animais , Elementos Facilitadores Genéticos/genética , Loci Gênicos , Humanos , Internet , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Patients with house dust mite (HDM) allergy or Ascariasis produce serum IgE specific to the antigens of HDM or nematode Ascaris, respectively. Although human IgE cross-reactivity has been reported between HDM and Ascaris antigens, it remains unclear whether it contributes to the pathogenesis of allergic diseases. We herein investigated the induction of cross-reactive antibodies and T cells in mice and effects of airway exposure to HDM antigens after preimmunization with Ascaris antigens. METHODS: Mice were intraperitoneally immunized with HDM or Ascaris antigens with Alum, followed by the intranasal administration of HDM antigens. Serum antigen-specific IgE and IgG were measured by ELISA. Cytokine release in splenocytes from Ascaris-immunized mice upon in vitro restimulation with HDM antigens were measured by ELISA. RESULTS: Immunization with Ascaris or HDM antigens induced cross-reactive IgG1. Splenocytes from Ascaris-immunized mice released IL-5 and IL-13 in response to the restimulation with HDM antigens. Subsequent airway exposure to HDM antigens promoted the induction of HDM-specific IgE and upregulation of HDM-specific IgG1 in Ascaris-immunized mice, whereas these responses were not detected or smaller without the Ascaris presensitization. CONCLUSIONS: We demonstrated that the immunization of naïve mice with Ascaris antigens induced production of antibodies and differentiation of Th2 cells, which were cross-reactive to HDM antigens, and accelerated induction of serum HDM-specific IgE upon subsequent airway exposure to HDM antigens in mice. These results suggest that sensitization to HDM towards IgE-mediated allergic diseases is faster in individuals with a previous history of Ascaris infection than in those without presensitization to Ascaris.
Assuntos
Antígenos de Dermatophagoides/imunologia , Antígenos de Helmintos/imunologia , Ascaris/imunologia , Hipersensibilidade/imunologia , Imunização , Imunoglobulina E/imunologia , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Hipersensibilidade/sangue , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Baço/citologia , Baço/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologiaRESUMO
Although many allergens bind endogenous molecules other than Abs in the human body, whether the interaction can modulate allergenicity has been unknown. Here, we investigated the effect of the interaction of recombinant major mite group 1 allergens (Der f 1 and Der p 1), which belong to the papain-like cysteine protease family, with an endogenous protease inhibitor, cystatin A, on their allergenicity. Cystatin A bound reduced forms of the allergens, in which the cysteine residue at the catalytic center of the protease activity was reduced by treatment with L-cysteine, but did not bind oxidized forms. Cystatin A partially inhibited the binding of IgE in mite-allergic volunteers' sera to the reduced forms, but unexpectedly enhanced the basophil histamine-releasing activity. A catalytic site-mutant of Der f 1 behaved in terms of histamine release, similarly to the reduced form. Molecular modeling showed that cystatin A interacts with the allergens within a narrow area. The results indicate that interaction with cystatin A reduces the limited number of IgE epitopes of the allergens but enhances their biological activity to release histamine, suggesting a new concept, that interaction between allergens and their endogenous ligands modulates the allergenicity even toward enhancement in the effector phase. On the other hand, i.p. immunization without alum of mice with cystatin A-treated reduced Der f 1 induced less serum Der f 1-specific IgE than immunization with reduced Der f 1 alone, suggesting that endogenous protease inhibitors suppress the induction of allergen-specific IgE, which is dependent on the enzymatic activity of cysteine protease-allergens, in the sensitization process.
Assuntos
Alérgenos/fisiologia , Antígenos de Dermatophagoides/imunologia , Cistatina A/fisiologia , Inibidores de Cisteína Proteinase/fisiologia , Dermatophagoides farinae/imunologia , Dermatophagoides pteronyssinus/imunologia , Alérgenos/administração & dosagem , Alérgenos/sangue , Animais , Antígenos de Dermatophagoides/administração & dosagem , Antígenos de Dermatophagoides/sangue , Proteínas de Artrópodes , Domínio Catalítico/imunologia , Cistatina A/administração & dosagem , Cistatina A/sangue , Cisteína/administração & dosagem , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase/administração & dosagem , Inibidores de Cisteína Proteinase/sangue , Dermatophagoides farinae/metabolismo , Dermatophagoides pteronyssinus/metabolismo , Feminino , Humanos , Imunoglobulina E/biossíntese , Imunoglobulina E/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos CBA , Oxirredução , Ligação Proteica/imunologia , Substâncias Redutoras/administração & dosagem , VacinaçãoRESUMO
Lipid-binding properties and/or involvement with host defense are often found in allergen proteins, implying that these intrinsic biological functions likely contribute to the allergenicity of allergens. The group 2 major mite allergens, Der f 2 and Der p 2, show structural homology with MD-2, the lipopolysaccharide (LPS)-binding component of the Toll-like receptor (TLR) 4 signalling complex. Elucidation of the ligand-binding properties of group 2 mite allergens and identification of interaction sites by structural studies are important to explore the relationship between allergenicity and biological function. Here, we report a ligand-fishing approach in which His-tagged Der f 2 was incubated with sonicated stable isotope-labelled Escherichia coli as a potential ligand source, followed by isolation of Der f 2-bound material by a HisTrap column and NMR analysis. We found that Der f 2 binds to LPS with a nanomolar affinity and, using fluorescence and gel filtration assays that LPS binds to Der f 2 in a molar ratio of 1 : 1. We mapped the LPS-binding interface of Der f 2 by NMR perturbation studies, which suggested that LPS binds Der f 2 between the two large beta-sheets, similar to its binding to MD-2, the LPS-binding component of the innate immunity receptor TLR4.
Assuntos
Alérgenos/metabolismo , Antígenos de Dermatophagoides/metabolismo , Lipopolissacarídeos/metabolismo , Animais , Antígenos de Dermatophagoides/química , Proteínas de Artrópodes , Sítios de Ligação , Cromatografia em Gel , Escherichia coli/metabolismo , Ligantes , Lipopolissacarídeos/química , Espectroscopia de Ressonância Magnética , Modelos MolecularesRESUMO
BACKGROUND: In studies on allergies in mouse models, IgE production is an essential parameter to be evaluated. Here, we examine the effect of commercially available immunoreaction enhancer solutions and different blocking reagents in enzyme-linked immunosorbent assay (ELISA) for total or antigen-specific murine IgE in order to improve the assays. METHODS: Sera from mice immunized with recombinant house dust mite major allergens, Der f 1 and Der p 1, were used for the assays. Total IgE was measured by sandwich ELISA using monoclonal antibodies against murine IgE. Antigen-specific IgE was assayed using allergen-coated plates. Sensitivity or signal intensity in ELISA was compared among conditions differing in the use of enhancer solutions, blocking reagents, or monoclonal antibodies, and incubation time. RESULTS: Use of enhancer solutions improved the sensitivity of ELISA for total IgE by approximately 30-fold of that using a conventional buffer. A blocking reagent caused more unwanted enhancement of the background signal in blank wells in ELISA for total IgE compared with another blocking reagent, however, improved signal intensity in ELISA for antigen-specific ELISA without significant enhancement of the background signal. Optimal assay conditions were determined. CONCLUSIONS: Enhancer solutions are effective in improving ELISAs for total and antigen-specific murine IgE. Selection of blocking reagents was important to decrease unwanted enhancement of background signals and was effective in enhancing signals for positive samples. The ELISAs improved in this study are useful for the study of allergies in mouse models.
Assuntos
Antígenos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Hipersensibilidade/imunologia , Imunoglobulina E/análise , Imunoglobulina E/imunologia , Alérgenos/genética , Alérgenos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos/genética , Antígenos de Dermatophagoides/genética , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes , Cisteína Endopeptidases , Feminino , Hipersensibilidade/diagnóstico , Imunoglobulina E/sangue , Imunoglobulina E/isolamento & purificação , Indicadores e Reagentes/química , Camundongos , Camundongos Endogâmicos CBA , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Temperatura , Fatores de Tempo , VacinaçãoRESUMO
The major proteolytic allergen derived from the house dust mite Dermatophagoides pteronyssinus, Der p1, is one of the most clinically relevant allergens worldwide. In the present study, we evaluate the contribution of the proteolytic activity and structure of a highly purified rDer p 1 to immune responses. Mice were i.p. immunized with three forms of rDer p 1 adsorbed to Alum: one enzymatically active, one treated with an irreversible cysteine protease-specific inhibitor, E-64, and one heat denatured. Immunization with E-64-treated or heat-denatured rDer p 1 elicited much less production of serum total IgE and not only rDer p 1-specific IgE but also IgGs compared with immunization with active rDer p 1. Assays for Ab-binding and its inhibition and structural analyses indicated that E-64-treated rDer p 1 retained its global structure and conformational B cell epitopes. A proliferative response and production of IL-5 by spleen cells restimulated with rDer p 1 were observed on immunization with the active rDer p 1 but not E-64-treated rDer p 1. The cells from mice immunized with heat-denatured rDer p 1 exhibited the highest levels of proliferation and production of IL-5 and IFN-gamma. The results indicate that the proteolytic activity of the highly purified rDer p 1 crucially commits to the sensitization process, including both IgE and IgG responses. Additionally, we demonstrated immunogenic differences by functional or structural manipulations of the rDer p 1. The findings have implications for sensitization to this relevant allergen in humans and for the design of modified allergen-vaccines for future allergen-specific immunotherapy.
Assuntos
Antígenos de Dermatophagoides/imunologia , Antígenos de Dermatophagoides/metabolismo , Cisteína Endopeptidases/metabolismo , Dermatophagoides pteronyssinus/imunologia , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Animais , Antígenos de Dermatophagoides/administração & dosagem , Proteínas de Artrópodes , Inibidores de Cisteína Proteinase/farmacologia , Feminino , Temperatura Alta , Hidrólise , Imunização/métodos , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Camundongos Endogâmicos CBA , Desnaturação Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/isolamento & purificação , Baço/citologia , Baço/imunologia , Baço/metabolismoRESUMO
Group 2 major mite allergens Der f 2 and Der p 2 are classified into the recently identified group of MD-2-related lipid-recognition (ML) proteins, but the ligands and biological functions of these allergens are unknown. We have obtained a high-quality NMR structure for Der f 2, and found that it is more similar to the crystal structure of NPC2, a distant homologue, than to that of Der p 2, in terms of the separation and angle between the two major beta-sheets. This made us propose that ML proteins undergo clamshell-like motions that change the sizes of ligand-binding spaces inside their immunoglobulin-fold beta-sandwich to accommodate lipid molecules. This type of motion in lipopolysaccaride recognition of MD-2 is suggested to be likely as well by structural models. We also report the applicability of NMR differential exchange broadening experiments for complexes of intact monoclonal antibodies and antigens; using this technique, we have detected the conformational epitopes for monoclonal antibodies 15E11 and 13A4 as two separate surface patches.
Assuntos
Antígenos de Dermatophagoides/química , Antígenos de Dermatophagoides/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Antígenos de Dermatophagoides/fisiologia , Antígenos de Superfície/química , Proteínas de Artrópodes , Proteínas de Transporte/química , Espectroscopia de Ressonância de Spin Eletrônica , Epitopos/imunologia , Lipopolissacarídeos/metabolismo , Antígeno 96 de Linfócito , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteínas de Transporte Vesicular/químicaRESUMO
We assessed the effect of multiple-mutations within one IgE-binding area on allergenicity of Der f 2. The triple-mutant of Der f 2, P34/95/99A, exhibited the most significant reduction of allergenicity and circular dichroism analysis showed that the global structure of Der f 2 was maintained in P34/95/99A. These results indicate that such a strategy is effective when designing allergen-vaccines, which achieve less allergenicity for a broad population of patients without disrupting the global structure. Structurally, Der f 2 is a member of the MD-2 related lipid-recognition proteins. The sites for the triple-mutation located on the characteristically charged entrance of a cavity and corresponded to the regions critical to ligand-binding in the Niemann-Pick type 2 disease protein and MD-2.
Assuntos
Antígenos de Dermatophagoides/química , Antígenos de Dermatophagoides/imunologia , Imunoglobulina E/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antígenos de Dermatophagoides/genética , Proteínas de Artrópodes , Basófilos/efeitos dos fármacos , Sítios de Ligação/genética , Clonagem Molecular , Dermatophagoides farinae/genética , Heparina/metabolismo , Humanos , Ligantes , Dados de Sequência Molecular , MutaçãoRESUMO
In the postgenomic era, many researchers and organizations have been engaged in structural and functional analyses of proteins. As a part of these efforts, searching for small organic compounds that bind specifically to target proteins is quite important. In this study, we have developed a rational strategy for ligand discovery based on the three-dimensional structures of target proteins, which were elucidated by X-ray crystallography and nuclear magnetic resonance spectroscopy. The strategy has three features: (i) rapid selection of candidate compounds by in silico screening, (ii) automated preparation of sample solutions with robotics, and (iii) reliable evaluation of the candidates with surface plasmon resonance. Applying the strategy to a protein, At2g24940 from Arabidopsis thaliana, we discovered four small ligands out of a commercially available library of about 150 000 compounds. Although these compounds had only weak affinities to the target protein, with dissociation constants ranging from 68 to 120 microM, they apparently possess common structural features. They would be leads for the development of specific inhibitors/drugs for At2g24940, and provide important clues toward elucidation of the protein function.
Assuntos
Simulação por Computador , Ligantes , Proteínas/química , Proteínas de Arabidopsis/química , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
A member of the PIAS (protein inhibitor of activated STAT) family of proteins, PIAS1, have been reported to serve as an E3-type SUMO ligase for tumor suppressor p53 and its own. It also was proposed that the N-terminal domain of PIAS1 interacts with DNA as well as p53. Extensive biochemical studies have been devoted recently to understand sumoylations and its biological implications, whereas the structural aspects of the PIAS family and the mechanism of its interactions with various factors are less well known to date. In this study, the three-dimensional structure of the N-terminal domain (residues 1-65) of SUMO ligase PIAS1 was determined by NMR spectroscopy. The structure revealed a unique four-helix bundle with a topology of an up-down-extended loop-down-up, a part of which the helix-extended loop-helix represented the SAP (SAF-A/B, Acinus, and PIAS) motif. Thus, this N-terminal domain may be referred to as a four-helix SAP domain. The glutathione S-transferase pull-down assay demonstrated that this domain possesses a binding ability to tumor suppressor p53, a target protein for sumoylation by PIAS1, whereas gel mobility assays showed that it has a strong affinity toward A/T-rich DNA. An NMR analysis of the four-helix SAP domain complexed with the 16-bp-long DNA demonstrated that one end of the four-helix bundle is the binding site and may fit into the minor groove of DNA. The three-dimensional structure and its binding duality are discussed in conjunction with the biological functions of PIAS1 as a SUMO ligase.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , DNA/química , DNA/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Transporte/genética , DNA/genética , Humanos , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Inibidoras de STAT Ativados , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína SUMO-1/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaAssuntos
Ligases/química , Doença de Parkinson/genética , Ubiquitina-Proteína Ligases , Ubiquitina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Ligases/genética , Ligases/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Doença de Parkinson/enzimologia , Estrutura Secundária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Small protein B (SmpB) is required for trans-translation, binding specifically to tmRNA. We show here the solution structure of SmpB from an extremely thermophilic bacterium, Thermus thermophilus HB8, determined by heteronuclear nuclear magnetic resonance methods. The core of the protein consists of an antiparallel beta-barrel twisted up from eight beta-strands, each end of which is capped with the second or third helix, and the first helix is located beside the barrel. Its C-terminal sequence (20 residues), which is rich in basic residues, shows a poorly structured form, as often seen in isolated ribosomal proteins. The results are discussed in relation to the oligonucleotide binding fold.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Soluções , Thermus thermophilusRESUMO
The three-dimensional structure of the N-terminal SH3 domain (residues 583-660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Proteínas/química , Proteínas Proto-Oncogênicas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína Adaptadora GRB2 , Isomerismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-vav , Soluções , Domínios de Homologia de srcRESUMO
The Gag polyprotein is key to the budding of retroviruses from host cells and is cleaved upon virion maturation, the N-terminal membrane-binding domain forming the matrix protein (MA). The 2.8-A resolution crystal structure of MA of equine infectious anemia virus (EIAV), a lentivirus, reveals that, despite showing no sequence similarity, more than half of the molecule can be superimposed on the MAs of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). However, unlike the structures formed by HIV-1 and SIV MAs, the oligomerization state observed is not trimeric. We discuss the potential of this molecule for membrane binding in the light of conformational differences between EIAV MA and HIV or SIV MA.