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1.
Artigo em Inglês | MEDLINE | ID: mdl-31437325

RESUMO

BACKGROUND: For patients with peanut allergy, there are currently no methods to predict who will develop sustained unresponsiveness (SU) after oral immunotherapy (OIT). OBJECTIVE: Assess IgE binding to peanut (PN), Ara h 2, and specific linear epitopes of Ara h 2 as predictors of the important clinical parameters: eliciting dose threshold and attainment of SU following OIT. METHODS: Samples and clinical data were collected from children undergoing OIT. PN- and Ara h 2-sIgE were quantified by ImmunoCAP® . IgE binding to linear peptides of Ara h 2 and Ara h 6 was measured with peptide microarrays. RESULTS: Lower values of PN-sIgE correlated with eliciting dose (P = .001) and with a higher likelihood of achieving SU (P < .0001), but these relationships were lost at higher values for PN-sIgE (≥14 kIU for eliciting dose and ≥35 kIU/L for SU). In subjects with PN-sIgE ≥ 14 kIU/L, binding of IgE to epitopes 5 and 6 of Ara h 2 was associated with a lower eliciting dose at baseline challenge (P < .001; Pc  < .02). In subjects with PN-sIgE ≥ 35 kIU/L, a combined model of IgE binding to epitopes 1, 5 and 6 with PN-sIgE was highly predictive of attainment of SU (AUC of 0.86; P = .0067). CONCLUSION: In young patients with peanut allergy, measurement of PN-sIgE and IgE binding to specific linear epitopes of Ara h 2 in baseline samples may allow stratification of patients regarding sensitivity to challenge and outcome of OIT.

2.
J Virol ; 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518655

RESUMO

Ebolaviruses Zaire (EBOV), Bundibugyo (BDBV) and Sudan (SUDV) cause human disease with high case fatality rates. Experimental monovalent vaccines, which all utilize the sole envelope glycoprotein (GP), do not protect against heterologous ebolaviruses. Human parainfluenza virus type 3-vectored vaccines offer benefits including needle-free administration and induction of mucosal responses in the respiratory tract. Multiple approaches were taken to induce a broad protection against the three ebolaviruses. While GP consensus-based antigens failed to elicit neutralizing antibodies, polyvalent immunization induced neutralizing responses to all three ebolaviruses and protected animals from death and disease caused by EBOV, SUDV and BDBV. As immunization with a cocktail of antigenically-related antigens can skew the responses and change the epitope hierarchy, we performed comparative analysis of antibody repertoire and Fc-mediated protective mechanisms in animals immunized with monovalent versus polyvalent vaccines. Compared to the monovalent vaccines, sera from trivalent vaccinated guinea pigs bound and neutralized EBOV and SUDV at equivalent levels and BDBV at only slightly reduced level. Peptide microarrays revealed a preponderance of binding to amino acids 389-403, 397-415 and 477-493, representing three linear epitopes in the mucin-like domain known to induce a protective antibody response. Competition binding assays with monoclonal antibodies isolated from human ebolavirus survivors demonstrated that the immune sera block binding of antibodies specific for the GP glycan cap, GP1-GP2 interface, the mucin-like domain, and the membrane-proximal external region. Thus, cocktail administration of three ebolavirus vaccines induces a desirable broad antibody response, without skewing of the response toward preferential recognition of a single virus.IMPORTANCE Symptoms of the disease caused by ebolaviruses Ebola, Bundibugyo and Sudan are similar, and their endemic areas overlap. However, because of the limited antigenic relatedness of ebolavirus glycoprotein (GP) used in all candidate vaccines against these viruses, they protect only against homologous but not heterologous ebolaviruses. Therefore, a broadly specific pan-ebolavirus vaccine is required, which might be achieved by administration of a cocktail of vaccines. The effects of cocktail administration of ebolavirus vaccines on the antibody repertoire remain unknown. Here in-depth analysis of the antibody responses to cocktail administration of human parainfluenza type 3-vectored vaccines against individual ebolaviruses was performed, which included analysis of binding to GP, neutralization of individual ebolaviruses, epitope specificity, Fc-mediated functions, and protection against the three ebolaviruses. The results demonstrated potent and balanced responses against individual ebolaviruses and no significant reduction of the responses, compared to that induced by individual vaccines.

3.
Mol Immunol ; 99: 1-8, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29627609

RESUMO

Quantitative guidelines to distinguish allergenic proteins from related, but non-allergenic ones are urgently needed for regulatory agencies, biotech companies and physicians. In a previous study, we found that allergenic proteins populate a relatively small number of protein families, as characterized by the Pfam database. However, these families also contain non-allergenic proteins, meaning that allergenic determinants must lie within more discrete regions of the sequence. Thus, new methods are needed to discriminate allergenic proteins within those families. Physical-Chemical Properties (PCP)-motifs specific for allergens within a Pfam class were determined for 17 highly populated protein domains. A novel scoring method based on PCP-motifs that characterize known allergenic proteins within these families was developed, and validated for those domains. The motif scores distinguished sequences of allergens from a large selection of 80,000 randomly selected non-allergenic sequences. The motif scores for the birch pollen allergen (Bet v 1) family, which also contains related fruit and nut allergens, correlated better than global sequence similarities with clinically observed cross-reactivities among those allergens. Further, we demonstrated that the average scores of allergen specific motifs for allergenic profilins are significantly different from the scores of non-allergenic profilins. Several of the selective motifs coincide with experimentally determined IgE epitopes of allergenic profilins. The motifs also discriminated allergenic pectate lyases, including Jun a 1 from mountain cedar pollen, from similar proteins in the human microbiome, which can be assumed to be non-allergens. The latter lacked key motifs characteristic of the known allergens, some of which correlate with known IgE binding sites.

4.
Sci Rep ; 7(1): 13940, 2017 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-29066768

RESUMO

Proteins are fundamental to life and exhibit a wide diversity of activities, some of which are toxic. Therefore, assessing whether a specific protein is safe for consumption in foods and feeds is critical. Simple BLAST searches may reveal homology to a known toxin, when in fact the protein may pose no real danger. Another challenge to answer this question is the lack of curated databases with a representative set of experimentally validated toxins. Here we have systematically analyzed over 10,000 manually curated toxin sequences using sequence clustering, network analysis, and protein domain classification. We also developed a functional sequence signature method to distinguish toxic from non-toxic proteins. The current database, combined with motif analysis, can be used by researchers and regulators in a hazard screening capacity to assess the potential of a protein to be toxic at early stages of development. Identifying key signatures of toxicity can also aid in redesigning proteins, so as to maintain their desirable functions while reducing the risk of potential health hazards.

5.
Bioinformatics ; 33(7): 1014-1020, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28062447

RESUMO

The phenomenon of cross-reactivity between allergenic proteins plays an important role to understand how the immune system recognizes different antigen proteins. Allergen proteins are known to cross-react if their sequence comparison shows a high sequence identity which also implies that the proteins have a similar 3D fold. In such cases, linear sequence alignment methods are frequently used to predict cross-reactivity between allergenic proteins. However, the prediction of cross-reactivity between distantly related allergens continues to be a challenging task. To overcome this problem, we developed a new structure-based computational method, Cross-React, to predict cross-reactivity between allergenic proteins available in the Structural Database of Allergens (SDAP). Our method is based on the hypothesis that we can find surface patches on 3D structures of potential allergens with amino acid compositions similar to an epitope in a known allergen. We applied the Cross-React method to a diverse set of seven allergens, and successfully identified several cross-reactive allergens with high to moderate sequence identity which have also been experimentally shown to cross-react. Based on these findings, we suggest that Cross-React can be used as a predictive tool to assess protein allergenicity and cross-reactivity. Availability and Implementation: : Cross-React is available at: http://curie.utmb.edu/Cross-React.html. Contact: ssnegi@utmb.edu.


Assuntos
Alérgenos/imunologia , Biologia Computacional/métodos , Reações Cruzadas/imunologia , Alérgenos/química , Sequência de Aminoácidos , Aminoácidos/química , Animais , Anticorpos/química , Anticorpos/imunologia , Abelhas , Bovinos , Cristalografia por Raios X , Bases de Dados de Proteínas , Epitopos/química , Epitopos/imunologia , Humanos , Proteínas/química , Proteínas/imunologia , Alinhamento de Sequência
6.
Sci Rep ; 6: 35415, 2016 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-27739488

RESUMO

The homeobox encodes a DNA-binding domain found in transcription factors regulating key developmental processes. The most notable examples of homeobox containing genes are the Hox genes, arranged on chromosomes in the same order as their expression domains along the body axis. The mechanisms responsible for the synchronous regulation of Hox genes and the molecular function of their colinearity remain unknown. Here we report the discovery of a conserved structural signature of the 180-base pair DNA fragment comprising the homeobox. We demonstrate that the homeobox DNA has a characteristic 3-base-pair periodicity in the hydroxyl radical cleavage pattern. This periodic pattern is significant in most of the 39 mammalian Hox genes and in other homeobox-containing transcription factors. The signature is present in segmented bilaterian animals as evolutionarily distant as humans and flies. It remains conserved despite the fact that it would be disrupted by synonymous mutations, which raises the possibility of evolutionary selective pressure acting on the structure of the coding DNA. The homeobox coding DNA may therefore have a secondary function, possibly as a regulatory element. The existence of such element may have important consequences for understanding how these genes are regulated.


Assuntos
Sequência Conservada , Genes Homeobox/genética , Animais , Drosophila , Evolução Molecular , Humanos , Camundongos , Motivos de Nucleotídeos , Fases de Leitura Aberta
7.
Clin Exp Allergy ; 46(8): 1120-1128, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27238146

RESUMO

BACKGROUND: Cross-linking of IgE antibody by specific epitopes on the surface of mast cells is a prerequisite for triggering symptoms of peanut allergy. IgE epitopes are frequently categorized as linear or conformational epitopes. Although linear IgE-binding epitopes of peanut allergens have been defined, little is known about conformational IgE-binding epitopes. OBJECTIVE: To identify clinically relevant conformational IgE epitopes of the two most important peanut allergens, Ara h 2 and Ara h 6, using phage peptide library. METHODS: A phage 12mer peptide library was screened with allergen-specific IgE from 4 peanut-allergic patients. Binding of the mimotopes to IgE from a total of 29 peanut-allergic subjects was measured by ELISA. The mimotope sequences were mapped on the surface areas of Ara h 2 and Ara h 6 using EpiSearch. RESULTS: Forty-one individual mimotopes were identified that specifically bind anti- Ara h 2/Ara h 6 IgE as well as rabbit anti-Ara h 2 and anti-Ara h 6 IgG. Sequence alignment showed that none of the mimotope sequences match a linear segment of the Ara h 2 or Ara h 6 sequences. EpiSearch analysis showed that all the mimotopes mapped to surface patches of Ara h 2 and Ara h 6. Eight of the mimotopes were recognized by more than 90% of the patients, suggesting immunodominance. Each patient had distinct IgE recognition patterns but the recognition frequency was not correlated to the concentration of peanut specific IgE or to clinical history. CONCLUSIONS: The mimotopes identified in this study represent conformational epitopes. Identification of similar surface patches on Ara h 2 and Ara h 6 further underscores the similarities between these two potent allergens.


Assuntos
Albuminas 2S de Plantas/química , Alérgenos/química , Antígenos de Plantas/química , Epitopos/química , Glicoproteínas/química , Imunoglobulina E/imunologia , Modelos Moleculares , Conformação Proteica , Albuminas 2S de Plantas/imunologia , Albuminas 2S de Plantas/metabolismo , Alérgenos/imunologia , Alérgenos/metabolismo , Sequência de Aminoácidos , Antígenos de Plantas/imunologia , Antígenos de Plantas/metabolismo , Arachis/imunologia , Sítios de Ligação , Técnicas de Visualização da Superfície Celular , Sequência Consenso , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Epitopos/metabolismo , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Humanos , Imunoglobulina E/química , Imunoglobulina E/metabolismo , Hipersensibilidade a Amendoim/imunologia , Biblioteca de Peptídeos , Ligação Proteica
8.
Proteins ; 84 Suppl 1: 323-48, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27122118

RESUMO

We present the results for CAPRI Round 30, the first joint CASP-CAPRI experiment, which brought together experts from the protein structure prediction and protein-protein docking communities. The Round comprised 25 targets from amongst those submitted for the CASP11 prediction experiment of 2014. The targets included mostly homodimers, a few homotetramers, and two heterodimers, and comprised protein chains that could readily be modeled using templates from the Protein Data Bank. On average 24 CAPRI groups and 7 CASP groups submitted docking predictions for each target, and 12 CAPRI groups per target participated in the CAPRI scoring experiment. In total more than 9500 models were assessed against the 3D structures of the corresponding target complexes. Results show that the prediction of homodimer assemblies by homology modeling techniques and docking calculations is quite successful for targets featuring large enough subunit interfaces to represent stable associations. Targets with ambiguous or inaccurate oligomeric state assignments, often featuring crystal contact-sized interfaces, represented a confounding factor. For those, a much poorer prediction performance was achieved, while nonetheless often providing helpful clues on the correct oligomeric state of the protein. The prediction performance was very poor for genuine tetrameric targets, where the inaccuracy of the homology-built subunit models and the smaller pair-wise interfaces severely limited the ability to derive the correct assembly mode. Our analysis also shows that docking procedures tend to perform better than standard homology modeling techniques and that highly accurate models of the protein components are not always required to identify their association modes with acceptable accuracy. Proteins 2016; 84(Suppl 1):323-348. © 2016 Wiley Periodicals, Inc.


Assuntos
Biologia Computacional/estatística & dados numéricos , Modelos Estatísticos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas/química , Software , Algoritmos , Motivos de Aminoácidos , Bactérias/química , Sítios de Ligação , Biologia Computacional/métodos , Humanos , Cooperação Internacional , Internet , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Termodinâmica
9.
J Virol ; 88(16): 9260-7, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24899192

RESUMO

UNLABELLED: Western equine encephalitis virus (WEEV) is an arbovirus from the genus Alphavirus, family Togaviridae, which circulates in North America between birds and mosquitoes, occasionally causing disease in humans and equids. In recent decades, human infection has decreased dramatically; the last documented human case in North America occurred in 1994, and the virus has not been detected in mosquito pools since 2008. Because limited information exists regarding the evolution of WEEV, we analyzed the genomic sequences of 33 low-passage-number strains with diverse geographic and temporal distributions and performed comprehensive phylogenetic analyses. Our results indicated that WEEV is a highly conserved alphavirus with only approximately 5% divergence in its most variable genes. We confirmed the presence of the previously determined group A and B lineages and further resolved group B into three sublineages. We also observed an increase in relative genetic diversity during the mid-20th century, which correlates with the emergence and cocirculation of several group B sublineages. The estimated WEEV population size dropped in the 1990s, with only the group B3 lineage being sampled in the past 20 years. Structural mapping showed that the majority of substitutions in the envelope glycoproteins occurred at the E2-E2 interface. We hypothesize that an event occurred in the mid-20th century that resulted in the increased genetic diversity of WEEV in North America, followed by genetic constriction due to either competitive displacement by the B3 sublineage or stochastic events resulting from a population decline. IMPORTANCE: Western equine encephalitis virus (WEEV) has caused several epidemics that resulted in the deaths of thousands of humans and hundreds of thousands of equids during the past century. During recent decades, human infection decreased drastically and the virus has not been found in mosquito pools since 2008. Because limited information exists regarding the evolution of WEEV, we analyzed 33 complete genome sequences and conducted comprehensive phylogenetic analyses. We confirmed the presence of two major lineages, one of which diverged into three sublineages. Currently, only one of those sublineages is found circulating in nature. Understanding the evolution of WEEV over the past century provides a unique opportunity to observe an arbovirus that is in decline and to better understand what factors can cause said decline.


Assuntos
Evolução Biológica , Vírus da Encefalite Equina do Oeste/genética , Genoma Viral/genética , Animais , Encefalomielite Equina/virologia , Variação Genética/genética , Cavalos , América do Norte , Filogenia , Análise de Sequência/métodos
10.
J Biol Chem ; 287(46): 38543-51, 2012 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-23007387

RESUMO

The measles virus (MV) fusion apparatus consists of a fusion protein and an attachment protein named hemagglutinin (H). After receptor-binding through its cuboidal head, the H-protein transmits the fusion-triggering signal through its stalk to the fusion protein. However, the structural basis of signal transmission is unclear because only structures of H-heads without their stalk have been solved. On the other hand, the entire ectodomain structure of the hemagglutinin-neuraminidase protein of another Paramyxovirus revealed a four-helix bundle stalk. To probe the structure of the 95-residue MV H-stalk we individually substituted head-proximal residues (positions 103-153) with cysteine, and biochemically and functionally characterized the resultant proteins. Our results indicate that most residues in the central segment (positions 103-117) can be cross-linked by engineered disulfide bonds, and thus may be engaged in a tetrameric structure. While covalent tetramerization disrupts fusion triggering function, disulfide bond reduction restores it in most positions except Asp-113. The next stalk segment (residues 123-138) also has high propensity to form covalent tetramers, but since these cross-links have little or no effect on function, it can conduct the fusion-triggering signal while remaining in a stabilized tetrameric configuration. This segment may act as a spacer, maintaining H-heads at an optimal height. Finally, the head-proximal segment (residues 139-154) has very limited propensity to trap tetramers, suggesting bifurcation into two flexible linkers clamped by inter-subunit covalent links formed by natural Cys-139 and Cys-154. We discuss the modular structure of the MV H-stalk in the context of membrane fusion triggering and cell entry by Paramyxoviruses.


Assuntos
Vírus do Sarampo/metabolismo , Fusão de Membrana , Sequência de Aminoácidos , Animais , Cercopithecus aethiops , Cisteína/química , Dissulfetos/química , Hemaglutininas/química , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Transfecção , Células Vero , Proteínas Virais de Fusão/química , Ligação Viral , Internalização do Vírus
11.
J Biol Chem ; 287(39): 33026-35, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22859308

RESUMO

The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses.


Assuntos
Vírus do Sarampo/química , Multimerização Proteica , Proteínas Virais de Fusão/química , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Vírus do Sarampo/genética , Vírus do Sarampo/metabolismo , Estrutura Quaternária de Proteína , Homologia Estrutural de Proteína , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo
12.
Proteins ; 80(5): 1308-15, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22274941

RESUMO

Use of atomic force microscopy (AFM) has recently led to a better understanding of the molecular mechanisms of the unfolding process by mechanical forces; however, the rational design of novel proteins with specific mechanical strength remains challenging. We have approached this problem from a new perspective that generates linear physical-chemical properties (PCP) motifs from a limited AFM data set. Guided by our linear sequence analysis, we designed and analyzed four new mutants of the titin I1 domain with the goal of increasing the domain's mechanical strength. All four mutants could be cloned and expressed as soluble proteins. AFM data indicate that at least two of the mutants have increased molecular mechanical strength. This observation suggests that the PCP method is useful to graft sequences specific for high mechanical stability to weak proteins to increase their mechanical stability, and represents an additional tool in the design of novel proteins besides steered molecular dynamics calculations, coarse grained simulations, and ϕ-value analysis of the transition state.


Assuntos
Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Dicroísmo Circular , Conectina , Fibronectinas/química , Fibronectinas/genética , Ligações de Hidrogênio , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Quinases/química , Proteínas Quinases/genética , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética
13.
Int Arch Allergy Immunol ; 157(4): 323-30, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22123204

RESUMO

BACKGROUND: Bla g 2, one of the major cockroach allergens, induces a strong IgE response against conformational epitopes, and on reexposure, sensitized individuals often display symptoms of allergic rhinitis and asthma. The aim of the current study was to perform a test of the efficacy of a modified phage display screening, characterization of selected phages and an automated algorithm, EpiSearch, in locating an important conformational epitope. METHODS: The monoclonal antibody 7C11, which partially inhibits the binding of patient IgE antibodies to Bla g 2, was used to screen a random peptide phage library. After 3 rounds of panning, 32 phage clones were isolated and the amino acid sequences of their peptides were determined. The relative affinity and specificity of the binding of these peptides to 7C11 were tested in ELISAs. The amino acid composition of these peptides was then matched with clusters of residues on the surface of the 3-dimensional (3D) structure of Bla g 2, using our EpiSearch algorithm. RESULTS: The amino acid sequences of the peptides on selected phages differed at only one position, occupied by 1 of 2 negatively charged residues. The two 12-mer sequences bound to 7C11 with similar avidity and specificity. There was good concordance between the residues in the 3D clusters identified from our phage display/computational method with the co-crystal structural analysis. CONCLUSION: Conformational epitopes may be mapped through screening of clones from random peptide phage display libraries and EpiSearch.


Assuntos
Algoritmos , Ácido Aspártico Endopeptidases/química , Baratas/imunologia , Mapeamento de Epitopos/métodos , Hipersensibilidade/diagnóstico , Biblioteca de Peptídeos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/imunologia , Biologia Computacional , Cristalização , Estudos de Viabilidade , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Conformação Proteica
14.
Regul Toxicol Pharmacol ; 54(3 Suppl): S11-9, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19121639

RESUMO

In many countries regulatory agencies have adopted safety guidelines, based on bioinformatics rules from the WHO/FAO and EFSA recommendations, to prevent potentially allergenic novel foods or agricultural products from reaching consumers. We created the Structural Database of Allergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP/) to combine data that had previously been available only as flat files on Web pages or in the literature. SDAP was designed to be user friendly, to be of maximum use to regulatory agencies, clinicians, as well as to scientists interested in assessing the potential allergenic risk of a protein. We developed methods, unique to SDAP, to compare the physicochemical properties of discrete areas of allergenic proteins to known IgE epitopes. We developed a new similarity measure, the property distance (PD) value that can be used to detect related segments in allergens with clinical observed cross-reactivity. We have now expanded this work to obtain experimental validation of the PD index as a quantitative predictor of IgE cross-reactivity, by designing peptide variants with predetermined PD scores relative to known IgE epitopes. In complementary work we show how sequence motifs characteristic of allergenic proteins in protein families can be used as fingerprints for allergenicity.


Assuntos
Alérgenos/imunologia , Epitopos/química , Proteínas/imunologia , Alérgenos/química , Alérgenos/classificação , Motivos de Aminoácidos , Sequência de Aminoácidos , Bases de Dados de Proteínas , Dados de Sequência Molecular , Conformação Proteica , Organização Mundial da Saúde
15.
Bioinform Biol Insights ; 3: 71-81, 2009 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20140073

RESUMO

BACKGROUND: Precise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs. RESULTS: We present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu), the antibody mAb Bo2C11 targeting the C(2) domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples the conformational epitopes as determined by the X-ray crystal structures of the antibody-antigen complexes, were found within the highest scoring patches of EpiSearch, covering in most cases more than 50% residues of experimental observed conformational epitopes. Input options of the program include mapping of a single peptide or a set of peptides on the antigen structure, and the results of the calculation can be visualized on our interactive web server. AVAILABILITY: Users can access the EpiSearch from our web server http://curie.utmb.edu/episearch.html.

16.
Mol Immunol ; 45(14): 3740-7, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18621419

RESUMO

Similarities in sequences and 3D structures of allergenic proteins provide vital clues to identify clinically relevant immunoglobulin E (IgE) cross-reactivities. However, experimental 3D structures are available in the Protein Data Bank for only 5% (45/829) of all allergens catalogued in the Structural Database of Allergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP). Here, an automated procedure was used to prepare 3D-models of all allergens where there was no experimentally determined 3D structure or high identity (95%) to another protein of known 3D structure. After a final selection by quality criteria, 433 reliable 3D models were retained and are available from our SDAP Website. The new 3D models extensively enhance our knowledge of allergen structures. As an example of their use, experimentally derived "continuous IgE epitopes" were mapped on 3 experimentally determined structures and 13 of our 3D-models of allergenic proteins. Large portions of these continuous sequences are not entirely on the surface and therefore cannot interact with IgE or other proteins. Only the surface exposed residues are constituents of "conformational IgE epitopes" which are not in all cases continuous in sequence. The surface exposed parts of the experimental determined continuous IgE epitopes showed a distinct statistical distribution as compared to their presence in typical protein-protein interfaces. The amino acids Ala, Ser, Asn, Gly and particularly Lys have a high propensity to occur in IgE binding sites. The 3D-models will facilitate further analysis of the common properties of IgE binding sites of allergenic proteins.


Assuntos
Alérgenos/química , Imunoglobulina E/química , Modelos Moleculares , Proteínas/química , Alérgenos/classificação , Alérgenos/genética , Alérgenos/imunologia , Sequência de Aminoácidos , Aminoácidos/química , Bases de Dados de Proteínas , Epitopos/química , Imunoglobulina E/classificação , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Dados de Sequência Molecular , Conformação Proteica , Proteínas/classificação , Proteínas/genética , Proteínas/imunologia , Homologia de Sequência de Aminoácidos
17.
J Mol Biol ; 377(1): 232-45, 2008 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-18241887

RESUMO

Structural plasticity of mammalian cytochromes P450 (CYP) has recently been explored in our laboratory and elsewhere to understand the ligand-binding promiscuity. CYP2B4 exhibits very different conformations and thermodynamic signatures in binding the small inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) versus the large bifonazole. Using four key active-site mutants (F296A, T302A, I363A, and V367L) that are involved in binding one or both inhibitors, we dissected the thermodynamic basis for the ability of CYP2B4 to bind substrates and inhibitors of different sizes and chemistry. In all cases, 1:1 binding stoichiometry was observed. The inhibitors 4-CPI, 1-(4-chlorophenyl)imidazole, and 1-(2-(benzyloxy)ethyl)imidazole bind to the mutants with a free energy difference (Delta Delta G) of approximately 0.5 to 1 kcal/mol compared with the wild type but with a large entropy-enthalpy compensation of up to 50 kcal/mol. The substrate testosterone binds to all four mutants with a Delta Delta G of approximately 0.5 kcal/mol but with as much as 40 kcal/mol of entropy-enthalpy compensation. In contrast, benzphetamine binding to V367L and F296A is accompanied by a Delta Delta G of approximately 1.5 and 3 kcal/mol, respectively. F296A, I363A, and V367L exhibit very different benzphetamine metabolite profiles, indicating the different substrate-binding orientations in the active site of each mutant. Overall, the findings indicate that malleability of the active site allows mammalian P450s to exhibit a high degree of thermodynamic fidelity in ligand binding.


Assuntos
Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/metabolismo , Inibidores Enzimáticos/farmacologia , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Benzfetamina/química , Benzfetamina/farmacologia , Sítios de Ligação , Calorimetria , Família 2 do Citocromo P450 , Inibidores Enzimáticos/química , Ligantes , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato/efeitos dos fármacos , Termodinâmica , Água/química
18.
J Struct Biol ; 161(1): 18-30, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17945509

RESUMO

In immunogold double-labeling of pea leaf thin sections with antibodies raised against ferredoxin-NADP reductase (EC 1.18.1.2, FNR) and antibodies directed against the A or B subunits of the NADP-linked glyceraldehyde-3-P dehydrogenase (GAPD) (EC 1.2.1.13), many small and large gold particles were found together over the chloroplasts. Nearest neighbor analysis of the distribution of the gold particles indicates that FNR and the NADP-linked GAPD are co-localized, in situ. This suggests that FNR might carry FADH2 or NADPH from the thylakoid membrane to GAPD, or that ferredoxin might carry electrons to FNR co-localized with GAPD in the stroma. Crystal structures of the spinach enzymes are available. When they are docked computationally, the proteins appear, as modeled, to be able to form at least two different complexes. One involves a single GAPD monomer and an FNR monomer (or dimer). The amino acid residues located at the putative interface are highly conserved on the chloroplastic forms of both enzymes. The other potential complex involves the GAPD A2B2 tetramer and an FNR monomer (or dimer). The interface residues are conserved in this model as well. Ferredoxin is able to interact with FNR in either complex.


Assuntos
Cloroplastos/enzimologia , Ferredoxina-NADP Redutase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Ervilhas/enzimologia , Folhas de Planta/enzimologia , Sequência de Aminoácidos , Cloroplastos/ultraestrutura , Simulação por Computador , Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/genética , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/genética , Immunoblotting , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
19.
Bioinformatics ; 23(24): 3397-9, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17933856

RESUMO

UNLABELLED: A new web server, InterProSurf, predicts interacting amino acid residues in proteins that are most likely to interact with other proteins, given the 3D structures of subunits of a protein complex. The prediction method is based on solvent accessible surface area of residues in the isolated subunits, a propensity scale for interface residues and a clustering algorithm to identify surface regions with residues of high interface propensities. Here we illustrate the application of InterProSurf to determine which areas of Bacillus anthracis toxins and measles virus hemagglutinin protein interact with their respective cell surface receptors. The computationally predicted regions overlap with those regions previously identified as interface regions by sequence analysis and mutagenesis experiments. AVAILABILITY: The InterProSurf web server is available at http://curie.utmb.edu/


Assuntos
Algoritmos , Internet , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Software , Sítios de Ligação , Ligação Proteica
20.
J Mol Model ; 13(11): 1157-67, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17828612

RESUMO

We have developed a fully automated method, InterProSurf, to predict interacting amino acid residues on protein surfaces of monomeric 3D structures. Potential interacting residues are predicted based on solvent accessible surface areas, a new scale for interface propensities, and a cluster algorithm to locate surface exposed areas with high interface propensities. Previous studies have shown the importance of hydrophobic residues and specific charge distribution as characteristics for interfaces. Here we show differences in interface and surface regions of all physical chemical properties of residues as represented by five quantitative descriptors. In the current study a set of 72 protein complexes with known 3D structures were analyzed to obtain interface propensities of residues, and to find differences in the distribution of five quantitative descriptors for amino acid residues. We also investigated spatial pair correlations of solvent accessible residues in interface and surface areas, and compared log-odds ratios for interface and surface areas. A new scoring method to predict potential functional sites on the protein surface was developed and tested for a new dataset of 21 protein complexes, which were not included in the original training dataset. Empirically we found that the algorithm achieves a good balance in the accuracy of precision and sensitivity by selecting the top eight highest scoring clusters as interface regions. The performance of the method is illustrated for a dimeric ATPase of the hyperthermophile, Methanococcus jannaschii, and the capsid protein of Human Hepatitis B virus. An automated version of the method can be accessed from our web server at http://curie.utmb.edu/prosurf.html.


Assuntos
Proteínas/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Algoritmos , Proteínas Arqueais/química , Automação , Bases de Dados de Proteínas , Dimerização , Mathanococcus/enzimologia , Modelos Moleculares , Conformação Molecular
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