RESUMO
Brucella lumazine synthase (BLS) is a homodecameric protein that activates dendritic cells via toll like receptor 4, inducing the secretion of pro-inflammatory cytokines and chemokines. We have previously shown that BLS has a therapeutic effect in B16 melanoma-bearing mice only when administered at early stages of tumor growth. In this work, we study the mechanisms underlying the therapeutic effect of BLS, by analyzing the tumor microenvironment. Administration of BLS at early stages of tumor growth induces high levels of serum IFN-γ, as well as an increment of hematopoietic immune cells within the tumor. Moreover, BLS-treatment increases the ratio of effector to regulatory cells. However, all treated mice eventually succumb to the tumors. Therefore, we combined BLS administration with anti-PD-1 treatment. Combined treatment increases the outcome of both monotherapies. In conclusion, we show that the absence of the therapeutic effect at late stages of tumor growth correlates with low levels of serum IFN-γ and lower infiltration of immune cells in the tumor, both of which are essential to delay tumor growth. Furthermore, the combined treatment of BLS and PD-1 blockade shows that BLS could be exploited as an essential immunomodulator in combination therapy with an immune checkpoint blockade to treat skin cancer.
Assuntos
Interferon gama/genética , Melanoma Experimental/tratamento farmacológico , Receptor de Morte Celular Programada 1/genética , Receptor 4 Toll-Like/genética , Animais , Quimiocinas/genética , Citocinas/genética , Células Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Fatores Imunológicos/farmacologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Receptor 4 Toll-Like/agonistas , Microambiente Tumoral/efeitos dos fármacosRESUMO
One of the most remarkable characteristics of Brucella lumazine synthase (BLS) is its versatility to undergo reversible dissociation and reassociation as a polymeric scaffold. We have proposed a mechanism of dissociation and unfolding of BLS. Using static light scattering (SLS) analysis, we were able to demonstrate that the decameric assembly dissociates into two different conditions [pH 5 or 2M guanidinium chloride (GdnHCl) pH 7] forming stable folded pentamers. The transition from folded pentamers to unfolded monomers by GdnHCl denaturation is highly cooperative and can be measured by different spectroscopic techniques. In this work, we show the successful insertion of an intrinsic probe to study in more detail the equilibria described in previous publications. For that purpose, we performed single-point mutations of Phe residues 121 and 127, located at the pentamer-pentamer and monomer-monomer interface, respectively, to Trp residues. These mutations produced only a marginal perturbation of the BLS structure. We analyzed the unfolding and stability of the mutants through different techniques: far-and near-UV CD, SLS, dynamic light scattering, and fluorescence spectroscopy. The introduced intrinsic probe could be used to gain insights into the detailed folding and assembly mechanism of this protein.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutação Puntual , Algoritmos , Proteínas de Bactérias/genética , Brucella/enzimologia , Guanidina , Concentração de Íons de Hidrogênio , Luz , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espalhamento de Radiação , Análise Espectral , Termodinâmica , TriptofanoRESUMO
The multiple display of protein domains on polymeric scaffolds is an emerging technology for many applications. BLS is a highly immunogenic protein that has an oligomeric structure formed by a 17.2 kDa subunit arranged as a dimer of pentamers. Here we describe the production as well as the structural, functional, and immunological properties of a 9 kDa double-stranded RNA-binding domain (RBD3) fused to the structure of BLS. We demonstrate that the BLS and RBD3 modules are stably and independently folded in the structure of the chimera and form a decameric structure of 255 kDa as the native BLS oligomers. The polymeric display of RBD3 in the structure of BLS increases the dsRNA binding strength of this domain both in vitro and in vivo, and also enhances its immunogenicity to the point that it breaks the tolerance of mice to the RBD3 self-antigen. Our results underscore the BLS display strategy as a powerful tool for biotechnological and therapeutic applications.
Assuntos
Complexos Multienzimáticos/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sítios de Ligação , Brucella/enzimologia , Dicroísmo Circular , Clonagem Molecular , Feminino , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Desnaturação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , TermodinâmicaRESUMO
Protein assemblies with a high degree of repetitiveness and organization are known to induce strong immune responses. For that reason they have been postulated for the design of subunit vaccines by means of protein engineering. The enzyme lumazine synthase from Brucella spp. (BLS) is highly immunogenic, presumably owing to its homodecameric arrangement and remarkable thermodynamic stability. Structural analysis has shown that it is possible to insert foreign peptides at the ten amino terminus of BLS without disrupting its general folding. These peptides would be displayed to the immune system in a highly symmetric three-dimensional array. In the present work, BLS has been used as a protein carrier of foreign peptides. We have established a modular system to produce chimeric proteins decorated with ten copies of a desired peptide as long as 27 residues and have shown that their folding and stability is similar to that of the wild-type protein. The knowledge about the mechanisms of dissociation and unfolding of BLS allowed the engineering of polyvalent chimeras displaying different predefined peptides on the same molecular scaffold. Moreover, the reassembly of mixtures of chimeras at different steps of the unfolding process was used to control the stoichiometry and spatial arrangement for the simultaneous display of different peptides on BLS. This strategy would be useful for vaccine development and other biomedical applications.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biblioteca de Peptídeos , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Biopolímeros/química , Biopolímeros/metabolismo , Brucella/enzimologia , Dicroísmo Circular , Expressão Gênica , Vetores Genéticos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismoRESUMO
By taking advantage of the extreme stability of a protein-DNA complex, we have obtained two highly specific monoclonal antibodies against a predetermined palindromic DNA sequence corresponding to the binding site of the E2 transcriptional regulator of the human papillomavirus (HPV-16). The purified univalent antibody fragments bind to a double-stranded DNA oligonucleotide corresponding to the E2 binding site in solution with dissociation constants in the low and subnanomolar range. This affinity matches that of the natural DNA binding domain and is severalfold higher than the affinity of a homologous bovine E2 C-terminal domain (BPV-1) for the same DNA. These antibodies discriminate effectively among a number of double- and single-stranded synthetic DNAs with factors ranging from 125- to 20,000-fold the dissociation constant of the specific DNA sequence used in the immunogenic protein-DNA complex. Moreover, they are capable of fine specificity tuning, since they both bind less tightly to another HPV-16 E2 binding site, differing in only 1 base pair in a noncontact flexible region. Beyond the relevance of obtaining a specific anti-DNA response, these results provide a first glance at how DNA as an antigen is recognized specifically by an antibody. The accuracy of the spectroscopic method used for the binding analysis suggests that a detailed mechanistic analysis is attainable.
Assuntos
Anticorpos Monoclonais/química , DNA/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sítios de Ligação de Anticorpos , Bovinos , DNA/imunologia , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Região Variável de Imunoglobulina/química , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Cold agglutinins (CAs) are IgM autoantibodies characterized by their ability to agglutinate in vitro RBC at low temperatures. These autoantibodies cause hemolytic anemia in patients with CA disease. Many diverse Ags are recognized by CAs, most frequently those belonging to the I/i system. These are oligosaccharides composed of repeated units of N:-acetyllactosamine, expressed on RBC. The three-dimensional structure of the Fab of KAU, a human monoclonal IgM CA with anti-I activity, was determined. The KAU combining site shows an extended cavity and a neighboring pocket. Residues from the hypervariable loops V(H)CDR3, V(L)CDR1, and V(L)CDR3 form the cavity, whereas the small pocket is defined essentially by residues from the hypervariable loops V(H)CDR1 and V(H)CDR2. This fact could explain the V(H)4-34 germline gene restriction among CA. The KAU combining site topography is consistent with one that binds a polysaccharide. The combining site overall dimensions are 15 A wide and 24 A long. Conservation of key binding site residues among anti-I/i CAs indicates that this is a common feature of this family of autoantibodies. We also describe the first high resolution structure of the human IgM C(H)1:C(L) domain. The structural analysis shows that the C(H)1-C(L) interface is mainly conserved during the isotype switch process from IgM to IgG1.
Assuntos
Aglutininas/química , Temperatura Baixa , Hemaglutininas/química , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina M/química , Anemia Hemolítica Autoimune/imunologia , Animais , Autoanticorpos/química , Simulação por Computador , Crioglobulinas , Cristalização , Humanos , Regiões Constantes de Imunoglobulina/química , Cadeias Pesadas de Imunoglobulinas/química , Isotipos de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/química , Camundongos , Modelos MolecularesRESUMO
The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-A resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.
Assuntos
Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Brucella abortus/enzimologia , Brucelose/diagnóstico , Lipoproteínas , Complexos Multienzimáticos/química , Animais , Vacina contra Brucelose , Brucelose/imunologia , Cromatografia de Afinidade , Cristalografia , Ensaio de Imunoadsorção Enzimática , Humanos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/químicaRESUMO
The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-A resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.