RESUMO
BACKGROUND: Mycobacterium tuberculosis, the etiological agent of tuberculosis, has at least four ATP-Binding Cassette (ABC) transporters dedicated to carbohydrate uptake: LpqY/SugABC, UspABC, Rv2038c-41c, and UgpAEBC. LpqY/SugABC transporter is essential for M. tuberculosis survival in vivo and potentially involved in the recycling of cell wall components. The three-dimensional structures of substrate-binding proteins (SBPs) LpqY, UspC, and UgpB were described, however, questions about how these proteins interact with the cognate transporter are still being explored. Components of these transporters, such as SBPs, show high immunogenicity and could be used for the development of diagnostic and therapeutic tools. In this work, we used a phylogenetic and structural bioinformatics approach to compare the four systems, in an attempt to predict functionally important regions. RESULTS: Through the analysis of the putative orthologs of the carbohydrate ABC importers in species of Mycobacterium genus it was shown that Rv2038c-41c and UgpAEBC systems are restricted to pathogenic species. We showed that the components of the four ABC importers are phylogenetically separated into four groups defined by structural differences in regions that modulate the functional activity or the interaction with domain partners. The regulatory region in nucleotide-binding domains, the periplasmic interface in transmembrane domains and the ligand-binding pocket of the substrate-binding proteins define their substrates and segregation in different branches. The interface between transmembrane domains and nucleotide-binding domains show conservation of residues and charge. CONCLUSIONS: The presence of four ABC transporters in M. tuberculosis dedicated to uptake and transport of different carbohydrate sources, and the exclusivity of at least two of them being present only in pathogenic species of Mycobacterium genus, highlights their relevance in virulence and pathogenesis. The significant differences in the SBPs, not present in eukaryotes, and in the regulatory region of NBDs can be explored for the development of inhibitory drugs targeting the bacillus. The possible promiscuity of NBDs also contributes to a less specific and more comprehensive control approach.
Assuntos
Mycobacterium tuberculosis , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboidratos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , FilogeniaRESUMO
Hereditary Xerocytosis (HX) is an autosomal dominantly inherited congenital hemolytic anemia associated with erythrocyte dehydration due to decreased intracellular potassium content resulting in increased mean corpuscular hemoglobin concentration. The affected members of HX families show compensated anemia with splenomegaly, hemosiderosis, and perinatal edema but are in large part transfusion independent. Functional studies show a link between mutations in mechanosensitive ion channel, encoded by PIEZO1 gene and the HX. We identified new PIEZO1 variants that are likely pathogenic in three phenotypically characterized multi-generational HX Brazilian families. Interestingly, one missense variant of the PIEZO1 gene identified, p.E2494V was associated in trans with the previously reported most frequent pathogenic duplication p.E2496ELE. The three-dimensional structure of the human protein modeled using structural coordinates of the mouse Piezo1 solved by cryo-electron microscopy (Cryo-ME) showed that the two identified variants, p.M2007L and p.T2014I, are localized to an important mechanosensitive transmembrane domain suggesting a conformational mechanism for altered channel's gating. The p.E2496ELE variant identified alters the extension of helix α1 bringing it much closer to the beam affecting the position of it structure at the end of the pore.
Assuntos
Anemia Hemolítica Congênita/genética , Hidropisia Fetal/genética , Canais Iônicos , Mutação de Sentido Incorreto , Adolescente , Adulto , Substituição de Aminoácidos , Animais , Criança , Feminino , Humanos , Recém-Nascido , Canais Iônicos/química , Canais Iônicos/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Conformação Proteica em alfa-Hélice , Domínios ProteicosRESUMO
INTRODUCTION AND OBJECTIVES: Direct antiviral agents (DAAs) are very efficient in inhibiting hepatitis C virus and might be used to treat infections caused by other flaviviruses whose worldwide detection has recently increased. The aim of this study was to verify the efficacy of DAAs in inhibiting yellow fever virus (YFV) by using drug repositioning (a methodology applied in the pharmaceutical industry to identify new uses for approved drugs). MATERIALS AND METHODS: Three DAAs were evaluated: daclatasvir, sofosbuvir and ledipasvir or their combinations. For in vitro assays, the drugs were diluted in 100% dimethyl sulfoxide. Vaccine strain 17D and a 17D strain expressing the reporter fluorescent protein were used in the assays. A fast and reliable cell-based screening assay using Vero cells or Huh-7 cells (a hepatocyte-derived carcinoma ell line) was carried out. Two patients who acquired yellow fever virus with acute liver failure were treated with sofosbuvir for one week as a compassionate use. RESULTS: Using a high-content screening assay, we verified that sofosbuvir presented the best antiviral activity against YFV. Moreover, after an off-label treatment with sofosbuvir, the two female patients diagnosed with yellow fever infection displayed a reduction in blood viremia and an improvement in the course of the disease, which was observed in the laboratory medical parameters related to disease evolution. CONCLUSIONS: Sofosbuvir may be used as an option for treatment against YFV until other drugs are identified and approved for human use. These results offer insights into the role of nonstructural protein 5 (NS5) in YFV inhibition and suggest that nonstructural proteins may be explored as drug targets for YFV treatment.
Assuntos
Antivirais/farmacologia , Benzimidazóis/farmacologia , Fluorenos/farmacologia , Imidazóis/farmacologia , Sofosbuvir/farmacologia , Febre Amarela/tratamento farmacológico , Vírus da Febre Amarela/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Carbamatos , Linhagem Celular Tumoral , Chlorocebus aethiops , Ensaios de Uso Compassivo , Reposicionamento de Medicamentos , Feminino , Humanos , Técnicas In Vitro , Falência Hepática Aguda/etiologia , Pirrolidinas , Sofosbuvir/uso terapêutico , Valina/análogos & derivados , Células Vero , Carga Viral/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Febre Amarela/complicaçõesRESUMO
The uptake and transport of sulfate in bacteria is mediated by an ATP-binding cassette transporter (ABC transporter) encoded by sbpcysUWA genes, whose importance has been widely demonstrated due to their relevance in cysteine synthesis and bacterial growth. In Xanthomonas citri, the causative agent of canker disease, the expression of components from this ABC transporter and others related to uptake of organic sulfur sources has been shown during in vitro growth cultures. In this work, based on gene reporter and proteomics analyses, we showed the activation of the promoter that controls the sbpcysUWA operon in vitro and in vivo and the expression of sulfate-binding protein (Sbp), a periplasmic-binding protein, indicating that this protein plays an important function during growth and that the transport system is active during Citrus sinensis infection. To characterize Sbp, we solved its three-dimensional structure bound to sulfate at 1.14 Å resolution and performed biochemical and functional characterization. The results revealed that Sbp interacts with sulfate without structural changes, but the interaction induces a significant increasing of protein thermal stability. Altogether, the results presented in this study show the evidence of the functionality of the ABC transporter for sulfate in X. citri and its relevance during infection.
Assuntos
Proteínas Periplásmicas de Ligação/metabolismo , Proteômica/métodos , Sulfatos/metabolismo , Xanthomonas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Citrus sinensis/microbiologia , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Doenças das Plantas/microbiologia , Ligação Proteica , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Sulfatos/química , Xanthomonas/genética , Xanthomonas/fisiologiaRESUMO
Esterases catalyze the cleavage and formation of ester bonds and are members of the diverse family of α/ß hydrolase fold. They are useful in industries from different sectors, such as food, detergent, fine chemicals, and biofuel production. In a previous work, 30 positive clones for lipolytic activity were identified from a metagenomic library of a microbial consortium specialized in diesel oil degradation. In this study, a putative gene encoding an esterase/lipase, denominated est8, has been cloned and the corresponding protein expressed recombinantly, purified to homogeneity and characterized functional and structurally. We show that the protein codified by est8 gene, denominated Est8, is an alkaline esterase with high catalytic efficiency against p-nitrophenyl acetate and stable in the presence of up to 10% dimethyl sulfoxide. The three-dimensional structure of Est8 was determined at 1.85-Ǻ resolution, allowing the characterization of the substrate-binding pocket and features that rationalize the preference of Est8 for short-chain substrates. In an attempt to increase the size of ligand-binding pocket and enzyme activity against distinct substrates of long chain, we mutated two residues (Met213 and Phe217) that block the substrate channel. A small increase in the reaction velocity for p-nitrophenyl butyrate and p-nitrophenyl valerate hydrolysis was observed. Activity against p-nitrophenyl acetate was reduced. The functional and structural characterization of Est8 is explored in comparison with orthologues.
Assuntos
Esterases/química , Esterases/metabolismo , Metagenômica , Consórcios Microbianos/genética , Butiratos/metabolismo , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Estabilidade Enzimática , Esterases/genética , Esterases/isolamento & purificação , Biblioteca Gênica , Concentração de Íons de Hidrogênio , Hidrólise , Lipase/genética , Lipase/isolamento & purificação , Lipase/metabolismo , Lipólise , Nitrofenóis/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Metagenomic libraries from diverse environments have been extensive sources of many lipases and esterases; nevertheless, most of these enzymes remain biochemically uncharacterized. We previously built a metagenomic fosmid library from a microbial consortium specialized for diesel oil degradation and tested it for lipolytic activity. In the present study, we identified the PL14.H10 clone that was subcloned and sequenced, which enabled the identification of the EST3 protein. This enzyme exhibited 74 % amino acid identity with the uncharacterized alpha/beta hydrolase from Parvibaculum lavamentivorans [GenBank: WP012110575.1] and was classified into lipolytic enzyme family IV. Biochemical characterization revealed that EST3 presents high activity in a wide range of temperature with highest activity from 41 to 45 °C. Also, this thermostable esterase acts from mild acidic to alkaline conditions with an optimum pH of 6.0. The enzyme exhibited activity against p-nitrophenyl esters of different chain lengths and highest catalytic efficiency against p-nitrophenyl caprylate. The activity of the protein was increased in the presence of 0.5 mM of Mn(+2), Li(+), EDTA, and 1 % of CTAB and exhibited half of the activity in the presence of 10 % methanol and ethanol. Moreover, the homology model of EST3 was built and compared to other esterases, revealing a substrate channel that should fit a wide range of substrates. Taken together, the data presented in this work reveal the unique and interesting characteristics of EST3 that might be explored for further use in biotechnological applications.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Esterases/química , Esterases/genética , Sequência de Aminoácidos , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Biotecnologia , Clonagem Molecular , Estabilidade Enzimática , Esterases/metabolismo , Biblioteca Gênica , Concentração de Íons de Hidrogênio , Metagenoma , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Especificidade por Substrato , TemperaturaRESUMO
BACKGROUND: The Xanthomonas citri pv. citri (X. citri) is a phytopathogenic bacterium that infects different species of citrus plants where it causes canker disease. The adaptation to different habitats is related to the ability of the cells to metabolize and to assimilate diverse compounds, including sulfur, an essential element for all organisms. In Escherichia coli, the necessary sulfur can be obtained by a set of proteins whose genes belong to the cys regulon. Although the cys regulon proteins and their importance have been described in many other bacteria, there are no data related to these proteins in X. citri or in the Xanthomonas genus. The study of the relevance of these systems in these phytopathogenic bacteria that have distinct mechanisms of infection is one essential step toward understanding their physiology. In this work, we used bioinformatics, molecular modeling and transcription analysis (RT-PCR) to identify and characterize the putative cys regulon genes in X. citri. RESULTS: We showed that the ATP Binding Cassette Transporter (ABC transporter) SbpCysUWA for sulfate uptake is conserved in X. citri and translated in presence of sulfate. On the other hand, differently from what is predicted in databases, according molecular modeling and phylogenetic analysis, X. citri does not show a proper taurine transporter, but two different ABC systems related to the alkanesulfonate/sulfonate transport that were recently acquired during evolution. RT-PCR analysis evidenced that these genes and their putative transcriptional regulator CysB are rather transcripted in XAM1, a medium with defined concentration of sulfate, than LB. CONCLUSIONS: The presence of at least three distinct systems for sulfate and sulfonates assimilation in X. citri evidenced the importance of these compounds for the bacterium. The transcription of genes involved with alkanesulfonate/sulfur compounds in XAM1 along to CysB suggests that despite the differences in the transporters, the regulation of these systems might be similar to the described for E. coli. Altogether, these results will serve as a foundation for further studies aimed to understanding the relevance of sulfur in growth, virulence and pathogenesis of X. citri and related bacteria.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Filogenia , Enxofre/metabolismo , Xanthomonas/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Alcanossulfonatos/metabolismo , Transporte Biológico/genética , Citrus/microbiologia , Escherichia coli/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Virulência/genética , Xanthomonas/genéticaRESUMO
PhoX is a high-affinity phosphate binding protein, present in Xanthomonas citri, a phytopathogen responsible for the citrus canker disease. Performing molecular dynamics simulations and different types of computational analyses, we study the molecular mechanisms at play in relation to phosphate binding, revealing the global functioning of the protein: PhoX naturally oscillates along its global normal modes, which allow it to explore both bound and unbound conformations, eventually attracting a nearby negative phosphate ion to the highly positive electrostatic potential on its surface, particularly close to the binding pocket. There, several hydrogen bonds are formed with the two main domains of the structure. Phosphate creates, in this way, a strong bridge that connects the domains, keeping itself between them, in a tight closed conformation, explaining its high binding affinity.
Assuntos
Proteínas de Bactérias , Simulação de Dinâmica Molecular , Fosfatos , Xanthomonas , Fosfatos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Ligação Proteica , Proteínas de Ligação a Fosfato/metabolismo , Ligação de Hidrogênio , Sítios de Ligação , Eletricidade EstáticaRESUMO
The role of the iron-sulfur [Fe-S] cluster transcriptional regulator IscR in maintaining [Fe-S] homeostasis in bacteria is still poorly characterized in many groups. Caulobacter crescentus and other Alphaproteobacteria have a single operon encoding [Fe-S] cluster biosynthesis enzymes. We showed that the expression of this operon increases in iron starvation, but not in oxidative stress, and is controlled mainly by IscR. Transcriptome analysis comparing an iscR null mutant strain with the wild-type (wt) strain identified 94 differentially expressed genes (DEGs), with 47 upregulated and 47 downregulated genes in the ΔiscR mutant. We determined the IscR binding sites in conditions of sufficient or scarce iron by Chromatin Immunoprecipitation followed by DNA sequencing (ChIP-seq), identifying two distinct putative DNA binding motifs. The estimated IscR regulon comprises 302 genes, and direct binding to several regulatory regions was shown by Electrophoresis Mobility Shift Assay (EMSA). The results showed that the IscR and Fur regulons partially overlap and that IscR represses the expression of the respiration regulator FixK, fine-tuning gene regulation in response to iron and redox balance.
RESUMO
Vaccination has played a critical role in mitigating COVID-19. Despite the availability of licensed vaccines, there remains a pressing need for improved vaccine platforms that provide high protection, safety, and versatility, while also reducing vaccine costs. In response to these challenges, our aim is to create a self-adjuvanted vaccine against SARS-CoV-2, utilizing Virus-Like Particles (VLPs) as the foundation. To achieve this, we produced bacteriophage (Qß) VLPs in a prokaryotic system and purified them using a rapid and cost-effective strategy involving organic solvents. This method aims to solubilize lipids and components of the cell membrane to eliminate endotoxins present in bacterial samples. For vaccine formulation, Receptor Binding Domain (RBD) antigens were conjugated using chemical crosslinkers, a process compatible with Good Manufacturing Practice (GMP) standards. Transmission Electron Microscopy (TEM) confirmed the expected folding and spatial configuration of the QßVLPs vaccine. Additionally, vaccine formulation assessment involved SDS-PAGE stained with Coomassie Brilliant Blue, Western blotting, and stereomicroscopic experiments. In vitro and in vivo evaluations of the vaccine formulation were conducted to assess its capacity to induce a protective immune response without causing side effects. Vaccine doses of 20 µg and 50 µg stimulated the production of neutralizing antibodies. In in vivo testing, the group of animals vaccinated with 50 µg of vaccine formulation provided complete protection against virus infection, maintaining stable body weight without showing signs of disease. In conclusion, the QßVLPs-RBD vaccine has proven to be effective and safe, eliminating the necessity for supplementary adjuvants and offering a financially feasible approach. Moreover, this vaccine platform demonstrates flexibility in targeting Variants of Concern (VOCs) via established conjugation protocols with VLPs.
Assuntos
Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Vacinas de Partículas Semelhantes a Vírus , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Animais , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Camundongos , Humanos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Feminino , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes de Vacinas , Vacinação/métodos , Camundongos Endogâmicos BALB CRESUMO
The periplasmic-binding proteins in ATP-binding cassette systems (ABC Transporters) are responsible for the capture and delivery of ligands to their specific transporters, triggering a series of ATP-driven conformational changes that leads to the transport of the ligand. Structurally consisting of two lobes, the proteins change conformation after interaction with the ligand. The structure of the molybdate-binding protein (ModA) from Xanthomonas citri, bound to molybdate, was previously solved by our group and an interdomain interaction, mediated by a salt bridge between K127 and D59, apparently supports the binding properties and keeps the domains closed. To determinate the importance of this interaction, we built two ModA mutants, K127S and D59A, and analysed their functional and structural properties. Based on a set of spectroscopic experiments, crystallisation trials, structure determination and molecular dynamics (MD) simulations, we showed that the salt bridge is essential to maintain the structure and binding properties. Additionally, the MD simulations revealed that this mutant adopted a more compact structure that packed down the ligand-binding pocket. From the closed bound to open structure, the positioning of the helices forming the dipole and the salt bridge are essential to induce an intermediate state.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Xanthomonas , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Permeabilidade da Membrana Celular , Dissulfetos/química , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Molibdênio/metabolismo , Mutação , Ligação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , TemperaturaRESUMO
The C-terminal portion of the E protein, known as stem, is conserved among flaviviruses and is an important target to peptide-based antiviral strategies. Since the dengue (DENV) and Zika (ZIKV) viruses share sequences in the stem region, in this study we evaluated the cross-inhibition of ZIKV by the stem-based DV2 peptide (419-447), which was previously described to inhibit all DENV serotypes. Thus, the anti-ZIKV effects induced by treatments with the DV2 peptide were tested in both in vitro and in vivo conditions. Molecular modeling approaches have demonstrated that the DV2 peptide interacts with amino acid residues exposed on the surface of pre- and postfusion forms of the ZIKA envelope (E) protein. The peptide did not have any significant cytotoxic effects on eukaryotic cells but efficiently inhibited ZIKV infectivity in cultivated Vero cells. In addition, the DV2 peptide reduced morbidity and mortality in mice subjected to lethal challenges with a ZIKV strain isolated in Brazil. Taken together, the present results support the therapeutic potential of the DV2 peptide against ZIKV infections and open perspectives for the development and clinical testing of anti-flavivirus treatments based on synthetic stem-based peptides.
Assuntos
Flavivirus , Infecção por Zika virus , Zika virus , Chlorocebus aethiops , Animais , Camundongos , Células Vero , Infecção por Zika virus/tratamento farmacológico , Peptídeos/farmacologia , Reações CruzadasRESUMO
Arboviruses are a major threat to public health in tropical regions, encompassing over 534 distinct species, with 134 capable of causing diseases in humans. These viruses are transmitted through arthropod vectors that cause symptoms such as fever, headache, joint pains, and rash, in addition to more serious cases that can lead to death. Among the arboviruses, dengue virus stands out as the most prevalent, annually affecting approximately 16.2 million individuals solely in the Americas. Furthermore, the re-emergence of the Zika virus and the recurrent outbreaks of chikungunya in Africa, Asia, Europe, and the Americas, with one million cases reported annually, underscore the urgency of addressing this public health challenge. In this manuscript we discuss the epidemiology, viral structure, pathogenicity and integrated control strategies to combat arboviruses, and the most used tools, such as vaccines, monoclonal antibodies, treatment, etc., in addition to presenting future perspectives for the control of arboviruses. Currently, specific medications for treating arbovirus infections are lacking, and symptom management remains the primary approach. However, promising advancements have been made in certain treatments, such as Chloroquine, Niclosamide, and Isatin derivatives, which have demonstrated notable antiviral properties against these arboviruses in vitro and in vivo experiments. Additionally, various strategies within vector control approaches have shown significant promise in reducing arbovirus transmission rates. These encompass public education initiatives, targeted insecticide applications, and innovative approaches like manipulating mosquito bacterial symbionts, such as Wolbachia. In conclusion, combatting the global threat of arbovirus diseases needs a comprehensive approach integrating antiviral research, vaccination, and vector control. The continued efforts of research communities, alongside collaborative partnerships with public health authorities, are imperative to effectively address and mitigate the impact of these arboviral infections on public health worldwide.
Assuntos
Febre de Chikungunya , Dengue , Infecção por Zika virus , Zika virus , Animais , Humanos , Febre de Chikungunya/epidemiologia , Febre de Chikungunya/prevenção & controle , Mosquitos Vetores , Infecção por Zika virus/epidemiologia , Infecção por Zika virus/prevenção & controle , Antivirais , Dengue/epidemiologia , Dengue/prevenção & controleRESUMO
Amongst the potential contribution of protein or peptide-display systems to study epitopes with relevant immunological features, the RAD display system stands out as a highly stable scaffold protein that allows the presentation of constrained target peptides. Here, we employed the RAD display system to present peptides derived from the SARS-CoV-2 Spike (S) protein as a tool to detect specific serum antibodies and to generate polyclonal antibodies capable of inhibiting SARS-CoV-2 infectivity in vitro. 44 linear S-derived peptides were genetically fused with the RAD scaffold (RAD-SCoV-epitopes) and screened for antigenicity with sera collected from COVID-19-infected patients. In a second step, selected RAD-SCoV-epitopes were used to immunize mice and generate antibodies. Phenotypic screening showed that some of these antibodies were able to recognize replicating viral particles in VERO CCL-81 and most notably seven of the RAD-SCoV-epitopes were able to induce antibodies that inhibited viral infection. Our findings highlight the RAD display system as an useful platform for the immunological characterization of peptides and a potentially valuable strategy for the design of antigens for peptide-based vaccines, for epitope-specific antibody mapping, and for the development of antibodies for diagnostic and therapeutic purposes.
Assuntos
COVID-19 , Pyrococcus furiosus , Humanos , Animais , Camundongos , Epitopos , Glicoproteína da Espícula de Coronavírus/metabolismo , Pyrococcus furiosus/metabolismo , Anticorpos Antivirais , Proteínas do Envelope Viral , SARS-CoV-2 , Peptídeos/química , Anticorpos NeutralizantesRESUMO
Envenoming by the contact of human skin with Lonomia obliqua caterpillars promotes a hemorrhagic syndrome characterized by a consumptive coagulopathy. Losac (Lonomia obliqua Stuart factor activator) is a component of the bristle of L. obliqua that is probably partially responsible for the observed syndrome because it activates factor X and is recognized by an effective antilonomic serum. Here we unveil the proteolytic activity of Losac and demonstrate the feasibility of its recombinant production. On the other hand, Losac has no homology to known proteases, but it can be inhibited by PMSF, a serine protease inhibitor. Instead, it shows closer homology to members of the hemolin family of proteins, a group of cell adhesion molecules. The recombinant protein (rLosac) shortened the coagulation time of normal and deficient plasmas, whereas it was ineffective in factor X-deficient plasma unless reconstituted with this protein. rLosac was able to activate factor X in a dose- and time-dependent manner but not γ-carboxyglutamic acid domainless factor X. Moreover, phospholipids and calcium ions increased rLosac activity. Also, rLosac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation. Linear double reciprocal plots indicate that rLosac follows a Michaelis-Menten kinetics. Cleavage of factor X by rLosac resulted in fragments that are compatible with those generated by RVV-X (a well known factor X activator). Together, our results validate Losac as the first protein from the hemolin family exhibiting procoagulant activity through selective proteolysis on coagulation factor X.
Assuntos
Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Mariposas/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Coagulação Sanguínea/efeitos dos fármacos , Simulação por Computador , Cisteína Endopeptidases/farmacologia , Fator X/metabolismo , Fator Xa/metabolismo , Imunoglobulinas/farmacologia , Proteínas de Insetos/farmacologia , Dados de Sequência Molecular , Mariposas/metabolismo , Proteínas de Neoplasias/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Heat-labile toxins (LTs) have ADP-ribosylation activity and induce the secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains in different mammalian hosts. LTs also act as adjuvants following delivery via mucosal, parenteral, or transcutaneous routes. Previously we have shown that LT produced by human-derived ETEC strains encompass a group of 16 polymorphic variants, including the reference toxin (LT1 or hLT) produced by the H10407 strain and one variant that is found mainly among bacterial strains isolated from pigs (LT4 or pLT). Herein, we show that LT4 (with six polymorphic sites in the A (K4R, K213E, and N238D) and B (S4T, A46E, and E102K) subunits) displays differential in vitro toxicity and in vivo adjuvant activities compared with LT1. One in vitro generated LT mutant (LTK4R), in which the lysine at position 4 of the A subunit was replaced by arginine, showed most of the LT4 features with an â¼10-fold reduction of the cytotonic effects, ADP-ribosylation activity, and accumulation of intracellular cAMP in Y1 cells. Molecular dynamic studies of the A subunit showed that the K4R replacement reduces the N-terminal region flexibility and decreases the catalytic site crevice. Noticeably, LT4 showed a stronger Th1-biased adjuvant activity with regard to LT1, particularly concerning activation of cytotoxic CD8(+) T lymphocytes when delivered via the intranasal route. Our results further emphasize the relevance of LT polymorphism among human-derived ETEC strains that may impact both the pathogenicity of the bacterial strain and the use of these toxins as potential vaccine adjuvants.
Assuntos
Toxinas Bacterianas/metabolismo , Escherichia coli Enterotoxigênica/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/metabolismo , Substituição de Aminoácidos , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , AMP Cíclico/genética , AMP Cíclico/imunologia , AMP Cíclico/metabolismo , Escherichia coli Enterotoxigênica/imunologia , Escherichia coli Enterotoxigênica/patogenicidade , Enterotoxinas/genética , Enterotoxinas/imunologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Feminino , Humanos , Camundongos , Mutação de Sentido Incorreto , Polimorfismo Genético , Especificidade da Espécie , Suínos , Células Th1/imunologia , Células Th1/metabolismoRESUMO
Oligogenic inheritance of autism spectrum disorder (ASD) has been supported by several studies. However, little is known about how the risk variants interact and converge on causative neurobiological pathways. We identified in an ASD proband deleterious compound heterozygous missense variants in the Reelin (RELN) gene, and a de novo splicing variant in the Cav3.2 calcium channel (CACNA1H) gene. Here, by using iPSC-derived neural progenitor cells (NPCs) and a heterologous expression system, we show that the variant in Cav3.2 leads to increased calcium influx into cells, which overactivates mTORC1 pathway and, consequently, further exacerbates the impairment of Reelin signaling. Also, we show that Cav3.2/mTORC1 overactivation induces proliferation of NPCs and that both mutant Cav3.2 and Reelin cause abnormal migration of these cells. Finally, analysis of the sequencing data from two ASD cohorts-a Brazilian cohort of 861 samples, 291 with ASD; the MSSNG cohort of 11,181 samples, 5,102 with ASD-revealed that the co-occurrence of risk variants in both alleles of Reelin pathway genes and in one allele of calcium channel genes confer significant liability for ASD. Our results support the notion that genes with co-occurring deleterious variants tend to have interconnected pathways underlying oligogenic forms of ASD.
Assuntos
Transtorno do Espectro Autista , Canais de Cálcio Tipo T , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Canais de Cálcio/genética , Canais de Cálcio Tipo T/genética , Predisposição Genética para Doença , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Herança MultifatorialRESUMO
Type 1 herpes virus (HSV-1) glycoprotein D (gD) enhances antigen-specific immune responses, particularly CD8(+) T cell responses, in mice immunized with DNA vaccines encoding hybrid proteins genetically fused with the target antigen at a site near the C-terminal end. These effects are attributed to the interaction of gD with the herpes virus entry mediator (HVEM) and the concomitant blockade of a coinhibitory mechanism mediated by the B- and T-lymphocyte attenuator (BTLA). However, questions concerning the requirement for endogenous synthesis of the antigen or the adjuvant/antigen fusion itself have not been addressed so far. In the present study, we investigated these points using purified recombinant gDs, genetically fused or not with type 16 papilloma virus (HPV-16) E7 oncoprotein. Soluble recombinant gDs, but not denatured forms, retained the ability to bind surface-exposed cellular receptors of HVEM-expressing U937 cells. In addition, in vivo administration of the recombinant proteins, particularly gD genetically fused with E7 (gDE7), promoted the activation of dendritic cells (DC) and antigen-specific cytotoxic CD8(+) T cells. More relevantly, mice immunized with the gDE7 protein developed complete preventive and partial therapeutic antitumor protection, as measured in mice following the implantation of TC-1 cells expressing HPV-16 oncoproteins. Collectively, these results demonstrate that the T cell adjuvant effects of the HSV-1 gD protein did not require endogenous synthesis and could be demonstrated in mice immunized with purified recombinant proteins.
Assuntos
Antineoplásicos/farmacologia , Herpesvirus Humano 1 , Papillomavirus Humano 16 , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/farmacologia , Linfócitos T/efeitos dos fármacos , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/genética , Animais , Antígenos CD8/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Linfócitos T/imunologia , Proteínas do Envelope Viral/farmacologiaRESUMO
ATP-Binding Cassette transporters (ABC transporters) are protein complexes involved in the import and export of different molecules, including ions, sugars, peptides, drugs, and others. Due to the diversity of substrates, they have large relevance in physiological processes such as virulence, pathogenesis, and antimicrobial resistance. In Xanthomonas citri subsp. citri, the phytopathogen responsible for the citrus canker disease, 20% of ABC transporters components are expressed under infection conditions, including the putative putrescine/polyamine ABC transporter, PotFGHI. Polyamines are ubiquitous molecules that mediate cell growth and proliferation and play important role in bacterial infections. In this work, we characterized the X. citri periplasmic-binding protein PotF (XAC2476) using bioinformatics, biophysical and structural methods. PotF is highly conserved in Xanthomonas sp. genus, and we showed it is part of a set of proteins related to the import and assimilation of polyamines in X. citri. The interaction of PotF with putrescine and spermidine was direct and indirectly shown through fluorescence spectroscopy analyses, and experiments of circular dichroism (CD) and small-angle X-ray scattering (SAXS), respectively. The protein showed higher affinity for spermidine than putrescine, but both ligands induced structural changes that coincided with the closing of the domains and increasing of thermal stability.
RESUMO
Mycobacterium tuberculosis (Mtb) has 11 Serine-Threonine Protein Kinases (STPK) that control numerous physiological processes, including cell growth, cell division, metabolic flow, and transcription. PknF is one of the 11 Mtb STPKs that has, among other substrates, two FHA domains (FHA-1 and FHA-2) of the ATP-Binding Cassette (ABC) transporter Rv1747. Phosphorylation in T152 and T210 located in a non-structured linker that connects Rv1747 FHA domains is considerate to be the regulatory mechanism of the transporter. In this work, we resolved the three-dimensional structure of the PknF catalytic domain (cPknF) in complex with the human kinase inhibitor IKK16. cPknF is conserved when compared to other STPKs but shows specific residues in the binding site where the inhibitor is positioned. In addition, using Small Angle X-Ray Scattering analysis we monitored the behavior of the wild type and three FHA-phosphomimetic mutants in solution, and measured the cPknF affinity for these domains. The kinase showed higher affinity for the non-phosphorylated wild type domain and preference for phosphorylation of T152 inducing the rapprochement of the domains and significant structural changes. The results shed some light on the process of regulating the transporter's activity by phosphorylation and arises important questions about evolution and importance of this mechanism for the bacillus.