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1.
Cell ; 161(4): 817-32, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25957687

RESUMO

Rod-derived cone viability factor (RdCVF) is an inactive thioredoxin secreted by rod photoreceptors that protects cones from degeneration. Because the secondary loss of cones in retinitis pigmentosa (RP) leads to blindness, the administration of RdCVF is a promising therapy for this untreatable neurodegenerative disease. Here, we investigated the mechanism underlying the protective role of RdCVF in RP. We show that RdCVF acts through binding to Basigin-1 (BSG1), a transmembrane protein expressed specifically by photoreceptors. BSG1 binds to the glucose transporter GLUT1, resulting in increased glucose entry into cones. Increased glucose promotes cone survival by stimulation of aerobic glycolysis. Moreover, a missense mutation of RdCVF results in its inability to bind to BSG1, stimulate glucose uptake, and prevent secondary cone death in a model of RP. Our data uncover an entirely novel mechanism of neuroprotection through the stimulation of glucose metabolism.


Assuntos
Proteínas do Olho/metabolismo , Glicólise , Tiorredoxinas/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Basigina/genética , Basigina/metabolismo , Proteínas do Olho/genética , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Humanos , Camundongos , Mutação de Sentido Incorreto , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Retinose Pigmentar/metabolismo , Tiorredoxinas/genética
2.
J Biol Chem ; 298(1): 101290, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34678315

RESUMO

The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Proteínas do Nucleocapsídeo/análise , SARS-CoV-2/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Cricetinae , Eletroforese em Gel de Poliacrilamida , Humanos , Limite de Detecção , Proteínas do Nucleocapsídeo/imunologia
3.
Cell Microbiol ; 21(2): e12949, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30171791

RESUMO

Pathogenic Leptospira bacteria are the causative agents of leptospirosis, a zoonotic disease affecting animals and humans worldwide. These pathogenic species have the ability to rapidly cross host tissue barriers by a yet unknown mechanism. A comparative analysis of pathogens and saprophytes revealed a higher abundance of genes encoding proteins with leucine-rich repeat (LRR) domains in the genomes of pathogens. In other bacterial pathogens, proteins with LRR domains have been shown to be involved in mediating host cell attachment and invasion. One protein from the pathogenic species Leptospira interrogans, LIC10831, has been previously analysed via X-ray crystallography, with findings suggesting it may be an important bacterial adhesin. Herein we show that LIC10831 elicits an antibody response in infected animals, is actively secreted by the bacterium, and binds human E- and VE-cadherins. These results provide biochemical and cellular evidences of LRR protein-mediated host-pathogen interactions and identify a new multireceptor binding protein from this infectious Leptospira species.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Leptospira interrogans/metabolismo , Proteínas/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Adesinas Bacterianas/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , Cobaias , Humanos , Leptospira interrogans/imunologia , Leptospirose/microbiologia , Proteínas de Repetições Ricas em Leucina
4.
Cell Microbiol ; 21(7): e13021, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30835870

RESUMO

Protozoan pathogens secrete nanosized particles called extracellular vesicles (EVs) to facilitate their survival and chronic infection. Here, we show the inhibition by Plasmodium berghei NK65 blood stage-derived EVs of the proliferative response of CD4+ T cells in response to antigen presentation. Importantly, these results were confirmed in vivo by the capacity of EVs to diminish the ovalbumin-specific delayed type hypersensitivity response. We identified two proteins associated with EVs, the histamine releasing factor (HRF) and the elongation factor 1α (EF-1α) that were found to have immunosuppressive activities. Interestingly, in contrast to WT parasites, EVs from genetically HRF- and EF-1α-deficient parasites failed to inhibit T cell responses in vitro and in vivo. At the level of T cells, we demonstrated that EVs from WT parasites dephosphorylate key molecules (PLCγ1, Akt, and ERK) of the T cell receptor signalling cascade. Remarkably, immunisation with EF-1α alone or in combination with HRF conferred a long-lasting antiparasite protection and immune memory. In conclusion, we identified a new mechanism by which P. berghei-derived EVs exert their immunosuppressive functions by altering T cell responses. The identification of two highly conserved immune suppressive factors offers new conceptual strategies to overcome EV-mediated immune suppression in malaria-infected individuals.


Assuntos
Biomarcadores Tumorais/genética , Vesículas Extracelulares/imunologia , Malária/genética , Fator 1 de Elongação de Peptídeos/genética , Animais , Apresentação de Antígeno/imunologia , Antígenos/genética , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Proliferação de Células/genética , Vesículas Extracelulares/genética , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Malária/parasitologia , Malária/patologia , Plasmodium berghei/genética , Plasmodium berghei/patogenicidade , Linfócitos T/imunologia , Linfócitos T/parasitologia , Proteína Tumoral 1 Controlada por Tradução
5.
Biochem J ; 475(1): 341-354, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29229758

RESUMO

In bacteria, one primary and multiple alternative sigma (σ) factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σS is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free σ factor remains elusive. The current model suggests that extensive interdomain contacts in a free σ factor result in a compact conformation that masks the DNA-binding determinants of σ, explaining why a free σ factor does not bind double-stranded promoter DNA efficiently. Here, we explored the solution conformation of σS using amide hydrogen/deuterium exchange coupled with mass spectrometry, NMR, analytical ultracentrifugation and molecular dynamics. Our data strongly argue against a compact conformation of free σS Instead, we show that σS adopts an open conformation in solution in which the folded σ2 and σ4 domains are interspersed by domains with a high degree of disorder. These findings suggest that E binding induces major changes in both the folding and domain arrangement of σS and provide insights into the possible mechanisms of regulation of σS activity by its chaperone Crl.


Assuntos
Proteínas de Bactérias/química , Regulação Bacteriana da Expressão Gênica , Holoenzimas/química , Proteínas Recombinantes de Fusão/química , Salmonella typhimurium/enzimologia , Fator sigma/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Medição da Troca de Deutério , Escherichia coli/enzimologia , Escherichia coli/genética , Holoenzimas/genética , Holoenzimas/metabolismo , Cinética , Simulação de Dinâmica Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Salmonella typhimurium/genética , Fator sigma/genética , Fator sigma/metabolismo , Solventes , Termodinâmica
6.
Biochem J ; 463(2): 215-24, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25056110

RESUMO

In many γ-proteobacteria, the RpoS/σS sigma factor associates with the core RNAP (RNA polymerase) to modify global gene transcription in stationary phase and under stress conditions. The small regulatory protein Crl stimulates the association of σS with the core RNAP in Escherichia coli and Salmonella enterica serovar Typhimurium, through direct and specific interaction with σS. The structural determinants of Crl involved in σS binding are unknown. In the present paper we report the X-ray crystal structure of the Proteus mirabilis Crl protein (CrlPM) and a structural model for Salmonella Typhimurium Crl (CrlSTM). Using a combination of in vivo and in vitro assays, we demonstrated that CrlSTM and CrlPM are structurally similar and perform the same biological function. In the Crl structure, a cavity enclosed by flexible arms contains two patches of conserved and exposed residues required for σS binding. Among these, charged residues that are likely to be involved in electrostatic interactions driving Crl-σS complex formation were identified. CrlSTM and CrlPM interact with domain 2 of σS with the same binding properties as with full-length σS. These results suggest that Crl family members share a common mechanism of σS binding in which the flexible arms of Crl might play a dynamic role.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteus mirabilis/metabolismo , Salmonella typhimurium/metabolismo , Fator sigma/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteus mirabilis/química , Proteus mirabilis/enzimologia , Proteus mirabilis/genética , Salmonella typhimurium/química , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Fator sigma/química , Fator sigma/genética
7.
J Bacteriol ; 195(24): 5583-91, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24123817

RESUMO

Pathogenic Leptospira species are the etiological agents of the widespread zoonotic disease leptospirosis. Most organisms, including Leptospira, require divalent cations for proper growth, but because of their high reactivity, these metals are toxic at high concentrations. Therefore, bacteria have acquired strategies to maintain metal homeostasis, such as metal import and efflux. By screening Leptospira biflexa transposon mutants for their ability to use Mn(2+), we have identified a gene encoding a putative orphan ATP-binding cassette (ABC) ATPase of unknown function. Inactivation of this gene in both L. biflexa and L. interrogans strains led to mutants unable to grow in medium in which iron was replaced by Mn(2+), suggesting an involvement of this ABC ATPase in divalent cation uptake. A mutation in this ATPase-coding gene increased susceptibility to Mn(2+) toxicity. Recombinant ABC ATPase of the pathogen L. interrogans exhibited Mg(2+)-dependent ATPase activity involving a P-loop motif. The structure of this ATPase was solved from a crystal containing two monomers in the asymmetric unit. Each monomer adopted a canonical two-subdomain organization of the ABC ATPase fold with an α/ß subdomain containing the Walker motifs and an α subdomain containing the ABC signature motif (LSSGE). The two monomers were arranged in a head-to-tail orientation, forming a V-shaped particle with all the conserved ABC motifs at the dimer interface, similar to functional ABC ATPases. These results provide the first structural and functional characterization of a leptospiral ABC ATPase.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Leptospira/enzimologia , Manganês/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Cristalografia por Raios X , Meios de Cultura/química , Elementos de DNA Transponíveis , Leptospira/efeitos dos fármacos , Leptospira/crescimento & desenvolvimento , Manganês/toxicidade , Mutagênese Insercional , Conformação Proteica
8.
J Pineal Res ; 54(1): 46-57, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22775292

RESUMO

Melatonin is a synchronizer of many physiological processes. Abnormal melatonin signaling is associated with human disorders related to sleep, metabolism, and neurodevelopment. Here, we present the X-ray crystal structure of human N-acetyl serotonin methyltransferase (ASMT), the last enzyme of the melatonin biosynthesis pathway. The polypeptide chain of ASMT consists of a C-terminal domain, which is typical of other SAM-dependent O-methyltransferases, and an N-terminal domain, which intertwines several helices with another monomer to form the physiologically active dimer. Using radioenzymology, we analyzed 20 nonsynonymous variants identified through the 1000 genomes project and in patients with neuropsychiatric disorders. We found that the majority of these mutations reduced or abolished ASMT activity including one relatively frequent polymorphism in the Han Chinese population (N17K, rs17149149). Overall, we estimate that the allelic frequency of ASMT deleterious mutations ranges from 0.66% in Europe to 2.97% in Asia. Mapping of the variants on to the 3-dimensional structure clarifies why some are harmful and provides a structural basis for understanding melatonin deficiency in humans.


Assuntos
Acetilserotonina O-Metiltransferasa/química , Acetilserotonina O-Metiltransferasa/genética , Acetilserotonina O-Metiltransferasa/metabolismo , Sequência de Aminoácidos , Povo Asiático/genética , Cristalografia por Raios X , Frequência do Gene , Humanos , Melatonina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Polimorfismo Genético , Alinhamento de Sequência
9.
Parasite Epidemiol Control ; 22: e00311, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37361928

RESUMO

Background: Porcine cysticercosis is an endemic parasitic zoonosis in many developing countries. The objective of this study was to estimate the seroprevalence of porcine cysticercosis in traditional pig farms in the departments of Dabou, Aboisso and Agboville. Methods: Blood samples were taken from pigs and analyzed by ELISA (IgG) and western blot. Data on farming practices and pig characteristics were collected. Multivariate logistic regression models were constructed to identify risk factors. Results: A total of 668 pigs were sampled from 116 farms and 639 samples were analyzed. The seroprevalence of cysticercosis was estimated at 13.2%. Overweight [OR = 2.6; 95%CI (1.3-4.9)] and fat pigs [OR = 2.3; 95%CI (1.0-4.8)] were twice as likely to be seropositive for cysticercosis. This risk was increased in farms using well water for drinking [OR = 2.5; 95%CI (1.0-6.3)] as well as those reporting veterinary care of the animals (OR = 2.9; 95%CI (1.2-7.3)). Conclusions: This study demonstrated the circulation of Taenia solium in pig farms in southern Côte d'Ivoire.

10.
Protein Expr Purif ; 75(1): 114-8, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20688166

RESUMO

N-acetyl serotonin methyl transferase (ASMT) is the last enzyme in the melatonin synthesis pathway. Evidence linking autism-related disorders with disorders of melatonin metabolism, and, more specifically, with mutations of the gene encoding ASMT, prompted us to investigate the properties and localization of this enzyme. As a first step, we undertook to overproduce the protein in a recombinant host. Early attempts to produce ASMT in recombinant Escherichia coli yielded only insoluble and heavily degraded material. However, recombinant ASMT (rASMT) could be produced in soluble, active form and purified in milligram amounts when the gene was cloned and expressed in Leishmania tarentolae.


Assuntos
Acetilserotonina O-Metiltransferasa/genética , Acetilserotonina O-Metiltransferasa/metabolismo , Expressão Gênica , Leishmania/genética , Acetilserotonina O-Metiltransferasa/isolamento & purificação , Clonagem Molecular , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade
11.
Microorganisms ; 9(8)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34442791

RESUMO

Cysticercosis is one of the main causes of secondary epilepsy in sub-Saharan Africa. To estimate the seroprevalence of cysticercosis among epileptic patients, we conducted a cross-sectional study of patients attending neurology consultation in Abidjan, Côte d'Ivoire. Methods: Patients' socio-demographic and lifestyle data were collected as well as blood samples for serological testing using ELISA and Western blot based on IgG antibodies detection. For qualitative variables comparison, Chi2 or Fisher tests were used; a Student's t-test was used to compare quantitative variables. A multivariate logistic regression model was fit to identify risks factors. Results: Among 403 epileptic patients included in the study, 55.3% were male; the median age was 16.9 years; 77% lived in Abidjan; 26.5% were workers. Most patients included in the study had tonic-clonic seizures (80%), and 11.2% had focal deficit signs. The seroprevalence of cysticercosis was 6.0%. The risk was higher in patients over 30 years old (aOR = 5.1 (1.3-20.0)) than in patients under 16. The risk was also considerably high in patients who reported epileptics in the family (aOR = 5 (1.7-14.6)). The risk was three-fold less in females than in males. Conclusions: This study highlighted the exposure of epileptic patients to Taenia solium larvae in an urban area. The risk of positive serology was increased with age, male gender, and family history of epilepsy.

12.
Sci Transl Med ; 12(559)2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32817357

RESUMO

It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Here, we performed a pilot study of four serological assays to assess the amounts of anti-SARS-CoV-2 antibodies in serum samples obtained from 491 healthy individuals before the SARS-CoV-2 pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. We used two ELISA assays that recognized the full-length nucleoprotein (N) or trimeric spike (S) protein ectodomain of SARS-CoV-2. In addition, we developed the S-Flow assay that recognized the S protein expressed at the cell surface using flow cytometry, and the luciferase immunoprecipitation system (LIPS) assay that recognized diverse SARS-CoV-2 antigens including the S1 domain and the carboxyl-terminal domain of N by immunoprecipitation. We obtained similar results with the four serological assays. Differences in sensitivity were attributed to the technique and the antigen used. High anti-SARS-CoV-2 antibody titers were associated with neutralization activity, which was assessed using infectious SARS-CoV-2 or lentiviral-S pseudotype virus. In hospitalized patients with COVID-19, seroconversion and virus neutralization occurred between 5 and 14 days after symptom onset, confirming previous studies. Seropositivity was detected in 32% of mildly symptomatic individuals within 15 days of symptom onset and in 3% of healthy blood donors. The four antibody assays that we used enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different subpopulations within one region.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19 , Teste para COVID-19 , Estudos de Coortes , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo/métodos , França/epidemiologia , Voluntários Saudáveis , Humanos , Imunoprecipitação/métodos , Luciferases , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , SARS-CoV-2 , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/imunologia , Pesquisa Translacional Biomédica , Adulto Jovem
13.
J Mol Biol ; 431(15): 2762-2776, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31132361

RESUMO

Listeria monocytogenes is riboflavin auxotrophic, but it has two genes envisaged to transform riboflavin into FMN and FAD after its uptaked by specialized transporters. One encodes a bifunctional type I FAD synthase (FADS, herein LmFADS-1), while the other produces a protein similar to type I at the FMN:ATP adenylyltransferase (FMNAT) site but with a shorter C-terminal that lacks any riboflavin kinase (RFK) motif. This second protein is rare among bacteria and has been named FADS type II (LmFADS-2). Here we present a biochemical and biophysical study of LmFADS-1 and LmFADS-2 by integrating kinetic and thermodynamic data together with sequence and structural prediction methods to evaluate their occurrence in Listeria, as well as their function and molecular properties. Despite LmFADS-1 similarities to other type I FADSs, (i) its RFK activity has not riboflavin substrate inhibition and occurs under reducing and oxidizing conditions, (ii) its FMNAT activity requires strong reducing environment, and (iii) binding of reaction products, but not substrates, favors binding of the second ligand. LmFADS-2 produces FAD under oxidizing and reducing environments, but its C-terminus module function remains unknown. Listeria species conserve both FADSs, being sequence identity high within L. monocytogenes strains. Our data exemplify alternative strategies for FMN and FAD biosynthesis and homeostasis, envisaging that in Listeria two FADSs might be required to fulfill the supply of flavin cofactors under niches that can go from saprophytism to virulence. As FADSs are attractive antimicrobial targets, understanding of FADSs traits in different species is essential to help in the discovery of specific antimicrobials.


Assuntos
Vias Biossintéticas , Flavinas/metabolismo , Listeria monocytogenes/metabolismo , Proteínas de Bactérias/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Listeriose/microbiologia , Modelos Moleculares , Nucleotidiltransferases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Especificidade por Substrato
14.
Sci Signal ; 12(601)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575732

RESUMO

The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) RIG-I, MDA5, and LGP2 stimulate inflammatory and antiviral responses by sensing nonself RNA molecules produced during viral replication. Here, we investigated how LGP2 regulates the RIG-I- and MDA5-dependent induction of type I interferon (IFN) signaling and showed that LGP2 interacted with different components of the RNA-silencing machinery. We identified a direct protein-protein interaction between LGP2 and the IFN-inducible, double-stranded RNA binding protein PACT. The LGP2-PACT interaction was mediated by the regulatory C-terminal domain of LGP2 and was necessary for inhibiting RIG-I-dependent responses and for amplifying MDA5-dependent responses. We described a point mutation within LGP2 that disrupted the LGP2-PACT interaction and led to the loss of LGP2-mediated regulation of RIG-I and MDA5 signaling. These results suggest a model in which the LGP2-PACT interaction regulates the inflammatory responses mediated by RIG-I and MDA5 and enables the cellular RNA-silencing machinery to coordinate with the innate immune response.


Assuntos
Antivirais/metabolismo , Proteína DEAD-box 58/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Chlorocebus aethiops , Proteína DEAD-box 58/genética , Enterovirus Humano B/genética , Enterovirus Humano B/fisiologia , Células HEK293 , Células HeLa , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Mengovirus/genética , Mengovirus/fisiologia , Ligação Proteica , RNA Helicases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Receptores Imunológicos , Transdução de Sinais/genética , Células Vero
15.
Sci Rep ; 7(1): 16129, 2017 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-29170442

RESUMO

PKR is a cellular kinase involved in the regulation of the integrative stress response (ISR) and pro-inflammatory pathways. Two N-terminal dsRNA Binding Domains (DRBD) are required for activation of PKR, by interaction with either dsRNA or PACT, another cellular DRBD-containing protein. A role for PKR and PACT in inflammatory processes linked to neurodegenerative diseases has been proposed and raised interest for pharmacological PKR inhibitors. However, the role of PKR in inflammation is subject to controversy. We identified the flavonoid luteolin as an inhibitor of the PKR/PACT interaction at the level of their DRBDs using high-throughput screening of chemical libraries by homogeneous time-resolved fluorescence. This was further validated using NanoLuc-Based Protein Complementation Assay. Luteolin inhibits PKR phosphorylation, the ISR and the induction of pro-inflammatory cytokines in human THP1 macrophages submitted to oxidative stress and toll-like receptor (TLR) agonist. Similarly, luteolin inhibits induction of pro-inflammatory cytokines in murine microglial macrophages. In contrast, luteolin increased activation of the inflammasome, in a PKR-independent manner. Collectively, these data delineate the importance of PKR in the inflammation process to the ISR and induction of pro-inflammatory cytokines. Pharmacological inhibitors of PKR should be used in combination with drugs targeting directly the inflammasome.


Assuntos
Inflamação/metabolismo , Proteínas de Ligação a RNA/metabolismo , eIF-2 Quinase/metabolismo , Células HEK293 , Humanos , Inflamação/imunologia , Fosforilação/genética , Fosforilação/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/genética , eIF-2 Quinase/genética
17.
J Clin Virol ; 69: 36-9, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26209375

RESUMO

BACKGROUND: Swine pasivirus (SPaV1) is a recently described enteric virus close to human parechoviruses and highly prevalent in pigs. Antibodies to Escherichia coli-expressed VP1 of SpaV1 have been found in a majority of humans in China. OBJECTIVES: The objectives were to estimate the antibody prevalence in a European country, to test if exposure to the virus was linked to pig products and if this exposure was a risk factor for the development of diabetes type 1. STUDY DESIGN: An ELISA test was developed and used to screen 842 healthy subjects with known exposure to pig products, 39 patients with diabetes type 1 and 20 controls. RESULTS: We identified a high seroprevalence (15.6%) reacting to VP1 of SPaV1 among healthy human subjects. Analysis of risk factors argues against cross-species transmission from pigs as the source of infection. Data also indicate that the presence of SPaV1 VP1-binding antibodies is not associated with diabetes type 1 in humans. CONCLUSION: Our results suggest that the seroreactivity frequently found in humans against SpaV1 is due to cross-reactivity with related antigen, perhaps a picornavirus, and that SpaV1 is not a zoonotic virus.


Assuntos
Anticorpos Antivirais/sangue , Picornaviridae/imunologia , Proteínas Estruturais Virais/imunologia , Adolescente , Adulto , Animais , Criança , Pré-Escolar , China , Reações Cruzadas , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/etiologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/virologia , Ensaio de Imunoadsorção Enzimática , Europa (Continente)/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Picornaviridae/epidemiologia , Infecções por Picornaviridae/transmissão , Infecções por Picornaviridae/veterinária , Fatores de Risco , Estudos Soroepidemiológicos , Suínos , Doenças dos Suínos/virologia , Adulto Jovem
18.
Sci Rep ; 5: 13564, 2015 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-26338235

RESUMO

In many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (S. Typhimurium), the sigma factor RpoS/σ(S) accumulates during stationary phase of growth, and associates with the core RNA polymerase enzyme (E) to promote transcription initiation of genes involved in general stress resistance and starvation survival. Whereas σ factors are usually inactivated upon interaction with anti-σ proteins, σ(S) binding to the Crl protein increases σ(S) activity by favouring its association to E. Taking advantage of evolution of the σ(S) sequence in bacterial species that do not contain a crl gene, like Pseudomonas aeruginosa, we identified and assigned a critical arginine residue in σ(S) to the S. Typhimurium σ(S)-Crl binding interface. We solved the solution structure of S. Typhimurium Crl by NMR and used it for NMR binding assays with σ(S) and to generate in silico models of the σ(S)-Crl complex constrained by mutational analysis. The σ(S)-Crl models suggest that the identified arginine in σ(S) interacts with an aspartate of Crl that is required for σ(S) binding and is located inside a cavity enclosed by flexible loops, which also contribute to the interface. This study provides the basis for further structural investigation of the σ(S)-Crl complex.


Assuntos
Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química , Pseudomonas aeruginosa/metabolismo , Salmonella/metabolismo , Fator sigma/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Modelos Químicos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Subunidades Proteicas , Fator sigma/metabolismo , Fator sigma/ultraestrutura , Especificidade da Espécie , Relação Estrutura-Atividade
19.
MAbs ; 2(4): 416-27, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20581462

RESUMO

Early diagnosis and appropriate treatment are key elements of malaria control programs in endemic areas. A major step forward in recent years has been the production and use of rapid diagnostic tests (RDTs) in settings where microscopy is impracticable. Many current RDTs target the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) released in the plasma of infected individuals. These RDTs have had an indisputably positive effect on malaria management, but still present several limitations, including the poor characterization of the commercial monoclonal antibodies (mAbs) used for PfHRP2 detection, variable sensitivity and specificity, and high costs. RDT use is further limited by impaired stability caused by temperature fluctuations during transport and uncontrolled storage in field-based facilities. To circumvent such drawbacks, an alternative could be the development of well-characterized, stabilized recombinant antibodies, with high binding affinity and specificity. Here, we report the characterization of the cDNA sequences encoding the Fab fragment of F1110 and F1546, two novels anti-PfHRP2 mAbs. FabF1546 was produced in the Escherichia coli periplasm. Its properties of binding to the parasite and to a recombinant PfHRP-2 antigen were similar to those of the parental mAb. As the affinity and stability of recombinant antibodies can be improved by protein engineering, our results open a novel approach for the development of an improved RDT for malaria diagnosis.


Assuntos
Anticorpos Antiprotozoários/genética , Escherichia coli/metabolismo , Fragmentos Fab das Imunoglobulinas/genética , Malária Falciparum/diagnóstico , Plasmodium falciparum/imunologia , Proteínas Recombinantes/genética , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/química , Antígenos de Protozoários/imunologia , DNA Complementar/análise , Diagnóstico Precoce , Escherichia coli/genética , Testes Hematológicos , Humanos , Malária Falciparum/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Engenharia de Proteínas , Proteínas de Protozoários/imunologia , Sensibilidade e Especificidade
20.
Appl Environ Microbiol ; 72(8): 5225-31, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16885269

RESUMO

A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.


Assuntos
Reatores Biológicos/microbiologia , Escherichia coli/crescimento & desenvolvimento , Sistemas On-Line , Proteínas Recombinantes/metabolismo , Automação , Biotecnologia/instrumentação , Meios de Cultura , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Oxigênio , Temperatura
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