Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Science ; 373(6554): 541-547, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34326236

RESUMO

Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities-rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Reposicionamento de Medicamentos , Lipidoses/induzido quimicamente , Fosfolipídeos/metabolismo , SARS-CoV-2/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , Antivirais/uso terapêutico , Antivirais/toxicidade , COVID-19/virologia , Cátions , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , SARS-CoV-2/fisiologia , Tensoativos/química , Tensoativos/farmacologia , Tensoativos/toxicidade , Células Vero , Replicação Viral/efeitos dos fármacos
2.
Mol Biol Evol ; 35(10): 2390-2400, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29955873

RESUMO

Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.


Assuntos
Afinidade de Anticorpos , Evolução Biológica , Aptidão Genética , Modelos Genéticos , Norovirus/genética , Animais , Anticorpos Neutralizantes , Proteínas do Capsídeo/fisiologia , Epistasia Genética , Camundongos , Dobramento de Proteína , Estabilidade Proteica , Seleção Genética
3.
Toxicol Sci ; 163(1): 265-278, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29432567

RESUMO

The FGF19- fibroblast growth factor receptor (FGFR4)-ßKlotho (KLB) pathway plays an important role in the regulation of bile acid (BA) homeostasis. Aberrant activation of this pathway has been described in the development and progression of a subset of liver cancers including hepatocellular carcinoma, establishing FGFR4 as an attractive therapeutic target for such solid tumors. FGF401 is a highly selective FGFR4 kinase inhibitor being developed for hepatocellular carcinoma, currently in phase I/II clinical studies. In preclinical studies in mice and dogs, oral administration of FGF401 led to induction of Cyp7a1, elevation of its peripheral marker 7alpha-hydroxy-4-cholesten-3-one, increased BA pool size, decreased serum cholesterol and diarrhea in dogs. FGF401 was also associated with increases of serum aminotransferases, primarily alanine aminotransferase (ALT), in the absence of any observable adverse histopathological findings in the liver, or in any other organs. We hypothesized that the increase in ALT could be secondary to increased BAs and conducted an investigative study in dogs with FGF401 and coadministration of the BA sequestrant cholestyramine (CHO). CHO prevented and reversed FGF401-related increases in ALT in dogs in parallel to its ability to reduce BAs in the circulation. Correlation analysis showed that FGF401-mediated increases in ALT strongly correlated with increases in taurolithocholic acid and taurodeoxycholic acid, the major secondary BAs in dog plasma, indicating a mechanistic link between ALT elevation and changes in BA pool hydrophobicity. Thus, CHO may offer the potential to mitigate elevations in serum aminotransferases in human subjects that are caused by targeted FGFR4 inhibition and elevated intracellular BA levels.


Assuntos
Alanina Transaminase/sangue , Ácidos e Sais Biliares/sangue , Resina de Colestiramina/farmacologia , Fígado/efeitos dos fármacos , Inibidores de Proteínas Quinases/toxicidade , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Alanina Transaminase/biossíntese , Animais , Ácidos e Sais Biliares/biossíntese , Cães , Relação Dose-Resposta a Droga , Feminino , Fígado/enzimologia , Masculino , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/sangue , Piridinas/farmacologia , Testes de Toxicidade , Toxicocinética
4.
Rev. bras. saúde matern. infant ; 15(4): 425-434, out.-dez. 2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-770024

RESUMO

To measure food expenditure for children living in a favela in Rio de Janeiro, and compare this expenditure to the cost of a healthy diet, based on local prices. Methods: panel study, with three collection dates – 2004, 2008 and 2012 – conducted in children (5 to 9 years old) in Manguinhos. Food prices were collected by way of a sample of local food stores in 2013 and deflated using indicators specific to food prices. Twenty-four hour diet recall, qualitative food frequency and the Brazilian food pyramid adequate for the age group were used to estimate the observed expenditure and the cost of a healthy diet. Results: in 2004, 49.2 percent of the families interviewed lived on less than US$1 per person/day and 9.7 percent in 2012.In the same period, the percentage of students eating free school meals dropped from 73 percent to 49 percent. Money spent on food was concentrated on sugary products (32.4 percent) and snacks (12.5 percent). The estimated monthly cost of a healthy diet (US$142) was lower than the observed expenditure (US$176). Conclusions: increased purchasing power has not led to healthier food choices. The common belief that poor people choose food based on prices was rejected by the present study. Other factors certainly play an important role in food purchasing decisions...


Mensurar os gastos com a alimentação de crianças moradoras de uma favela no Rio de Janeiro e comparar com os custos de uma dieta saudável, com base em preços praticados localmente. Métodos: estudo de painel, realizado em crianças, (5-9 anos), residentes em Manguinhos, com coletas em 2004, 2008, 2012. Os preços foram coletados em amostra de mercados locais em 2013, e deflacionados usando indicadores específicos de alimentos. Recordatório de 24 horas, frequência alimentar e pirâmide alimentar foram utilizadas na estimativa do gasto observado e custo da dieta saudável. Resultados: em 2004, 49,2 por cento das famílias entrevistadas vivia com menos de um US$1 por pessoa/dia, 9,7 por cento em 2012. A merenda escolar era consumida por 73 por cento e passou a 49 por cento. O gasto com alimentos concentrou- se em produtos açucarados (32,4 por cento) e lanches (12,5 por cento). O custo mensal estimado da dieta saudável (US$142) foi menor do que a despesa efetivamente observada (US$176). Conclusões: o aumento do poder de compra não levou a escolhas alimentares mais saudáveis. A crença comum de que as pessoas pobres escolhem alimentos com base nos preços foi rejeitada nesse estudo. Certamente outros fatores desempenham um papel importante nas decisões de compra de alimentos...


Assuntos
Humanos , Criança , Nutrição da Criança , Alimentos, Dieta e Nutrição , Ingestão de Alimentos , Economia dos Alimentos , Alimentos Infantis , Brasil , Comportamento Alimentar , Áreas de Pobreza , Alimentação Escolar
5.
Neurotoxicol Teratol ; 50: 1-10, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25968237

RESUMO

Pyridostigmine bromide (PB) is an FDA-approved drug for the treatment of myasthenia gravis and a prophylactic pre-treatment for organophosphate nerve agent poisoning. Current methods for evaluating nerve agent treatments include enzymatic studies and mammalian models. Rapid whole animal screening tools for assessing the effects of nerve agent pre-treatment and post-exposure drugs represent an underdeveloped area of research. We used zebrafish as a model for acute and chronic developmental exposure to PB and two related carbamate acetylcholinesterase (AChE) inhibitors, neostigmine bromide (NB) and physostigmine (PS). Lethal doses and gross morphological phenotypes resulting from exposure to sub-lethal doses of these compounds were determined. Quantitative analyses of motility impairment and AChE enzyme inhibition were used to determine optimal dosing conditions for evaluation of the effects of carbamate exposures on neuronal development; ~50% impairment of response to startle stimuli and >50% inhibition of AChE activity were observed at 80 mMPB, 20 mM NB and 0.1 mM PS. PB induced stunted somite length, but no other phenotypic effects were observed. In contrast, NB and PS induced more severe phenotypic morphological defects than PB as well as neurite outgrowth mislocalization. Additionally, NB induced mislocalization of nicotinic acetylcholine receptors, resulting in impaired synapse formation. Taken together, these data suggest that altered patterns of neuronal connectivity contribute to the developmental neurotoxicity of carbamates and demonstrate the utility of the zebrafish model for distinguishing subtle structure-based differential effects of AChE inhibitors, which include nerve agents, pesticides and drugs.


Assuntos
Carbamatos/toxicidade , Inibidores da Colinesterase/toxicidade , Agentes Neurotóxicos/toxicidade , Peixe-Zebra/embriologia , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Embrião não Mamífero/efeitos dos fármacos , Neostigmina/toxicidade , Neurogênese/efeitos dos fármacos , Fisostigmina/toxicidade , Brometo de Piridostigmina/toxicidade , Peixe-Zebra/metabolismo
6.
J Virol Methods ; 213: 111-7, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25522923

RESUMO

High mutation rates and short replication times lead to rapid evolution in RNA viruses. New tools for high-throughput culture and analysis of viral phenotypes will enable more effective studies of viral evolutionary processes. A water-in-oil drop microfluidic system to study virus-cell interactions at the single event level on a massively parallel scale is described here. Murine norovirus (MNV-1) particles were co-encapsulated with individual RAW 264.7 cells in 65 pL aqueous drops formed by flow focusing in 50 µm microchannels. At low multiplicity of infection (MOI), viral titers increased greatly, reaching a maximum 18 h post-encapsulation. This system was employed to evaluate MNV-1 escape from a neutralizing monoclonal antibody (clone A6.2). Further, the system was validated as a means for testing escape from antibody neutralization using a series of viral point mutants. Finally, the replicative capacity of single viral particles in drops under antibody stress was tested. Under standard conditions, many RNA virus stocks harbor minority populations of genotypic and phenotypic variants, resulting in quasispecies. These data show that when single cells are encapsulated with single viral particles under antibody stress without competition from other virions, the number of resulting infectious particles is nearly equivalent to the number of viral genomes present. These findings suggest that lower fitness virions can infect cells successfully and replicate, indicating that the microfluidics system may serve as an effective tool for isolating mutants that escape evolutionary stressors.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Virologia/métodos , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Macrófagos/virologia , Camundongos , Norovirus/fisiologia , Carga Viral , Cultura de Vírus/métodos , Replicação Viral
7.
J Virol ; 88(8): 4543-57, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24501415

RESUMO

UNLABELLED: New human norovirus strains emerge every 2 to 3 years, partly due to mutations in the viral capsid that allow escape from antibody neutralization and herd immunity. To understand how noroviruses evolve antibody resistance, we investigated the structural basis for the escape of murine norovirus (MNV) from antibody neutralization. To identify specific residues in the MNV-1 protruding (P) domain of the capsid that play a role in escape from the neutralizing monoclonal antibody (MAb) A6.2, 22 recombinant MNVs were generated with amino acid substitutions in the A'B' and E'F' loops. Six mutations in the E'F' loop (V378F, A382K, A382P, A382R, D385G, and L386F) mediated escape from MAb A6.2 neutralization. To elucidate underlying structural mechanisms for these results, the atomic structure of the A6.2 Fab was determined and fitted into the previously generated pseudoatomic model of the A6.2 Fab/MNV-1 virion complex. Previously, two distinct conformations, A and B, of the atomic structures of the MNV-1 P domain were identified due to flexibility in the two P domain loops. A superior stereochemical fit of the A6.2 Fab to the A conformation of the MNV P domain was observed. Structural analysis of our observed escape mutants indicates changes toward the less-preferred B conformation of the P domain. The shift in the structural equilibrium of the P domain toward the conformation with poor structural complementarity to the antibody strongly supports a unique mechanism for antibody escape that occurs via antigen flexibility instead of direct antibody-antigen binding. IMPORTANCE: Human noroviruses cause the majority of all nonbacterial gastroenteritis worldwide. New epidemic strains arise in part by mutations in the viral capsid leading to escape from antibody neutralization. Herein, we identify a series of point mutations in a norovirus capsid that mediate escape from antibody neutralization and determine the structure of a neutralizing antibody. Fitting of the antibody structure into the virion/antibody complex identifies two conformations of the antibody binding domain of the viral capsid: one with a superior fit and the other with an inferior fit to the antibody. These data suggest a unique mode of antibody neutralization. In contrast to other viruses that largely escape antibody neutralization through direct disruption of the antibody-virus interface, we identify mutations that acted indirectly by limiting the conformation of the antibody binding loop in the viral capsid and drive the antibody binding domain into the conformation unable to be bound by the antibody.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Caliciviridae/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Norovirus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/genética , Linhagem Celular , Humanos , Evasão da Resposta Imune , Camundongos , Camundongos Knockout , Testes de Neutralização , Norovirus/química , Norovirus/genética
8.
Rio de Janeiro; s.n; 2014. 109 p. mapas, tab, graf.
Tese em Português | LILACS | ID: lil-756742

RESUMO

A mortalidade infantil é um indicador de risco altamente sensível às condições de moradia, renda, alimentação e acesso a serviços. Seu estudo pemite a identificação disparidades e o monitoramento de mudanças em seus padrões é uma medida válida de avaliação e planejamento da saúde pública. O objetivo geral desse estudo foi analisar o padrão espacial do risco e as principais variáveis associadas ao óbito infantil segundo local de residência da mãe de nascidos vivos, no período de 2008 a 2012. Foi construído um estudo caso-controle espacial, na proporção de um para seis, selecionados entre nascidos vivos nos anos de 2008 a 2012. Realizamos análises exploratórias e espaciais, buscando investigar fatores associados à mortalidade infantil sobre quatro segmentos diferentes: menores de um ano, óbitos neonatais, óbitos pós-neonatais e óbitos evitáveis. Dentre os quatro segmentos, o espaço mostrou contribuição significativa para o óbito infantil. Para cada segmento, as variáveis foram ajustadas em modelos de regressão aditivos generalizados (GAMs), onde se observou maior contribuição das variáveis: APGAR no 5° minuto, peso ao nascer, número de consultas de pré-natal e presença de anomalia identificada ao nascer - tradicionalmente estabelecidas como mais proximais ao desfecho. Sabendo-se da diferença de riscos entre as diversas áreas do território, faz-se necessário a tomada de medidas especificas no combate a mortalidade infantil nesses locais...


Infant mortality is a risk index highly susceptible to living conditions, income, food and access to services. Its study identificatifies disparities among the general population and monitoring changes in its patterns is a validy measure for evaluation and planning in public health. The main objective of this study was to analyze the spatial pattern of risk and list variables associated with infant deaths, according to residence of the mother, among live births in the 2008 to 2012 period. For that, we built a spatial case-control study, selecting six controls for each case, and conducted exploratory and spatial analysis in order to investigate factors associated with infant mortality on four different segments: less than one year, neonatal deaths, post-neonatal deaths and preventable deaths. Among the four segments, the space showed significant contribution for infant death. For each segment, the variables were adjusted in a generalized additive models (GAMs), where greater contribution were observed in 5th minute APGAR, birth weight, number of prenatal appointments and presence of anomalies identified at birth – all of them traditionally established as proximal to the outcome. With the knowledge of the difference found in the risk pattern for specific areas of neighborhoods, it's possible to plan and apply specific measures to combat infant mortality in these locations...


Assuntos
Humanos , Mortalidade Infantil , Nascido Vivo , Análise Espacial , Sistemas de Informação/estatística & dados numéricos , Estudos de Casos e Controles , Atestado de Óbito
9.
Toxicon ; 75: 101-7, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23396042

RESUMO

Clostridium botulinum neurotoxin (BoNT) is a multi-domain protein made up of the approximately 100 kDa heavy chain (HC) and the approximately 50 kDa light chain (LC). The HC can be further subdivided into two halves: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). We have investigated the minimal requirements for channel activity and LC translocation. We utilize a cellular protection assay and a single channel/single molecule LC translocation assay to characterize in real time the channel and chaperone activities of BoNT/A truncation constructs in Neuro 2A cells. The unstructured, elongated belt region of the TD is demonstrated to be dispensable for channel activity, although may be required for productive LC translocation. We show that the RBD is not necessary for channel activity or LC translocation, however it dictates the pH threshold of channel insertion into the membrane. These findings indicate that each domain functions as a chaperone for the others in addition to their individual functions, working in concert to achieve productive intoxication.


Assuntos
Toxinas Botulínicas/química , Chaperonas Moleculares/metabolismo , Neurotoxinas/química , Animais , Armas Biológicas , Linhagem Celular , Clostridium botulinum/química , Concentração de Íons de Hidrogênio , Camundongos , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína
10.
Curr Top Microbiol Immunol ; 364: 115-37, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23239351

RESUMO

Clostridium botulinum neurotoxin (BoNT) is a multidomain protein in which the individual modules work in synchronized cooperative action in order to enter into neurons and inhibit synaptic transmission. The di-chain protein is made up of the ~50 kD light chain and the ~100 kD heavy chain. The HC can be further subdivided into the N-terminal translocation domain (H(N)) and the C-terminal Receptor Binding Domain (H(C)). BoNT entry into neurons requires the toxin to utilize the host cell's endocytosis pathway where it exploits the acidic environment of the endosome. Within the endosome the H(C) triggers the H(N) to change conformation from a soluble protein to a membrane inserted protein-conducting channel in precise timing with LC refolding. The LC must partially unfold to a translocation competent conformation in order to be translocated by the H(N) channel in an N to C terminal direction. Upon completion of translocation, the LC is released from the HC and allowed to interact with its substrate SNARE protein. This article discusses the individual functions of each module as well as the mechanisms by which each domain serves as a chaperone for the others, working in concert to achieve productive intoxication.


Assuntos
Toxinas Botulínicas/metabolismo , Chaperonas Moleculares/metabolismo , Neurônios/metabolismo , Neurotoxinas/metabolismo , Animais , Botulismo/metabolismo , Botulismo/microbiologia , Membrana Celular/metabolismo , Clostridium botulinum/metabolismo , Clostridium botulinum/patogenicidade , Citosol/metabolismo , Fenômenos Eletrofisiológicos , Endocitose , Endossomos/metabolismo , Endossomos/fisiologia , Ativação Enzimática , Concentração de Íons de Hidrogênio , Canais Iônicos/metabolismo , Bicamadas Lipídicas/metabolismo , Neurônios/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Desdobramento de Proteína , Proteólise , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/fisiologia , Proteínas SNARE/metabolismo
11.
J Biol Chem ; 287(3): 1657-61, 2012 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-22158863

RESUMO

Botulinum neurotoxin, the causative agent of the paralytic disease botulism, is an endopeptidase composed of a catalytic domain (or light chain (LC)) and a heavy chain (HC) encompassing the translocation domain (TD) and receptor-binding domain. Upon receptor-mediated endocytosis, the LC and TD are proposed to undergo conformational changes in the acidic endocytic environment resulting in the formation of an LC protein-conducting TD channel. The mechanism of channel formation and the conformational changes in the toxin upon acidification are important but less well understood aspects of botulinum neurotoxin intoxication. Here, we have identified a minimum channel-forming truncation of the TD, the "beltless" TD, that forms transmembrane channels with ion conduction properties similar to those of the full-length TD. At variance with the holotoxin and the HC, channel formation for both the TD and the beltless TD occurs independent of a transmembrane pH gradient. Furthermore, acidification in solution induces moderate secondary structure changes. The subtle nature of the conformational changes evoked by acidification on the TD suggests that, in the context of the holotoxin, larger structural rearrangements and LC unfolding occur preceding or concurrent to channel formation. This notion is consistent with the hypothesis that although each domain of the holotoxin functions individually, each domain serves as a chaperone for the others.


Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Canais Iônicos/metabolismo , Chaperonas Moleculares/metabolismo , Força Próton-Motriz , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/genética , Botulismo/genética , Botulismo/metabolismo , Linhagem Celular , Humanos , Canais Iônicos/química , Canais Iônicos/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mapeamento de Peptídeos/métodos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
12.
Proc Natl Acad Sci U S A ; 108(40): 16577-81, 2011 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-21949363

RESUMO

Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation.


Assuntos
Antígenos de Bactérias/química , Toxinas Bacterianas/química , Proteínas de Transporte/metabolismo , Eletrofisiologia/métodos , Bicamadas Lipídicas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/química , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Transporte Proteico/fisiologia
13.
J Biol Chem ; 285(11): 8130-7, 2010 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-20061382

RESUMO

Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. There is evidence that the side chains of the Phe(427) residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes the translocation process. In this report we describe the effects of replacing Phe(427) in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity >or=1,000-fold in our standard assay and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment. Our findings are discussed in relation to the role of the Phe clamp in a Brownian ratchet model of translocation.


Assuntos
Antígenos de Bactérias , Toxinas Bacterianas , Endossomos/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fenilalanina/genética , Substituição de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Glutamina/química , Histamina/química , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Potenciais da Membrana , Mutagênese , Estrutura Quaternária de Proteína , Transporte Proteico
14.
PLoS One ; 4(7): e6280, 2009 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-19609431

RESUMO

BACKGROUND: A key step of anthrax toxin action involves the formation of a protein-translocating pore within the endosomal membrane by the Protective Antigen (PA) moiety. Formation of this transmembrane pore by PA involves interaction of the seven 2beta2-2beta3 loops of the heptameric precursor to generate a 14-strand transmembrane beta barrel. METHODOLOGY/PRINCIPAL FINDINGS: We examined the effects on pore formation, protein translocation, and cytotoxicity, of mutating two phenylalanines, F313 and F314, that lie at the tip the beta barrel, and a third one, F324, that lies part way up the barrel. CONCLUSIONS/SIGNIFICANCE: Our results show that the function of these phenylalanine residues is to mediate membrane insertion and formation of stable transmembrane channels. Unlike F427, a key luminal residue in the cap of the pore, F313, F314, and F324 do not directly affect protein translocation through the pore. Our findings add to our knowledge of structure-function relationships of a key virulence factor of the anthrax bacillus.


Assuntos
Antígenos de Bactérias/química , Toxinas Bacterianas/química , Fenilalanina/fisiologia , Conformação Proteica
15.
Protein Sci ; 18(9): 1882-95, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19609933

RESUMO

Proteolytically activated Protective Antigen (PA) moiety of anthrax toxin self-associates to form a heptameric ring-shaped oligomer (the prepore). Acidic pH within the endosome converts the prepore to a pore that serves as a passageway for the toxin's enzymatic moieties to cross the endosomal membrane. Prepore is stable in solution under mildly basic conditions, and lowering the pH promotes a conformational transition to an insoluble pore-like state. N-tetradecylphosphocholine (FOS14) was the only detergent among 110 tested that prevented aggregation without dissociating the multimer into its constituent subunits. FOS14 maintained the heptamers as monodisperse, insertion-competent 440-kDa particles, which formed channels in planar phospholipid bilayers with the same unitary conductance and ability to translocate a model substrate protein as channels formed in the absence of detergent. Electron paramagnetic resonance analysis detected pore-like conformational changes within PA on solubilization with FOS14, and electron micrograph images of FOS14-solubilized pore showed an extended, mushroom-shaped structure. Circular dichroïsm measurements revealed an increase in alpha helix and a decrease in beta structure in pore formation. Spectral changes caused by a deletion mutation support the hypothesis that the 2beta2-2beta3 loop transforms into the transmembrane segment of the beta-barrel stem of the pore. Changes caused by selected point mutations indicate that the transition to alpha structure is dependent on residues of the luminal 2beta11-2beta12 loop that are known to affect pore formation. Stabilizing the PA pore in solution with FOS14 may facilitate further structural analysis and a more detailed understanding of the folding pathway by which the pore is formed.


Assuntos
Antígenos de Bactérias/química , Bacillus anthracis/química , Toxinas Bacterianas/química , Detergentes/química , Antígenos de Bactérias/isolamento & purificação , Toxinas Bacterianas/isolamento & purificação , Dicroísmo Circular , Bicamadas Lipídicas/química , Micelas , Modelos Moleculares , Conformação Proteica , Solubilidade
16.
Proc Natl Acad Sci U S A ; 106(5): 1330-5, 2009 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-19164566

RESUMO

Clostridium botulinum neurotoxin (BoNT) is the causative agent of botulism, a neuroparalytic disease. We describe here a semisynthetic strategy to identify inhibitors based on toosendanin, a traditional Chinese medicine reported to protect from BoNT intoxication. Using a single molecule assay of BoNT serotypes A and E light chain (LC) translocation through the heavy chain (HC) channel in neurons, we discovered that toosendanin and its tetrahydrofuran analog selectively arrest the LC translocation step of intoxication with subnanomolar potency, and increase the unoccluded HC channel propensity to open with micromolar efficacy. The inhibitory profile on LC translocation is accurately recapitulated in 2 different BoNT intoxication assays, namely the mouse protection and the primary rat spinal cord cell assays. Toosendanin has an unprecedented dual mode of action on the protein-conducting channel acting as a cargo-dependent inhibitor of translocation and as cargo-free channel activator. These results imply that the bimodal modulation by toosendanin depends on the dynamic interactions between channel and cargo, highlighting their tight interplay during the progression of LC transit across endosomes.


Assuntos
Toxinas Botulínicas/antagonistas & inibidores , Animais , Toxinas Botulínicas/metabolismo , Células Cultivadas , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Feminino , Camundongos , Técnicas de Patch-Clamp , Transporte Proteico , Ratos , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiologia
17.
PLoS Pathog ; 4(12): e1000245, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19096517

RESUMO

Clostridium botulinum neurotoxin (BoNT) causes flaccid paralysis by disabling synaptic exocytosis. Intoxication requires the tri-modular protein to undergo conformational changes in response to pH and redox gradients across endosomes, leading to the formation of a protein-conducting channel. The approximately 50 kDa light chain (LC) protease is translocated into the cytosol by the approximately 100 kDa heavy chain (HC), which consists of two modules: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). Here we exploited the BoNT modular design to identify the minimal requirements for channel activity and LC translocation in neurons. Using the combined detection of substrate proteolysis and single-channel currents, we showed that a di-modular protein consisting only of LC and TD was sufficient to translocate active protease into the cytosol of target cells. The RBD is dispensable for cell entry, channel activity, or LC translocation; however, it determined a pH threshold for channel formation. These findings indicate that, in addition to its individual functions, each module acts as a chaperone for the others, working in concert to achieve productive intoxication.


Assuntos
Toxinas Botulínicas Tipo A/genética , Toxinas Botulínicas Tipo A/metabolismo , Peptídeo Hidrolases/metabolismo , Toxinas Botulínicas Tipo A/química , Células Cultivadas , Sistemas de Liberação de Medicamentos/métodos , Ativação Enzimática , Deleção de Genes , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Canais Iônicos/fisiologia , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiologia , Ligação Proteica/genética , Engenharia de Proteínas , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Internalização do Vírus
18.
J Biol Chem ; 283(7): 3997-4003, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-18032388

RESUMO

Clostridial botulinum neurotoxin (BoNT) causes a neuroparalytic condition recognized as botulism by arresting synaptic vesicle exocytosis. Although the crystal structures of full-length BoNT/A and BoNT/B holotoxins are known, the molecular architecture of the five other serotypes remains elusive. Here, we present the structures of BoNT/A and BoNT/E using single particle electron microscopy. Labeling of the particles with three different monoclonal antibodies raised against BoNT/E revealed the positions of their epitopes in the electron microscopy structure, thereby identifying the three hallmark domains of BoNT (protease, translocation, and receptor binding). Correspondingly, these antibodies selectively inhibit BoNT translocation activity as detected using a single molecule assay. The global structure of BoNT/E is strikingly different from that of BoNT/A despite strong sequence similarity. We postulate that the unique architecture of functionally conserved modules underlies the distinguishing attributes of BoNT/E and contributes to differences with BoNT/A.


Assuntos
Toxinas Botulínicas/química , Microscopia Eletrônica/métodos , Anticorpos Monoclonais/imunologia , Toxinas Botulínicas/imunologia , Eletroforese em Gel de Poliacrilamida , Conformação Proteica
20.
J Biol Chem ; 282(40): 29604-11, 2007 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-17666397

RESUMO

Clostridial botulinum neurotoxins (BoNTs) exert their neuroparalytic action by arresting synaptic exocytosis. Intoxication requires the disulfide-linked, di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol. Here, we investigate the role of the disulfide bridge in the dynamics of protein translocation. We utilize a single channel/single molecule assay to characterize in real time the BoNT channel and chaperone activities in Neuro 2A cells under conditions that emulate those prevalent across endosomes. We show that the disulfide bridge must remain intact throughout LC translocation; premature reduction of the disulfide bridge after channel formation arrests translocation. The disulfide bridge must be on the trans compartment to achieve productive translocation of LC; disulfide disruption on the cis compartment or within the bilayer during translocation aborts it. We demonstrate that a peptide linkage between LC and HC in place of a disulfide bridge is insufficient for productive LC translocation. The disulfide linkage, therefore, dictates the outcome of translocation: productive passage of cargo or abortive channel occlusion by cargo. Based on these and previous findings we suggest a sequence of events for BoNT LC translocation to be HC insertion, coupled LC unfolding, and protein conduction through the HC channel in an N to C terminus orientation and ultimate release of the LC from the HC by reduction of the disulfide bridge concomitant with LC refolding in the cytosol.


Assuntos
Toxinas Botulínicas/química , Peptídeo Hidrolases/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Clostridium botulinum/metabolismo , Citosol/metabolismo , Dissulfetos/química , Endopeptidases/química , Humanos , Modelos Biológicos , Técnicas de Patch-Clamp , Desnaturação Proteica , Dobramento de Proteína , Transporte Proteico , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...