RESUMO
In the present study, we provide the results of a detailed genomic analysis and the growth characteristics of a colistin-resistant KPC-3-producing Klebsiella pneumoniae sequence type 512 (ST512) isolate (the colR-KPC3-KP isolate) with a mutated pmrB and isogenic isolates of colR-KPC3-KP with mcr-1.2 isolated from an immunocompromised patient. From 2014 to 2017, four colR-KPC3-KP isolates were detected in rectal swab samples collected from a pediatric hematology patient at the Azienda Ospedaliero-Universitaria Pisana in Pisa, Italy. Whole-genome sequencing was performed by MiSeq sequencing (Illumina). Growth experiments were performed using different concentrations of colistin. The growth lag phases both of an isolate harboring a deletion in pmrB and of clonal variants with mcr-1.2 were assessed by the use of real-time light-scattering measurements. In the first isolate (isolate 1000-pmrBΔ, recovered in September 2014), a 17-nucleotide deletion in pmrB was detected. In subsequent isolates, the mcr-1.2 gene associated with the plasmid pIncX4-AOUP was found, while pmrB was intact. Additionally, plasmid pIncQ-AOUP, harboring aminoglycoside resistance genes, was detected. The growth curves of the first three isolates were identical without colistin exposure; however, at higher concentrations of colistin, the growth curves of the isolate with a deletion in pmrB showed longer lag phases. We observed the replacement of mutated colR-KPC3-KP pmrB by isogenic isolates with multiple resistance plasmids, including mcr-1.2-carrying pIncX4, probably due to coselection under gentamicin treatment in a patient with prolonged colR-KPC3-KP carriage. The carriage of these isolates persisted in follow-up cultures. Coselection and the advantages in growth characteristics suggest that the plasmid-mediated resistance conferred by mcr has fewer fitness costs in colR-KPC3-KP than mutations in chromosomal pmrB, contributing to the success of this highly resistant hospital-adapted epidemiological lineage.IMPORTANCE Our study shows a successful prolonged human colonization by a colistin-resistant Klebsiella pneumoniae isolate harboring mcr-1.2 An intense antibiotic therapy contributed to the maintenance of this microorganism through the acquisition of new resistance genes. The isolates carrying mcr-1.2 showed fewer fitness costs than isogenic isolates with a pmrB mutation in the chromosome. Coselection and reduced fitness costs may explain the replacement of isolates with the pmrB mutation by other isolates and the ability of the microorganism to persist despite antibiotic treatment.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Colistina/farmacologia , Farmacorresistência Bacteriana , Etanolaminofosfotransferase/metabolismo , Klebsiella pneumoniae/crescimento & desenvolvimento , Proteínas Mutantes/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Etanolaminofosfotransferase/genética , Aptidão Genética , Hospitais , Humanos , Itália , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Proteínas Mutantes/genética , Deleção de Sequência , Fatores de Transcrição/genética , Sequenciamento Completo do Genoma , beta-Lactamases/metabolismoRESUMO
Kinesin force generation involves ATP-induced docking of the neck linker (NL) along the motor core. However, the roles of the proposed steps of NL docking, cover-neck bundle (CNB) and asparagine latch (N-latch) formation, during force generation are unclear. Furthermore, the necessity of NL docking for transport of membrane-bound cargo in cells has not been tested. We generated kinesin-1 motors impaired in CNB and/or N-latch formation based on molecular dynamics simulations. The mutant motors displayed reduced force output and inability to stall in optical trap assays but exhibited increased speeds, run lengths, and landing rates under unloaded conditions. NL docking thus enhances force production but at a cost to speed and processivity. In cells, teams of mutant motors were hindered in their ability to drive transport of Golgi elements (high-load cargo) but not peroxisomes (low-load cargo). These results demonstrate that the NL serves as a mechanical element for kinesin-1 transport under physiological conditions.
Assuntos
Trifosfato de Adenosina/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Cinesinas/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
BACKGROUND: Langerhans cell histiocytosis (LCH) is a histiocytic disorder driven by a constitutive activation of the MAPK signaling pathway in myeloid cells. In 50-60% of cases, it is caused by the BRAFV600E mutation. There is evidence that levels of BRAFV600E in the peripheral blood of patients with LCH correlate with disease burden and could be used as marker for disease extent and response to therapy. However, there is currently no consensus on how testing for minimal disseminated disease should be performed. METHODS: Different approaches to determine the mutation load in patients with LCH were assessed and longitudinal evaluation of patient DNA during treatment with chemotherapy and/or the RAF inhibitor vemurafenib was performed. DNA was isolated from whole blood, different leukocyte subsets, and circulating cell-free DNA (ccf-DNA). RESULTS: We show that determining BRAF levels from whole blood is superior to using ccfDNA. Furthermore, it is important to identify the clinically relevant BRAF-mutated cellular subpopulations such as CD14+ monocytes or CD1c+ DCs, since other blood cells can also harbor the mutation and therefore confound whole blood or ccfDNA measurements. CONCLUSION: Our data support the view that single-agent treatment with an RAF inhibitor reduces disease activity but does not cure LCH.
Assuntos
Histiocitose de Células de Langerhans/sangue , Histiocitose de Células de Langerhans/genética , Proteínas Mutantes/sangue , Proteínas Mutantes/genética , Proteínas Proto-Oncogênicas B-raf/sangue , Proteínas Proto-Oncogênicas B-raf/genética , Substituição de Aminoácidos , Biomarcadores/sangue , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Pré-Escolar , DNA/sangue , DNA/genética , Análise Mutacional de DNA , Feminino , Marcadores Genéticos , Histiocitose de Células de Langerhans/tratamento farmacológico , Humanos , Lactente , Estudos Longitudinais , Masculino , Proteínas Mutantes/antagonistas & inibidores , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Vemurafenib/uso terapêuticoRESUMO
ScFv is emerging as a therapeutic alternative to the full-length monoclonal antibodies due to its small size and low production cost, but its low solubility remains a limiting factor toward wider use. Here, we increased the solubility of an Anti-epidermal growth factor receptor ScFv (Anti-EGFR ScFv) by attaching, a short 12-residue solubility enhancing peptide (SEP) tag at its C terminus. We first estimated the solubility increase by running 500-ns Brownian dynamics (BD) simulations. We then experimentally evaluated the predictions by producing recombinant Anti-EGFR ScFv with and without a SEP tag (called C9R) in E. coli. At 20⯰C, â¼85% of Anti-EGFR ScFv-C9R expressed in the soluble fraction, whereas all of the Anti-EGFR ScFv remained in the insoluble fraction. The total yield of Anti-EGFR ScFv-C9R was 17.15â¯mg which was â¼3 times higher than that of Anti-EGFR ScFv refolded from the insoluble fraction. Static and dynamic light scattering demonstrated the higher solubility of the purified Anti-EGFR ScFv-C9R, and Circular Dichroism (CD) indicated its high thermal stability, whereas the untagged protein aggregated at 37⯰C and pH 6. Finally, the binding activity of Anti-EGFR ScFv-C9R to EGFR was confirmed by surface plasmon resonance (SPR). Altogether, these results illustrate the improved biophysical and biochemical characteristics of Anti-EGFR ScFv-C9R and emphasize the potentials of SEP-tags for enhancing the solubility of aggregation-prone antibody fragments.
Assuntos
Receptores ErbB/imunologia , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Difusão Dinâmica da Luz , Receptores ErbB/química , Receptores ErbB/isolamento & purificação , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Mutação/genética , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/isolamento & purificação , Solubilidade , Ressonância de Plasmônio de SuperfícieRESUMO
Polylactide (PLA) is a bio-based plastic commonly synthesized by chemical catalytic reaction using lactic acid (LA) as a substrate. Here, novel LA-containing terpolyesters, namely, P[LA-co-3-hydroxybutyrate (3HB)-co-3-hydroxypropionate (3HP)], short as PLBP, were successfully synthesized for the first time by a recombinant Escherichia coli harbouring polyhydroxyalkanoate (PHA) synthase from Pseudomonas stutzeri (PhaC1Ps ) with 4-point mutations at E130D, S325T, S477G and Q481K, and 3-hydroxypropionyl-CoA (3HP-CoA) synthesis pathway from glycerol, 3-hydroxybutyryl-CoA (3HB-CoA) as well as lactyl-CoA (LA-CoA) pathways from glucose. Combining these pathways with the PHA synthase mutant phaC1Ps (E130D S325T S477G Q481K), the random terpolyester P(LA-co-3HB-co-3HP), or PLBP, was structurally confirmed by nuclear magnetic resonance to consist of 2 mol% LA, 90 mol% 3HB, and 8 mol% 3HP respectively. Remarkably, the PLBP terpolyester was produced from low-cost sustainable glycerol and glucose. Monomer ratios of PLBP could be regulated by ratios of glycerol to glucose. Other terpolyester thermal and mechanical properties can be manipulated by adjusting the monomer ratios. More PLBP applications are to be expected.
Assuntos
Aciltransferases/metabolismo , Escherichia coli/metabolismo , Engenharia Metabólica , Poliésteres/metabolismo , Pseudomonas stutzeri/enzimologia , Proteínas Recombinantes/metabolismo , Acil Coenzima A/metabolismo , Aciltransferases/genética , Custos e Análise de Custo , Escherichia coli/enzimologia , Escherichia coli/genética , Glucose/metabolismo , Glicerol/metabolismo , Espectroscopia de Ressonância Magnética , Redes e Vias Metabólicas/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Mutação Puntual , Pseudomonas stutzeri/genética , Proteínas Recombinantes/genéticaRESUMO
Brown and beige adipose tissues can dissipate chemical energy as heat through thermogenic respiration, which requires uncoupling protein 1 (UCP1). Thermogenesis from these adipocytes can combat obesity and diabetes, encouraging investigation of factors that control UCP1-dependent respiration in vivo. Here we show that acutely activated thermogenesis in brown adipose tissue is defined by a substantial increase in levels of mitochondrial reactive oxygen species (ROS). Remarkably, this process supports in vivo thermogenesis, as pharmacological depletion of mitochondrial ROS results in hypothermia upon cold exposure, and inhibits UCP1-dependent increases in whole-body energy expenditure. We further establish that thermogenic ROS alter the redox status of cysteine thiols in brown adipose tissue to drive increased respiration, and that Cys253 of UCP1 is a key target. UCP1 Cys253 is sulfenylated during thermogenesis, while mutation of this site desensitizes the purine-nucleotide-inhibited state of the carrier to adrenergic activation and uncoupling. These studies identify mitochondrial ROS induction in brown adipose tissue as a mechanism that supports UCP1-dependent thermogenesis and whole-body energy expenditure, which opens the way to improved therapeutic strategies for combating metabolic disorders.
Assuntos
Cisteína/química , Metabolismo Energético , Canais Iônicos/química , Canais Iônicos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Termogênese , Tecido Adiposo Marrom/química , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Animais , Respiração Celular , Cisteína/genética , Cisteína/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Canais Iônicos/deficiência , Canais Iônicos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxirredução , Compostos de Sulfidrila/metabolismo , Termogênese/efeitos dos fármacos , Proteína Desacopladora 1RESUMO
Tyrosine kinases are enzymes playing a critical role in cellular signaling. Mutations causing increased in kinase activity are often associated with cancer and various pathologies. One example in Src tyrosine kinases is offered by the substitution of the highly conserved tryptophan 260 by an alanine (W260A), which has been shown to cause an increase in activity. Here, molecular dynamics simulations based on atomic models are carried out to characterize the conformational changes in the linker region and the catalytic (kinase) domain of Src kinase to elucidate the impact of the W260A mutation. Umbrella sampling calculations show that the conformation of the linker observed in the assembled down-regulated state of the kinase is most favored when the kinase domain is in the inactive state, whereas the conformation of the linker observed in the re-assembled up-regulated state of the kinase is favored when the kinase domain is in the unphosphorylated active-like state. The calculations further indicate that there are only small differences between the WT and W260A mutant. In both cases, the intermediates states are very similar and the down-regulated inactive conformation is the most stable state. However, the calculations also show that the free energy cost to reach the unphosphorylated active-like conformation is slightly smaller for the W260A mutant compared with WT. A simple kinetic model is developed and submitted to a Bayesian Monte Carlo analysis to illustrate how such small differences can contribute to accelerate the trans-autophosphorylation reaction and yield a large increase in the activity of the mutant as observed experimentally.
Assuntos
Alanina/metabolismo , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Quinases da Família src/genética , Quinases da Família src/metabolismo , Alanina/genética , Ativação Enzimática/genética , Cinética , Método de Monte Carlo , Proteínas Mutantes/genética , Quinases da Família src/químicaRESUMO
A computational protein design method is extended to allow Monte Carlo simulations where two ligands are titrated into a protein binding pocket, yielding binding free energy differences. These provide a stringent test of the physical model, including the energy surface and sidechain rotamer definition. As a test, we consider tyrosyl-tRNA synthetase (TyrRS), which has been extensively redesigned experimentally. We consider its specificity for its substrate l-tyrosine (l-Tyr), compared to the analogs d-Tyr, p-acetyl-, and p-azido-phenylalanine (ac-Phe, az-Phe). We simulate l- and d-Tyr binding to TyrRS and six mutants, and compare the structures and binding free energies to a more rigorous "MD/GBSA" procedure: molecular dynamics with explicit solvent for structures and a Generalized Born + Surface Area model for binding free energies. Next, we consider l-Tyr, ac- and az-Phe binding to six other TyrRS variants. The titration results are sensitive to the precise rotamer definition, which involves a short energy minimization for each sidechain pair to help relax bad contacts induced by the discrete rotamer set. However, when designed mutant structures are rescored with a standard GBSA energy model, results agree well with the more rigorous MD/GBSA. As a third test, we redesign three amino acid positions in the substrate coordination sphere, with either l-Tyr or d-Tyr as the ligand. For two, we obtain good agreement with experiment, recovering the wildtype residue when l-Tyr is the ligand and a d-Tyr specific mutant when d-Tyr is the ligand. For the third, we recover His with either ligand, instead of wildtype Gln.
Assuntos
Termodinâmica , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/metabolismo , Tirosina/química , Tirosina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Ligantes , Simulação de Dinâmica Molecular , Método de Monte Carlo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Tirosina-tRNA Ligase/genéticaRESUMO
The evaluation of free energy differences between specific states of a system is of fundamental interest in the study of (bio)chemical systems. Herein, we examine the use of the recently introduced confinement method (CM) to evaluate relative free energy changes upon protein/peptide mutations. CM is a path-independent technique that involves the transformation of a configurational state of the system into an ideal crystal permitting the direct computation of free energy differences. We illustrate the method by evaluating the differential stabilities between native and mutant sequences of a model peptide that has been extensively characterized by experimental approaches, the GB1 hairpin. We show a good correlation between calculated and experimental relative stabilities and discuss other possible applications of this method in the context of complex molecular conversions.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Mutação , Peptídeos/química , Peptídeos/genética , Modelos Moleculares , Proteínas Mutantes/genética , Estabilidade Proteica , Teoria Quântica , TermodinâmicaRESUMO
α-Synuclein has been implicated in the development of neural plaques in Parkinson's Disease and Lewy-Body Dementia. This paper reports on the structural phase change behavior exhibited over a relevant range of temperatures in canonical protein Monte Carlo simulations for wild-type α-synuclein and three of its familial variants. We performed and analyzed these simulations to determine residue occupancy variations above and below this phase transition. From this analysis, we found regions above the phase transition temperature that consistently exhibited increased propensity for formation of long-chain beta-sheets, suggesting a possible role in α-synuclein aggregation.
Assuntos
Método de Monte Carlo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação , Transição de Fase , alfa-Sinucleína/química , alfa-Sinucleína/genética , Estrutura Secundária de Proteína , Temperatura de TransiçãoRESUMO
The mechanochemical protein dynamin is the prototype of the dynamin superfamily of large GTPases, which shape and remodel membranes in diverse cellular processes. Dynamin forms predominantly tetramers in the cytosol, which oligomerize at the neck of clathrin-coated vesicles to mediate constriction and subsequent scission of the membrane. Previous studies have described the architecture of dynamin dimers, but the molecular determinants for dynamin assembly and its regulation have remained unclear. Here we present the crystal structure of the human dynamin tetramer in the nucleotide-free state. Combining structural data with mutational studies, oligomerization measurements and Markov state models of molecular dynamics simulations, we suggest a mechanism by which oligomerization of dynamin is linked to the release of intramolecular autoinhibitory interactions. We elucidate how mutations that interfere with tetramer formation and autoinhibition can lead to the congenital muscle disorders Charcot-Marie-Tooth neuropathy and centronuclear myopathy, respectively. Notably, the bent shape of the tetramer explains how dynamin assembles into a right-handed helical oligomer of defined diameter, which has direct implications for its function in membrane constriction.
Assuntos
Dinaminas/antagonistas & inibidores , Dinaminas/química , Multimerização Proteica , Doença de Charcot-Marie-Tooth , Cristalografia por Raios X , Dinaminas/genética , Dinaminas/metabolismo , Humanos , Cadeias de Markov , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Miopatias Congênitas Estruturais , Nucleotídeos , Multimerização Proteica/genética , Relação Estrutura-AtividadeRESUMO
Current HIV-1 gene therapy approaches aim at stopping the viral life cycle at its earliest steps, such as entry or immediate postentry events. Among the most widely adopted strategies are CCR5 downregulation/knockout and the use of broadly neutralizing antibodies. However, the long-term efficacy and side effects are still unclear. TRIM5α is an interferon-stimulated restriction factor that can intercept incoming retroviruses within one hour of cytosolic entry and potently inhibit the infectivity of restriction-sensitive viruses. The human TRIM5α (TRIM5αhu) generally does not efficiently target HIV-1, but point mutations in its capsid-binding domain can confer anti-HIV-1 activity. Although the mechanisms by which TRIM5αhu mutants inhibit HIV-1 are relatively well understood, their characterization as potential transgenes for gene therapy is lacking. Additionally, previous reports of general immune activation by overexpression of TRIM5α have hindered its broad adoption as a potential transgene. Here we demonstrate the ability of the R332G-R335G TRIM5αhu mutant to efficiently restrict highly divergent HIV-1 strains, including Group O, as well as clinical isolates bearing cytotoxic T lymphocyte escape mutations. R332G-R335G TRIM5αhu efficiently protected human lymphocytes against HIV-1 infection, even when expressed at relatively low levels following lentiviral transduction. Most importantly, under these conditions Rhesus macaque TRIM5α (TRIM5αRh) and TRIM5αhu (wild-type or mutated) had no major effects on the NF-κB pathway. Transgenic TRIM5α did not modulate the kinetics of IκBα, JunB, and TNFAIP3 expression following TNF-α treatment. Finally, we show that human lymphocytes expressing R332G-R335G TRIM5αhu have clear survival advantages over unmodified parental cells in the presence of pathogenic, replication-competent HIV-1. These results support the relevance of R332G-R335G and other mutants of TRIM5αhu as candidate effectors for HIV-1 gene therapy.
Assuntos
Proteínas de Transporte/genética , Terapia Genética , Infecções por HIV/genética , HIV-1/genética , Proteínas Mutantes/genética , Animais , Fatores de Restrição Antivirais , Proteínas de Transporte/uso terapêutico , Infecções por HIV/terapia , Infecções por HIV/virologia , HIV-1/patogenicidade , Humanos , Lentivirus/genética , Linfócitos/patologia , Linfócitos/virologia , Macaca mulatta , Proteínas Mutantes/uso terapêutico , Mutação , Ligação Proteica , Transgenes , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
Cry8Ka5 is a mutant protein from Bacillus thuringiensis (Bt) that has been proposed for developing transgenic plants due to promising activity against coleopterans, like Anthonomus grandis (the major pest of Brazilian cotton culture). Thus, an early food safety assessment of Cry8Ka5 protein could provide valuable information to support its use as a harmless biotechnological tool. This study aimed to evaluate the food safety of Cry8Ka5 protein following the two-tiered approach, based on weights of evidence, proposed by ILSI. Cry1Ac protein was used as a control Bt protein. The history of safe use revealed no convincing hazard reports for Bt pesticides and three-domain Cry proteins. The bioinformatics analysis with the primary amino acids sequence of Cry8Ka5 showed no similarity to any known toxic, antinutritional or allergenic proteins. The mode of action of Cry proteins is well understood and their fine specificity is restricted to insects. Cry8Ka5 and Cry1Ac proteins were rapidly degraded in simulated gastric fluid, but were resistant to simulated intestinal fluid and heat treatment. The LD50 for Cry8Ka5 and Cry1Ac was >5000 mg/kg body weight when administered by gavage in mice. Thus, no expected relevant risks are associated with the consumption of Cry8Ka5 protein.
Assuntos
Proteínas de Bactérias/efeitos adversos , Endotoxinas/efeitos adversos , Inocuidade dos Alimentos , Proteínas Hemolisinas/efeitos adversos , Proteínas Mutantes/efeitos adversos , Testes de Toxicidade Aguda/métodos , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Sequência de Aminoácidos , Animais , Aspartato Aminotransferases/sangue , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Contagem de Células Sanguíneas , Colesterol/sangue , Biologia Computacional , Creatinina/sangue , Endotoxinas/genética , Feminino , Proteínas Hemolisinas/genética , Insetos , Inseticidas , Dose Letal Mediana , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/genética , Tamanho do Órgão/efeitos dos fármacos , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Triglicerídeos/sangue , Ureia/sangueRESUMO
After their disappearance from the human population in 1968, influenza H2 viruses have continued to circulate in the natural avian reservoir. The isolation of this virus subtype from multiple bird species as well as swine highlights the need to better understand the potential of these viruses to spread and cause disease in humans. Here we analyzed the virulence, transmissibility and receptor-binding preference of two avian influenza H2 viruses (H2N2 and H2N3) and compared them to a swine H2N3 (A/swine/Missouri/2124514/2006 [swMO]), and a human H2N2 (A/England/10/1967 [Eng/67]) virus using the ferret model as a mammalian host. Both avian H2 viruses possessed the capacity to spread efficiently between cohoused ferrets, and the swine (swMO) and human (Eng/67) viruses transmitted to naïve ferrets by respiratory droplets. Further characterization of the swMO hemagglutinin (HA) by x-ray crystallography and glycan microarray array identified receptor-specific adaptive mutations. As influenza virus quasispecies dynamics during transmission have not been well characterized, we sequenced nasal washes collected during transmission studies to better understand experimental adaptation of H2 HA. The avian H2 viruses isolated from ferret nasal washes contained mutations in the HA1, including a Gln226Leu substitution, which is a mutation associated with α2,6 sialic acid (human-like) binding preference. These results suggest that the molecular structure of HA in viruses of the H2 subtype continue to have the potential to adapt to a mammalian host and become transmissible, after acquiring additional genetic markers.
Assuntos
Adaptação Biológica , Vírus da Influenza A/fisiologia , Vírus da Influenza A/patogenicidade , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Ligação Viral , Animais , Aves , Cristalografia por Raios X , Modelos Animais de Doenças , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Análise em Microsséries , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Infecções por Orthomyxoviridae/patologia , Polissacarídeos/análise , Ligação Proteica , Suínos , VirulênciaRESUMO
UNLABELLED: The HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexes in vitro and permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes. IMPORTANCE: The HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral infectivity. Replication of the PF74-resistant HIV-1 mutant was impaired in macrophages, likely owing to altered interactions with host cell factors. Our results suggest that HIV-1 resistance to capsid-targeting inhibitors will be limited by functional constraints on the viral capsid protein. Therefore, this work enhances the attractiveness of the HIV-1 capsid as a therapeutic target.
Assuntos
Substituição de Aminoácidos , Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Proteína do Núcleo p24 do HIV/genética , HIV-1/fisiologia , Indóis/farmacologia , Fenilalanina/análogos & derivados , Replicação Viral , Células Cultivadas , Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Macrófagos/virologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Fenilalanina/farmacologia , Seleção Genética , Supressão GenéticaRESUMO
Thermostability is an important property of enzymes utilized for practical applications because it allows long-term storage and use as catalysts. In this study, we constructed an error-prone strain of the thermophile Geobacillus kaustophilus HTA426 and investigated thermoadaptation-directed enzyme evolution using the strain. A mutation frequency assay using the antibiotics rifampin and streptomycin revealed that G. kaustophilus had substantially higher mutability than Escherichia coli and Bacillus subtilis. The predominant mutations in G. kaustophilus were A · TâG · C and C · GâT · A transitions, implying that the high mutability of G. kaustophilus was attributable in part to high-temperature-associated DNA damage during growth. Among the genes that may be involved in DNA repair in G. kaustophilus, deletions of the mutSL, mutY, ung, and mfd genes markedly enhanced mutability. These genes were subsequently deleted to construct an error-prone thermophile that showed much higher (700- to 9,000-fold) mutability than the parent strain. The error-prone strain was auxotrophic for uracil owing to the fact that the strain was deficient in the intrinsic pyrF gene. Although the strain harboring Bacillus subtilis pyrF was also essentially auxotrophic, cells became prototrophic after 2 days of culture under uracil starvation, generating B. subtilis PyrF variants with an enhanced half-denaturation temperature of >10°C. These data suggest that this error-prone strain is a promising host for thermoadaptation-directed evolution to generate thermostable variants from thermolabile enzymes.
Assuntos
Enzimas/metabolismo , Geobacillus/enzimologia , Geobacillus/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Estabilidade Enzimática/efeitos da radiação , Enzimas/química , Enzimas/genética , Deleção de Genes , Genética Microbiana , Geobacillus/genética , Biologia Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Taxa de Mutação , Estabilidade Proteica/efeitos da radiação , Seleção Genética , TemperaturaRESUMO
AIMS: The diagnosis of adult-type granulosa cell tumours of the ovary (aGCT) is based on histomorphology aided by immunohistochemical staining for sex cord markers. Recently a single, recurrent somatic point mutation (402CâG) in FOXL2 was described in aGCT. We have investigated the impact of FOXL2 mutation testing in a large cohort of aGCT diagnosed previously by conventional histology and immunohistochemistry. METHODS AND RESULTS: Formalin-fixed, paraffin-embedded tissue cores from a cohort of 52 aGCT diagnosed previously by expert gynaecopathologists were analysed immunohistologically. FOXL2 mutation status was determined by Sanger sequencing and high-sensitivity TaqMan allelic discrimination assay. Histomorphology was reassessed by two expert gynaecopathologists. FOXL2 mutation analyses could be performed successfully in 46 cases, 40 of which were positive for the c.402C>G mutation, confirming a diagnosis of aGCT. In the six cases negative for the c.402C>G mutation, one case was confirmed on review as FOXL2 wild-type aGCT, whereas in the remaining five cases diagnoses other than aGCT were made. CONCLUSION: In cases where a diagnosis of aGCT is a consideration and unequivocal diagnosis is not possible based on morphology and routine immunostains, FOXL2 mutation testing can help to confirm the diagnosis. It is particularly relevant for accurate subclassification within the group of sex cord-stromal tumours.
Assuntos
Fatores de Transcrição Forkhead/genética , Tumor de Células da Granulosa/genética , Tumor de Células da Granulosa/patologia , Proteínas Mutantes/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Adulto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Estudos de Coortes , Análise Mutacional de DNA , DNA de Neoplasias/genética , Feminino , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/metabolismo , Tumor de Células da Granulosa/metabolismo , Humanos , Imuno-Histoquímica , Proteínas Mutantes/metabolismo , Neoplasias Ovarianas/metabolismo , Mutação Puntual , Estudos RetrospectivosRESUMO
Propagation of influenza A(H3N2) viruses in MDCK cells has been associated with the emergence of neuraminidase (NA) variants carrying a change at residue 151. In this study, the pyrosequencing assay revealed that â¼90% of A(H3N2) virus isolates analyzed (n=150) contained more than one amino acid variant (D/G/N) at position 151. Susceptibilities of the virus isolates to zanamivir and oseltamivir were assessed using the chemiluminescent and fluorescent NA inhibition (NI) assays. In the chemiluminescent assay, which utilizes NA-Star® substrate, up to 13-fold increase in zanamivir-IC50 was detected for isolates containing a high proportion (>50%) of the G151 NA variant. However, an increase in zanamivir-IC50s was not seen in the fluorescent assay, which uses MUNANA as substrate. To investigate this discrepancy, recombinant NAs (rNAs) were prepared and tested in both NI assays. Regardless of the assay used, the zanamivir-IC50 for the rNA G151 was much greater (>1500-fold) than that for rNA D151 wild-type. However, zanamivir resistance conferred by the G151 substitution was masked in preparations containing the D151 NA which had much greater activity, especially against MUNANA. In conclusion, the presence of NA D151G variants in cell culture-grown viruses interferes with drug susceptibility assessment and therefore measures need to be implemented to prevent their emergence.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/enzimologia , Mutação de Sentido Incorreto , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Animais , Cães , Farmacorresistência Viral , Fluorometria , Humanos , Vírus da Influenza A Subtipo H3N2/genética , Concentração Inibidora 50 , Medições Luminescentes , Células Madin Darby de Rim Canino , Testes de Sensibilidade Microbiana , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neuraminidase/metabolismo , Oseltamivir/farmacologia , Seleção Genética , Cultura de Vírus , Zanamivir/farmacologiaRESUMO
BACKGROUND: In 2001, the World Health Organization (WHO) has recommended the use of artemisinin-based combination therapy (ACT) as the first-line treatment of uncomplicated malaria cases, as monotherapies had become ineffective in many parts of the world. As a result, the Democratic Republic of Congo (DRC) withdrew chloroquine (CQ) from its malaria treatment policy in 2002 and an artesunate (AS)-amodiaquine (AQ) combination became the ACT of choice in DRC in 2005. AQ-resistance (AQR) has been reported in several parts of the world and mutations in codons 72-76 of the Plasmodium falciparum chloroquine-resistance transporter (pfcrt) gene have been strongly correlated with resistance, especially mutations encoding the SVMNT haplotype. This haplotype was first identified in Southeast Asia and South America but was recently reported in two African countries neighbouring DRC. These facts raised two questions: the first about the evolution of CQ resistance (CQR) in DRC and the second about the presence of the SVMNT haplotype, which would compromise the use of AQ as a partner drug for ACT. METHODS: A total of 213 thick blood films were randomly collected in 2010 from a paediatric clinic in Kinshasa, DRC. Microscopy controls and real-time polymerase chain reaction (RT-PCR) were performed for Plasmodium species identification. Haplotypes of the pfcrt gene were determined by sequencing. RESULTS: The K76T mutation was detected in 145 out of 198 P. falciparum-positive samples (73.2%). In these 145 resistant strains, only the CVIET haplotype was detected. CONCLUSIONS: This study is the first to assess the molecular markers of resistance to CQ and AQ after the introduction of ACT in DRC. The results suggest first that CQR is decreasing, as wild-type pfcrt haplotypes were found in only 26.8% of the samples and secondly that the SVMNT haplotype is not yet present in Kinshasa, suggesting that AQ remains valid as a partner drug for ACT in this region.
Assuntos
Antimaláricos/uso terapêutico , Cloroquina/uso terapêutico , Resistência a Medicamentos , Haplótipos , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Pré-Escolar , DNA de Protozoário/química , DNA de Protozoário/genética , República Democrática do Congo/epidemiologia , Feminino , Frequência do Gene , Humanos , Lactente , Recém-Nascido , Malária Falciparum/epidemiologia , Masculino , Proteínas Mutantes/genética , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
The formation of human islet amyloid polypeptide (hIAPP) is implicated in the loss of pancreatic ß-cells in type II diabetes. Rat amylin, which differs from human amylin at six residues, does not lead to formation of amyloid fibrils. Pramlintide is a synthetic analog of human amylin that shares three proline substitutions with rat amylin. Pramlintide has a much smaller propensity to form amyloid aggregates and has been widely prescribed in amylin replacement treatment. It is known that the three prolines attenuate ß-sheet formation. However, the detailed effects of these proline substitutions on full-length hIAPP remain poorly understood. In this work, we use molecular simulations and bias-exchange metadynamics to investigate the effect of proline substitutions on the conformation of the hIAPP monomer. Our results demonstrate that hIAPP can adopt various ß-sheet conformations, some of which have been reported in experiments. The proline substitutions perturb the formation of long ß-sheets and reduce their stability. More importantly, we find that all three proline substitutions of pramlintide are required to inhibit ß conformations and stabilize the α-helical conformation. Fewer substitutions do not have a significant inhibiting effect.