RESUMO
Alzheimer's disease (AD) is the leading cause of disabilities in old age and a rapidly growing condition in the elderly population. AD brings significant burden and has a devastating impact on public health, society and the global economy. Thus, developing new therapeutics to combat AD is imperative. Human glutaminyl cyclase (hQC), which catalyzes the formation of neurotoxic pyroglutamate (pE)-modified ß-amyloid (Aß) peptides, is linked to the amyloidogenic process that leads to the initiation of AD. Hence, hQC is an essential target for developing anti-AD therapeutics. Here, we systematically screened and identified hQC inhibitors from natural products by pharmacophore-driven inhibitor screening coupled with biochemical and biophysical examinations. We employed receptor-ligand pharmacophore generation to build pharmacophore models and Phar-MERGE and Phar-SEN for inhibitor screening through ligand-pharmacophore mapping. About 11 and 24 hits identified from the Natural Product and Traditional Chinese Medicine databases, respectively, showed diverse hQC inhibitory abilities. Importantly, the inhibitors TCM1 (Azaleatin; IC50 = 1.1 µM) and TCM2 (Quercetin; IC50 = 4.3 µM) found in foods and plants exhibited strong inhibitory potency against hQC. Furthermore, the binding affinity and molecular interactions were analyzed by surface plasmon resonance (SPR) and molecular modeling/simulations to explore the possible modes of action of Azaleatin and Quercetin. Our study successfully screened and characterized the foundational biochemical and biophysical properties of Azaleatin and Quercetin toward targeting hQC, unveiling their bioactive potential in the treatment of AD.
Assuntos
Doença de Alzheimer , Aminoaciltransferases , Inibidores Enzimáticos , Idoso , Humanos , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Farmacóforo , Quercetina/isolamento & purificação , Quercetina/farmacologia , Aminoaciltransferases/antagonistas & inibidores , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêuticoRESUMO
Streptococcus pneumoniae (S. pneumoniae) is a major Gram-positive opportunistic pathogen that causes pneumonia, bacteremia, and other fatal infections. This bacterium is responsible for more deaths than any other single pathogen in the world. Inexplicably, these symptoms persist despite the administration of effective antibiotics. Targeting pneumolysin (PLY) and sortase A (SrtA), the major virulence factors of S. pneumoniae, this study uncovered a novel resistance mechanism to S. pneumoniae infection. Using protein phenotype assays, we determined that the small molecule inhibitor alnustone is a potent drug that inhibits both PLY and SrtA. As essential virulence factors of S. pneumoniae, PLY and SrtA play a significant role in the occurrence of infection. Furthermore, evaluation using PLY-mediated hemolysis assay demonstrated alunstone had the potential to interrupt the haemolytic activity of PLY with treatment alunstone (4 µg/ml). Co-incubation of S. pneumoniae D39 SrtA with small-molecule inhibitors decreases cell wall-bound Nan A (pneumococcal-anchored surface protein SrtA), inhibits biofilm formation, and reduces biomass significantly. The protective effect of invasive pneumococcal disease (IPD) on murine S. pneumoniae was demonstrated further. Our study proposes a comprehensive bacteriostatic mechanism for S. pneumoniae and highlights the significant translational potential of targeting both PLY and SrtA to prevent pneumococcal infections. Our findings indicate that the antibacterial strategy of directly targeting PLY and SrtA with alnustone is a promising treatment option for S. pneumoniae and that alnustone is a potent inhibitor of PLY and SrtA.
Assuntos
Infecções Pneumocócicas , Streptococcus pneumoniae , Aminoaciltransferases , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias , Cisteína Endopeptidases , Hemólise , Camundongos , Estrutura Molecular , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/microbiologia , Estreptolisinas , Virulência , Fatores de Virulência/farmacologia , Fatores de Virulência/uso terapêuticoRESUMO
Understating how antibiotic tolerance impacts subsequent resistance development in the clinical setting is important to identifying effective therapeutic interventions and prevention measures. This study describes a patient case of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia which rapidly developed resistance to three primary MRSA therapies and identifies genetic and metabolic changes selected in vivo that are associated with rapid resistance evolution. Index blood cultures displayed susceptibility to all (non-beta-lactam) antibiotics with the exception of trimethoprim/ sulfamethoxazole. One month after initial presentation, during the same encounter, blood cultures were again positive for MRSA, now displaying intermediate resistance to vancomycin and ceftaroline and resistance to daptomycin. Two weeks later, blood cultures were positive for a third time, still intermediate resistant to vancomycin and ceftaroline and resistant to daptomycin. Mutations in mprF and vraT were common to all multidrug resistant isolates whereas mutations in tagH, agrB and saeR and secondary mprF mutation emerged sequentially and transiently resulting in distinct in vitro phenotypes. The baseline mutation rate of the patient isolates was unremarkable ruling out the hypermutator phenotype as a contributor to the rapid emergence of resistance. However, the index isolate demonstrated pronounced tolerance to the antibiotic daptomycin, a phenotype that facilitates the subsequent development of resistance during antibiotic exposure. This study exemplifies the capacity of antibiotic-tolerant pathogens to rapidly develop both stable and transient genetic and phenotypic changes, over the course of a single patient encounter.
Assuntos
Antibacterianos/farmacologia , Bacteriemia/microbiologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Infecções Estafilocócicas/microbiologia , Idoso , Aminoaciltransferases/genética , Antibacterianos/classificação , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Evolução Molecular , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mutação , Infecções Estafilocócicas/tratamento farmacológico , Fatores de Transcrição/genéticaRESUMO
Drug-resistant pathogenic Staphylococcus aureus (S. aureus) has severely threatened human health and arouses widespread concern. Sortase A (SrtA) is an essential virulence factor of S. aureus, which is responsible for the covalent anchoring of a variety of virulence-related proteins to the cell wall. SrtA has always been regarded as an ideal pharmacological target against S. aureus infections. In this research, we have determined that orientin, a natural compound isolated from various medicinal plants, can effectively inhibit the activity of SrtA with an IC50 of 50.44 ± 0.51 µM. We further demonstrated that orientin inhibited the binding of S. aureus to fibrinogen and diminished biofilm formation and the attaching of Staphylococcal protein A (SpA) to the cell wall in vitro. Using the fluorescence quenching assay, we demonstrated a direct interaction between orientin and SrtA. Further mechanistic studies revealed that the residues Glu-105, Thr-93, and Cys-184 were the key sites for the binding of SrtA to orientin. Importantly, we demonstrated that treatment with orientin attenuated S. aureus virulence of in vivo and protected mice against S. aureus-induced lethal pneumonia. These findings indicate that orientin is a potential drug to counter S. aureus infections and limit the development of drug resistance.
Assuntos
Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Flavonoides/farmacologia , Glucosídeos/farmacologia , Pneumonia Bacteriana , Infecções Estafilocócicas , Aminoaciltransferases/genética , Animais , Cisteína Endopeptidases , Staphylococcus aureus Resistente à Meticilina , Camundongos , Pneumonia Bacteriana/prevenção & controle , Infecções Estafilocócicas/prevenção & controleRESUMO
The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.
Assuntos
Aminoaciltransferases/química , Periodontite/microbiologia , Porphyromonas gingivalis/enzimologia , Prevotella intermedia/enzimologia , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/genética , Aminoaciltransferases/ultraestrutura , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Periodontite/tratamento farmacológico , Periodontite/genética , Porphyromonas gingivalis/patogenicidade , Prevotella intermedia/patogenicidade , Estrutura Terciária de Proteína/efeitos dos fármacos , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/metabolismo , Tannerella forsythia/enzimologia , Tannerella forsythia/patogenicidadeRESUMO
Staphylococcus aureus is still one of the leading causes of both hospital- and community-acquired infections. Due to the very high percentage of drug-resistant strains, the participation of drug-tolerant biofilms in pathological changes, and thus the limited number of effective antibiotics, there is an urgent need to search for alternative methods of prevention or treatment for S. aureus infections. In the present study, biochemically characterized (HPLC/UPLC-QTOF-MS) acetonic, ethanolic, and water extracts from fruits and bark of Viburnum opulus L. were tested in vitro as diet additives that potentially prevent staphylococcal infections. The impacts of V. opulus extracts on sortase A (SrtA) activity (Fluorimetric Assay), staphylococcal protein A (SpA) expression (FITC-labelled specific antibodies), the lipid composition of bacterial cell membranes (LC-MS/MS, GC/MS), and biofilm formation (LIVE/DEAD BacLight) were assessed. The cytotoxicity of V. opulus extracts to the human fibroblast line HFF-1 was also tested (MTT reduction). V. opulus extracts strongly inhibited SrtA activity and SpA expression, caused modifications of S. aureus cell membrane, limited biofilm formation by staphylococci, and were non-cytotoxic. Therefore, they have pro-health potential. Nevertheless, their usefulness as diet supplements that are beneficial for the prevention of staphylococcal infections should be confirmed in animal models in the future.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Fibroblastos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Extratos Vegetais/farmacologia , Viburnum/química , Aminoaciltransferases/biossíntese , Antibacterianos/química , Proteínas de Bactérias/biossíntese , Linhagem Celular , Cisteína Endopeptidases/biossíntese , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Fibroblastos/patologia , Frutas/química , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Casca de Planta/química , Extratos Vegetais/químicaRESUMO
Thirteen coumarins (1-13), including five new compounds (1-5), were isolated from the folk medicinal plant Poncirus trifoliata. Combined spectroscopic analyses revealed that coumarins 1-4 are bis-isoprenylated coumarins with diverse oxidation patterns, while 5 is an enantiomeric di-isoprenylated coumarin. The absolute configurations of the stereogenic centers in the isoprenyl chains were assigned through MTPA and MPA methods, and those of the known compounds triphasiol (6) and ponciol (7) were also assigned using similar methods. These coumarins inhibited significantly Staphylococcus aureus-derived sortase A (SrtA), a transpeptidase responsible for anchoring surface proteins to the peptidoglycan cell wall in Gram-positive bacteria. The present results obtained indicated that the bioactivity and underlying mechanism of action of these coumarins are associated with the inhibition of SrtA-mediated S. aureus adhesion to eukaryotic cell matrix proteins including fibrinogen and fibronectin, thus potentially serving as SrtA inhibitors.
Assuntos
Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Cumarínicos/farmacologia , Plantas Medicinais , Poncirus , Cisteína Endopeptidases , Fibrinogênio , Fibronectinas , Bactérias Gram-Positivas , Proteínas de Membrana , Estrutura Molecular , Infecções Estafilocócicas , Staphylococcus aureusRESUMO
BACKGROUND: Incomplete fracture healing may lead to chronic nonunion; thus, determining fracture healing is the primary issue in the clinical treatment. However, there are no validated early diagnostic biomarkers for assessing chronic nonunion. In this study, bioinformatics analysis combined with an experimental verification strategy was used to identify blood biomarkers for chronic nonunion. METHODS: First, differentially expressed genes in chronic nonunion were identified by microarray data analysis. Second, Dipsaci Radix (DR), a traditional Chinese medicine for fracture treatment, was used to screen the drug target genes. Third, the drug-disease network was determined, and biomarker genes were obtained. Finally, the potential blood biomarkers were verified by ELISA and qPCR methods. RESULTS: Fifty-five patients with open long bone fractures (39 healed and 16 nonunion) were enrolled in this study, and urgent surgical debridement and the severity of soft tissue injury had a significant effect on the prognosis of fracture. After the systems pharmacology analysis, six genes, including QPCT, CA1, LDHB, MMP9, UGCG, and HCAR2, were chosen for experimental validation. We found that all six genes in peripheral blood mononuclear cells (PBMCs) and serum were differentially expressed after injury, and five genes (QPCT, CA1, MMP9, UGCG, and HCAR2) were significantly lower in nonunion patients. Further, CA1, MMP9, and QPCT were markedly increased after DR treatment. CONCLUSION: CA1, MMP9, and QPCT are biomarkers of nonunion patients and DR treatment targets. However, HCAR2 and UGCG are biomarkers of nonunion patients but not DR treatment targets. Therefore, our findings may provide valuable information for nonunion diagnosis and DR treatment. TRIAL REGISTRATION: ISRCTN, ISRCTN13271153. Registered 05 April 2020-Retrospectively registered.
Assuntos
Biomarcadores/sangue , Fraturas não Consolidadas/sangue , Fraturas não Consolidadas/diagnóstico , Adulto , Aminoaciltransferases/sangue , Anticorpos/sangue , Doença Crônica , Biologia Computacional , Feminino , Consolidação da Fratura , Fraturas não Consolidadas/terapia , Humanos , Lactato Desidrogenases/sangue , Masculino , Metaloproteinase 9 da Matriz/sangue , Pessoa de Meia-Idade , Proteínas de Transporte de Monossacarídeos/sangue , Receptores Acoplados a Proteínas G/sangue , Resultado do Tratamento , Adulto JovemRESUMO
Eisenia fetida earthworm is an ecotoxicologically important test species to monitor various pollutants. However, there is a little knowledge about the effects of cadmium (Cd) on earthworms at the transcriptional level. Firstly, we exposed E. fetida to soils supplemented with different concentrations (10, 30, 60â¯mg/kg soil) of Cd. Moreover, we depicted the characterization of gene expressions with E. fetida using high-throughput profiling of gene expression. In addition, a comparison of the gene expression profiles between each Cd treatment group and the control group suggested that differential expressional genes (DEGs) mainly enriched in enzyme activity, metabolism, oxidative stress, regeneration and apoptosis pathways. 8 DEGs from these pathways had been selected randomly to confirm the data of RNA-seq. Among these DEGs, six genes (metallothionein-2, phytochelatin synthase 1a, CuZn superoxide dismutase, sex determining region Y-box 2, sex determining region Y-box 4b, TP53-regulated inhibitor of apoptosis 1-like) up-regulated and 2 genes (beta-1,4-endoglucanase, apoptosis-stimulating of p53 protein 2-like) down-regulated in response to Cd exposure. The alteration of them indicated that earthworms could reduce the toxicity and bioavailability of Cd in polluted soil ecosystems through different pathways. This work lays an important foundation for linking earthworm transcriptional level with the ecological risk of Cd in soil ecosystem.
Assuntos
Cádmio/toxicidade , Oligoquetos/fisiologia , Poluentes do Solo/toxicidade , Aminoaciltransferases , Animais , Disponibilidade Biológica , Cádmio/análise , Ecossistema , Perfilação da Expressão Gênica , Metalotioneína/genética , Metalotioneína/metabolismo , Oligoquetos/efeitos dos fármacos , Oligoquetos/genética , Estresse Oxidativo/efeitos dos fármacos , Solo , Poluentes do Solo/análise , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Human glutaminyl cyclase (hQC) appeared as a promising new target with its inhibitors attracted much attention for the treatment of Alzheimer's disease (AD) in recent years. But so far, only a few compounds have been reported as hQC inhibitors. To find novel and potent hQC inhibitors, a high-specificity ZBG (zinc-binding groups)-based pharmacophore model comprising customized ZBG feature was first generated using HipHop algorithm in Discovery Studio software for screening out hQC inhibitors from the SPECS database. After purification by docking studies and drug-like ADMET properties filters, four potential hit compounds were retrieved. Subsequently, these hit compounds were subjected to 30-ns molecular dynamic (MD) simulations to explore their binding modes at the active side of hQC. MD simulations demonstrated that these hit compounds formed a chelating interaction with the zinc ion, which was consistent with the finding that the electrostatic interaction was the major driving force for binding to hQC confirmed with MMPBSA energy decomposition. Higher binding affinities of these compounds were also verified by the binding free energy calculations comparing with the references. Thus, these identified compounds might be potential hQC candidates and could be used for further investigation.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Simulação de Dinâmica Molecular , Doença de Alzheimer/enzimologia , Aminoaciltransferases/química , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Termodinâmica , Interface Usuário-ComputadorRESUMO
Antimicrobial resistance resulting in ineffective treatment of infectious diseases is an increasing global problem, particularly in infections with pathogenic bacteria. In some bacteria, such as Streptococcus pyogenes, the pathogenicity is strongly linked to the attachment of virulence factors. Their attachment to the cellular membrane is a transpeptidation reaction, catalyzed by sortase enzymes. As such, sortases pose an interesting target for the development of new antivirulence strategies that could yield novel antimicrobial drugs. Using the substitution-oriented fragment screening (SOS) approach, we discovered a potent and specific inhibitor (C10) of sortase A from S. pyogenes. The inhibitor C10 showed high specificity towards S. pyogenes sortase A, with an IC50 value of 10⯵M and a Kd of 60⯵M. We envision that this inhibitor could be employed as a starting point for further exploration of sortase's potential as therapeutic target for antimicrobial drug development.
Assuntos
Aminoaciltransferases/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Streptococcus pyogenes/efeitos dos fármacos , Aminoaciltransferases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Cinética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Streptococcus pyogenes/enzimologia , Relação Estrutura-AtividadeRESUMO
N-myristoylation is the covalent addition of a 14-carbon saturated fatty acid (myristate) to the N-terminal glycine of specific protein substrates by N-myristoyltransferase (NMT) and plays an important role in protein regulation by controlling localization, stability, and interactions. We developed a novel method for whole-proteome profiling of free N-terminal glycines through labeling with S. Aureus sortase A (SrtA) and used it for assessment of target engagement by an NMT inhibitor. Analysis of the SrtA-labeling pattern with an engineered biotinylated depsipeptide SrtA substrate (Biotin-ALPET-Haa, Haa = 2-hydroxyacetamide) enabled whole proteome identification and quantification of de novo generated N-terminal Gly proteins in response to NMT inhibition by nanoLC-MS/MS proteomics, and was confirmed for specific substrates across multiple cell lines by gel-based analyses and ELISA. To achieve optimal signal over background noise we introduce a novel and generally applicable improvement to the biotin/avidin affinity enrichment step by chemically dimethylating commercial NeutrAvidin resin and combining this with two-step LysC on-bead/trypsin off-bead digestion, effectively eliminating avidin-derived tryptic peptides and enhancing identification of enriched peptides. We also report SrtA substrate specificity in whole-cell lysates for the first time, confirming SrtA promiscuity beyond its recognized preference for N-terminal glycine, and its usefulness as a tool for unbiased labeling of N-terminal glycine-containing proteins. Our new methodology is complementary to metabolic tagging strategies, providing the first approach for whole proteome gain-of signal readout for NMT inhibition in complex samples which are not amenable to metabolic tagging.
Assuntos
Aciltransferases/metabolismo , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Glicina/metabolismo , Proteômica/métodos , Staphylococcus aureus/enzimologia , Linhagem Celular Tumoral , Cromatografia Líquida , Células HeLa , Humanos , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
Uranium phytoextraction is a promising technology, however, facing difficult that limited plant biomass due to nutrient deficiency in the contaminated sites. The aim of this study is to evaluate the potential of a symbiotic associations of a legume Sesbania rostrata, rhizobia and arbuscular mycorrhiza fungi (AMF) for reclamation of uranium contaminated soils. Results showed AMF and rhizobia had a mutual beneficial relations in the triple symbiosis, which significantly increased plant biomass and uranium accumulation in S. rostrata plant. The highest uranium removal rates was observed in plant-AMF-rhizobia treated soils, in which 50.5-73.2% had been extracted, whereas 7.2-23.3% had been extracted in plant-treated soil. Also, the S. rostrata phytochelatin synthase (PCS) genes expression were increased in AMF and rhizobia plants compared with the plants. Meantime, content of malic acid, succinic acid and citric acid were elevated in S. rostrata root exudates of AMF and rhizobia inoculated plants. The facts suggest that the mutual interactions in the triple symbiosis help to improve phytoremediation efficiency of uranium by S. rostrata.
Assuntos
Biodegradação Ambiental , Micorrizas/metabolismo , Rhizobium/metabolismo , Sesbania/metabolismo , Simbiose , Urânio/farmacocinética , Aminoaciltransferases/metabolismo , Biomassa , Fabaceae/metabolismo , Raízes de Plantas/metabolismo , Sesbania/enzimologia , Sesbania/microbiologia , Poluentes do Solo/análise , Poluentes do Solo/farmacocinéticaRESUMO
The identification and quantification of low-abundant proteins are always impeded by high-abundant proteins in proteomic analysis because of the extreme complexity of peptide mixtures and wide dynamic range of protein abundances. Here, we developed a novel approach to enrich and quantify N-terminal glycine peptides through sortase A mediated ligation. This strategy was based on the formation of a covalent bond between the sortase A recognition motif LPXTG and a N-terminal glycine residue. Also, the quantification was achieved by introducing isotopically labeled threonine in the motif LPXTG. In this strategy, both the enrichment of N-terminal glycine peptides and the stable isotope labeling were achieved in a single step. We applied this approach for the proteome analysis of MCF-7 cell line. It was demonstrated a significant reduction in sample complexity via highly selective and efficient enrichment of N-terminal glycine peptides, thereby detecting lots of less abundant proteins and enhancing proteome coverage. In comparison to the untreated sample, an increase of 34% of proteins was additionally identified. Furthermore, 97% of proteins were successfully quantified with high accuracy. In summary, this quantitative N-terminal glycine peptides enrichment strategy is expected for high-throughput qualitative and quantitative proteomic analysis as a complementary approach to conventional shotgun proteomics.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Glicina/química , Peptídeos/química , Peptídeos/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Humanos , Células MCF-7RESUMO
Staphylococcus aureus (S. aureus) causes a broad variety of diseases. The spread of multidrugresistant S. aureus highlights the need to develop new ways to combat S. aureus infections. Sortase A (SrtA) can anchor proteins containing LPXTG binding motifs to the bacteria surface and plays a key role in S. aureus infections, making it a promising antivirulence target. In the present study, we used aSrtA activity inhibition assay to discover that isovitexin, a Chinese herbal product, can inhibit SrtA activity with an IC50 of 28.98 µg/ml. Using a fibrinogenbinding assay and a biofilm formation assay, we indirectly proved the SrtA inhibitory activity of isovitexin. Additionally, isovitexin treatment decreased the amount of staphylococcal protein A (SpA) on the surface of the cells. These data suggest that isovitexin has the potential to be an anti-infective drug against S. aureus via the inhibition of sortase activity.
Assuntos
Aminoaciltransferases/antagonistas & inibidores , Anti-Infecciosos/farmacologia , Apigenina/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Biofilmes/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Aminoaciltransferases/genética , Aminoaciltransferases/isolamento & purificação , Aminoaciltransferases/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade , Fatores de Virulência/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Hymenocallis littoralis (Jacq.) Salisb. has been referred as beach spider lily and commonly known for its rich phytochemical diversity. Phytochemicals such as alkaloids, volatile constituents, phenols, flavonoids, flavonols extracted from different parts of these plants like bulbs, flowers, leaf, stem and root had been used in folk medicines from ancient times because of their excellent antimicrobial and antioxidant properties. The leaf and bulb extract of H. littoralis plant was traditionally used for wound healing. Alkaloids extracted from bulb of this plant possess anti-viral, anti-neoplastic and cytotoxic properties. However, these phytochemicals have also shown antibiofilm activity, which is considered as one of the important factor accountable for the drug resistance in microorganisms. Thus, the investigation of medicinal properties of H. littoralis could be useful to control biofilm producing pathogens. AIM OF THE STUDY: Explore antimicrobial, antibiofilm and antioxidant potentials of H. littoralis against pathogenic microorganisms using experimental and computational biology approach. MATERIALS AND METHODS: Phytochemical extraction from dried powder of H. littoralis leaves was done by solvent extraction using methanol. Antimicrobial and antibiofilm activities of leaves extract were carried out using agar well diffusion method, growth curve, minimum inhibitory concentration (MIC) and Scanning Electron Microscopy (SEM). Liquid Chromatography and Mass Spectroscopy (LCMS) technique was used for the identification of phytochemicals. Molecular docking studies of antibiofilm agents with adhesin proteins were performed using Autodock 4.2. Antioxidant activity of extract was carried out by FRAP assay. The noxious effect of extract was investigated by histological studies on rat skin. RESULTS: The preliminary phytochemical analysis of methanolic leaves extract revealed the presence of alkaloids, flavonoids, terpenoid, glycosides, terpene, terpenoids and phenolics. The various phytochemicals such as Apigenin 7-(4'', 6'' diacetylalloside)-4'- alloside, Catechin 7-O- apiofuranoside, Emodic acid, Epicatechin 3-O- ß-D-glucopyranoside, 4 - Methylesculetin, Methylisoeugenol, Quercetin 5,7,3',4'-tetramethyl ether 3-rutinoside, 4 - Methylumbelliferyl ß-D- glucuronide were extracted, characterized and recognized from the leaves extract of H. littoralis. The identification of these phytochemicals was performed using LC-MS. The antimicrobial property of H. littoralis leaf extract was investigated against different pathogenic microorganisms. Out of these tested microorganisms, promising antibiofilm and antimicrobial activities were confirmed against S. aureus NCIM 2654 and C. albicans NCIM 3466 by using growth curve and SEM analysis. MIC of this leaf extract was identified as 45⯵g/ml and 70⯵g/ml for S. aureus NCIM 2654 and C. albicans NCIM 3466 respectively. The leaves extract also showed good antioxidant activity due to presence of phenols and flavonoids. Molecular docking of these identified antibiofilm components interacts with the active site residues of adhesin proteins, Sortase A and Als3 from S. aureus and C. albicans respectively. Histological studies of extracted phytochemicals revealed non-noxious effects on rat skin. CONCLUSION: Thus, the present study revealed that the leaves extract of H. littoralis contains various phytochemicals having good extent of antimicrobial, antibiofilm and antioxidant properties. The in-vitro and in-silico results would be useful to design new lead compounds against biofilm producing pathogenic microorganisms.
Assuntos
Amaryllidaceae , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Extratos Vegetais/farmacologia , Adesinas Bacterianas/metabolismo , Aminoaciltransferases/metabolismo , Animais , Anti-Infecciosos/análise , Antioxidantes/análise , Antioxidantes/farmacologia , Bactérias/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Candida albicans/crescimento & desenvolvimento , Candida albicans/fisiologia , Cisteína Endopeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Masculino , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Simulação de Acoplamento Molecular , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/análise , Folhas de Planta , Ratos Wistar , Pele/efeitos dos fármacosRESUMO
Strict L-chiral rejection through Gly-cisPro motif during chiral proofreading underlies the inability of D-aminoacyl-tRNA deacylase (DTD) to discriminate between D-amino acids and achiral glycine. The consequent Gly-tRNAGly 'misediting paradox' is resolved by EF-Tu in the cell. Here, we show that DTD's active site architecture can efficiently edit mischarged Gly-tRNAAla species four orders of magnitude more efficiently than even AlaRS, the only ubiquitous cellular checkpoint known for clearing the error. Also, DTD knockout in AlaRS editing-defective background causes pronounced toxicity in Escherichia coli even at low-glycine levels which is alleviated by alanine supplementation. We further demonstrate that DTD positively selects the universally invariant tRNAAla-specific G3â¢U70. Moreover, DTD's activity on non-cognate Gly-tRNAAla is conserved across all bacteria and eukaryotes, suggesting DTD's key cellular role as a glycine deacylator. Our study thus reveals a hitherto unknown function of DTD in cracking the universal mechanistic dilemma encountered by AlaRS, and its physiological importance.
Assuntos
Alanina-tRNA Ligase/antagonistas & inibidores , Aminoaciltransferases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Glicina/metabolismoRESUMO
Surface proteins in Gram-positive bacteria are incorporated into the cell wall through a peptide ligation reaction catalyzed by transpeptidase sortase. Six main classes (A-F) of sortase have been identified of which class A sortase is meant for housekeeping functions. The prototypic housekeeping sortase A (SaSrtA) from Staphylococcus aureus cleaves LPXTG-containing proteins at the scissile T-G peptide bond and ligates protein-LPXT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan lipid II precursor. Sortase-mediated ligation ("sortagging") of LPXTG-containing substrates and Gly-terminated nucleophiles occurs in vitro as well as in cellulo in the presence of Ca2+ and has been applied extensively for protein conjugations. Although the majority of applications emanate from SaSrtA, low catalytic efficiency, LPXTG specificity restriction, and Ca2+ requirement (particularly for in cellulo applications) remain a drawback. Given that Gram-positive bacteria genomes encode a variety of sortases, natural sortase mining can be a viable complementary approach akin to engineering of wild-type SaSrtA. Here, we describe the structure and specificity of a new class E sortase (SavSrtE) annotated to perform housekeeping roles in Streptomyces avermitilis Biochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca2+-independent activity, and provide insights about contrasting functional characteristics of SavSrtE and SaSrtA. Crystal structure, substrate docking, and mutagenesis experiments have identified a critical residue that dictates the preference for a non-canonical LAXTG recognition motif over LPXTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca2+-independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.
Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Streptomyces/enzimologia , Motivos de Aminoácidos , Cálcio/química , Domínio Catalítico , Parede Celular/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Genoma Bacteriano , Peptídeos/química , Peptidil Transferases/metabolismo , Proteínas Recombinantes/química , Staphylococcus aureus/enzimologia , Streptomyces/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Sortase A (SrtA) has long been recognized as an ideal drug target for therapeutic agents against Gram-positive pathogens. However, the SrtA of Streptococcus suis (Ss-SrtA), an important zoonotic agent, has not been studied. In this study, the enzymatic properties of Ss-SrtA were investigated, and inhibition of Ss-SrtA by natural products was evaluated. Ss-SrtA was expressed and purified. The purified recombinant Ss-SrtA had maximal activity at pH 6.0-7.5, 45°C, and showed a Km of 6.7 µM for the hydrolysis of substrate abz-LPATG-dnp. Different from Staphylococcus aureus SrtA (Sa-SrtA) which is stimulated by Ca2+, Ss-SrtA was observed to be Ca2+ independent. Structural analysis showed that salt bridges formed between K111 and D180 in Ss-SrtA replaced the function of Ca2+ in Sa-SrtA to stabilize the substrate-binding cleft. Site-directed mutagenesis identified H126, C192 and R200 as the key residues of Ss-SrtA active site. To discover potential inhibitors, the percent inhibition of sortase activity by natural products was measured. Among these selected natural products, acteoside, isoquercitrin and baicalin were discovered as novel SrtA inhibitors, with IC50 values of 36.3 ± 1.3 µM, 100.0 ± 1.3 µM and 85.4 ± 1.5 µM, respectively. The inhibitory effects of these three natural products were further confirmed on endogenous Sa-SrtA. Using a previously established S. aureus model with a fluorescent-labeled Sa-SrtA substrate, acteoside, isoquercitrin, and baicalin showed 86%, 28% and 45% inhibition on endogenous Sa-SrtA activity, respectively. Overall, these findings shed new light on enzymatic properties, Ca2+-independent catalytic mechanism and potential inhibitors of Ss-SrtA.
Assuntos
Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Glucosídeos/farmacologia , Fenóis/farmacologia , Quercetina/análogos & derivados , Streptococcus suis/enzimologia , Sequência de Aminoácidos , Aminoaciltransferases/química , Aminoaciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cálcio/metabolismo , Domínio Catalítico , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Avaliação Pré-Clínica de Medicamentos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Quercetina/farmacologia , TemperaturaRESUMO
Sortases are a family of Gram-positive transpeptidases responsible for anchoring surface protein virulence factors to the peptidoglycan cell wall layer. In Staphylococcus aureus (S. aureus), deletion of sortase isoform results in a significant reduction in virulence and infection potential. Twenty flavonoids were isolated from the stem of the folk medicinal plant Spatholobus suberectus Dunn. These compounds were tested against S. aureus-derived sortase A (SrtA), a key transpeptidase for bacterial virulence. Among these active flavonoids, 7-hydroxy-6-methoxy-flavanone (3) and formononetin (10) were identified as compounds with promising SrtA inhibitory activity. These compounds also exhibited inhibitory activity against S. aureus cell clumping to fibrinogen. The suppression of cell-clumping activity indicates the potential of these compounds in the treatment of S. aureus infections via the inhibition of SrtA.