RESUMO
Controlling human pathogenic bacteria is a worldwide problem due to increasing bacterial resistance. This has prompted a number of studies investigating peptides isolated from marine animals as a possible alternative for control of human pathogen infections. Clavanins are antimicrobial peptides isolated from the marine tunicate Styela clava, showing 23 amino acid residues in length, cationic properties, and also high bactericidal activity. In spite of clear benefits from the use of peptides, currently 95% of peptide properties have limited pharmaceutical applicability, such as low solubility and short half-life in the circulatory system. Here, nanobiotechnology was used to encapsulate clavanin A in order to develop nanoantibiotics against bacterial sepsis. Clavanin was nanostructured using EUDRAGIT(®) L 100-55 and RS 30 D solution (3:1 w:w). Atomic force, scanning electron microscopy and dynamic light scattering showed nanoparticles ranging from 120 to 372 nm in diameter, with a zeta potential of -7.16 mV and a polydispersity index of 0.123. Encapsulation rate of 98% was assessed by reversed-phase chromatography. In vitro bioassays showed that the nanostructured clavanin was partially able to control development of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Furthermore, nanostructures did not show hemolytic activity. In vivo sepsis bioassays were performed using C57BL6 mice strain inoculated with a polymicrobial suspension. Assays led to 100% survival rate under sub-lethal sepsis assays and 40% under lethal sepsis assays in the presence of nanoformulated clavanin A until the seventh day of the experiment. Data here reported indicated that nanostructured clavanin A form shows improved antimicrobial activity and has the potential to be used to treat polymicrobial infections.
Assuntos
Antibacterianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Bacteriemia/tratamento farmacológico , Proteínas Sanguíneas/administração & dosagem , Metacrilatos/administração & dosagem , Nanopartículas/química , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Bactérias/efeitos dos fármacos , Proteínas Sanguíneas/química , Linhagem Celular Tumoral , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Metacrilatos/química , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Nanotecnologia , Urocordados/químicaRESUMO
Recently, defense peptides that are able to act against several targets have been characterized. The present work focuses on structural and functional evaluation of the peptide analogue Pa-MAP, previously isolated as an antifreeze peptide from Pleuronectes americanus. Pa-MAP showed activities against different targets such as tumoral cells in culture (CACO-2, MCF-7 and HCT-116), bacteria (Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 25923), viruses (HSV-1 and HSV-2) and fungi (Candida parapsilosis ATCC 22019, Trichophyton mentagrophytes (28d&E) and T. rubrum (327)). This peptide did not show toxicity against mammalian cells such as erythrocytes, Vero and RAW 264.7 cells. Molecular mechanism of action was related to hydrophobic residues, since only the terminal amino group is charged at pH 7 as confirmed by potentiometric titration. In order to shed some light on its structure-function relations, in vitro and in silico assays were carried out using circular dichroism and molecular dynamics. Furthermore, Pa-MAP showed partial unfolding of the peptide changes in a wide pH (3 to 11) and temperature (25 to 95°C) ranges, although it might not reach complete unfolding at 95°C, suggesting a high conformational stability. This peptide also showed a conformational transition with a partial α-helical fold in water and a full α-helical core in SDS and TFE environments. These results were corroborated by spectral data measured at 222 nm and by 50 ns dynamic simulation. In conclusion, data reported here show that Pa-MAP is a potential candidate for drug design against pathogenic microorganisms due to its structural stability and wide activity against a range of targets.
Assuntos
Alanina/química , Linguado/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Animais , Células CACO-2 , Candida/efeitos dos fármacos , Linhagem Celular , Eritrócitos/efeitos dos fármacos , Células HCT116 , Humanos , Staphylococcus aureus/efeitos dos fármacos , Trichophyton/efeitos dos fármacosRESUMO
The flavivirus NS5 protein is one of the most important proteins of the replication complex, and cellular proteins can interact with it. This study shows for the first time that the yellow fever virus (YFV) NS5 protein is able to interact with U1A, a protein involved in splicing and polyadenylation. We confirmed this interaction by GST-pulldown assay and by co-immunoprecipitation in YFV-infected cells. A region between amino acids 368 and 448 was identified as the site of interaction of the NS5 protein with U1A. This region was conserved among some flaviviruses of medical importance. The implications of this interaction for flavivirus replication are discussed.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Proteínas não Estruturais Virais/metabolismo , Vírus da Febre Amarela , Sequência de Aminoácidos , Animais , Sítios de Ligação , Chlorocebus aethiops , Sequência Conservada , Células HeLa , Humanos , Imunoprecipitação , Reação em Cadeia da Polimerase , Ligação Proteica , RNA Viral , Ribonucleoproteína Nuclear Pequena U1/química , Técnicas do Sistema de Duplo-Híbrido , Células Vero , Proteínas não Estruturais Virais/química , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/metabolismoRESUMO
BACKGROUND: Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins. METHODOLOGY/PRINCIPAL FINDINGS: Here, we performed yeast two-hybrid screens with human septins 1-10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments. CONCLUSIONS/SIGNIFICANCE: If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the "group rule", i.e. members of the same group (e.g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.
Assuntos
Proteínas de Transporte/metabolismo , Septinas/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ligação Competitiva , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Humanos , Família Multigênica , Ligação Proteica , Septinas/genéticaRESUMO
Beetle luciferases evolved from AMP/CoA-ligases. However, it is unclear how the new luciferase activity evolved. In order to clarify this question, we compared the luminescence and catalytic properties of a recently cloned luciferase-like enzyme from Zophobas mealworm, an AMP/CoA-ligase displaying weak luminescence activity, with those of cloned luciferases from the three main families of luminescent beetles: Phrixthrix hirtus railroad worm; Pyrearinus termitilluminans click beetle and Photinus pyralis firefly. The catalytic constant of the mealworm enzyme was 2-4 orders of magnitude lower than that of beetle luciferases, but 3 orders of magnitude above the non-catalyzed chemiluminescence of luciferyl-adenylate in buffer. Studies with D- and L-luciferin and their adenylates show that the luminescence reaction of the luciferase-like enzyme and beetle luciferases are stereoselective for D-luciferin and its adenylate, and that the selectivity is determined mainly at the adenylation step. Modelling studies showed that the luciferin binding site cavity of this enzyme is smaller and more hydrophobic than that of beetle luciferases. Therefore Zophobas mealworm enzyme displays true luciferase activity, keeping the attributes of an ancient protoluciferase. These results suggest that stereoselectivity for D-luciferin may have been a key event for the origin of oxygenase/luciferase activity in AMP/CoA-ligases, and that efficient luciferase activity may have further evolved mainly by increasing the catalytic constant of the oxidative reaction and the quantum yield of bioluminescence.
Assuntos
Proteínas de Insetos/metabolismo , Luciferases/metabolismo , Oxigenases/metabolismo , Tenebrio/enzimologia , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biocatálise , Simulação por Computador , Luciferina de Vaga-Lumes/química , Luciferina de Vaga-Lumes/metabolismo , Proteínas de Insetos/química , Luciferases/química , Substâncias Luminescentes/química , Substâncias Luminescentes/metabolismo , Medições Luminescentes , Dados de Sequência Molecular , Oxirredução , Homologia de Sequência de Aminoácidos , EstereoisomerismoRESUMO
Structures of digestive lysozymes 1 and 2 from housefly (MdL1 and MdL2) show that S106-T107 delimit a polar pocket around E32 (catalytic acid/base) and N46 contributes to the positioning of D50 (catalytic nucleophile), whereas those residues are replaced by V109-A110 and D48 in the non-digestive lysozyme from hen egg-white (HEWL). Further analyses revealed that MdL1 and MdL2 surfaces are less positively charged than HEWL surface. To verify the relevance of these differences to the acidic pH optimum of digestive lysozymes it was determined that pKas of the catalytic residues of the triple mutant MdL2 (N46D-S106V-T107A) are similar to HEWL pKas and higher than those for MdL2. In agreement, triple mutant MdL2 and HEWL exhibits the same pH optimum upon methylumbelliferylchitotrioside. In addition to that, the introduction of six basic residues on MdL1 surface increased by 1unit the pH optimum for the activity upon bacterial walls. Thus, the acidic pH optimum for MdL2 and MdL1 activities upon methylumbelliferylchitotrioside is determined by the presence of N46, S106 and T107 in the environment of their catalytic residues, which favors pKas reduction. Conversely, acidic pH optimum upon bacterial walls is determined by a low concentration of positive charges on the MdL2 and MdL1 surfaces.
Assuntos
Moscas Domésticas/enzimologia , Proteínas de Insetos/química , Muramidase/química , Animais , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Proteínas de Insetos/metabolismo , Cinética , Muramidase/metabolismo , Conformação Proteica , Dobramento de Proteína , Propriedades de SuperfícieRESUMO
Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors.
Assuntos
Besouros/enzimologia , Luciferases/química , Luciferases/metabolismo , Solventes/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Concentração de Íons de Hidrogênio , Luciferases/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Sensibilidade e Especificidade , Alinhamento de Sequência , Espectrometria de FluorescênciaRESUMO
The YaeQ family of proteins are found in many Gram-negative and a few Gram-positive bacteria. We have determined the first structure of a member of the YaeQ family by X-ray crystallography. Comparisons with other structures indicate that YaeQ represents a new compact protein fold built around a variation of the PD-(D/E)XK nuclease motif found in type II endonucleases and enzymes involved in DNA replication, repair, and recombination. We show that catalytically important residues in the PD-(D/E)XK nuclease superfamily are spatially conserved in YaeQ and other highly conserved YaeQ residues may be poised to interact with nucleic acid structures.
Assuntos
Proteínas de Bactérias/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Xanthomonas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Bases de Dados de Proteínas , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Selenometionina/química , Homologia de Sequência de AminoácidosRESUMO
Apobec1 edits the ApoB mRNA by deaminating nucleotide C(6666), which results in a codon change from Glutamate to stop, and subsequent expression of a truncated protein. Apobec1 is regulated by ACF (Apobec1 complementation factor) and hnRNPQ, which contains an N-terminal "acidic domain" (AcD) of unknown function, three RNA recognition motifs, and an Arg/Gly-rich region. Here, we modeled the structure of AcD using the bacterial protein Barstar as a template. Furthermore, we demonstrated by in vitro pull-down assays that 6xHis-AcD alone is able to interact with GST-Apobec1. Finally, we performed in silico phosphorylation of AcD and molecular dynamics studies, which indicate conformational changes in the phosphorylated form. The results of the latter studies were confirmed by in vitro phosphorylation of 6xHis-AcD by protein kinase C, mass spectrometry, and spectroscopic analyses. Our data suggest hnRNPQ interactions via its AcD with Apobec1 and that this interaction is regulated by the AcD phosphorylation.
Assuntos
Citidina Desaminase/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/química , Desaminase APOBEC-1 , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação , Dicroísmo Circular , Simulação por Computador , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Histidina/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Alinhamento de Sequência , Serina/metabolismo , Espectrometria de Fluorescência , Homologia Estrutural de ProteínaRESUMO
Esse artigo descreve realizações do Programa SMolBNet (Rede de Biologia Molecular Estrutural) do Estado de São Paulo, apoiado pela FAPESP (Fundação de Apoio à Pesquisa do Estado de São Paulo). Ele reúne vinte grupos de pesquisa e é coordenado pelos pesquisadores do Laboratório Nacional de Luz Síncrotron (LNLS), em Campinas. O Programa SMolBNet tem como metas: Elucidar a estrutura tridimensional de proteínas de interesse aos grupos de pesquisa componentes do Programa; Prover os grupos com treinamento em todas as etapas de determinação de estrutura: clonagem gênica, expressão de proteínas, purificação de proteínas, cristalização de proteínas e elucidação de suas estruturas. Tendo começado em 2001, o Programa alcançou sucesso em ambas as metas. Neste artigo, quatro dos grupos descrevem suas participações, e discutem aspectos estruturais das proteínas que eles selecionaram para estudos.