RESUMO
Micro Exon Gene (MEG) proteins are thought to play major roles in the infection and survival of parasitic Schistosoma mansoni worms in host organisms. Here, the physical chemical properties of two small MEG proteins found in the genome of S. mansoni, named MEG-24 and MEG-27, were examined by a combination of biophysical techniques such as differential scanning calorimetry, tensiometry, circular dichroism, fluorescence, and electron spin resonance spectroscopies. The proteins are surface active and structurally arranged as cationic amphipathic α-helices that can associate with lipid membranes and cause their disruption. Upon adsorption to lipid membranes, MEG-27 strongly affects the fluidity of erythrocyte ghost membranes, whereas MEG-24 forms pores in erythrocytes without modifying the ghost membrane fluidity. Whole-mount in situ hybridization experiments indicates that MEG-27 and MEG-24 transcripts are located in the parasite esophagus and subtegumental cells, respectively, suggesting a relevant role of these proteins in the host-parasite interface. Taken together, these characteristics lead us to propose that these MEG proteins may interact with host cell membranes and potentially modulate the immune process using a similar mechanism as that described for α-helical membrane-active peptides.
Assuntos
Éxons/genética , Membranas/química , Schistosoma mansoni/genética , Sequência de Aminoácidos , Animais , Varredura Diferencial de Calorimetria/métodos , Dicroísmo Circular/métodos , Peptídeos/química , Conformação Proteica em alfa-Hélice , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/genética , Esquistossomose mansoni/metabolismoRESUMO
Antimicrobial peptides are a large group of natural compounds which present promising properties for the pharmaceutical and food industries, such as broad-spectrum activity, potential for use as natural preservatives, and reduced propensity for development of bacterial resistance. Plantaricin 149 (Pln149), isolated from Lactobacillus plantarum NRIC 149, is an intrinsically disordered peptide with the ability to inhibit bacteria from the Listeria and Staphylococcus genera, and which is capable of promoting inhibition and disruption of yeast cells. In this study, the interactions of Pln149 with model membranes composed of zwitterionic and/or anionic phospholipids were investigated using a range of biophysical techniques, including isothermal titration calorimetry, surface tension measurements, synchrotron radiation circular dichroism spectroscopy, oriented circular dichroism spectroscopy, and optical microscopy, to elucidate these peptides' mode of interactions and provide insight into their functional roles. In anionic model membranes, the binding of Pln149 to lipid bilayers is an endothermic process and induces a helical secondary structure in the peptide. The helices bind parallel to the surfaces of lipid bilayers and can promote vesicle disruption, depending on peptide concentration. Although Pln149 has relatively low affinity for zwitterionic liposomes, it is able to adsorb at their lipid interfaces, disturbing the lipid packing, assuming a similar parallel helix structure with a surface-bound orientation, and promoting an increase in the membrane surface area. Such findings can explain the intriguing inhibitory action of Pln149 in yeast cells whose cell membranes have a significant zwitterionic lipid composition.
Assuntos
Bacteriocinas/química , Bacteriocinas/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Adsorção , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Ligação Proteica , Tensão Superficial , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
The biotechnological and industrial uses of thermostable and organic solvent-tolerant enzymes are extensive and the investigation of such enzymes from microbiota present in oil reservoirs is a promising approach. Searching sequence databases for esterases from such microbiota, we have identified in silico a potentially secreted esterase from Acetomicrobium hydrogeniformans, named AhEst. The recombinant enzyme was produced in E. coli to be used in biochemical and biophysical characterization studies. AhEst presented hydrolytic activity on short-acyl-chain p-nitrophenyl ester substrates. AhEst activity was high and stable in temperatures up to 75 °C. Interestingly, high salt concentration induced a significant increase of catalytic activity. AhEst still retained ~ 50% of its activity in 30% concentration of several organic solvents. Synchrotron radiation circular dichroism and fluorescence spectroscopies confirmed that AhEst displays high structural stability in extreme conditions of temperature, salinity, and organic solvents. The enzyme is a good emulsifier agent and is able to partially reverse the wettability of an oil-wet carbonate substrate, making it of potential interest for use in enhanced oil recovery. All the traits observed in AhEst make it an interesting candidate for many industrial applications, such as those in which a significant hydrolytic activity at high temperatures is required.
Assuntos
Proteínas de Bactérias/metabolismo , Esterases/metabolismo , Ambientes Extremos , Desnaturação Proteica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Esterases/química , Esterases/genética , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salinidade , Solventes/química , Especificidade por SubstratoRESUMO
BACKGROUND: The Micro-Exon Gene-14 (MEG-14) displays a remarkable structure that allows the generation of antigenic variation in Schistosomes. Previous studies showed that the soluble portion of the MEG-14 protein displays features of an intrinsically disordered protein and is expressed exclusively in the parasite esophageal gland. These features indicated a potential for interaction with host proteins present in the plasma and cells from ingested blood. METHODS: A yeast two-hybrid experiment using as bait the soluble domain of Schistosoma mansoni MEG-14 (sMEG-14) against a human leukocyte cDNA library was performed. Pull-down and surface plasmon resonance (SPR) experiments were used to validate the interaction between sMEG-14 and human S100A9. Synchrotron radiation circular dichroism (SRCD) were used to detect structural changes upon interaction between sMEG-14 and human S100A9. Feeding of live parasites with S100A9 attached to a fluorophore allowed the tracking of the fate of this protein in the parasite digestive system. RESULTS: S100A9 interacted with sMEG-14 consistently in yeast two-hybrid assay, pull-down and SPR experiments. SRCD suggested that MEG-14 acquired a more regular structure as a result of the interaction with S100A9. Accumulation of recombinant S100A9 in the parasite's esophageal gland, when ingested by live worms suggests that such interaction may occur in vivo. CONCLUSION: S100A9, a protein previously described to be involved in modulation of inflammatory response, was found to interact with sMEG-14. GENERAL SIGNIFICANCE: Our results allow proposing a mechanism involving MEG-14 for the parasite to block inflammatory signaling, which would occur upon release of S100A9 when ingested blood cells are lysed.
Assuntos
Esôfago/metabolismo , Inflamação/patologia , Proteínas de Protozoários/metabolismo , Proteínas S100/metabolismo , Schistosoma mansoni/metabolismo , Processamento Alternativo/genética , Animais , Dicroísmo Circular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Humanos , Ligação Proteica , Estrutura Secundária de Proteína , Ressonância de Plasmônio de Superfície , Técnicas do Sistema de Duplo-HíbridoRESUMO
Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/ß protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.
Assuntos
Proteínas de Bactérias/metabolismo , Esterases/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , 1-Propanol/química , Proteínas de Bactérias/química , Estabilidade Enzimática , Esterases/química , Etanol/química , Concentração de Íons de Hidrogênio , Dobramento de Proteína , Especificidade por Substrato , Ureia/químicaRESUMO
PIP: This paper highlights the challenges involved in integrating AIDS control programs into existing health care services. The Belize Red Cross Association has integrated its AIDS control program into its extensive child survival programs in Belize. The program involved training of volunteers who would provide AIDS related information at the household level. Trained traditional birth attendant and community leaders were also well represented in the program. On evaluation of the year's field experience, it was concluded that integration had worked, since a large percentage of the target population used condoms and 74% of the female population sought it as their responsibility to initiate the use of condoms. The success of this project was primarily attributed to the determination of the participants, and to the fact that the existing health services had already established a target population which was accessible to the health workers.^ieng