RESUMO
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
Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.
Assuntos
Diacilglicerol O-Aciltransferase/química , Lipossomas Unilamelares/química , Sítios de Ligação , Diacilglicerol O-Aciltransferase/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ligação Proteica , Eletricidade Estática , Lipossomas Unilamelares/metabolismoRESUMO
Mo-CBP3 is a chitin-binding protein purified from Moringa oleifera Lam. seeds that displays inhibitory activity against phytopathogenic fungi. This study investigated the structural properties and the antifungal mode of action of this protein. To this end, circular dichroism spectroscopy, antifungal assays, measurements of the production of reactive oxygen species and microscopic analyses were utilized. Mo-CBP3 is composed of 30.3% α-helices, 16.3% ß-sheets, 22.3% turns and 30.4% unordered forms. The Mo-CBP3 structure is highly stable and retains its antifungal activity regardless of temperature and pH. Fusarium solani was used as a model organism for studying the mechanisms by which this protein acts as an antifungal agent. Mo-CBP3 significantly inhibited spore germination and mycelial growth at 0.05 mg.mL-1. Mo-CBP3 has both fungistatic and fungicidal effects, depending on the concentration used. Binding of Mo-CBP3 to the fungal cell surface is achieved, at least in part, via electrostatic interactions, as salt was able to reduce its inhibitory effect. Mo-CBP3 induced the production of ROS and caused disorganization of both the cytoplasm and the plasma membrane in F. solani cells. Based on its high stability and specific toxicity, with broad-spectrum efficacy against important phytopathogenic fungi at low inhibitory concentrations but not to human cells, Mo-CBP3 has great potential for the development of new antifungal drugs or transgenic crops with enhanced resistance to phytopathogens.
Assuntos
Antifúngicos/química , Quitina/metabolismo , Moringa oleifera/química , Proteínas de Plantas/química , Antifúngicos/farmacologia , Colletotrichum/efeitos dos fármacos , Fusarium/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Ligação Proteica , Estabilidade Proteica , Sementes/química , Esporos Fúngicos/efeitos dos fármacosRESUMO
Plantaricin149a (Pln149a) is a cationic antimicrobial peptide, which was suggested to cause membrane destabilization via the carpet mechanism. The mode of action proposed to this antimicrobial peptide describes the induction of an amphipathic α-helix from Ala7 to Lys20, while the N-terminus residues remain in a coil conformation after binding. To better investigate this assumption, the purpose of this study was to determine the contributions of the Tyr1 in Pln149a in the binding to model membranes to promote its destabilization. The Tyr to Ser substitution increased the dissociation constant (KD) of the antimicrobial peptide from the liposomes (approximately three-fold higher), and decreased the enthalpy of binding to anionic vesicles from -17.2 kcal/mol to -10.2 kcal/mol. The peptide adsorption/incorporation into the negatively charged lipid vesicles was less effective with the Tyr1 substitution and peptide Pln149a perturbed the liposome integrity more than the analog, Pln149S. Taken together, the peptide-lipid interactions that govern the Pln149a antimicrobial activity are found not only in the amphipathic helix, but also in the N-terminus residues, which take part in enthalpic contributions due to the allocation at a lipid-aqueous interface.
Assuntos
Bacteriocinas/química , Lipossomos/química , Fosfolipídeos/química , Tirosina/metabolismo , Sequência de Aminoácidos , Anti-Infecciosos/farmacologia , Calorimetria , Dicroísmo Circular , Hemólise/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Naftalenos/metabolismo , Peptídeos/síntese química , Peptídeos/química , Peptídeos/isolamento & purificação , Fosfatidilgliceróis/química , Compostos de Piridínio/metabolismo , Espectrometria de Fluorescência , Relação Estrutura-Atividade , TriptofanoRESUMO
Recent results from our laboratory have previously shown the purification of a small serine proteinase inhibitor (PI), named CaTI1, from Capsicum annuum seeds. This work demonstrated the characterization of CaTI now named CaTI1, and the identification of two other small serine PIs, named CaTI2 and CaTI3, also present in these seeds. CaTI1 presented molecular mass of 6 kDa and pI value of â¼9.0. CaTI1 inhibited both trypsin and chymotrypsin with inhibition constants (Ki and Ki') of 14 and 2.8 nM for trypsin and 4.3 and 0.58 nM for chymotrypsin, respectively. Circular dichroism analysis suggested the predominance of both disordered and ß-strands regions in the secondary structure. CaTI1 presented striking physico-chemical stability. In an attempt to get the entire sequence of CaTI1 we found another PI called CaTI2. The discussion of this finding is in the main text. A degenerate primer was designed based on the sequence of trypsin inhibitor CaTI1 in an attempt to achieve the cloning of this PI. Surprisingly, the alignment of the predicted peptide derived from the cDNA with the protein database showed similarity with other C. annuun PIs, and thus it was called CaTI3.
Assuntos
Capsicum , DNA Complementar , Sequência de Aminoácidos , Clonagem Molecular , Dados de Sequência Molecular , Sementes/química , Tripsina/metabolismo , Inibidores da Tripsina/químicaRESUMO
BACKGROUND: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). METHODS: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. RESULTS: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.564.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% ß-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx. GENERAL SIGNIFICANCE: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.
Assuntos
Antioxidantes/metabolismo , Cisteína/química , Fabaceae/metabolismo , Chaperonas Moleculares/metabolismo , Peroxirredoxinas/isolamento & purificação , Peroxirredoxinas/metabolismo , Folhas de Planta/metabolismo , Sequência de Aminoácidos , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Fabaceae/crescimento & desenvolvimento , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Dados de Sequência Molecular , Oxirredução , Folhas de Planta/crescimento & desenvolvimento , Homologia de Sequência de Aminoácidos , Espectrometria de Massas em TandemRESUMO
Frutalin is a homotetrameric alpha-d-galactose (d-Gal)-binding lectin that activates natural killer cells in vitro and promotes leukocyte migration in vivo. Because lectins are potent lymphocyte stimulators, understanding the interactions that occur between them and cell surfaces can help to the action mechanisms involved in this process. In this paper, we present a detailed investigation of the interactions of frutalin with phospho- and glycolipids using Langmuir monolayers as biomembrane models. The results confirm the specificity of frutalin for d-Gal attached to a biomembrane. Adsorption of frutalin was more efficient for the galactose polar head lipids, in contrast to the one for sulfated galactose, in which a lag time is observed, indicating a rearrangement of the monolayer to incorporate the protein. Regarding ganglioside GM1 monolayers, lower quantities of the protein were adsorbed, probably due to the farther apart position of d-galactose from the interface. Binary mixtures containing galactocerebroside revealed small domains formed at high lipid packing in the presence of frutalin, suggesting that lectin induces the clusterization and the forming of domains in vitro, which may be a form of receptor internalization. This is the first experimental evidence of such lectin effect, and it may be useful to understand the mechanism of action of lectins at the molecular level.
Assuntos
Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Adsorção , Artocarpus/química , Sítios de Ligação , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Elasticidade , Gangliosídeo G(M1)/química , Gangliosídeo G(M1)/metabolismo , Galactose/metabolismo , Humanos , Técnicas In Vitro , Fluidez de Membrana , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Membranas Artificiais , Modelos Biológicos , Ligação ProteicaRESUMO
Lectins have been classified into a structurally diverse group of proteins that bind carbohydrates and glycoconjugates with high specificity. They are extremely useful molecules in the characterization of saccharides, as drug delivery mediators, and even as cellular surface makers. In this study, we present camptosemin, a new lectin from Camptosema ellipticum. It was characterized as an N-acetyl-D-galactosamine-binding homo-tetrameric lectin, with a molecular weight around 26 kDa/monomers. The monomers were stable over a wide range of pH values and exhibited pH-dependent oligomerization. Camptosemin promoted adhesion of breast cancer cells and hemagglutination, and both activities were inhibited by its binding of sugar. The stability and unfolding/folding behavior of this lectin was characterized using fluorescence and far-UV circular dichroism spectroscopies. The results indicate that chemical unfolding of camptosemin proceeds as a two-state monomer-tetramer process. In addition, small-angle X-ray scattering shows that camptosemin behaves as a soluble and stable homo-tetramer molecule in solution.
Assuntos
Fabaceae/química , Lectinas de Plantas/química , Acetilgalactosamina/química , Neoplasias da Mama/química , Carboidratos/química , Adesão Celular , Linhagem Celular Tumoral , Dicroísmo Circular , Feminino , Gleiquênias , Hemaglutinação , Humanos , Concentração de Íons de Hidrogênio , Peso Molecular , Lectinas de Plantas/genética , Lectinas de Plantas/isolamento & purificação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Raios Ultravioleta , Difração de Raios XRESUMO
Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final and dedicated step in the synthesis of triacylglycerol, which is believed to involve the lipids oleoyl coenzyme A (OCoA) and dioleoyl-sn-glycerol (DOG) as substrates. In this work we investigated the interaction of a specific peptide, referred to as SIT2, on the C-terminal of DGAT1 (HKWCIRHFYKP) with model membranes made with OCoA and DOG in Langmuir monolayers and liposomes. According to the circular dichroism and fluorescence data, conformational changes on SIT2 were seen only on liposomes containing OCoA and DOG. In Langmuir monolayers, SIT2 causes the isotherms of neat OCoA and DOG monolayers to be expanded, but has negligible effect on mixed monolayers of OCoA and DOG. This synergistic interaction between SIT2 and DOG+OCoA may be rationalized in terms of a molecular model in which SIT2 may serve as a linkage between the two lipids. Our results therefore provide molecular-level evidence for the interaction between this domain and the substrates OCoA and DOG for the synthesis of triacylglycerol.
Assuntos
Acil Coenzima A/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Diglicerídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Acil Coenzima A/química , Animais , Bovinos , Dicroísmo Circular , Diacilglicerol O-Aciltransferase/química , Diglicerídeos/química , Lipossomos , Fragmentos de Peptídeos/química , Conformação ProteicaRESUMO
The action of a synthetic antimicrobial peptide analog of Plantaricin 149 (Pln149a) against Saccharomyces cerevisiae and its interaction with biomembrane model systems were investigated. Pln149a was shown to inhibit S. cerevisiae growth by more than 80% in YPD medium, causing morphological changes in the yeast wall and remaining active and resistant to the yeast proteases even after 24 h of incubation. Different membrane model systems and carbohydrates were employed to better describe the Pln149a interaction with cellular components using circular dichroism and fluorescence spectroscopies, adsorption kinetics and surface elasticity in Langmuir monolayers. These assays showed that Pln149a does not interact with either mono/polysaccharides or zwitterionic LUVs, but is strongly adsorbed to and incorporated into negatively charged surfaces, causing a conformational change in its secondary structure from random-coil to helix upon adsorption. From the concurrent analysis of Pln149a adsorption kinetics and dilatational surface elasticity data, we determined that 2.5 muM is the critical concentration at which Pln149a will disrupt a negative DPPG monolayer. Furthermore, Pln149a exhibited a carpet-like mechanism of action, in which the peptide initially binds to the membrane, covering its surface and acquiring a helical structure that remains associated to the negatively charged phospholipids. After this electrostatic interaction, another peptide region causes a strain in the membrane, promoting its disruption.
Assuntos
Bacteriocinas/farmacologia , Membrana Celular/efeitos dos fármacos , Bicamadas Lipídicas/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/metabolismo , Lipossomos/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Tensão SuperficialRESUMO
Two aspartyl proteases activities were identified and isolated from Trypanosoma cruzi epimastigotes: cruzipsin-I (CZP-I) and cruzipsin-II (CZP-II). One was isolated from a soluble fraction (CZP-II) and the other was solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CZP-I). The molecular mass of both proteases was estimated to be 120 kDa by HPLC gel filtration and the activity of the enzymes was detected in a doublet of bands (56 and 48 kDa) by substrate-sodium dodecyl sulphate-polyacrylamide-gelatin gel electrophoresis. Substrate specificity studies indicated that the enzymes consistently hydrolyze the cathepsin D substrate Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu-O4MP but failed to hydrolyze serine and other protease substrates. Both proteases activities were strongly inhibited by the classic inhibitor pepstatin-A (> or =68%) and the aspartic active site labeling agent, 1,2-epoxy-3-(phenyl-nitrophenoxy) propane (> or =80%). These findings show that both proteases are novel T. cruzi acidic proteases. The physiological function of these enzymes in T. cruzi has under investigation.
Assuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/isolamento & purificação , Trypanosoma cruzi/enzimologia , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Cromatografia Líquida de Alta Pressão , Compostos Cromogênicos/metabolismo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Peso Molecular , Especificidade por SubstratoRESUMO
The proline-rich N-terminal domain of gamma-zein has been reported in relevant processes, which include its ability to cross the cell membranes. Evidences indicate that synthetic hexapeptide (PPPVHL), naturally found in N-terminal portion of gamma-zein, can adopt the polyproline II (PPII) conformation in aqueous solution. The secondary structure of gamma-zein in maize protein bodies had been analyzed by solid state Fourier transform infrared and nuclear magnetic resonance spectroscopies. However, it was not possible to measure PPII content in physiological environment since the beta-sheet and PPII signals overlap in both solid state techniques. Here, the secondary structure of gamma-zein has been analyzed by circular dichroism in SDS aqueous solution with and without ditiothreitol (DTT), and in 60% of 2-propanol and water with DTT. The results show that gamma-zein has high helical content in all solutions. The PPII conformation was present at about 7% only in water/DTT solution.
Assuntos
Dicroísmo Circular , Zea mays/química , Zeína/química , Zeína/isolamento & purificação , Motivos de Aminoácidos , Dicroísmo Circular/métodos , Ditiotreitol/química , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/química , Prolina , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Dodecilsulfato de Sódio/química , Soluções/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Zea mays/classificaçãoRESUMO
The major core protein of equine infectious anemia virus (EIAV), p26, is one of the primary immunogenic structural proteins during a persistent infection of horses and is highly conserved among antigenically variants of viral isolates. In order to investigate its immune profile in more detail for a better diagnostic, an epitope mapping was carried out by means of two libraries of overlapping peptide fragments prepared by simultaneous and parallel SPPS on derivatized cellulose membranes (SPOT synthesis). Polyclonal equine sera from infected horses were used for the biological assay. Particularly two promising continuous epitopes (NAMRHL and MYACRD) were localized on the C-terminal extreme of p26, region 194-222. A cyclic synthetic fragment of 29 amino acid residues containing the identified epitopes was designed and studied. A significant conformational change towards a helical structure was observed when the peptide was cyclized by a bridge between Cys198 and Cys218. This observation correlated with an improvement of its ability to be recognized by specific antibodies in an EIA (Enzyme-linked Immunosorbent assay). These results suggest that the conformationally restricted synthetic antigen adequately mimics the native structure of this region of p26 core protein.
Assuntos
Mapeamento de Epitopos/métodos , Proteínas do Core Viral/análise , Proteínas do Core Viral/imunologia , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Análise Serial de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismoRESUMO
A trypsin inhibitor was purified from Calliandra selloi Macbride seeds (CSTI). SDS-PAGE under non-reducing conditions showed a single band of approximately 20,000 Da, while under reducing conditions two bands of 16,000 and 6000 Da were observed, indicating that CSTI consists of two polypeptide chains. Molecular masses of 20,078 and 20,279 were obtained by mass spectrometry, although only one pI of 4.0 was observed and one peak was obtained by reversed phase chromatography. Amino-terminal sequence analysis showed homology to Kunitz-type inhibitors. CSTI was able to inhibit trypsin (Ki 2.21 x 10(-7)M), alpha-chymotrypsin (Ki 4.95 x 10(-7)M) and kallikrein (Ki 4.20 x 10(-7)M) but had no effect on elastase. Trypsin inhibitory activity was stable over a wide range of pH and temperature. CSTI was particularly susceptible to DTT treatment, followed by addition of iodoacetamide. Far-UV circular dichroism measurements revealed that CSTI is a beta-II protein. Thermal unfolding showed a two-state transition with a midpoint at 68 degrees C. Far-UV CD spectra of CSTI at pH extremes showed little changes, while more pronounced differences in near-UV CD spectra were detected. Remarkably, treatment with 1mM DTT caused very slight changes in the far-UV CD spectrum, and only after carbamidomethylation was there was a marked loss observed in secondary structure.
Assuntos
Fabaceae , Sementes/enzimologia , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificação , Animais , Células Cultivadas , Eritrócitos/efeitos dos fármacos , Temperatura Alta , Concentração de Íons de Hidrogênio , Peso Molecular , Coelhos , Espectrometria de Fluorescência , Inibidores da Tripsina/farmacologiaRESUMO
Plantaricin-149 is a bacteriocin produced by Lactobacillus plantarum NRIC 149 (a LAB isolated from pineapple), which consists of a peptidic chain made up of 22 amino acid residues [Kato et al. J. Ferment. Bioeng. 1994; 77: 277-282]. In this work, a synthetic C-terminal amidated peptide analog denoted Pln149a was prepared by SPPS-Fmoc chemistry and the antagonistic activity against gram-positive and gram-negative bacteria was tested. The secondary structure was studied by circular dichroism (CD) and the vicinity of the tyrosine residue by fluorescence spectroscopy under different conditions. We report the results of the interaction of Pln149a with reverse micelles prepared from the amphiphilic AOT in cyclohexane. Synthetic plantaricin was active against one strain of Staphylococcus aureus and four strains of Listeria genus at pH 5.5 and 7.4 and, like its natural variant, inhibited L. plantarum ATCC 8014. The data derived from spectroscopic measurements in presence of AOT reverse micelles suggest that the secondary structure of the peptide upon interaction is an alpha-helix. In this membrane model, the hydrophobic side of the alpha-helix is inserted into the micelles, leaving the lysines exposed to the solvent and interacting with the polar moieties of AOT. The fluorescence data point out that the N-terminal tyrosine residue is close to the micellar interface.
Assuntos
Bacteriocinas/farmacologia , Lactobacillus plantarum/efeitos dos fármacos , Listeria/efeitos dos fármacos , Estrutura Secundária de Proteína , Staphylococcus aureus/efeitos dos fármacos , Dicroísmo Circular , Microbiologia de Alimentos , Micelas , Testes de Sensibilidade Microbiana , Modelos Moleculares , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The ubiquitin system represents a selective mechanism for intracellular proteolysis in eukaryotic cells that involves the sequential activity of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). The identification of these proteins and their cellular targets, as well as structural data, are essential to understanding how this system operates in the eukaryotic cell. In the present study, the open reading frame of the human ubiquitin-conjugating enzyme UBE2G2 was isolated from a human brain cDNA panel, cloned into pET28a vector and expressed in Escherichia coli. The His-tagged protein was then purified through nickel-affinity chromatography and subjected to structural and functional studies using circular dichroism (CD) and an in vitro ubiquitin-binding assay, respectively. Our results showed that the production of the HISUBE2G2 protein in bacteria, carried out with 0.1 mM of IPTG at 30 degrees C, was successfully achieved, rendering high concentrations of soluble, pure and stable enzyme after a single purification step. The recombinant protein was able to bind ubiquitin molecules when exposed to a HeLa cell extract during the ubiquitin assay. Moreover, the fact that HISUBE2G2 was expressed in its active form is supported by the typical alpha/beta secondary structure specific to other class I E2 enzymes displayed during the CD assay.
Assuntos
Enzimas de Conjugação de Ubiquitina , Dicroísmo Circular , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Células HeLa , Humanos , Fases de Leitura Aberta , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/isolamento & purificação , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Two novel chitin-binding lectins from seeds of Artocarpus genus were described in this paper, one from A. integrifolia (jackfruit) and one from A. incisa (breadfruit). They were purified from saline crude extract of seeds using affinity chromatography on chitin column, size-exclusion chromatography and reverse-phase chromatography on the C-18 column. Both are 14 kDa proteins, made up of 3 chains linked by disulfide bonds. The partial amino acid sequences of the two lectins showed they are homologous to each other but not to other plant chitin-binding proteins. Thus, they cannot be classified in any known plant chitin-binding protein family, particularly because of their inter-chain covalent bonds. Their circular dichroism spectra and deconvolution showed a secondary structure content of beta-sheet and unordered elements. The lectins were thermally stable until 80 degrees C and structural changes were observed below pH 6. Both lectins inhibited the growth of Fusarium moniliforme and Saccharomyces cerevisiae, and presented hemagglutination activity against human and rabbit erythrocytes. These lectins were denoted jackin (from jackfruit) and frutackin (from breadfruit).
Assuntos
Antifúngicos/química , Artocarpus/química , Lectinas de Plantas/química , Sequência de Aminoácidos , Animais , Antifúngicos/farmacologia , Aspergillus niger/efeitos dos fármacos , Quitina/química , Dicroísmo Circular , Fusarium/efeitos dos fármacos , Testes de Hemaglutinação , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Lectinas de Plantas/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Coelhos , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase ( K i(app) 5.3 nM), porcine pancreatic elastase ( K i(app) 40 nM), cathepsin G ( K i(app) 160 nM) and the cysteine proteinases cruzipain ( K i(app) 1.2 nM), cruzain ( K i(app) 0.3 nM) and cathepsin L ( K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein ( K i(app) 2.4 nM) and plasmin ( K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the beta-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.
Assuntos
Inibidores de Cisteína Proteinase/genética , Proteínas de Plantas/genética , Inibidores de Serina Proteinase/genética , Sequência de Aminoácidos , Sequência de Bases , Bauhinia , Sítios de Ligação , Catepsina L , Catepsinas/antagonistas & inibidores , Dicroísmo Circular , Clonagem Molecular , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , DNA Complementar/isolamento & purificação , DNA de Plantas/análise , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Peptídeos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Proteínas de Protozoários , RNA de Plantas/análise , Proteínas Recombinantes/farmacologia , Sementes/química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Inibidores de Serina Proteinase/farmacologiaRESUMO
Pulchellin is a type 2 ribosome-inactivating protein isolated from seeds of the Abrus pulchellus tenuiflorus plant. This study aims to obtain active and homogeneous protein for structural and biological studies that will clarify the functional aspects of this toxin. The DNA fragment encoding pulchellin A-chain was cloned and inserted into pGEX-5X to express the recombinant pulchellin A-chain (rPAC) as a fusion protein in Escherichia coli. The deduced amino acid sequence analyses of the rPAC presented a high sequential identity (> 86%) with the A-chain of abrin-c. The ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. In order to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reduced/oxidized system, yielding an active heterodimer (rPAB). The rPAB showed an apparent molecular mass of approximately 60 kDa, similar to the native pulchellin. The toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection of different dilutions into mice. The rPAB was able to kill 50% of the tested mice with doses of 45 microg x kg(-1). Our results indicated that the heterodimer showed toxic activity and a conformational pattern similar to pulchellin. In addition, rPAC produced in this heterologous system might be useful for the preparation of immunoconjugates with potential as a therapeutic agent.
Assuntos
Abrus/genética , Proteínas de Plantas/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribossomos/metabolismo , Abrus/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA de Plantas/genética , DNA de Plantas/metabolismo , Injeções Intraperitoneais , Camundongos , Dados de Sequência Molecular , N-Glicosil Hidrolases/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/toxicidade , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/toxicidade , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/toxicidade , Saccharomyces cerevisiae/metabolismo , Sementes/química , Homologia de Sequência de AminoácidosRESUMO
The study of interactions between biological molecules and model membranes is essential for the understanding of a number of physiological mechanisms involved in viral infections and dissemination. In this paper, the analysis of the interaction between a peptide from the p24 protein of Human Immunodeficiency Virus type 1 (HIV-1) and a phospholipid monolayer has pointed to a cooperative response in which very small amounts of peptide p24-1 (e.g. 0.05 mol%) can lead to measurable effects. Monolayer surface pressure and surface potential isotherms were affected for peptide concentrations as low as 0.05 mol%, with saturation at 0.5 mol%. The expansion effect from p24-1 is confirmed by changes in morphology of the monolayers using Brewster angle microscopy. Even though p24-1 is disordered in aqueous solutions, the interaction with dipalmitoyl phosphatidylcholine (DPPC) causes it to adopt an alpha-helix structure, as shown by circular dichroism (CD) data for multilamellar vesicles (MLV). The expansion of the phospholipid monolayer in a cooperative way may imply that p24-1 has potential antiviral activity, by participating in the cell rupture, with no need of specific receptors in the membrane.