RESUMO
Venoms from three poneromorph ant species (Paraponera clavata, Ectatomma quadridens and Ectatomma tuberculatum) were investigated for the growth inhibition of Gram-positive and Gram-negative bacteria. It was shown that the venom of E. quadridens and its peptide fraction in particular possess marked antibacterial action. Three linear antimicrobial peptides sharing low similarity to the well-known ponericin peptides were isolated from this ant venom by means of size-exclusion and reversed-phase chromatography. The peptides showed antimicrobial activity at low micromolar concentrations. Their primary structure was established by direct Edman sequencing in combination with mass spectrometry. The most active peptide designated ponericin-Q42 was chemically synthesized. Its secondary structure was investigated in aqueous and membrane-mimicking environment, and the peptide was shown to be partially helical already in water, which is unusual for short linear peptides. Analysis of its activity on different bacterial strains, human erythrocytes and chronic myelogenous leukemia K562 cells revealed that the peptide shows broad spectrum cytolytic activity at micromolar and submicromolar concentrations. Ponericin-Q42 also possesses weak toxic activity on flesh fly larvae with LD50 of â¼105 µg/g.
Assuntos
Venenos de Formiga/química , Anti-Infecciosos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Venenos de Formiga/farmacologia , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Dicroísmo Circular , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Células K562/efeitos dos fármacos , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , Estrutura Secundária de ProteínaRESUMO
In addition to the conventional neurotoxins and cytotoxins, venom of the lynx spider Oxyopes takobius was found to contain two-domain modular toxins named spiderines: OtTx1a, 1b, 2a and 2b. These toxins show both insecticidal activity (a median lethal dose against flesh fly larvae of 75 µg·g(-1)) and potent antimicrobial effects (minimal inhibitory concentrations in the range 0.1-10 µm). Full sequences of the purified spiderines were established by a combination of Edman degradation, mass spectrometry and cDNA cloning. They are relatively large molecules (~ 110 residues, 12.0-12.5 kDa) and consist of two distinct modules separated by a short linker. The N-terminal part (~ 40 residues) contains no cysteine residues, is highly cationic, forms amphipathic α-helical structures in a membrane-mimicking environment, and shows potent cytolytic effects on cells of various origins. The C-terminal part (~ 60 residues) is disulfide-rich (five S-S bonds), and contains the inhibitor cystine knot (ICK/knottin) signature. The N-terminal part of spiderines is very similar to linear cytotoxic peptides found in various organisms, whereas the C-terminal part corresponds to the usual spider neurotoxins. We synthesized the modules of OtTx1a and compared their activity to that of full-length mature toxin produced recombinantly, highlighting the importance of the N-terminal part, which retained full-length toxin activity in both insecticidal and antimicrobial assays. The unique structure of spiderines completes the range of two-domain spider toxins.
Assuntos
Anti-Infecciosos/farmacologia , Dissulfetos/química , Venenos de Aranha/farmacologia , Aranhas/fisiologia , Staphylococcus aureus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Sequência de Bases , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Dissulfetos/metabolismo , Células HeLa , Hemólise/efeitos dos fármacos , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Fragmentos de Peptídeos/farmacologia , Estrutura Terciária de Proteína , Sarcofagídeos/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Venenos de Aranha/química , Venenos de Aranha/isolamento & purificaçãoRESUMO
A unique 30-residue cationic peptide oxyopinin 4a (Oxt 4a) was identified in the venom of the lynx spider Oxyopes takobius (Oxyopidae). Oxt 4a contains a single N-terminally located disulfide bond, Cys4-Cys10, and is structurally different from any spider toxin studied so far. According to NMR findings, the peptide is disordered in water, but assumes a peculiar torpedo-like structure in detergent micelles. It features a C-terminal amphipathic α-helical segment (body; residues 12-25) and an N-terminal disulfide-stabilized loop (head; residues 1-11), and has an unusually high density of positive charge in the head region. Synthetic Oxt 4a was produced and shown to possess strong and broad-spectrum cytolytic and antimicrobial activity. cDNA cloning showed that the peptide is synthesized in the form of a conventional prepropeptide with an acidic prosequence. Unlike other arachnid toxins, Oxt 4a exhibits striking similarity with defense peptides from the skin of ranid frogs that contain the so-called Rana-box motif (a C-terminal disulfide-enclosed loop). Parallelism or convergence is apparent on several levels: the structure, function and biosynthesis of a lynx spider toxin are mirrored by those of Rana-box peptides from frogs.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Inseticidas/farmacologia , Pele/metabolismo , Venenos de Aranha/química , Venenos de Aranha/farmacologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/síntese química , Bactérias/efeitos dos fármacos , Dicroísmo Circular , Clonagem Molecular , Cristalografia por Raios X , Hemólise/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Ranidae , Homologia de Sequência de Aminoácidos , AranhasRESUMO
Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a-c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.
Assuntos
Peptídeos/química , Venenos de Aranha/química , Venenos de Aranha/classificação , Aranhas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Dados de Sequência Molecular , Junção Neuromuscular/efeitos dos fármacos , Peptídeos/genética , Peptídeos/farmacologia , Estrutura Secundária de Proteína , Ranidae , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Venenos de Aranha/genética , Venenos de Aranha/farmacologia , Aranhas/anatomia & histologiaRESUMO
In silico structural analyses of sets of alpha-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Hemólise , Estrutura Secundária de Proteína , Venenos de Aranha , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Dados de Sequência Molecular , Mutação , Venenos de Aranha/química , Venenos de Aranha/genética , Venenos de Aranha/metabolismoRESUMO
Cytochrome c is one of the key proteins involved in the programmed cell death, and lysine 72 is known to be required for its apoptogenic activity. We have engineered a number of horse and murine cytochrome c single-point mutants with various substitutions at position 72 and compared quantitatively their proapoptotic activity in living cells. Apoptosis was activated by transferring exogenous cytochrome c into the cytoplasm of cells via a nontraumatic electroporation procedure. All mutant proteins studied exhibited significantly reduced proapoptotic activities in comparison with those for the wild type cytochromes. Relative activity of the horse (h(K72X)) and murine (m(K72W)) mutant proteins diminished in the order: h(K72R) > h(K72G) > h(K72A) > h(K72E) > h(K72L) >> h(K72W) > m(K72W). As estimated, the horse and murine K72W mutants were at least 200- and 500-fold less active than corresponding wild type proteins. Thus, the K72W-substituted cytochrome c can serve as an adequate candidate for knock-in studies of cytochrome c-mediated apoptosis. The proapoptotic activity of wild-type cytochrome c from different species in murine monocytic WEHI-3 cells reduced in the order: murine cytochrome c > human cytochrome c approximately horse cytochrome c, thus indicating that apoptotic effect of cytochrome c depends on the species compatibility.
Assuntos
Apoptose/efeitos dos fármacos , Citocromos c/genética , Citocromos c/farmacologia , Proteínas Mutantes/farmacologia , Substituição de Aminoácidos/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células HL-60 , Cavalos , Humanos , Células K562 , Lisina/genética , Camundongos , Especificidade da EspécieRESUMO
Eight linear cationic peptides with cytolytic and insecticidal activity, designated cyto-insectotoxins (CITs), were identified in Lachesana tarabaevi spider venom. The peptides showed antibiotic activity towards Gram-positive and Gram-negative bacteria at micromolar concentrations as well as toxicity to insects. The primary structures of the toxins were established by direct Edman sequencing in combination with enzymatic and chemical polypeptide degradation and MS. CITs represent a novel class of cytolytic molecules and spider venom toxins. They are the first example of molecules showing equally potent antimicrobial and insecticidal effects. Analysis of L. tarabaevi venom gland expressed sequence tag database revealed the primary structures of the protein precursors; eight peptides homologous with the purified toxins were additionally predicted. CIT precursors share a conventional prepropeptide structure with an acidic prosequence and a processing motif common to most spider toxin precursors. The most abundant peptide, CIT 1a, was chemically synthesized, and its lytic activity on different bacterial strains, human erythrocytes and lymphocytes, insect cells, planar lipid bilayers and lipid vesicles was characterized. The spider L. tarabaevi is suggested to have evolved to rely on a unique set of linear cytolytic toxins, as opposed to the more common disulfide-containing spider neurotoxins.
Assuntos
Peptídeos/química , Peptídeos/farmacologia , Venenos de Aranha/química , Venenos de Aranha/farmacologia , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/classificação , Alinhamento de Sequência , Venenos de Aranha/classificaçãoRESUMO
Seven novel short linear antimicrobial and cytolytic peptides named latarcins were purified from the venom of the spider Lachesana tarabaevi. These peptides were found to produce lytic effects on cells of diverse origin (Gram-positive and Gram-negative bacteria, erythrocytes, and yeast) at micromolar concentrations. In addition, five novel peptides that share considerable structural similarity with the purified latarcins were predicted from the L. tarabaevi venom gland expressed sequence tag data base. Latarcins were shown to adopt amphipathic alpha-helical structure in membrane-mimicking environment by CD spectroscopy. Planar lipid bilayer studies indicated that the general mode of action was scaled membrane destabilization at the physiological membrane potential consistent with the "carpet-like" model. Latarcins represent seven new structural groups of lytic peptides and share little homology with other known peptide sequences. For every latarcin, a precursor protein sequence was identified. On the basis of structural features, latarcin precursors were split into three groups: simple precursors with a conventional prepropeptide structure; binary precursors with a typical modular organization; and complex precursors, which were suggested to be cleaved into mature chains of two different types.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos/química , Venenos de Aranha/química , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Dicroísmo Circular , Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Moscas Domésticas , Bicamadas Lipídicas/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Homologia de Sequência de Aminoácidos , Venenos de Aranha/metabolismo , Venenos de Aranha/farmacologia , AranhasRESUMO
Reactive oxygen species generated by photosensitizers are efficacious remedy for tumor eradication. Eleven cycloimide derivatives of bacteriochlorin p (CIBCs) with different N-substituents at the fused imide ring and various substituents replacing the 3-acetyl group were evaluated as photosensitizers with special emphasis on structure-activity relationships. The studied CIBCs absorb light within a tissue transparency window (780-830 nm) and possess high photostability at prolonged light irradiation. The most active derivatives are 300-fold more phototoxic toward HeLa and A549 cells than the clinically used photosensitizer Photogem due to the substituents that improve intracellular accumulation (distribution ratio of 8-13) and provide efficient photoinduced singlet oxygen generation (quantum yields of 0.54-0.57). The substituents predefine selective CIBC targeting to lipid droplets, Golgi apparatus, and lysosomes or provide mixed lipid droplets and Golgi apparatus localization in cancer cells. Lipid droplets and Golgi apparatus are critically sensitive to photoinduced damage. The average lethal dose of CIBC-generated singlet oxygen per volume unit of cell was estimated to be 0.22 mM. Confocal fluorescence analysis of tissue sections of tumor-bearing mice revealed the features of tissue distribution of selected CIBCs and, in particular, their ability to accumulate in tumor nodules and surrounding connective tissues. Considering the short-range action of singlet oxygen, these properties of CIBCs are prerequisite to efficient antitumor photodynamic therapy.
Assuntos
Leucemia P388 , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacocinética , Porfirinas/farmacocinética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Feminino , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Humanos , Dose Letal Mediana , Leucemia P388/tratamento farmacológico , Leucemia P388/metabolismo , Leucemia P388/patologia , Lipídeos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Oxigênio Singlete/metabolismo , Distribuição TecidualRESUMO
Cytotoxins from cobra venom are known to manifest cytotoxicity in various cell types. It is widely accepted that the plasma membrane is a target of cytotoxins, but the mechanism of their action remains obscure. Using the confocal spectral imaging technique, we show for the first time that cytotoxins from cobra venom penetrate readily into living cancer cells and accumulate markedly in lysosomes. Cytotoxins CT1 and CT2 from Naja oxiana, CT3 from Naja kaouthia and CT1 from Naja haje are demonstrated to possess this property with respect to human lung adenocarcinoma A549 and promyelocytic leukaemia HL60 cells. Immobilized plasma membrane binding accompanies the internalization of CT3 from Naja kaouthia in the HL60 cells, but it is very weak for other cytotoxins. Detectable membrane binding is not a property of any of the cytotoxins tested in A549 cells. The kinetics and concentration-dependence of cytotoxin accumulation in lysosomes correlate well with their cytotoxic effects. On the basis of the results obtained, we propose that lysosomes are a primary target of the lytic action of cytotoxins. Plasma membrane permeabilization seems to be a downstream event relative to lysosome rupture. Direct damage to the plasma membrane may be a complementary mechanism, but its relative contribution to the cytotoxic action depends on the cytotoxin structure and cell type.
Assuntos
Citotoxinas/química , Citotoxinas/farmacologia , Venenos Elapídicos/química , Lisossomos/efeitos dos fármacos , Adenocarcinoma/metabolismo , Animais , Transporte Biológico Ativo , Morte Celular , Linhagem Celular Tumoral , Membrana Celular/química , Citotoxinas/metabolismo , Humanos , Leucemia Promielocítica Aguda/metabolismo , Neoplasias Pulmonares/metabolismo , Lisossomos/metabolismo , Ligação Proteica , Fatores de TempoRESUMO
Lipid rafts are specialized micro-domains of the plasma membrane enriched in glycosphingolipid and cholesterol that play important role in signal transduction, membrane trafficking, and cell adhesion. A distinct feature of lipid rafts is their resistance to solubilization with non-ionic detergent Triton X-100 (TX-100). In this study, we used flow cytometry to evaluate TX-100 resistance of 74 cell membrane molecules expressed on normal human peripheral blood lymphocytes (PBL), thymocytes, and 12 lymphoid cell lines. Resistance of membrane molecules to solubilization with TX-100 was determined by comparing the intensities of fluorescence of cells treated with TX-100 or left untreated. The majority of antigens analyzed were easily solubilized with TX-100 that resulted in decreased fluorescence intensity. However, a group of antigens showed TX-100 resistance in the range of 20-100%. These included all glycosylphosphatidylinositol (GPI)-anchored antigens under study, as well as some glycolipid and trans-membrane antigens. With the few exceptions, antigen resistance to solubilization with TX-100 was stable parameter, which did not depend on cell type in which it was analyzed. There was a good correspondence between the antigens showing resistance to solubilization with TX-100 as evaluated by our flow cytometry method, and the antigens that were previously demonstrated in detergent-resistant membranes using a more standard method of physical fractionation. Taken collectively, our data suggest that flow cytometry is a useful method for rapid evaluation of the possible association of a membrane antigen with lipid rafts.
Assuntos
Citometria de Fluxo , Linfócitos/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Octoxinol/farmacologia , Tensoativos/farmacologia , Anticorpos/imunologia , Antígenos de Superfície/efeitos dos fármacos , Antígenos de Superfície/imunologia , Biomarcadores , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Imunofluorescência , Humanos , Microscopia Confocal , SolubilidadeRESUMO
Translocation into detergent-insoluble microdomains (rafts) represents one of the earliest events in the process of cell activation, which follows the binding of surface receptor with natural ligand or mimicking antibody. In this study, the antibody-induced TX-100 resistance of surface antigens has been studied utilizing flow cytometry on TX-100 extracted cells. TX-100 resistance was evaluated by the ratio of antigen retained on the cells after detergent extraction compared with mAb-pretreated and untreated cells. All the antigens under study except CD98 demonstrated antibody-induced TX-100 resistance if the cells were treated with monoclonal antibodies and further cross-linked with secondary antibodies prior to lysis. CD20, CD5, and sIgM molecules were capable of transferring into TX-100-insoluble state in the absence of additional cross-linking. The experiments on modification of raft and cytoskeletal components of the cell, as well as the data on co-localization of TX-100-resistant antigens with raft and cytoskeletal markers strongly indicate that antibody-induced TX-100 resistance of antigens is mainly related to the translocation of antigens into lipid microdomains.
Assuntos
Anticorpos/imunologia , Antígenos de Superfície/metabolismo , Octoxinol/metabolismo , Animais , Antígenos de Superfície/efeitos dos fármacos , Antígenos de Superfície/imunologia , Linhagem Celular , Cetomacrogol/farmacologia , Colesterol/deficiência , Citometria de Fluxo , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Camundongos , Octoxinol/farmacologia , Tensoativos/farmacologiaRESUMO
asCP, the unique green fluorescent protein-like nonfluorescent chromoprotein from the sea anemone Anemonia sulcata, becomes fluorescent ("kindles") upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be "quenched" instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr(66) in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.