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1.
BMC Complement Med Ther ; 23(1): 132, 2023 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-37098530

RESUMEN

BACKGROUND: Apitherapy is an emerging field in cancer research, particularly in developing communities. The potency of Melittin (MEL), a major constituent in bee venom is accounted for the cytotoxic capacity against cancer cells. It is postulated that the genotype of bees and the time of venom collection influences its specific activity against certain types of cancer. METHOD: Hereby, Jordanian crude bee venom (JCBV) was collected during different seasons of the year, specifically spring, summer and autumn and investigated for in vitro antitumour effects. Venom collected during springtime comprised the highest quantity of MEL in comparison to venom collected some other time. Springtime-collected JCBV extract and MEL were tested on an immortal myelogenous leukaemia cell line, namely K562 leukemic cells. Treated cells were examined for cell modality via flow cytometry analysis and cell death mediating gene expressions. RESULTS: Springtime-collected JCBV extract and MEL showed an IC50 of 3.7 ± 0.37 µg/ml and 1.84 ± 0.75 µg/ml, respectively. In comparison to JCBV and positive control, MEL-treated cells exhibited late apoptotic death with a moderate cellular arrest at G0/G1 and an increase of cell number at G2/M phase. Expression of NF-κB/MAPK14 axis was inhibited in MEL and JCBV-treated cells, as well as expression of c-MYC and CDK4. Moreover, marked upregulation in ABL1, JUN and TNF was observed. In conclusion, springtime-collected JCBV showed the highest content of MEL while both JCBV and pure MEL showed apoptotic, necrotic, and cell cycle arrest efficiency against K562 leukemic cells. CONCLUSION: Integration of bee venom in chemotherapy needs more investigation and should be carefully translated into clinical use. During such translation, the correlation of bee genotype, collection time and concentration of MEL in CBV should be profiled.


Asunto(s)
Venenos de Abeja , Leucemia , Humanos , Abejas , Animales , Meliteno/farmacología , Meliteno/química , Meliteno/genética , Venenos de Abeja/farmacología , Células K562 , Péptidos , Leucemia/tratamiento farmacológico
2.
Toxins (Basel) ; 14(12)2022 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-36548715

RESUMEN

The venom of honeybees is composed of numerous peptides and proteins and has been used for decades as an anti-inflammatory and anti-cancer agent in traditional medicine. However, the bioactivity of specific biomolecular components has been evaluated for the predominant constituent, melittin. So far, only a few melittin-like peptides from solitary bee species have been investigated, and the molecular mechanisms of bee venoms as therapeutic agents remain largely unknown. Here, the preclinical pharmacological activities of known and proteo-transcriptomically discovered new melittin variants from the honeybee and more ancestral variants from phylogenetically older solitary bees were explored in the context of cancer and inflammation. We studied the effects of melittin peptides on cytotoxicity, second messenger release, and inflammatory markers using primary human cells, non-cancer, and cancerous cell lines. Melittin and some of its variants showed cytotoxic effects, induced Ca2+ signaling and inhibited cAMP production, and prevented LPS-induced NO synthesis but did not affect the IP3 signaling and pro-inflammatory activation of endothelial cells. Compared to the originally-described melittin, some phylogenetically more ancestral variants from solitary bees offer potential therapeutic modalities in modulating the in vitro inflammatory processes, and hindering cancer cell viability/proliferation, including aggressive breast cancers, and are worth further investigation.


Asunto(s)
Antiinflamatorios , Antineoplásicos , Venenos de Abeja , Abejas , Meliteno , Animales , Humanos , Venenos de Abeja/farmacología , Venenos de Abeja/química , Células Endoteliales , Meliteno/química , Meliteno/aislamiento & purificación , Meliteno/farmacología , Antiinflamatorios/química , Antiinflamatorios/aislamiento & purificación , Antiinflamatorios/farmacología , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Línea Celular Tumoral
3.
Amino Acids ; 54(9): 1275-1285, 2022 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-35779173

RESUMEN

The emergence of multidrug-resistant (MDR) bacteria is a major challenge for antimicrobial chemotherapy. Concerning this issue, antimicrobial peptides (AMPs) have been presented as novel promising antibiotics. Our previous de novo designed melittin-derived peptides (MDP1 and MDP2) indicated their potential as peptide drug leads. Accordingly, this study was aimed to evaluate the kinetics of activity, toxicity, and stability of MDP1 and MDP2 as well as determination of their structures. The killing kinetics of MDP1 and MDP2 demonstrate that all bacterial strains were rapidly killed. MDP1 and MDP2 were ca. 100- and 26.6-fold less hemolytic than melittin and found to be respectively 72.9- and 41.6-fold less cytotoxic than melittin on the HEK293 cell line. MDP1 and MDP2 showed 252- and 132-fold improvement in their therapeutic index in comparison to melittin. MDP1 and MDP2 sustained their activities in the presence of human plasma and were found to be ca. four to eightfold more stable than melittin. Spectropolarimetry analysis of MDP1 and MDP2 indicates that the peptides adopt an alpha-helical structure predominantly. According to the fast killing kinetics, significant therapeutic index, and high stability of MDP1, it could be considered as a drug lead in a mouse model of septicemia infections.


Asunto(s)
Péptidos Antimicrobianos , Meliteno , Animales , Antibacterianos/química , Células HEK293 , Humanos , Cinética , Meliteno/química , Meliteno/farmacología , Ratones , Pruebas de Sensibilidad Microbiana , Péptidos/química , Índice Terapéutico
4.
ACS Nano ; 13(11): 12638-12652, 2019 11 26.
Artículo en Inglés | MEDLINE | ID: mdl-31625721

RESUMEN

Photodynamic therapy (PDT) is a clinical cancer treatment modality based on the induction of therapeutic reactive oxygen species (ROS), which can trigger immunogenic cell death (ICD). With the aim of simultaneously improving both PDT-mediated intracellular ROS production and ICD levels, we designed a serum albumin (SA)-coated boehmite ("B"; aluminum hydroxide oxide) organic-inorganic scaffold that could be loaded with chlorin e6 (Ce6), a photosensitizer, and a honey bee venom melittin (MLT) peptide, denoted Ce6/MLT@SAB. Ce6/MLT@SAB was anchored by a boehmite nanorod structure and exhibited particle size of approximately 180 nm. Ce6/MLT@SAB could significantly reduce hemolysis relative to that of free MLT, while providing MLT-enhanced PDT antitumor effects in vitro. Compared with Ce6@SAB, Ce6/MLT@SAB improved Ce6 penetration of cancer cells both in vitro and in vivo, thereby providing enhanced intracellular ROS generation with 660 nm light treatment. Following phototreatment, Ce6/MLT@SAB-treated cells displayed significantly improved levels of ICD and abilities to activate dendritic cells. In the absence of laser irradiation, multidose injection of Ce6/MLT@SAB could delay the growth of subcutaneous murine tumors by more than 60%, compared to controls. When combined with laser irradiation, a single injection and phototreatment with Ce6/MLT@SAB eradicated one-third of subcutaneous tumors in treated mice. The addition of an immune checkpoint blockade to Ce6/MLT@SAB phototreatment further augmented antitumor effects, generating increased numbers of CD4+ and CD8+ T cells in tumors with concomitant reduction of myeloid-derived suppressor cells.


Asunto(s)
Antineoplásicos , Inmunoterapia/métodos , Meliteno , Nanopartículas/química , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Femenino , Muerte Celular Inmunogénica/efectos de los fármacos , Meliteno/química , Meliteno/farmacocinética , Meliteno/farmacología , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/patología , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacocinética , Fármacos Fotosensibilizantes/farmacología
5.
ACS Appl Mater Interfaces ; 9(31): 25755-25766, 2017 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-28714303

RESUMEN

The design of biocompatible and efficacious anticancer biomaterials to achieve relatively low tumor recurrence rates is the main pursuit of cancer photothermal therapy (PTT). RADA16-I is a synthetic amphiphilic peptide with the sequence RADARADARADARADA that can self-assemble into a peptide nanofiber hydrogel. In this study, we synthesized a novel melittin-RADA32-indocyanine green (ICG) hydrogel ("MRI hydrogel"), which contains melittin in the peptide hydrogel backbone and ICG in the hydrogel matrix, for enhanced PTT of glioblastomas. The MRI hydrogel exhibited physiologic characteristics similar to those of the RADA16 hydrogel, while displaying concentration-dependent cytotoxicity to C6 glioma cells and photothermal effects. The in vivo biodistribution of the MRI hydrogel was visualized by near-infrared fluorescence and photoacoustic imaging. More importantly, in vivo PTT provided by the MRI hydrogel significantly reduced the tumor size and the tumor recurrence rate compared with the RADR16-ICG hydrogel and other controls, suggesting a synergistic effect of MRI hydrogel-carried melittin and ICG-based PTT treatment. Thus, MRI provides an alternative tool for the safe and efficient PTT treatment of tumors.


Asunto(s)
Meliteno/química , Glioblastoma , Humanos , Hidrogeles , Fototerapia , Distribución Tisular
6.
Cancer Lett ; 402: 16-31, 2017 08 28.
Artículo en Inglés | MEDLINE | ID: mdl-28536009

RESUMEN

Melittin (MEL), a major peptide component of bee venom, is an attractive candidate for cancer therapy. This agent has shown a variety of anti-cancer effects in preclinical cell culture and animal model systems. Despite a convincing efficacy data against variety of cancers, its applicability to humans has met with challenges due to several issues including its non-specific cytotoxicity, degradation and hemolytic activity. Several optimization approaches including utilization of nanoparticle based delivery of MEL have been utilized to circumvent the issues. Here, we summarize the current understanding of the anticancer effects of bee venom and MEL on different kinds of cancers. Further, we also present the available information for the possible mechanism of action of bee venom and/or MEL.


Asunto(s)
Antineoplásicos/uso terapéutico , Meliteno/uso terapéutico , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/química , Portadores de Fármacos , Composición de Medicamentos , Estabilidad de Medicamentos , Humanos , Meliteno/efectos adversos , Meliteno/análogos & derivados , Meliteno/química , Nanopartículas , Nanotecnología/métodos , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal/efectos de los fármacos
7.
Eur J Pharm Sci ; 99: 209-218, 2017 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-28024889

RESUMEN

Prostate cancer is the second leading cause of death due to cancer in men. Owing to shortcomings in the current treatments, other therapies are being considered. Toxic gene delivery is one of the most effective methods for cancer therapy. Cationic polymers are able to form stable nanoparticles via interaction with nucleic acids electrostatically. Branched polyethylenimine that contains amine groups has notable buffering capacity and the ability to escape from endosome through the proton sponge effect. However, the cytotoxicity of this polymer is high, and modification is one of the applicable strategies to overcome this problem. In this study, PEI was targeted with chlorotoxin (CTX) via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) cross-linker. CTX can bind specifically to matrix metalloproteinase-2 that is overexpressed in certain cancers. Melittin as the major component of bee venom has been reported to have anti-cancer activity. This was thus selected to deliver to PC3 cell line. Flow cytometry analysis revealed that transfection efficiency of targeted nanoparticles is significantly higher compared to non-targeted nanoparticles. Targeted nanoparticles carrying the melittin gene also decreased cell viability of PC3 cells significantly while no toxic effects were observed on NIH3T3 cell line. Therefore, CTX-targeted nanoparticles carrying the melittin gene could serve as an appropriate gene delivery system for prostate and other MMP-2 positive cancer cells.


Asunto(s)
Meliteno/administración & dosificación , Meliteno/química , Nanopartículas/química , Neoplasias de la Próstata/terapia , Venenos de Escorpión/administración & dosificación , Venenos de Escorpión/química , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Células 3T3 NIH , Polietileneimina/química , Polímeros/química , Transfección/métodos
9.
Zhongguo Zhong Yao Za Zhi ; 39(22): 4324-8, 2014 Nov.
Artículo en Chino | MEDLINE | ID: mdl-25850260

RESUMEN

The stability of melittin in different solvents (water, deoxygenated water, physiological saline, PBS, 50% ethanol, ethanol, glycerol)was studied and the results showed that the stability of melittin is not influenced by light, temperature and pH in 50% ethanol, which melittin can be completed dissolved when compared with ethanol and glycerol, in such, 50% ethanol was chosen as solvent storage when measured content of melittin. Then the effect of different concentrations of PBS, the pH of PBS and rat skin ho- mogenates were tested, and the results showed that melittin was degraded rapidly at low concentration solution and low ionic strength. Increasing pH of PBS and rat skin homogenate can accelerate the degradation of melittin. These researches provide an experimental ba- sis for further study of melittin.


Asunto(s)
Meliteno/química , Solventes/química , Animales , Estabilidad de Medicamentos , Etanol/química , Concentración de Iones de Hidrógeno , Ratas , Piel/efectos de los fármacos , Temperatura
10.
J Colloid Interface Sci ; 346(1): 127-35, 2010 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-20226468

RESUMEN

Cryo-transmission electron microscopy was used in combination with turbidity and leakage measurements to explore and compare the membrane perturbing effects of melittin and alamethicin on POPC-based liposomes of varying composition. The results show that the two peptides, despite their differences in physico-chemical properties and proposed mode of action, induce similar structural effects on the liposomes. Importantly, whereas low peptide concentrations leave pure POPC liposomes intact and seemingly unperturbed, POPC liposomes supplemented with 40 mol.% cholesterol change their shape, rupture and fuse in response to the addition of both melittin and alamethicin. In the case of alamethicin, but not melittin, fusion is effectively prevented by inclusion of 10 mol.% POPG in the liposome membranes. By means of a competitive binding assay we furthermore show that alamethicin, in line with earlier findings for melittin, possess high affinity for positively curved lipid surfaces. Moreover, results from the present study show that magainin 2 has a similar preference for curved surfaces.


Asunto(s)
Alameticina/química , Liposomas/química , Meliteno/química , Fosfatidilcolinas/química , Estructura Molecular , Tamaño de la Partícula , Propiedades de Superficie
11.
J Phys Chem B ; 113(10): 3227-34, 2009 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-19708168

RESUMEN

The interactions of ions with a model peptide (a single melittin alpha-helix) in solutions of tetrapropylammonium sulfate or guanidinium chloride were examined by molecular dynamics simulations. The tetrapropylammonium cation shares the geometrical property of essentially flat faces with the previously examined guanidinium cation, and it was found that that this geometry leads to a strong preference for tetrapropylammonium to interact in a similar stacking-type fashion with flat nonpolar groups such as the indole side chain of tryptophan. In contrast to guanidinium, however, tetrapropylammonium does not exhibit strong ion pairing or clustering with sulfate counterions in the solution. Sulfate was found to interact almost exclusively and strongly with the cationic groups of the peptide, such that, already in a 0.1 m solution of tetrapropylammonium sulfate, the 6+ charge of the peptide is effectively locally neutralized. In combination with previous simulations, neutron scattering studies, and experiments on the conformational stability of model peptides, the present results suggest that the Hofmeister series can be explained in higher detail by splitting ions according to the effect they have on hydrogen bonding, salt bridges, and hydrophobic interactions in the protein and how these effects are altered by the counterion.


Asunto(s)
Cloruros/química , Guanidina/química , Iones , Compuestos de Amonio Cuaternario/química , Sulfatos/química , Animales , Biofisica/métodos , Biología Computacional/métodos , Simulación por Computador , Enlace de Hidrógeno , Meliteno/química , Conformación Molecular , Péptidos/química , Unión Proteica , Soluciones
12.
Int Immunopharmacol ; 7(8): 1092-101, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17570326

RESUMEN

Bee venom (BV), well known as a traditional Oriental medicine, has been shown to exhibit anti-arthritic and anti-carcinogenic effects. However, the molecular mechanisms responsible for the anti-inflammatory activity of BV have not been elucidated in microglia. In the present study, we investigated the anti-inflammatory effect of BV and its major component, melittin (MEL), on lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results indicate that BV and MEL suppress LPS-induced nitric oxide (NO) and inducible NO synthase (iNOS) expression in a dose-dependent manner, without causing cytotoxicity in BV2 microglia. Moreover, BV and MEL suppressed LPS-induced activation of nuclear factor kappa B (NF-kappaB) by blocking degradation of IkappaBalpha and phosphorylation of c-Jun N-terminal kinase (JNK) and Akt, which resulted in inhibition of iNOS expression. Our data also indicate that BV and MEL exert anti-inflammatory effects by suppressing the transcription of cyclooxygenase (COX)-2 genes and proinflammatory cytokines, such as interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha. BV and MEL also attenuated the production of prostaglandin E(2) (PGE(2)). These results demonstrate that BV and MEL possess a potent suppressive effect on proinflammatory responses of BV2 microglia and suggest that these compounds may offer substantial therapeutic potential for treatment of neurodegenerative diseases that are accompanied by microglial activation.


Asunto(s)
Venenos de Abeja/farmacología , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , Meliteno/farmacología , Microglía/efectos de los fármacos , Animales , Antioxidantes/farmacología , Venenos de Abeja/química , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Quinasa I-kappa B/metabolismo , Immunoblotting , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Meliteno/química , Microglía/citología , Microglía/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Prolina/análogos & derivados , Prolina/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiocarbamatos/farmacología
13.
J Pept Sci ; 13(4): 253-62, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17394119

RESUMEN

Synthesis and conformational studies of a cecropin-melittin hybrid pentadecapeptide CA(1-7)MEL(2-9), and its three alpha, beta-dehydrophenylalanine (DeltaPhe) containing analogs in water-TFE mixtures are described. DeltaPhe is placed at strategic positions in order to preserve the amphipathicity of the molecule. The wild type CAMEL0 and its three analogs, containing one, two and three DeltaPhe residues namely CAMELDeltaPhe1, CAMELDeltaPhe2 and CAMELDeltaPhe3 respectively were synthesized in solid phase and their conformation determined by CD and NMR. CAMELDeltaPhe2 and CAMELDeltaPhe3 peptides exhibit the presence of 3(10)-helix and beta-turns in the former and only turns in the latter. CAMELDeltaPhe1 peptide was found to have a largely extended conformation. Antibacterial and hemolytic activities of the peptides were also evaluated. CAMELDeltaPhe2 peptide is maximally potent against both Staphylococcus aureus ATCC 259230 and Escherichia coli ATCC 11303. CAMELDeltaPhe1 with a single DeltaPhe at the center shows minimal hemolysis.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Meliteno/química , Fenilalanina/análogos & derivados , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Evaluación Preclínica de Medicamentos/métodos , Hemolíticos/química , Hemolíticos/farmacología , Espectroscopía de Resonancia Magnética , Ratones , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Fenilalanina/química , Conformación Proteica , Relación Estructura-Actividad
14.
Proteins ; 58(3): 518-27, 2005 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-15609352

RESUMEN

A recently developed implicit membrane model (IMM1) is supplemented with a Gouy-Chapman term describing counterion-screened electrostatic interactions of a solute with negatively charged membrane lipids. The new model is tested on peptides that bind to anionic membranes. Pentalysine binds just outside the plane of negative charge, whereas Lys-Phe peptides insert their aromatic rings into the hydrophobic core. Melittin and magainin 2 bind more strongly to anionic than to neutral membranes and in both cases insert their hydrophobic residues into the hydrocarbon core. The third domain of Antennapedia homeodomain (penetratin) binds as an alpha-helix in the headgroup region. Cardiotoxin II binds strongly to anionic membranes but marginally to neutral ones. In all cases, the location and configuration of the peptides are consistent with experimental data, and the effective energy changes upon binding compare favorably with experimental binding free energies. The model opens the way to exploring the effect of membrane charge on the location, conformation, and dynamics of a large variety of biologically active peptides on membranes.


Asunto(s)
Membrana Celular/metabolismo , Lípidos/química , Solventes/química , Animales , Aniones , Péptidos Catiónicos Antimicrobianos/química , Proteínas Cardiotóxicas de Elápidos/química , Simulación por Computador , Iones , Lisina/química , Magaininas , Meliteno/química , Modelos Químicos , Modelos Moleculares , Modelos Estadísticos , Modelos Teóricos , Distribución Normal , Péptidos/química , Fenilalanina/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Programas Informáticos , Electricidad Estática , Termodinámica , Proteínas de Xenopus/química
15.
Biochim Biophys Acta ; 1667(1): 26-37, 2004 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-15533303

RESUMEN

We have examined the kinetics of the adsorption of melittin, a secondary amphipathic peptide extracted from bee venom, on lipid membranes using three independent and complementary approaches. We probed (i) the change in the polarity of the 19Trp of the peptide upon binding, (ii) the insertion of this residue in the apolar core of the membrane, measuring the 19Trp-fluorescence quenching by bromine atoms attached on lipid acyl chains, and (iii) the folding of the peptide, by circular dichroism (CD). We report a tight coupling of the insertion of the peptide with its folding as an alpha-helix. For all the investigated membrane systems (cholesterol-containing, phosphoglycerol-containing, and pure phosphocholine bilayers), the decrease in the polarity of 19Trp was found to be significantly faster than the increase in the helical content of melittin. Therefore, from a kinetics point of view, the formation of the alpha-helix is a consequence of the insertion of melittin. The rate of melittin folding was found to be influenced by the lipid composition of the bilayer and we propose that this was achieved by the modulation of the kinetics of insertion. The study reports a clear example of the coupling existing between protein penetration and folding, an interconnection that must be considered in the general scheme of membrane protein folding.


Asunto(s)
Membrana Dobles de Lípidos/química , Meliteno/química , Proteínas de la Membrana/química , Pliegue de Proteína , Animales , Abejas , Colesterol , Cinética , Fosfatidilcolinas , Fosfatidilgliceroles , Unión Proteica , Estructura Secundaria de Proteína , Triptófano
16.
Biochem J ; 317 ( Pt 3): 747-54, 1996 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-8760358

RESUMEN

The ability of the peptides melittin, [Ala-14]melittin (P14A) and whole bee venom to lyse red blood cells (RBC) and to cause shape transformation, binding, partitioning and changes in volume of the cells during haemolysis, as well as the action of the bivalent cations Zn2+ and Ca2+, chlorpromazine, albumin and plasma on the peptide-induced haemolysis of RBC in high ionic-strength solution, have been investigated. The protective effect of all inhibitors depends on whether they have been added to the media before or after the cells. When added before the cells they reduced significantly the rate of peptide-induced haemolysis and shape transformation. The effect was maximal when agents acted simultaneously after introduction of the cells into the media containing both inhibitors and peptides. Incubation of the cells in isotonic solution before the addition of peptides enhanced 2-3-fold the RBC susceptibility (i.e. rate of haemolysis) to lytic action of the same amount of peptides, and increased the order of the haemolytic reaction, although the power law coefficient did not exceed a value of 2 for all peptides, suggesting that haemolysis is attributable to the monomeric or dimeric forms of the peptides. Partition coefficients were of the order of approximately 10(6) M-1, and P14A possessed a value 3-fold larger compared with melittin and bee venom, which correlated with its enhanced haemolytic activity. The protective action of inhibitors against peptide-induced haemolysis has been explained on the basis of their ability to compete with peptide binding at an early stage of peptide-membrane interaction, and not as a result of inhibition of a pre-existing peptide-induced pore. Whereas melittin increased the volume of RBC during haemolysis, P14A, melittin in the presence of phospholipase A2 or bee venom, reduced the volume in a concentration-dependent manner. The present data reveal the significant role of the initial stage of peptide-membrane interaction and peptide structure in the mechanism of haemolysis. These data are not consistent with a lipid-based mechanism of peptide-induced haemolysis, indicating that the mode of peptide-protein interaction is an important and decisive step in the haemolytic mechanism. It should be noted that data (in the form of three additional Tables) on the ability of inhibitors to protect cells from haemolysis when inhibitor and peptide act simultaneously are available. They are reported in Supplementary Publication SUP 50178, which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1996) 313, 9.


Asunto(s)
Albúminas/farmacología , Venenos de Abeja/farmacología , Cationes Bivalentes/farmacología , Clorpromazina/farmacología , Hemólisis/efectos de los fármacos , Meliteno/farmacología , Calcio/farmacología , Tamaño de la Célula/efectos de los fármacos , Membrana Eritrocítica/efectos de los fármacos , Humanos , Cinética , Meliteno/antagonistas & inhibidores , Meliteno/química , Fragmentos de Péptidos/farmacología , Zinc/farmacología
17.
Biophys J ; 70(5): 2223-38, 1996 May.
Artículo en Inglés | MEDLINE | ID: mdl-9172746

RESUMEN

Mixed micelles of the 26-residue, lytic peptide melittin (MLT) and 1-myristoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MMPC) in aqueous solution at 25 degrees C were investigated by (13)C- and (31)P-NMR spectroscopy. (13)C alpha chemical shifts of isotopically labeled synthetic MLT revealed that MLT in the micelle is predominantly alpha-helical and that the peptide secondary structure is stable from pH 4 to pH 11. Although the helical transformation of MLT as determined from NMR is evident at lipid:peptide molar ratios as low as 1:2, tryptophan fluorescence measurements demonstrate that well-defined micellar complexes do not predominate until lipid:peptide ratios exceed 30:1. (31)P linewidth measurements indicate that the interaction between phosphate ions in solution and cationic groups on MLT is pH dependent, and that the phosphoryl group of MMPC senses a constant charge, most likely +2, on MLT from pH 4 to pH 10. (13)C-NMR relaxation data, analyzed using the model-free formalism, show that the peptide backbone of MLT is partially, but not completely, immobilized in the mixed micelles. Specifically, order parameters (S(2)) of C alpha-H vectors averaged 0.7 and were somewhat larger for residues in the N-terminal half of the molecule. The amino terminal glycine had essentially the same range of motion as the backbone carbons. Likewise, order parameters for the trp side chain were similar to those found for the peptide C alpha moieties, as was verified by trp fluorescence anisotropy decay data. In contrast, the motion of the lysine side chains was less restricted, the average S(2) values for the C epsilon-H vectors being 0.19, 0.30, and 0.44 for lys-7, 21, and 23, respectively, for MLT in the mixed micelles. Values of the effective correlation time of the local motion tau e were in the motional narrowing limit and usually longer for side-chain atoms than for those in the backbone. The dynamics were independent of pH from pH 4 to pH 9, but at pH 11 the correlation time for the rotational motion of the mixed micelles as a whole increased from 10 ns to 16 ns, and S(2) for the lys side chains increased. Overall it appears that the MLT helix lies near the surface of the micelle at low to neutral pH, but at higher pH its orientation changes, accompanied by deeper penetration of the lysine side chains into the micelle interior. It is apparent, however, that the MLT-lipid interaction is not dependent on deprotonation of any of the titratable cationic groups in the peptide in the pH 4-10 range, and that there is substantial backbone and side-chain mobility in micelle-bound MLT.


Asunto(s)
Lisofosfatidilcolinas , Meliteno/química , Micelas , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Isótopos de Carbono , Polarización de Fluorescencia , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Modelos Teóricos , Datos de Secuencia Molecular , Fósforo , Conformación Proteica , Espectrometría de Fluorescencia
18.
Biochim Biophys Acta ; 1234(2): 235-43, 1995 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-7696299

RESUMEN

Light scattering and 31P-NMR have been used to monitor the effect of the bee-toxin, melittin, on phosphatidylcholine (PC) bilayers of variable acyl chain length (from C16:0 to C20:0). Melittin interacts with all lipids provided the interaction is initiated in the lipid fluid phase. For low-to-moderate amounts of toxin (lipid-peptide molar ratios, Ri > or = 15), the system takes the form of large spheroidal vesicles, in the fluid phase, whose radius increases from 750 A with dipalmitoyl-PC (DPPC) to 1500 A with diarachinoyl-PC (DAPC). These vesicles fragment into small discoids of 100-150 A radius when the system is cooled down below Tc (the gel-to-fluid phase transition temperature). Little chain length dependence is observed for the small objects. Small structures are also detected independently of the physical state of lipids (gel or fluid) when Ri < or = 5 and provided the interaction has been made above Tc. Small discs clearly characterized for DPPC and distearoyl-PC (DSPC) lipids are much less stable with DAPC. However in the long term, all these small structures fuse into large lipid lamellae. Discs are thermodynamically unstable and kinetics of disappearance of the small lipid-toxin complexes increases as the chain length increases in the sense: DAPC >> DSPC > DPPC. Kinetics of fusion of the small discs into extended bilayers is described by a pseudo-first-order law involving a lag time after which fusion starts. Increasing the chain length decreases the lag time and increases the rate of fusion. Formation of both the large vesicles in the fluid phase and the small discs in the gel phase as well as their stability is discussed in terms of relative shapes and dynamics of both lipids and toxin.


Asunto(s)
Membrana Dobles de Lípidos , Meliteno/química , Fosfatidilcolinas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Estabilidad de Medicamentos , Cinética , Luz , Espectroscopía de Resonancia Magnética , Conformación Molecular , Fósforo , Dispersión de Radiación , Relación Estructura-Actividad , Termodinámica
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