RESUMEN
Gramicidin A (gA) is a linear antimicrobial peptide that can form a channel and specifically conduct monovalent cations such as H+ across the lipid membrane. The antimicrobial activity of gA is associated with the formation of hydroxyl free radicals and the imbalance of NADH metabolism, possibly a consequence caused by the conductance of cations. The ion conductivity of gramicidin A can be blocked by Ca2+ ions. However, the effect of Ca2+ ions on the antimicrobial activity of gA is unclear. To unveil the role of Ca2+ ions, we examined the effect of Ca2+ ions on the antimicrobial activity of gramicidin A against Staphylococcus aureus (S. aureus). Results showed that the antimicrobial mechanism of gA and antimicrobial activity by Ca2+ ions are concentration-dependent. At the low gA concentration (≤1 µM), the antimicrobial mechanism of gA is mainly associated with the hydroxyl free radical formation and NADH metabolic imbalance. Under this mode, Ca2+ ions can significantly inhibit the hydroxyl free radical formation and NADH metabolic imbalance. On the other hand, at high gA concentration (≥5 µM), gramicidin A acts more likely as a detergent. Gramicidin A not only causes an increase in hydroxyl free radical levels and NAD+/NADH ratios but also induces the destruction of the lipid membrane composition. At this condition, Ca2+ ions can no longer reduce the gA antimicrobial activity but rather enhance the bacterial killing ability of gramicidin A.
Asunto(s)
Antibacterianos , Calcio , Gramicidina , Staphylococcus aureus , Antibacterianos/química , Antibacterianos/farmacología , Calcio/metabolismo , Cationes Bivalentes , Membrana Celular/metabolismo , Gramicidina/química , Gramicidina/farmacología , Lípidos de la Membrana/metabolismo , NAD/metabolismo , Staphylococcus aureus/efectos de los fármacosRESUMEN
The pathological role of vitamin K2 in Alzheimer's disease (AD) involves a definite link between impaired cognitive functions and decreased serum vitamin K levels. Vitamin K2 supplementation may have a protective effect on AD. However, the mechanism underlying vitamin K2 protection has not been elucidated. With the amyloid-ß (Aß) cascade hypothesis, we constructed a clone containing the C-terminal fragment of amyloid precursor protein (ß-CTF/APP), transfected in astroglioma C6 cells and used this cell model (ß-CTF/C6) to study the protective effect of vitamin K2 against Aß cytotoxicity. Both cellular and biochemical assays, including cell viability and reactive oxygen species (ROS), assays assay, and Western blot and caspase activity analyses, were used to characterize and unveil the protective role and mechanism of vitamin K2 protecting against Aß-induced cytotoxicity. Vitamin K2 treatment dose-dependently decreased the death of neural cells. The protective effect of vitamin K2 could be abolished by adding warfarin, a vitamin K2 antagonist. The addition of vitamin K2 reduced the ROS formation and inhibited the caspase-3 mediated apoptosis induced by Aß peptides, indicating that the mechanism underlying the vitamin K2 protection is likely against Aß-mediated apoptosis. Inhibitor assay and Western blot analyses revealed that the possible mechanism of vitamin K2 protection against Aß-mediated apoptosis might be via regulating phosphatidylinositol 3-kinase (PI3K) associated-signaling pathway and inhibiting caspase-3-mediated apoptosis. Our study demonstrates that vitamin K2 can protect neural cells against Aß toxicity.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Citoprotección , Vitamina K 2/farmacología , Animales , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Radicales Libres/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/efectos de los fármacos , Warfarina/farmacología , Proteína Letal Asociada a bcl/metabolismoRESUMEN
Gramicidin A (gA) forms several convertible conformations in different environments. In this study, we investigated the effect of calcium halides on the molecular state and antimicrobial activity of gramicidin A. The molecular state of gramicidin A is highly affected by the concentration of calcium salt and the type of halide anion. Gramicidin A can exist in two states that can be characterized by circular dichroism (CD), mass, nuclear magnetic resonance (NMR) and fluorescence spectroscopy. In State 1, the main molecular state of gramicidin A is as a dimer, and the addition of calcium salt can convert a mixture of four species into a single species, which is possibly a left-handed parallel double helix. In State 2, the addition of calcium halides drives gramicidin A dissociation and denaturation from a structured dimer into a rapid equilibrium of structured/unstructured monomer. We found that the abilities of dissociation and denaturation were highly dependent on the type of halide anion. The dissociation ability of calcium halides may play a vital role in the antimicrobial activity, as the structured monomeric form had the highest antimicrobial activity. Herein, our study demonstrated that the molecular state was correlated with the antimicrobial activity.
Asunto(s)
Antibacterianos/farmacología , Compuestos de Calcio/química , Gramicidina/farmacología , Antibacterianos/química , Bromuros/química , Cloruro de Calcio/química , Dicroismo Circular , Gramicidina/química , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Espectrometría de Fluorescencia , Staphylococcus aureus/efectos de los fármacosRESUMEN
The glycine zipper motif at the C-terminus of the ß-amyloid (Aß) peptide have been shown to strongly influence the formation of neurotoxic aggregates. A previous study showed that the G37L mutation dramatically reduces the Aß toxicity in vivo and in vitro. However, the primary cause and mechanism of the glycine zipper motif on Aß properties remain unknown. To gain molecular insights into the impact of glycine zipper on Aß properties, we substituted the residue 37 of Glycine by Valine and studied the structural and biochemical properties of G37V mutation, Aß42(37V), by using in vitro and in silico approaches. Unlike G37L mutation, the G37V mutation reduced toxicity substantially but did not significantly accelerate the aggregation rate or change the content of secondary structures. Further TEM analyses showed that the G37V mutation formed an ellipse-like aggregate rather than a network-like fibril as wild type or G37L mutation of Aß42 form. This different aggregation morphology may be highly linked with the reduction of toxicity. To gain the insight for the different properties of Aß42(37V), we studied the structure of Aß42 and G37V mutation using the replica exchange molecular dynamics simulation. Our results demonstrate that although the overall secondary structure population is similar with Aß42 and Aß42(G37V), Aß42(G37V) shows an increase in the ß-turn and ß-hairpin at residues 36-37 and the flexibility of the Asp23-Lys28 salt bridge. These unique structural features may be the possible reason to account for the ellipse-like morphology.
Asunto(s)
Sustitución de Aminoácidos , Péptidos beta-Amiloides/genética , Mutación Missense , Fragmentos de Péptidos/genética , Mutación Puntual , Agregado de Proteínas , Secuencias de Aminoácidos , Amiloide/química , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Línea Celular Tumoral , Supervivencia Celular , Simulación por Computador , Humanos , Técnicas In Vitro , Modelos Moleculares , Simulación de Dinámica Molecular , Neuroblastoma/patología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , Agregación Patológica de Proteínas/genética , Estructura Secundaria de ProteínaRESUMEN
Alzheimer's disease (AD) is the most common form of dementia caused by the formation of Aß aggregates. So far, no effective medicine for the treatment of AD is available. Many efforts have been made to find effective medicine to cope with AD. Curcumin is a drug candidate for AD, being a potent anti-amyloidogenic compound, but the results of clinical trials for it were either negative or inclusive. In the present study, we took advantages from accumulated knowledge about curcumin and have screened out four compounds that have chemical and structural similarity with curcumin more than 80% from all FDA-approved oral drugs. Using all-atom molecular dynamics simulation and the free energy perturbation method we showed that among predicted compounds anti-arrhythmic medication propafenone shows the best anti-amyloidogenic activity. The in vitro experiment further revealed that it can inhibit Aß aggregation and protect cells against Aß induced cytotoxicity to almost the same extent as curcumin. Our results suggest that propafenone may be a potent drug for the treatment of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/química , Antiarrítmicos/farmacología , Simulación por Computador , Fragmentos de Péptidos/química , Propafenona/farmacología , Agregado de Proteínas/efectos de los fármacos , Administración Oral , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Antiarrítmicos/metabolismo , Antiarrítmicos/farmacocinética , Antiarrítmicos/uso terapéutico , Sitios de Unión , Disponibilidad Biológica , Supervivencia Celular/efectos de los fármacos , Curcumina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Radicales Libres/metabolismo , Enlace de Hidrógeno , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fragmentos de Péptidos/metabolismo , Propafenona/metabolismo , Propafenona/farmacocinética , Propafenona/uso terapéutico , Estructura Secundaria de Proteína , Electricidad Estática , TermodinámicaRESUMEN
Many properties of Aß such as toxicity, aggregation and ROS formation are modulated by Cu2+. Previously, the coordination configuration and interaction of Cu2+ with the Aß N-terminus has been extensively studied. However, the effect of Aß C-terminal residues on related properties is still unclear. In the present study, several C-terminus-truncated Aß peptides, including Aß1-40, Aß1-35, Aß1-29, Aß1-24 and Aß1-16, were synthesized to characterize the effect of Aß C-terminal residues on Cu2+ binding affinity, structure, aggregation ability and ROS formation. Results show that the Aß C-terminal residues have effect on Cu2+ binding affinity, aggregation ability and inhibitory ability of ROS formation. Compared to the key residues responsible for Aß aggregation and structure in the absence of Cu2+, it is more likely that residues 36-40, rather than residues 17-21 and 30-35, play a key role on the related properties of Aß in the presence of Cu2+.