RESUMEN
Here a new method to determine the oligomeric state and orientation of coiled-coil peptide motifs is described. Peptides K and E, which are designed to form a parallel heterodimeric complex in aqueous solution, were labeled with the aromatic amino acids tryptophan and tyrosine on the C-terminus respectively as 'fingerprint' residues. One of the peptides was also labeled with the paramagnetic probe MTSL. One dimensional proton NMR spectroscopy was used to study the peptide quaternary structure by monitoring the signal suppression of the aromatic labels due to proximity of the nitroxyl radical. 1D-NMR confirmed that the peptides K and E form a heterodimeric coiled coil with a parallel orientation. In addition, fluorescence emission quenching of the aromatic labels due to electron exchange with a nitroxyl radical confirmed the parallel coiled coil orientation. Thus, paramagnetic nitroxide and aromatic fluorophore labeling of peptides yields valuable information regarding the quaternary structure from 1D-NMR and steady-state fluorescence measurements. This convenient method is useful not only to investigate coiled coil assembly, but can also be applied to any defined supramolecular assembly.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Péptidos/química , Secuencia de Aminoácidos , Dimerización , Datos de Secuencia Molecular , Óxidos de Nitrógeno/química , Estructura Secundaria de ProteínaRESUMEN
Coiled-coil peptide motifs were used as thermo-responsive valves for mesoporous silica nanoparticles (MSNs). The controlled release of a model drug as a function of temperature was demonstrated.
Asunto(s)
Nanopartículas/química , Péptidos/química , Dióxido de Silicio/química , Temperatura , Preparaciones de Acción Retardada , Fluoresceína/química , Modelos Biológicos , Porosidad , Estructura Terciaria de ProteínaRESUMEN
We have determined the pharmacological profile of the new serotonin 5-HT(7) receptor agonist N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211). Radioligand binding assays were performed on a panel of 5-HT receptor subtypes. The compound was also evaluated in vivo by examining its effect on body temperature regulation in mice lacking the 5-HT(7) receptor (5-HT(7)(-/-)) and their 5-HT(7)(+/+) sibling controls. Disposition studies were performed in mice of both genotypes. It was found that LP-211 was brain penetrant and underwent metabolic degradation to 1-(2-diphenyl)piperazine (RA-7). In vitro binding assays revealed that RA-7 possessed higher 5-HT(7) receptor affinity than LP-211 and a better selectivity profile over a panel of 5-HT receptor subtypes. In vivo it was demonstrated that LP-211, and to a lesser degree RA-7, induced hypothermia in 5-HT(7)(+/+) but not in 5-HT(7)(-/-) mice. Our results suggest that LP-211 can be used as a 5-HT(7) receptor agonist in vivo.
Asunto(s)
Encéfalo/metabolismo , Fenilcarbamatos/metabolismo , Fenilcarbamatos/farmacología , Piperazinas/metabolismo , Piperazinas/farmacología , Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/farmacología , Uretano/análogos & derivados , Animales , Temperatura Corporal/efectos de los fármacos , Temperatura Corporal/genética , Encéfalo/anatomía & histología , Encéfalo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Ratones , Ratones Noqueados , Fenilcarbamatos/química , Piperazinas/química , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Receptores de Serotonina/deficiencia , Agonistas de Receptores de Serotonina/química , Uretano/química , Uretano/metabolismo , Uretano/farmacologíaRESUMEN
A group of novel tricyclic Delta(2)-isoxazolines (4b, 5b, 7a-b, and 8a-b) and 3-oxo-isoxazolidines (6a-b and 9a-b), structurally related to cytisine or norferruginine, was prepared through 1,3-dipolar cycloadditions involving suitable olefins and bromonitrile oxide. The target compounds were assayed at alpha4beta2 and alpha7 neuronal acetylcholine receptors (nAChRs). The results of competition binding experiments indicated for the new derivatives a reduction of the affinity at the alpha4beta2 subtype in comparison with the reference molecules, coupled with an overall negligible affinity at the alpha7 subtype. The binding mode of the bromo-Delta(2)-isoxazolines 4b and 7b, which were the highest affinity ligands in the series (K(i)=0.92 and 0.75 microM, respectively), was analyzed by applying a recently developed model of the alpha4beta2 nAChRs.
Asunto(s)
Compuestos Heterocíclicos con 3 Anillos/química , Isoxazoles/química , Neuronas/metabolismo , Receptores Nicotínicos/química , Alcaloides/química , Animales , Azocinas/química , Sitios de Unión , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Heterocíclicos con 3 Anillos/síntesis química , Compuestos Heterocíclicos con 3 Anillos/farmacología , Isoxazoles/síntesis química , Isoxazoles/farmacología , Ligandos , Modelos Moleculares , Unión Proteica , Quinolizinas/química , Ratas , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Human sirtuins are a family of seven conserved proteins (SIRT1-7). The most investigated is the silent mating type information regulation-2 homolog (SIRT1, NM_012238), which was associated with neuroprotection in models of polyglutamine toxicity or Alzheimer's disease (AD) and whose activation by the phytocompound resveratrol (RES) has been described. We have examined the neuroprotective role of RES in a cellular model of oxidative stress, a common feature of neurodegeneration. RES prevented toxicity triggered by hydrogen peroxide or 6-hydroxydopamine (6-OHDA). This action was likely mediated by SIRT1 activation, as the protection was lost in the presence of the SIRT1 inhibitor sirtinol and when SIRT1 expression was down-regulated by siRNA approach. RES was also able to protect SK-N-BE from the toxicity arising from two aggregation-prone proteins, the AD-involved amyloid-beta (1-42) peptide (Abeta42) and the familiar Parkinson's disease linked alpha-synuclein(A30P) [alpha-syn(A30P)]. Alpha-syn(A30P) toxicity was restored by sirtinol addition, while a partial RES protective effect against Abeta42 was found even in presence of sirtinol, thus suggesting a direct RES effect on Abeta42 fibrils. We conclude that SIRT1 activation by RES can prevent in our neuroblastoma model the deleterious effects triggered by oxidative stress or alpha-syn(A30P) aggregation, while RES displayed a SIRT1-independent protective action against Abeta42.