RESUMO
The amino acid sequence of protein Gln3 in yeast Saccharomyces cerevisiae has a region enriched with Gln (Q) and Asn (N) residues. In this study, we analyzed the effects of overexpression of Gln3 and its Q/N-rich fragment fused with yellow fluorescent protein (YFP). Being overexpressed, full-length Gln3-YFP does not form aggregates, inhibits vegetative growth, and demonstrates nuclear localization, while the Q/N-rich fragment (Gln3QN) fused with YFP forms aggregates that do not colocalize with the nucleus and do not affect growth of the cells. Although detergent-resistant aggregates of Gln3QN are formed in the absence of yeast prions, the aggregation of Gln3QN significantly increases in the presence of [PIN(+)] prion, while in the presence of two prions, [PSI(+)] and [PIN(+)], the percentage of cells with Gln3QN aggregates is significantly lower than in the strain bearing only [PIN(+)]. Data on colocalization demonstrate that this effect is mediated by interaction between Gln3QN aggregates and [PSI(+)] and [PIN(+)] prions.
Assuntos
Asparagina/metabolismo , Glutamina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Núcleo Celular/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/metabolismo , Príons/química , Príons/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The amyloid-beta peptide (Abeta) can generate cytotoxic oligomers, and their accumulation is thought to underlie the neuropathologic changes found in Alzheimer's disease. Known inhibitors of Abeta polymerization bind to undefined structures and can work as nonspecific aggregators, and inhibitors that target conformations that also occur in larger Abeta assemblies may even increase oligomer-derived toxicity. Here we report on an alternative approach whereby ligands are designed to bind and stabilize the 13-26 region of Abeta in an alpha-helical conformation, inspired by the postulated Abeta native structure. This is achieved with 2 different classes of compounds that also reduce Abeta toxicity to cells in culture and to hippocampal slice preparations, and that do not show any nonspecific aggregatory properties. In addition, when these inhibitors are administered to Drosophila melanogaster expressing human Abeta(1-42) in the central nervous system, a prolonged lifespan, increased locomotor activity, and reduced neurodegeneration is observed. We conclude that stabilization of the central Abeta alpha-helix counteracts polymerization into toxic assemblies and provides a strategy for development of specific inhibitors of Abeta polymerization.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Amiloide/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila melanogaster , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de ProteínaRESUMO
The effect of dimethyl sulfoxide (DMSO) and its nearest homologue diethyl sulfoxide (DESO) as a polar cosolvents on the binding of vitamin E to water + DMSO (DESO) containing reversed micelles of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) has been investigated by a spectrophotometric method. The results suggest that compare with water without organic cosolvent-containing reversed micelles in this case an increase of binding constant of vitamin E in reversed micelles takes place. The results obtained shown that with the addition of DMSO and DESO it will be possible to monitoring a penetration of vitamin E into micellar core.
Assuntos
Dimetil Sulfóxido/química , Micelas , Sulfóxidos/química , Vitamina E/química , Espectrofotometria UltravioletaRESUMO
G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatable alpha-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to cross-linked fragments of Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an alpha-factor analog with the photoaffinity group p-benzoyl-l-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 of alpha-factor and a region of Ste2p covering residues 251-294.
