RESUMO
Five new benzophenone derivatives named tenellones Dâ»H (1â»5), sharing a rare naturally occurring aldehyde functionality in this family, and a new eremophilane derivative named lithocarin A (7), together with two known compounds (6 and 8), were isolated from the deep marine sediment-derived fungus Phomopsis lithocarpus FS508. All of the structures for these new compounds were fully characterized and established on the basis of extensive spectroscopic interpretation and X-ray crystallographic analysis. Compound 5 exhibited cytotoxic activity against HepG-2 and A549 cell lines with IC50 values of 16.0 and 17.6 µM, respectively.
Assuntos
Aldeídos/farmacologia , Antineoplásicos/farmacologia , Organismos Aquáticos/química , Ascomicetos/química , Benzofenonas/farmacologia , Células A549 , Aldeídos/química , Aldeídos/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Benzofenonas/química , Benzofenonas/isolamento & purificação , Cristalografia por Raios X , Sedimentos Geológicos/química , Células Hep G2 , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Oceanos e MaresRESUMO
The results of cell and animal model studies demonstrate that molecular chaperones play an important role in controlling the processes of protein misfolding and amyloid formation in vivo. In addition, chaperones are involved in the appearance, propagation and clearance of prion phenotypes in yeast. The effect of chaperones on amyloid formation has been studied in great detail in recent years in order to elucidate the underlying mechanisms. An important approach is the direct study of effects of chaperones on amyloid fibril formation in vitro. This review introduces the methods and techniques that are commonly used to control and monitor the time course of fibril formation, and to detect interactions between chaperones and fibril-forming proteins. The techniques we address include thioflavin T binding fluorescence and filter retardation assays, size-exclusion chromatography, dynamic light scattering, and biosensor assays. Our aim in this review is to provide guidance on how to embark on study of the effect of chaperones on amyloid fibril formation, and how to avoid common problems that may be encountered, using examples and experience from the authors' lab and from the wider literature.
Assuntos
Amiloide/química , Chaperonas Moleculares/química , Benzotiazóis , Cromatografia em Gel , Filtração/métodos , Fluorometria/métodos , Humanos , Luz , Filtros Microporos , Técnicas de Microbalança de Cristal de Quartzo/métodos , Espalhamento de Radiação , Ressonância de Plasmônio de Superfície/métodos , Tiazóis/químicaRESUMO
Previous results suggest that methylotrophic yeasts may contain factors that modulate prion stability. Alcohol oxidase (AOX), a key enzyme in methanol metabolism, is an abundant protein that is specific to methylotrophic yeasts. We examined the effect of Pichia pastoris AOX1 on prion phenotypes in Saccharomyces cerevisiae. The S. cerevisiae prion states [PSI(+)] and [URE3] arise from aggregation of the proteins Sup35p and Ure2p respectively, and correlate with the ability of Sup35p and Ure2p to form amyloid-like fibrils in vitro. We found that expression of P. pastoris AOX1 in S. cerevisiae had no effect on propagation of the [PSI(+)] prion, but inhibited propagation of [URE3]. Addition of AOX1 early in the time-course of fibril formation inhibits Ure2p fibril formation in vitro. AOX1 has not previously been identified as an ATPase. However, we discovered that in addition to its flavin adenine dinucleotide-dependent AOX activity, AOX1 possesses ATPase activity. This study identifies AOX1 as a novel prion inhibitory factor and a potential ATPase.
Assuntos
Adenosina Trifosfatases/metabolismo , Oxirredutases do Álcool/metabolismo , Proteínas Fúngicas/metabolismo , Pichia/enzimologia , Príons/metabolismo , Adenosina Trifosfatases/genética , Oxirredutases do Álcool/genética , Proteínas Fúngicas/genética , Glutationa Peroxidase , Fatores de Terminação de Peptídeos , Fenótipo , Pichia/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Ure2p is the protein determinant of the Saccharomyces cerevisiae prion state [URE3]. Constitutive overexpression of the HSP70 family member SSA1 cures cells of [URE3]. Here, we show that Ssa1p increases the lag time of Ure2p fibril formation in vitro in the presence or absence of nucleotide. The presence of the HSP40 co-chaperone Ydj1p has an additive effect on the inhibition of Ure2p fibril formation, whereas the Ydj1p H34Q mutant shows reduced inhibition alone and in combination with Ssa1p. In order to investigate the structural basis of these effects, we constructed and tested an Ssa1p mutant lacking the ATPase domain, as well as a series of C-terminal truncation mutants. The results indicate that Ssa1p can bind to Ure2p and delay fibril formation even in the absence of the ATPase domain, but interaction of Ure2p with the substrate-binding domain is strongly influenced by the C-terminal lid region. Dynamic light scattering, quartz crystal microbalance assays, pull-down assays and kinetic analysis indicate that Ssa1p interacts with both native Ure2p and fibril seeds, and reduces the rate of Ure2p fibril elongation in a concentration-dependent manner. These results provide new insights into the structural and mechanistic basis for inhibition of Ure2p fibril formation by Ssa1p and Ydj1p.