RESUMEN
RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. Here, we identify two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signaling pathways with distinct isoform and mutant profiles. Affimer K6 binds in the SI/SII pocket, whilst Affimer K3 is a non-covalent inhibitor of the SII region that reveals a conformer of wild-type RAS with a large, druggable SII/α3 pocket. Competitive NanoBRET between the RAS-binding Affimers and known RAS binding small-molecules demonstrates the potential to use Affimers as tools to identify pharmacophores. This work highlights the potential of using biologics with small interface surfaces to select unseen, druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.
Asunto(s)
Productos Biológicos/farmacología , Técnicas de Visualización de Superficie Celular/métodos , Descubrimiento de Drogas/métodos , Neoplasias/tratamiento farmacológico , Proteínas ras/antagonistas & inhibidores , Sitio Alostérico , Productos Biológicos/química , Humanos , Neoplasias/química , Neoplasias/enzimología , Transducción de Señal , Proteínas ras/metabolismoRESUMEN
The formation of amyloid fibres is a hallmark of amyloid disorders. Nevertheless, the lack of correlation between fibre load and disease as observed, for example, in Alzheimer's disease, means that fibres are considered secondary contributors to the onset of cellular dysfunction. Instead, soluble intermediates of amyloid assembly are often described as the agents of toxicity. Here, we discuss recent experimental discoveries which suggest that amyloid fibres should be considered as disease-relevant species that can mediate a range of pathological processes. These include disruption of biological membranes, secondary nucleation, amyloid aggregate transmission, and the disruption of protein homeostasis (proteostasis). Thus, a greater understanding of amyloid fibre biology could enhance prospects of developing therapeutic interventions against this devastating class of protein-misfolding disorders.
Asunto(s)
Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Deficiencias en la Proteostasis/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Amiloide/química , Animales , Humanos , Agregación Patológica de Proteínas , Deficiencias en la Proteostasis/tratamiento farmacológicoRESUMEN
Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using ß2-microglobulin (ß2m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused by the molecular shedding of membrane-active oligomers in a process that is dependent on pH. Using thioflavin T (ThT) fluorescence, NMR, EM and fluorescence correlation spectroscopy (FCS), we show that fibril disassembly at pH 6.4 results in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. Chemical cross-linking or interaction with hsp70 increases the kinetic stability of fibrils and decreases their capacity to cause membrane disruption and cellular dysfunction. The results demonstrate how pH can modulate the deleterious effects of preformed amyloid aggregates and suggest why endocytic trafficking through acidic compartments may be a key factor in amyloid disease.
Asunto(s)
Amiloide/química , Amiloidosis/metabolismo , Benzotiazoles , Endosomas/química , Proteínas HSP70 de Choque Térmico/química , Humanos , Concentración de Iones de Hidrógeno , Cinética , Lisosomas/química , Monocitos/metabolismo , Muramidasa/química , Unión Proteica , Proteínas Recombinantes/química , Espectrometría de Fluorescencia , Tiazoles/química , Microglobulina beta-2/químicaRESUMEN
Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of ß2-microglobulin (ß2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented ß2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented ß2m fibrils did not, however, cause cell death. Instead, fragmented ß2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.
Asunto(s)
Amiloide/fisiología , Lisosomas/metabolismo , Proteolisis , Microglobulina beta-2/fisiología , Línea Celular Tumoral , Supervivencia Celular , Humanos , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Nanopartículas/metabolismo , Oxidación-Reducción , Permeabilidad , Transporte de ProteínasRESUMEN
Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of ß2-microglobulin (ß2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which ß2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of ß2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that ß2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between ß2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of ß2m amyloid-associated osteoarticular tissue destruction in DRA.
Asunto(s)
Amiloide/química , Endosomas/química , Membranas Intracelulares/química , Microglobulina beta-2/química , Amiloide/genética , Amiloide/metabolismo , Amiloidosis/etiología , Amiloidosis/genética , Amiloidosis/metabolismo , Endosomas/genética , Endosomas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Membranas Artificiales , Diálisis Renal/efectos adversos , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMEN
Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer's disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of ß2-microglobulin (ß2m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of ß2m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates.