RESUMEN
Parkinson's disease (PD) is one of the most common neurodegenerative disorders, and both genetic and histopathological evidence have implicated the ubiquitous presynaptic protein α-synuclein (αSyn) in its pathogenesis. Recent work has investigated how disrupting αSyn's interaction with membranes triggers trafficking defects, cellular stress, and apoptosis. Special interest has been devoted to a series of mutants exacerbating the effects of the E46K mutation (associated with autosomal dominant PD) through homologous Glu-to-Lys substitutions in αSyn's N-terminal region (i.e. E35K and E61K). Such E46K-like mutants have been shown to cause dopaminergic neuron loss and severe but L-DOPA-responsive motor defects in mouse overexpression models, presenting enormous translational potential for PD and other "synucleinopathies." In this work, using a variety of biophysical techniques, we characterize the molecular pathology of E46K-like αSyn mutants by studying their structure and membrane-binding and remodeling abilities. We find that, although a slight increase in the mutants' avidity for synaptic vesicle-like membranes can be detected, most of their deleterious effects are connected to their complete disruption of αSyn's curvature selectivity. Indiscriminate binding can shift αSyn's subcellular localization away from its physiological interactants at the synaptic bouton toward trafficking vesicles and organelles, as observed in E46K-like cellular and murine models, as well as in human pathology. In conclusion, our findings suggest that a loss of curvature selectivity, rather than increased membrane affinity, could be the critical dyshomeostasis in synucleinopathies.
Asunto(s)
Membrana Celular/patología , Ácido Glutámico/química , Lípidos/análisis , Lisina/química , Proteínas Mutantes/metabolismo , Mutación , alfa-Sinucleína/metabolismo , Membrana Celular/metabolismo , Ácido Glutámico/genética , Humanos , Lípidos/química , Lisina/genética , Proteínas Mutantes/genética , alfa-Sinucleína/genéticaRESUMEN
α-Synuclein (αS) is an abundant neuronal protein which has been implicated, among others, in the pathogenesis of neurodegenerative diseases like Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In fact, αS is the major constituent of Lewy bodies, the primarily proteinaceous inclusions found in the nervous tissue of PD and DLB patients. While its physiological role is unclear, it is believed to be involved in the regulation of synaptic vesicle exocytosis. However, in a disease state, αS will "misfold" and aggregate, leading to neuronal dysfunction and death. The study of the molecular events underlying pathogenesis, especially with biophysical and biochemical approaches, requires highly pure untagged αS. In this protocol we describe a method to purify untagged recombinant αS, which can be used for binding, folding, and aggregation studies. The purification method includes a cell lysis step, followed by two chromatography steps: ion-exchange chromatography first, and size-exclusion chromatography for polishing.
Asunto(s)
Expresión Génica , alfa-Sinucleína/genética , alfa-Sinucleína/aislamiento & purificación , Cromatografía Liquida , Vectores Genéticos/genética , Humanos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/químicaRESUMEN
α-Synuclein (αSyn) is a key player in the pathogenesis of Parkinson's disease and other synucleinopathies. Here, we report the existence of a novel soluble α-helical conformer of αSyn, obtained through transient interaction with lipid interfaces, and propose dynamic oligomerization as the mechanism underlying its stability. The conformational space of αSyn appears to be highly context-dependent, and lipid bilayers might thus play crucial roles as molecular chaperones in a cellular environment.
