Quantitative analysis of ?-synuclein solubility in living cells using split GFP complementation.

ABSTRACT: Presently incurable, Parkinson's disease (PD) is the most common neurodegenerative movement disorder and affects 1% of the population over 60 years of age. The hallmarks of PD pathogenesis are the loss of dopaminergic neurons in the substantia nigra pars compacta, and the occurrence of proteinaceous cytoplasmic inclusions (Lewy bodies) in surviving neurons. Lewy bodies are mainly composed of the pre-synaptic protein alpha-synuclein (?syn), an intrinsically unstructured, misfolding-prone protein with high propensity to aggregate. Quantifying the pool of soluble ?syn and monitoring ?syn aggregation in living cells is fundamental to study the molecular mechanisms of ?syn-induced cytotoxicity and develop therapeutic strategies to prevent ?syn aggregation. In this study, we report the use of a split GFP complementation assay to quantify ?syn solubility. Particularly, we investigated a series of naturally occurring and rationally designed ?syn variants and showed that this method can be used to study how ?syn sequence specificity affects its solubility. Furthermore, we demonstrated the utility of this assay to explore the influence of the cellular folding network on ?syn solubility. The results presented underscore the utility of the split GFP assay to quantify ?syn solubility in living cells.

SUBMITTER: Kothawala A 

PROVIDER: S-EPMC3425482 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature