title: Quantitative analysis of α-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 (αsyn), an intrinsically unstructured, misfolding-prone protein with high propensity to aggregate. Quantifying the pool of soluble αsyn and monitoring αsyn aggregation in living cells is fundamental to study the molecular mechanisms of αsyn-induced cytotoxicity and develop therapeutic strategies to prevent αsyn aggregation. In this study, we report the use of a split GFP complementation assay to quantify αsyn solubility. Particularly, we investigated a series of naturally occurring and rationally designed αsyn variants and showed that this method can be used to study how αsyn sequence specificity affects its solubility. Furthermore, we demonstrated the utility of this assay to explore the influence of the cellular folding network on αsyn solubility. The results presented underscore the utility of the split GFP assay to quantify αsyn solubility in living cells.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas, United States of America.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Kothawala
authors.forename: Kiri
authors.initials: K
authors.lastname: Kilpatrick
authors.forename: Jose Andres
authors.initials: JA
authors.lastname: Novoa
authors.forename: Laura
authors.initials: L
authors.lastname: Segatori
mesh_terms: D002470:Cell Survival; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D055503:Protein Multimerization; D059748:Proteolysis / Q000187:drug effects; D012995:Solubility / Q000187:drug effects; D013050:Spectrometry, Fluorescence / Q000379:methods*; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D051844:alpha-Synuclein; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex