Citation

  • Authors: Kothawala, A., Kilpatrick, K., Novoa, J. A., Segatori, L.
  • Year: 2012
  • Journal: PLoS One 7 e43505
  • Applications: in vitro / DNA / jetPRIME
  • Cell types:
    1. Name: H4
      Description: Human neuroglioma cells
    2. Name: HeLa
      Description: Human cervix epitheloid carcinoma cells

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 (alphasyn), an intrinsically unstructured, misfolding-prone protein with high propensity to aggregate. Quantifying the pool of soluble alphasyn and monitoring alphasyn aggregation in living cells is fundamental to study the molecular mechanisms of alphasyn-induced cytotoxicity and develop therapeutic strategies to prevent alphasyn aggregation. In this study, we report the use of a split GFP complementation assay to quantify alphasyn solubility. Particularly, we investigated a series of naturally occurring and rationally designed alphasyn variants and showed that this method can be used to study how alphasyn sequence specificity affects its solubility. Furthermore, we demonstrated the utility of this assay to explore the influence of the cellular folding network on alphasyn solubility. The results presented underscore the utility of the split GFP assay to quantify alphasyn solubility in living cells.

Pubmed