quantitative analysis of α-synuclein solubility in living cells using split gfp complementation定量分析α-synuclein溶解度的活细胞分裂gfp互补.pdfVIP

Quantitative Analysis of a-Synuclein Solubility in Living Cells Using Split GFP Complementation 1 1 1 1,2,3 Ahmed Kothawala , Kiri Kilpatrick , Jose Andres Novoa , Laura Segatori * 1 Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas, United States of America, 2 Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, United States of America, 3 Department of Bioengineering, Rice University, Houston, Texas, United States of America 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 (asyn), an intrinsically unstructured, misfolding-prone protein with high propensity to aggregate. Quantifying the pool of soluble asyn and monitoring asyn aggregation in living cells is fundamental to study the molecular mechanisms of asyn-induced cytotoxicity and develop therapeutic strategies to prevent asyn aggregation. In this study, we report the use of a split GFP complementation assay to quantify asyn solubility. Particularly, we investigated a series of naturally occurring and rationally designed asyn variants and showed that this method can be used to study how asyn sequence specificity affects its solubility. Furthermore, we demonstrated the utility of this assay to explore the influence of the cellular folding network on asyn solubility. The results presented underscor