273351 A Non-Invasive, Quantitative Method to Monitor Misfolding/Aggregation of Protein in Mammalian Cells

Tuesday, October 30, 2012: 1:42 PM
Washington (Westin )
Simpson Gregoire, Joseph Costanzo, Shaojie Zhang, Erik J. Fernandez and Inchan Kwon, Department of Chemical Engineering, University of Virginia, Charlottesville, VA

Protein misfolding and aggregation are often causative to the onset of numerous neurodegenerative diseases such as Alzheimer's disease, Parkinson’s disease, Huntington’s disease, and Amyotrophic lateral sclerosis (ALS).   Therefore, quantitative analysis of protein misfolding and aggregation in mammalian cells will facilitate enhanced understanding of the molecular mechanisms for various human diseases. We have developed a non-invasive assay that quantifies the extent of deviation from the correctly folded structure of proteins expressed in mammalian cells. In order to validate the assay, we chose wild-type human copper/zinc superoxide dismutase (SOD1) and its mutants (A4V, A4V/C57S, and A4V/C111S) because of its ability to form intracellular aggregates in a short period of time, its variation in aggregation propensity and misfolding and its implications in familial amyotrophic lateral sclerosis (fALS). ALS is a progressive neurodegenerative disorder that inevitably leads to the death of motor neurons. In particular, the A4V mutation is the most prevalent mutation in fALS patients in the United States and leads to death within a year after diagnosis. It is believed that the development of SOD1_A4V aggregates in the spinal cord and brain of fALS patients is pathologically linked to fALS. We have begun screening variants of SOD1_A4V in order to identify less aggregation-prone SOD1_A4V using the aforementioned monitoring system. Biophysical properties of the less aggregation-prone mutants identified will be characterized in vitro.

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