Friday, October 12, 2012: 5:20 PM
Hall 4E/F (WSCC)
The accumulation and aggregation of misfolded proteins is a common hallmark of a number of age-related neurodegenerative diseases, including Parkinson’s and Alzheimer’s diseases. Accumulation of misfolded proteins, in turn, leads to impairment of proteasome activity and further affects the ability of the cell to degrade misfolded proteins. Therefore, we hypothesize that enhancing the cellular degradation capacity by engineering proteasome activity will alleviate toxicity induced by accumulation and aggregation of misfolded proteins. To identify endogenous proteins that can regulate proteasome activity, we are developing a high-throughput screening method based on the yeast protein Ura3p. URA3 converts 5-fluoroorotic acid (5-FOA) into a toxic product, ultimately leading to cell death. We engineered URA3 to create a model substrate of proteasomal degradation by fusing degron tags – short peptides that confer susceptibility to proteasomal degradation – to the C-terminal of Ura3p, thereby generating yeast strains that survive on medium containing 5-FOA when proteasomal activity is enhanced. Two degron tags were used to generate proteasome substrates that are degraded through ubiquitin-dependent and ubiquitin-independent proteasomal degradation. We report studies to correlate the proteasome activity with cell growth and optimize the concentration of 5-FOA in the culturing medium. Results from this work will generate a reliable assay to screen chemical and genetic libraries and isolate compounds and endogenous proteins that enhance proteasome activity, thereby providing potential targets for the development of therapeutics for protein misfolding diseases.