(110g) A Platform for the Discovery of Proteasomal Degradation Activators

Protein misfolding and aggregation currently comprise one of the main challenges of the fields of bioengineering and biotechnology. Protein folding in the crowded cellular milieu leads to the formation of natively folded as well as off-pathway misfolded species. Misfolded proteins are intrinsically prone to aggregation and, since aggregates are toxic to cells, they must be immediately degraded. In eukaryotic cells, the degradation of misfolded proteins is catalyzed by the ubiquitin proteasome system (UPS). However, aberrant accumulation of misfolded proteins often overloads the UPS capacity and causes the formation of insoluble aggregates. Aggregation is a key impediment to the high expression of recombinant proteins not only in bacteria but also in eukaryotic cells and limits the high-yield production of proteins for research, diagnostic and industrial applications. Protein misfolding and aggregation are also hallmarks of pathogenesis in a number of human diseases, such as Parkinson’s and Alzheimer’s. Enhancement of proteasomal degradation is hypothesized to facilitate intracellular clearance of misfolded proteins, thus restoring protein homeostasis. Compounds that inhibit proteasomal degradation are widely used in research and have transitioned to clinical therapeutics. Although the mechanism of proteasomal inhibition is understood, we currently do not know how to enhance proteasomal degradation. Our laboratory developed a technology for detecting proteasomal degradation enhancement based on coupling the increase in degradation of a UPS model substrate to an easily detectable fluorescent output. We employed a synthetic biology approach to generate a cellular circuit in which the expression of a fluorescent protein is under the transcriptional repression of an UPS model substrate. This novel strategy overcomes limitations of current methods, which are based on detecting a decrease in concentration of a degradation-prone protein and thus are prone to artifactual results. We also generated a computational model to predict how fine-tuning this cellular circuit and, particularly, inducing self-activation of the UPS model substrate’s expression affect the intensity of cell fluorescence. The results of this simulation led to assay optimization for high-throughput screening applications. We report here the development of this innovative technology and its use for the discovery of molecular probes that enhance the cell’s innate ability to dispose of misfolded proteins.