(217b) The Deg-on System: A Mammalian Synthetic Gene Circuit for Sensitive Detection of Proteasomal Activation

Protein homoeostasis in eukaryotic cells relies on the quality control function of the ubiquitin proteasome system (UPS), a complex macromolecular machinery that regulates protein turnover and catalyzes degradation of misfolded or damaged proteins. UPS dysfunction has been linked to the pathogenesis of several diseases, ranging from cancer to cardiovascular and neurodegenerative disorders, implicating the UPS as a molecular target for therapeutic intervention in a diverse range of human diseases. Small molecules and peptides that inhibit the peptidase activities of the proteasome have been widely used to characterize the role of the UPS in maintaining protein homoeostasis and transitioned to the clinic for treatment of cancer and inflammatory diseases. On the other hand, pharmacological agents that increase UPS activity remain largely unexplored, although this mode of regulation has great potential to ameliorate the phenotypes associated with the development of protein deposition diseases, such as Parkinson’s and Huntington’s. To develop a mammalian cell-based platform to quantify proteasomal degradation that is amenable to high-throughput screening of UPS activators, we created a genetic inverter, the Deg-On system. This orthogonal genetic circuit translates proteasomal degradation of an engineered version of the transcriptional regulator TetR into a fluorescent signal, thereby linking UPS activity to an easily detectable output. The output signal can be finely tuned using tetracycline, enabling calibration of the inverter for the detection of different degrees of activation of UPS function. Predictive modeling was used to improve the sensitivity and dynamic range of the Deg-On system and lead to the design of the ‘enhanced Deg-On’ (eDeg-On) system, a genetic inverter relying on a positive feedback loop that enables self-amplification of engineered TetR. Introducing an artificial positive feedback loop as a genetic amplifier, the eDeg-On system may exhibit bistability, thereby resulting in hysteresis or ‘cellular memory’ in response to tetracycline dosage and UPS activity. The input–output behavior of the eDeg-On system was also confirmed in the context of stable cell lines generated by genetically integrating the relevant parts of the circuits. By providing a cell-based platform to quantify modulation of UPS activity, this eDeg-On cell line is used in the screening for compounds that enhance UPS activity, which will open the way to the development of therapeutic strategies to reduce the accumulation of aberrant proteins under conditions of proteotoxic stress.