Sensitive detection of proteasomal degradation using orthogonal gene circuits

The ubiquitin proteasome system (UPS) has been linked to the development of a diverse range of diseases characterized by protein misexpression and accumulation of aberrant proteins. Evidence of the molecular mechanisms that could be potentially targeted to enhance UPS activity have begun to emerge, including the upregulation of UPS components and modulation of the conformational changes that are rate-limiting for peptidase activity. These studies clearly suggest that upregulation of UPS activity holds potential for the treatment of protein misfolding diseases in which the formation of nonnative proteinaceous inclusion bodies is associated with neurodegeneration. However, the molecular mechanisms controlling proteasomal activity are not sufficiently understood to rationally design compounds to upregulate UPS activity. Pharmacologic agents that enhance UPS activity are rare and hard to discover because current in vitro technologies
to screen compounds that enhance UPS activity fail to recapitulate the complexity of the quality control system within the context of a cell. Moreover, existing cellular assays are based on reporter proteins engineered to function as model UPS substrates, which are poorly suited to screen for enhancement of proteasomal degradation, since a loss of signal in these assays can arise from disruptions of many basic cellular processes.

To develop a mammalian cell-based platform to quantify proteasomal degradation that can overcome these challenges, we explored a series of orthogonal genetic circuits that interface with the natural proteasomal degradation machinery to convert modulation of UPS activity into an easily detectable fluorescent output signal. We developed a platform (Deg-On system) based on an engineered tetracycline repressor (TetR-Deg) that functions as UPS substrate and as repressor of GFP expression. As a result, TetR-Deg serves as a signal inverter that converts enhancement of proteasomal degradation (and thus reduced, potentially undetectable cellular concentration of a proteasome substrate) into increase in GFP output (Nature Communications, accepted). Guided by predictive modeling, we enhanced the circuit’s signal sensitivity and dynamic range by introducing a feedback loop that enables self-amplification of TetR. By linking UPS activity to a simple and tunable fluorescence output, these genetic inverters will enable a variety of applications, including screening for UPS activating molecules and selecting for mammalian cells with different levels of proteasome activity.

To introduce memory of the output signal we are also currently exploring alternative circuit architectures, including an UPS sensitive toggle switch. We report here the use of synthetic biology tools for the design and implementation of tunable cell-based platforms to quantify the enhancement of proteasomal degradation in tissue culture that will be generally useful for discovering and characterizing natural and synthetic proteasome activators that target degradation in a wide range of cell types and for different therapeutic applications.