From detection of proteasomal degradation to targeted control of protein depletion
Doctor of Philosophy
The ubiquitin proteasome system (UPS) regulates protein turnover and catalyzes degradation of misfolded or damaged proteins, thus playing an important role in maintaining protein homoeostasis in eukaryotic cells. The UPS has emerged as a drug target for diverse diseases characterized by altered protein homeostasis. However, while proteasome inhibitors are widely used in research and have transitioned to clinical settings, pharmacological agents that enhance proteasomal degradation are rare and poorly characterized. In addition, approaches aimed at controlling the expression levels of cellular proteins by modulating proteasomal degradation are typically based on cumbersome modifications of the target protein. To generate a tool to monitor increase in proteasomal degradation, I developed a genetic inverter (Deg-On system) that translates proteasomal degradation of a transcriptional regulator engineered to function as a UPS substrate into increase in expression of a fluorescent reporter (GFP), thereby linking enhancement of UPS activity to an easily detectable output. I demonstrate that this genetic inverter responds to modulation of UPS activity. Guided by predictive modeling, I modified this genetic inverter by introducing a positive feedback loop that allows self-amplification of the transcriptional regulator, thereby enhancing the output signal sensitivity and dynamic range. This technology for monitoring proteasomal activation will be useful for a variety of applications, including the discovery of UPS activators and the selection of cell lines with different levels of proteasome activity. To achieve fast, targeted, and predictable control of cellular protein levels without genetic manipulation of the target, I developed a technology for post-translational depletion based on a bifunctional molecule (NanoDeg) consisting of the antigen-binding fragment from the Camelidae sp. heavy-chain antibody engineered to mediate degradation through the proteasome. Guided by predictive modeling, I show that customizing the NanoDeg rate of synthesis, rate of degradation, or mode of degradation allows to achieve quantitatively predictable control over the target’s levels. Integrating a GFP-specific NanoDeg within the Deg-On system results in enhanced dynamic range and resolution of the output. By providing predictable control over cellular proteins’ levels, the NanoDeg could be used for systems-level analyses of cellular protein function and to improve the design of mammalian gene circuits.
Ubiquitin-Proteasome System; Protein Degradation; Control protein level; Synthetic Biology