External control over synthetic-biological systems or generally cellular processes can be exercised at various levels – transcription, translation, protein function, or protein stability. Recently, there has been growing interest in using protein degradation to control protein stability for research and therapeutic purposes. Developing systems for the degradation of target proteins requires diverse approaches to enable the interaction of target proteins with endogenous protein degradation machinery.
The primary pathway for cellular protein degradation is via the ubiquitin-proteasomal system, where ubiquitin ligases, or E3 ligase complexes, play a key role in recognizing and marking proteins for degradation. This work aimed to explore the use of various E3 ligase subunits as degrons for the degradation of cellular proteins, to investigate their properties, to develop methods to regulate their activity, and to test their usefulness for degrading various substrates. In designing such degrons, we relied on structural data from the SCF-Skp2 E3 ligase complex, composed of Skp2, Skp1, Cul1, Rbx1 proteins, and also explored the use of CDC34 E2 protein as a degron. We showed that all subunits of the SCF-Skp2 E3 ligase can be used as designed degrons, enabling efficient degradation of protein substrates and that their effectiveness could be enhanced by considering their interactions with other proteins. Degradation of substrates fused with degrons was very effective, comparable to the best-performing degrons in the literature.
The value of a synthetic biology tool depends on its modularity, adaptability, and robustness of operation. We described several factors that affect the efficiency of degradation with designed degrons, which researchers must consider when implementing them in their own environment. We demonstrated that degradation efficiency can be modulated by changing the affinity between the substrate and degron and influencing the interaction surface of the degron. We developed several methods of control over the activation or repression of degradation, based on the use of viral proteases and chemically inducible dimerization. To enable the degradation of substrates in different cellular compartments, we utilized the concatenation of degrons. The concatenated degron proved to be very robust, enabling the degradation of cytosolic, nuclear, and membrane proteins, with rapid kinetics and good activity in various cell lines.
Our results provide insights into the use of subunits of natural protein degradation systems in systems for control the expression of target proteins and strategies for modulation and approaches to control their activity.
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