Developing a tool to temporally degrade proteins

Abstract

To investigate the effects of proteins on cellular processes, researchers employ a variety of strategies to modulate protein expression levels. Current methods include genomic approaches like CRISPR-Cas9 knockouts, post-transcriptional approaches such as RNA interference, and protein-level approaches using chemical inhibitors and inducible degron systems. However, these strategies cannot control protein expression levels during specific cell cycle stages. To address this limitation, we aimed to develop a novel inducible system for temporal protein degradation from anaphase onset until G1-phase by using the ubiquitinase activity of the anaphase-promoting complex/cyclosome. Inspired by the fluorescent cell cycle reporter (Fucci) system, we linked geminin (hGem), a direct substrate of APC/C, to proteins of interest. Two constructs were developed: (I) hGem(1:60) linked to eGFP and FRB (the degron), and (II) two FKBP domains linked to mCherry or mRuby and the protein of interest (the prey). Live-cell imaging demonstrated that the degron construct degrades after anaphase onset. Fixed-cell imaging demonstrated recruitment of degron counterparts to prey constructs, while live-cell imaging revealed proteasomal degradation of the degron but not the prey after anaphase onset. Colocalization analysis indicated dimerization in only 20% of cells, by which degron constructs are protected from degradation. We hypothesize that the low efficiency of rapalog-induced dimerization and subsequent protection from degradation are distinct challenges, both of which could be explained by individual steps in the experimental design. This thesis lays the foundation for the temporal perturbation of proteins from anaphase until G1-phase and identifies challenges for the development of this system.