Weak and transient protein-protein interactions are essential for cellular homeostasis but are challenging to study and therefore incompletely understood. Many of these interactions involve globular domains that recognize a short linear motif (SLiM) in disordered regions of interacting proteins. E3 ubiquitin ligases frequently use this principle of interaction to recognize their substrates. The conjugation of ubiquitin chains to a substrate often serves as a signal for degradation by the proteasome, and the recognized SLiM is therefore referred to as a degron. The ubiquitin system has a well-established critical function in genome stability maintenance, which is of fundamental importance for avoiding severe diseases such as cancer. A number of E3 ubiquitin ligases have key roles in promoting DNA damage signaling and repair pathways, but mechanistic insights into how these ubiquitin ligases recognize their substrates are lacking. This is due in large part to an absence of sensitive methods for discovery and characterization of transient E3 ligase-degron interactions. Here, I propose to apply and combine two novel methods in an innovative workflow to remedy this knowledge gap. I will use a powerful and highly sensitive novel protein-protein interaction mapping technique (SPARK2) to screen peptides derived from the disordered proteome for their interaction with E3 ligases important for genome stability maintenance. Detailed characterization of identified potential SLiMs interacting with DNA damage-responsive E3 ligases will subsequently be performed using MRBLE:pep, a novel multiplexing approach to simultaneously quantify dozens of protein-peptide affinities that I recently helped to develop. This in-depth characterization of E3 ligase degrons, together with functional studies of validated novel E3 ligase-substrate interactions, will enable unprecedented mechanistic insights into regulatory networks underlying genome stability maintenance.
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