Deciphering the regulatory logic of the ubiquitin system

As the primary route through which eukaryotic cells achieve selective protein degradation, the ubiquitin-proteasome system (UPS) plays a key role in virtually all critical cellular processes. A major unresolved question concerns how the ubiquitin system attains such high selectivity towards its myriad of substrates. The main specificity determinants are the E3 ubiquitin ligases, which recognise cognate ‘degron’ motifs found in substrate proteins. However, for the majority of the ~600 E3 ligases encoded in the human genome we still have little or no knowledge as to their substrates, and our understanding of degron motifs remains limited. This knowledge gap prevents us from appreciating how key cellular processes are regulated and impedes the development of small molecules capable of either inhibiting or hijacking E3 ligases for therapeutic benefit. To complement the biochemical and proteomic techniques that have served as the primary route to discovery in the field, we seek to exploit functional genetic approaches to understand how E3 ligases recognise their substrates. Leveraging an expression screening platform that enables proteome-wide stability profiling, our specific goals are to: (1) identify physiological substrates regulated via degrons lying at their extreme C-termini, (2) define novel mechanisms through which conditional protein degradation is achieved via phospho-degrons, and (3) characterise the spectrum of degradative pathways responsible for the instability of hundreds of the most short-lived cellular proteins. For each goal, we will first provide global insight by systematically interrogating the proteome for relevant substrates; for the most interesting candidates, we will then seek detailed mechanistic understanding to illuminate new biology. Successful completion of this work will transform our understanding of how specificity is achieved within the UPS and greatly facilitate its future therapeutic manipulation.