(3ab) Harnessing Degron-Based Substrates As Tools for the Development and Evaluation of Novel Chemotherapeutics Targeting the Ubiquitin Proteasome System

Aberrant protein expression can lead to dysregulation of essential cell signaling pathways culminating in a variety of cancers.  These abnormal protein levels are the result of improper activity of the ubiquitin proteasome system (UPS), specifically the family of E3 ubiquitin ligases which are responsible for the recognition and degradation of target proteins.  E3 ligases, the final component of a cascade of E1, E2, and E3 enzymes, recognize a specific amino acid sequence, or degron, on a target protein which facilitates their binding and, ultimately, the transfer of the small protein ubiquitin to a proximal lysine residue.  Ubiquitin polymerization results in target protein recognition by the proteasome, where it is degraded into small peptide fragments.  The large number of E3 ligases (>600) confer the specificity necessary for this degradation pathway and, as such, offer an attractive target for potential chemotherapeutics.  For example, blocking expression of the E3 ligase Hdm2 has been shown to stabilize p53, a powerful tumor suppressor.  Further, in recent years it has been demonstrated that improper E3 ligase signaling results in defects in a variety of cellular responses such as the cell cycle, cell migration, epithelial-mesenchymal transition, intracellular trafficking, and DNA damage repair. 

In my research I propose to harness small (~25-30 amino acid) degron-based substrates as novel tools for understanding the intracellular dynamics of E3 ligase/degron binding. While current research mainly focuses on the identification of new E3 ligases and degron targets, I propose to conduct an in-depth study of E3 ligase signaling dynamics across a wide range of cells, protein-protein interactions, and different classes of degrons (i.e. phospho-degrons, oxygen-dependent degrons, and the N-end rule pathway) to identify potential targets for reporters and inhibitors.  As reporters, the degron-based substrates could be utilized to evaluate a cellular response to current E3 ligase-based chemotherapeutics (such as MLN-4924).  As inhibitors, I believe that degron-based substrates could provide a new method for competitive inhibition of unregulated E3 ligases.  As previously mentioned, the binding of the E3 ligase to the target protein is the essential step for the targeted degradation of a protein which can result in improper protein clearance.  By competitively targeting the E3 ligase, I aim to abrogate this protein clearance while simultaneously minimizing off-target effects commonly associated with small molecule based therapeutics.  Many of the current UPS-targeted chemotherapeutics mainly focus on E3 ligase/degron combinations involved in the cell cycle (i.e. APC/C, p53, and Cdc4); however an increasing amount of evidence suggests that uncontrolled E3 ligase activity plays an important role in the directed migration of cells in response to extracellular gradients of chemoattractants, or chemotaxis.  Recent work has identified potential E3 ligases such as c-Cbl, SMURF1/2, and Hdm2 as regulators of cell migration; although many of these studies use bulk, qualitative metrics such as the scratch assay or the Boyden chamber.  I intend to expand on this work by taking advantage of the degron-based substrates as both reporters and inhibitors of E3 ligases involved in cell migration.  Finally, to evaluate the spatial and temporal dynamics of E3 ligases on chemotaxis I will develop novel microfluidic devices designed to deliver controllable gradients of chemoattractants to both mesenchymal and amoeboid cell migration.  Similar to the chemotherapeutics developed against kinases to specifically target cancer, I believe that specifically targeting the E3 ligase/degron interaction will open the door to a new level of chemotherapy drugs targeting an array of cancers.