Researcher(s)
- Jillian Shelly, Biochemistry, University of Delaware
Faculty Mentor(s)
- Zhihao Zhuang, Chemistry and Biochemistry, University of Delaware
Abstract
Ubiquitin (Ub), a small, highly conserved 76-amino acid protein, plays several roles in post-translational modification and cell regulation such as DNA damage repair and protein degradation. Ubiquitination occurs through a three enzyme cascade in which ubiquitin is conjugated to a lysine residue on a protein of interest (POI) by its C-terminal glycine which can be followed by subsequent ubiquitination steps on one of Ub’s seven known accessible lysine residues to establish polyUb chains1. This process is observed in one of Ub’s heavily studied pathways, proteasomal protein degradation via the 26S proteasome2. This process, however, can be counteracted by deubiquitinating enzymes (DUBs) that cleave the isopeptide bonds of these polyUb chains, effectively removing the Ub moiety/chain from the POI. Dysregulated DUB activity has been linked to cancer and certain neurodegenerative diseases, making their inhibition an area of interest for therapies3. Efforts have been made to find DUB specific inhibitors for the library of unique DUBs that have been identified. The focus of this project, however, is the application of the proteolysis targeting chimera (PROTAC) strategy for targeted DUB degradation as a new class of potent therapeutics for regulation of their harmful activity. PROTAC-mediated protein degradation involves the targeted degradation of a protein of interest by ubiquitination. Construction of a PROTAC activity-based probe includes a pomalidomide ubiquitin ligase recruiting ligand and Ub equipped with a propargyl amine warhead DUB recruiting ligand conjugated by Sortase A. In theory, this probe should allow for potent DUB ubiquitination and targeted degradation in whole cells with the ligation of cell-permeable peptide cR10, granting the probe permeability whose activity could be profiled by western blot.