|Challenge: Human cells eliminate non-functional proteins using a sophisticated degradation pathway named the ubiquitin proteasome system (UPS), in which UPS enzymes attach a small protein called ubiquitin to damaged target proteins to tag them for degradation. However, abnormalities in protein degradation are frequently observed in many diseases, including cancer where aberrant control of protein degradation can lead to uncontrolled cell growth. As such, UPS enzymes represent novel targets for drug development but unfortunately, the paucity of selective molecules which modulate their function has severely hampered attempts to manipulate them for therapeutic benefit.
Solution: The goal of this project was to apply protein engineering methods to design highly specific and potent ubiquitin-like molecules that associate tightly with UPS enzymes to affect their catalytic function, and thereby enlarge the spectrum of tractable targets associated with cancer.
Achievements/Impact: A library of ubiquitin variants (UbVPs) was generated to modulate activity and function of five specific UPS enzymes termed RING E3 ligases. Such ligases are involved in the ubiquitin conjugation process of histone H2A, a protein critical for transcription and DNA repair and to the stability of the p53 tumor suppressor protein. After conducting an in vitro screening of the UbVPs library on the selected targets, eight UbVPs affecting enzyme activity were successfully identified. In mammalian cell culture, an enhanced ubiquitination state of p53 was observed in presence of one ubiquitin variant. Furthermore, it was demonstrated that following the expression of selected UbVPs, there was a notable increase in the level of histone H2A ubiquitination.
These results indicate that it is now possible to generate potent and specific modulators of this family of enzymes, a finding that will help in guiding the development of novel protein therapeutics to treat proteasome associated diseases such as cancer.