Abstract
Ubiquitylation is a type of post-translational modification that regulates many cellular functions. The attachment of ubiquitin depends on a cascade of three enzymes, which includes ubiquitin conjugating enzymes that catalyse the second step of the cascade and help determine the type of polyubiquitin chains built on substrate proteins. Ube2R1, also known as cell cycle dependent 34 (CDC34), is a ubiquitin conjugating enzyme that specifically catalyses the synthesis of lysine 48-linked polyubiquitin chains that trigger proteasomal degradation of substrate proteins. Substrates of Ube2R1 include the important cell cycle regulators p21(Cip1), p27(Kip1) and cyclin E. Deregulation of Ube2R1 has been found in various types of cancer and neurodegenerative diseases making it an appealing drug target. However, no Ube2R1-targeting compounds have been approved for clinical use. The goal of the project is to develop tools that could help study Ube2R1 function in cells and might serve as a starting point for drug development.
This project investigated two peptide-based modulators of Ube2R1 that were previously discovered by phage display. Peptide 1 is a 12-residue linear peptide, whereas peptide 2 is a 10-residue circular peptide connected by a disulfide bond. By using in vitro ubiquitylation assays, peptide 2 was found to quench ubiquitin chain building by Ube2R1. The mechanism of inhibition requires more investigation, but peptide 2 may act as a preferred substrate for Ube2R1 ubiquitylation. Biophysical techniques were used to assess the affinity of peptide-Ube2R1 interactions. Peptide 1 was shown to have a low micromolar affinity for Ube2R1 while the interaction with peptide 2 was not detected.
To have detailed understandings of peptide-Ube2R1 interactions, attempts were made to crystallise peptide-Ube2R1 complexes. Although crystals were obtained, bound peptide was not found in the electron density. Since the peptide-Ube2R1 complex structure has not been solved experimentally, computational biology tools including AlphaFold and a peptide-protein docking algorithm were used. The computational tools provided interesting peptide-Ube2R1 binding poses but did not explain the experimental data and further analysis is required.
In conclusion, this project demonstrated that peptide 1 binds to Ube2R1 but does not alter its activity, whereas peptide 2 acts as a preferred substrate of ubiquitylation and prevents ubiquitin chain building by Ube2R1. Future experiments will focus on determining the structures of peptide-Ube2R1 complexes and optimising peptide-Ube2R1 binding. It is hoped that peptide-Ube2R1 complex structures will identify new druggable surfaces that could inform fragment-based drug discovery. In addition, strongly binding peptides may form a framework for developing proteolysis targeting chimeras (PROTACs) to selectively degrade pathogenic proteins.