Mechanisms of USPs, and Sequential Cleavage of Ubiquitin Chains on PCNA by USP1/UAF1

In every cell in our body, proteins are the tiny molecular machines that carry out all the different functions of the cell, virtually performing every single active process in the human body. A single protein can perform multiple roles within the cell, which needs to be tightly regulated so that we do not develop disease. By attaching ubiquitin to a protein, the cell can change the fate of that protein: switching between pathways or directing it towards degradation. In this thesis, we study the molecular process in which ubiquitin is removed from other proteins in a process called ‘deubiquitination’, with a focus on the mechanisms of deubiquitinating enzymes that belong to the USP family. The results reveal novel insights in the function and regulation of different USPs. Importantly, it shows that individual USPs distinguish themselves in important ways on a molecular level, despite their many similarities. We show that USP1/UAF1 uses a unique ubiquitin-chain cleaving mechanism unlike any other USP. USP48 is revealed to have a C-terminal peptide, party similar to that of other USPs, which distinguishes itself in important way. Finally, we show that the mechanism of catalysis itself, thought to be highly conserved in USPs, actually varies between different USPs and is more complex than we realized. These biochemical and structural studies of this thesis allow us to gain detailed insights into the nuances of proteins, which can guide and aid us in better understanding the cell.