Structural and Functional Characterization of Ubiquitin Variants and Ubiquitin Binding Proteins

The ubiquitin (Ub) system is an essential post-translational modification pathway found throughout eukaryotic cells and consists of proteins responsible for adding Ub to substrates (E1, E2, and E3 enzymes), removing Ub from substrates (deubiquitinases), and reading the Ub encoded signal found on substrates. The Ub system regulates cellular functions such as DNA repair and cell-cycle progression and as such, its dysregulation can contribute to disease states. Due to its prevalent nature in regulating critical cellular functions therapeutic modulation of the Ub system has become a promising area for therapeutic development. The first step towards modulating any cellular system is target identification, which requires a detailed understanding of the system’s components and how they function. Towards this end, Dr. Sidhu and his lab have developed a set of protein-based tools termed Ub variants (UbV). These reagents are engineered variants of Ub that contain substitutions that allow them to selectively and tightly bind to specific Ub binding proteins in the cell, leading to the inhibition of and in some cases activation of the targeted protein’s function. Consequently, researchers can use UbVs to understand the role of the targeted proteins in vivo and their potential as therapeutic targets. In this thesis, I aid our understanding of the Ub system through the use of biochemical and structural biology approaches by: (i) helping further the development of UbVs tools by identifying how they bind with high specificity and affinity to their target proteins and (ii) furthering our understanding of how the deubiquitinase USP37 uses a unique insertion containing Ub interacting motifs to cleave ubiquitin chains.Ph.D