This multidisciplinary project will provide a fundamental understanding of the mechanism of action of a new class of enzymes. It focuses on one member of this class, the human enzyme USP7, that plays a central role in fundamental cellular processes such as DNA regulation, stress response and protein stability control. The aim of this project is to understand how the activity of USP7 is regulated and how it recognizes its diverse targets. To fill a critical gap in knowledge of USP7 function, an interdisciplinary approach will be employed that combines solution Nuclear Magnetic Resonance (NMR) spectroscopy, cryo-Electron Microscopy, Small-Angle X-ray Scattering (SAXS), a variety of biophysical and biochemical methods, and a range of in vitro and in vivo assays. This project will create a platform for teaching, training and learning, including outreach to underrepresented minorities to promote understanding of basic science. This project will also promote teaching and training in the K-12 environment by creating several short-term educational and training opportunities.
The goal of this project is to elucidate the molecular mechanisms of activation and substrate specificity of the deubiquitinating enzyme USP7, which rescues a number of diverse cellular and viral proteins from proteasomal degradation. Its substrates include a number of transcription factors, E3 ubiquitin ligases and epigenetic modifiers. USP7 exemplifies a newly recognized subgroup of deubiquitinating enzymes that is characterized by the presence of multiple ubiquitin-like domains (UBLs). These UBL domains are not catalytically active, but can greatly influence the activity of the enzyme by a yet unknown mechanism. The mechanisms of USP7 activation will be addressed by determining the arrangement of individual USP7 domains within the full-length enzyme. The molecular basis of USP7 specificity towards its substrates will be addressed by identifying and mapping distinct sites on C-USP7 responsible for recognition of two transcription factors, p53 and FOX(O)4. An array biophysical and spectroscopic methods will be employed in vitro and in vivo experiments. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.
