Wednesday, April 2, 2025
4/2/2025
UBIQUITIN-PROTEASOME PATHWAY in HUMAN DISEASE - ABSTRACT Ubiquitin-Proteasome Pathway (UPP) is a tightly regulated machinery that controls the degradation and turnover of 80-90% of proteins in human cells. Its malfunction is linked to a plethora of human diseases, from neurodevelopmental disorders and viral infections to cancer. The overarching goal of our research program is to develop a detailed understanding of how UPP components recognize their substrates and execute their function, which is of paramount importance for the development of new therapeutic strategies in the future. This MIRA proposal aims to extend our successful UPP research program with a vision to structurally and functionally characterize critical ubiquitinating and de-ubiquitinating enzymes implicated in human diseases. Previously, we have explored protein regulators of the p53 pathway most commonly affected in cancer with a focus on the deubiquitinating enzyme USP7. As a result of this work, we have shown that FL-USP7 is a dynamic enzyme that adopts multiple conformations with transient inter-domain interactions in solution, characterized conformational dynamics of the catalytic domain of USP7 and showed that its active site switches between inactive and low- populated “excited” active states. Together, our data suggest that structural plasticity might be a key feature of USP7 required for the regulation of its activity and might even represent a common mechanism shared by other deubiquitinating enzymes with conserved catalytic sites. This proposal will take advantage of our recent discovery of USP7 mutants with enhanced enzymatic activity identified among patients suffering from Hao-Fountain syndrome – a novel and rare neurodevelopmental disease caused by mutations of the Usp7 gene. Hyperactive mutants are rare in enzymes and provide a unique opportunity to investigate mechanisms of enzymatic activity regulation. In Project 1 we will address the question of the role of conformational dynamics in the regulation of USP7 activity by comparing the dynamics and structures of the hyperactive mutants to those of the WT USP7. A combination of enzymatic assays, NMR relaxation experiments, and X-ray crystallography will be used. Project 2 will focus on the structural characterization of a complex between USP7 and its substrate implicated in Hao-Fountain syndrome development, MAGE-L2 using sequence analyses, quantitative binding assayas, NMR, and X-ray crystallography. In Project 3 cell-based assays and proteomic analyses of Hao- Fountain patient cells will be used to identify major cellular substrates/pathways affected by USP7 malfunction. The anticipated results will provide fundamental molecular mechanisms of action of clinically relevant deubiquitinating enzyme and will help guide future efforts to develop novel therapeutic strategies to treat human diseases associated with UPP malfunction.
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