BME PhD Dissertation Defense: Quan Le

Title: "Development Of Drug-Stabilizable Molecular Tools For Modulating Protein Activity And Intracellular Signaling"

Advisory Committee: John T. Ngo, PhD – BME (Research Advisor) Ahmad (Mo) Khalil, PhD – BME (Chair) Mary Dunlop, Ph.D. – BME Zeba Wunderlich, Ph.D. – Biology Liangliang Hao, PhD – BME

Abstract: Nature has evolved elegant mechanisms to regulate the activities of biomolecules that make up sophisticated cellular processes. In order to untangle complex biological systems, researchers have developed a wide variety of molecular tools that allow them to study individual biological processes in isolation, such as small-molecule dimerizers that are used to precisely determine when protein interactions occur. There remains a constant need for new orthogonal biological parts to simulate cellular processes for basic biology and therapeutic applications. In this Thesis, we develop a variety of protein-based tools using NS3, a protease derived from hepatitis C virus. Due to its high substrate specificity and availability of clinically approved inhibitors, NS3 has been used in previous works to make drug-controllable tools. We first utilize NS3 to convert nanobodies into single-chain binders with drug-controllable binding activity. We demonstrate that this engineering strategy is generalizable to multiple nanobodies, and that these engineered nanobodies can be used to control endogenous signaling activity. We then develop a genetically encoded inhibitor of Notch signaling with drug-dependent stability using NS3. We show that this inhibitor can nearly fully repress Notch-mediated signaling in a drug-dependent manner and can target multiple Notch paralogous receptors. Finally, we explore a budding approach to targeted protein degradation that directly engages the proteasome and bypasses ubiquitination. We demonstrate that our design, which consists of a proteasome-binding nanobody and NS3, can be used to degrade proteins in a drug-dependent manner. We also show various ways that this nanobody can be used to make new tools. With this work, we hope to not only contribute to the molecular toolbox, but also to devise new protein engineering strategies that will inform future tool designs.