BME PhD Dissertation: Christopher Kuffner

Title: "REGULATED PROTEIN DEGRADATION STRATEGIES FOR INVESTIGATING AND ENGINEERING MAMMALIAN CELL BIOLOGY"

Advisory Committee: Stephen Blacklow, M.D., Ph.D - HMS Biological Chemistry and Molecular Pharmacology Michael Economo, Ph.D - BME (Chair) Alex Green, Ph.D - BME John Ngo, Ph.D - BME (Advisor) Zeba Wunderlich, Ph.D - Biology

Abstract: Cells conduct precise protein quality control programs to finely regulate where, when, and in what state proteins exist. Work in synthetic biology has shown that these natural mechanisms can be adapted to engineer sensitive tools for the study and control of cells. In this thesis, I describe new engineered proteins that enable detailed observation and manipulation of mammalian cellular processes with increased spatial, temporal, and molecular precision.  In Part I, I discuss novel cell biology tools based on regulated protein degradation and preservation. First, I describe the use of a degron-sequestration strategy to engineer proteins with RNA-binding-dependent stability. These can be used to visualize single RNA molecules in live mammalian cells with enhanced clarity. I additionally describe new cooperative RNA-binding proteins that can regulate and respond to RNA. Part II of this thesis describes the design of SynNotch-based receptors with expanded sense-and response capabilities, including receptors capable of detecting extracellular protease activity or the folding state of extracellular matrix (ECM) components. Finally, strategies for elaborating the output modality of SynNotch are described.