Engineering Bioorthogonal Protein Tools for Studying Cell Signaling

Abstract

Chemically controlled genetic tools allow the dose- and time-dependent manipulation of intracellular protein function. Among these, systems built around the hepatitis C virus protease NS3a and clinically approved antiviral drugs are particularly attractive because they are bioorthogonal to mammalian cells, multi-input, and modular. In this dissertation, I develop and apply NS3a-based bioorthogonal tools to probe cell signaling. In Chapter 1, I describe a transcriptional reporter assay to screen a comprehensive single-substitution library of the NS3a-binding peptide ANR for variants with enhanced intracellular engagement. Combining the most effective substitutions yields an affinity-optimized ANR that enables tunable drug-dependent transcriptional control and supports the construction of autoinhibited chemically inducible activator of RAS (NS3a-CIAR) switches with low basal signaling. In Chapter 2, I apply this optimized NS3a-CIAR to dissect how the architecture of the epidermal growth factor receptor (EGFR) shapes RAS-MAPK signaling. By embedding NS3a-CIAR within the EGFR transmembrane scaffold, I show that membrane localization is necessary but not sufficient for receptor-tethered activators to drive downstream signaling: chimeras lacking the EGFR extracellular region (ECR) or carrying a constitutively dimeric Fc replacement remain properly membrane-integrated yet fail to activate ERK. Using oncogenic EGFR truncations to systematically dissect the ECR, I find that most of the extracellular region is dispensable but that a short 11-amino-acid segment—specifically three cysteine residues within it—is required for productive coupling between RAS activation and MAPK signaling. Direct measurement of RAS-GTP formation using a live-cell FRET biosensor further reveals that this ECR feature acts not at the level of RAS activation itself but at the propagation of that signal through the MAPK cascade. Together, this dissertation establishes optimized NS3a-based bioorthogonal tools and demonstrates their utility as precise probes of receptor-proximal control of mammalian signal transduction.