ProNotch converts extracellular protease activity into programmable transcriptional outputs

Synthetic receptors that convert extracellular protease activity into programmable transcriptional outputs would expand the toolkit of mammalian cell biology and cell engineering, yet modular platforms for directly coupling extracellular proteolysis to gene expression remain limited. Here we introduce ProNotch, a receptor architecture that harnesses protease-gated derepression of a mutant Notch1 negative regulatory region (NRR) to drive user-defined gene expression. ProNotch tethers destabilized NRR mutants to inhibitory anti-NRR single-chain variable fragments (scFvs) via protease-cleavable linkers. NRR engagement by high-affinity scFvs simultaneously rescues surface trafficking of mutant receptors and suppresses basal signaling until linker cleavage releases the inhibitory scFv module, permitting the destabilized NRR to initiate ligand-independent signaling. Linker substitution reprogrammed protease specificity across diverse enzymes, and tandem substrate repeats enhanced sensitivity without increasing basal activity. Single-chain receptor designs enabled OR and AND logic gates, allowing integration of multi-protease inputs into a single transcriptional output. ProNotch detected endogenous MMP-14 activity from cancer cell lines in cis and in trans and drove protease-dependent cell-state transitions in C3H/10T1/2 fibroblasts. The scFv-NRR module also functioned as a protease-activated pro-antibody that conditionally inhibited DLL4-dependent signaling and ligand-independent activation of mutant NOTCH1 in the T cell acute lymphoblastic leukemia cell line HPB-ALL. Together, these results establish ProNotch as a modular platform for engineering protease-responsive cells and demonstrate that its regulatory module can be extended to a soluble, conditionally activated inhibitor of NOTCH1 signaling.