ProNotch converts extracellular protease activity into programmable transcriptional outputs.

Area of Science:

• Synthetic biology
• Molecular and Cell Biology
• Biotechnology

Background:

• Modular platforms for coupling extracellular protease activity to gene expression are limited.
• Synthetic receptors are needed to expand the toolkit of mammalian cell biology and engineering.

Purpose of the Study:

• To introduce ProNotch, a novel receptor architecture for protease-gated gene expression.
• To demonstrate ProNotch's modularity, protease specificity, and logic gate capabilities.
• To validate ProNotch's function in detecting endogenous protease activity and controlling cell-state transitions.

Main Methods:

• ProNotch utilizes protease-cleavable linkers to release inhibitory single-chain variable fragments (scFvs) from mutant Notch1 negative regulatory regions (NRRs).
• Linker substitution allowed reprogramming of protease specificity.
• Single-chain receptor designs enabled OR and AND logic gates for integrating multiple protease inputs.

Main Results:

• ProNotch successfully detected endogenous matrix metalloproteinase-14 (MMP-14) activity from cancer cell lines.
• The system drove protease-dependent cell-state transitions in fibroblasts.
• The ProNotch regulatory module functioned as a protease-activated pro-antibody, conditionally inhibiting NOTCH1 signaling.

Conclusions:

• ProNotch is a versatile, modular platform for engineering protease-responsive cells.
• The system enables programmable transcriptional outputs based on extracellular protease activity.
• ProNotch technology can be extended to develop soluble, conditionally activated inhibitors for therapeutic applications, particularly in NOTCH1 signaling pathways.