Programmable Protein Circuits in Living Cells

Circuits of interacting proteins enable cells to sense stimuli, process information, and actuate cellular responses. Rational design of synthetic protein circuits could provide powerful new capabilities to cells, but a general purpose system for design of programmable protein circuits has remained elusive. The key limitation has been a lack of composable protein components that can regulate one another to generate diverse circuit architectures. Here, we introduce a system of engineered viral proteases and show that it can implement diverse functions including regulatory cascades, binary logic gates, and dynamic, analog signal processing. In this system, termed CHOMP (Circuits of Hacked Orthogonal Modular Proteases), an input protease can dock with a target protease using modular leucine zippers and cleave it at a specific site to inhibit its function. Using these components, we then engineered and rationally optimized a circuit that activates caspase-mediated cell death in response to activation of the Ras oncogene. We demonstrated that multi-component CHOMP circuits can be encoded compactly as single transcripts and delivered without genomic integration, avoiding mutagenesis and transcriptional interference. Furthermore, because the operation of CHOMP components does not depend on how they are expressed, they can be optimized using transient transfections, accelerating the overall design-build-test cycle. CHOMP circuits thus offer a composable and scalable platform for programming diverse protein-level functionality in mammalian cells, facilitating applications in regenerative medicine, cell-based therapies, and other biotechnologies.