Figure 5.
Modular protease- and coiled-coil-based molecular circuits
(A) Designed molecular circuits that can sense different input signals, process information, and trigger complex cellular responses.
(B) Split-protease-cleavable orthogonal-CC-based (SPOC). Strategically positioned protease cleavage sites and CCs enabled different levels of binding and competition. This allowed the creation of a range of two-input Boolean logic circuits.
(C) Logic and circuits of hacked orthogonal modular proteases (CHOMP). Similar to SPOCK, CHOMP relies on protease-induced regulation; however, an additional type of regulation is added through the implementation of degrons.
(D) Engineered inducible secretion of proteins from the endoplasmic reticulum (ER), lumER, RELEASE, and membER. CC-operated split protease system applied to an inducible protein secretion system enables regulated secretion of therapeutic proteins by an array of inputs.
(E) CC-based two-part transmembrane synthetic receptor termed DocTAR. One part carries a protease and the other contains a protease-responsive split transcription factor fused to an autoinhibited antiparallel CC. Ligand-based bridging enables the release of a split transcription factor that is reconstituted in the cytosol by a third part of the system via CC interaction.
(F) CC modified proteolytically engineered activators of calcium channels (PACE). The system is composed of a CAD protein domain, which can activate Orai Ca2+ channels, and a CC pair that renders the domain inactive. Activation was achieved by strategically incorporating protease cleavage sites positioned at the end of the CC inhibitory region.