Synthetic Signal Sensing and Transduction Systems Based on Autoinhibited Proteases

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Viktor Stein, Kirill Alexandrov

University of Queensland, Australia

A synthetic signaling system has been developed based on engineered viral proteases. The signaling platform mimics the principles of natural signaling systems as signal transducers are formed by artificially autoinhibited proteases which act as molecular switches able to receive, convert, transmit and amplify biomolecular signals. The system is highly engineerable featuring a high degree of orthogonality and modularity as viral proteases with stringent and well-defined substrate specificities are recombined with engineered autoinhibition-domains and other functional elements to create different types of molecular switches in a plug-and-play fashion. Functionality is primarily conferred by structural and functional features in the linker connecting the transducer protease with the autoinhibition-domain. For instance, highly specific protease biosensors for biomedical applications were constructed by incorporating cleavage sites for several clinically important proteases into the linker. In the presence of the target protease, the autoinhibition-domain is then irreversibly separated from the transducer protease causing its activity to increase 150-200-fold. The specificity of the assay could be further improved by affinity targeting of the biosensor to its target protease, either directly through antibody-like fragments or indirectly though monoclonal antibodies effectively adopting the latter as synthetic signaling scaffolds. Using a second autoinhibited transducer protease with substrate specificities orthogonal to the first stage, it subsequently became possible to amplify protease signals through linear amplification cascades and positive feedback loops enhancing sensitivity and shortening response times. Moreover, the components of a basic autoinhibited protease unit could be rapidly repurposed to create protease-based ligand sensors. To this end, an allosteric binding scaffold was incorporated into the linker yielding an allosterically regulated protease whose activity could be modulated 15-fold following addition of the peptide ligand. The ligand sensor could then be readily connected to protease-based amplification motives forming an integrated signal sensing and amplification network.