Cellular signal transduction is predominantly based on protein interactions and their posttranslational modifications, which enable a fast response to input signals. Due to difficulties in designing new unique protein-protein interactions, designed cellular logic has focused on transcriptional regulation; however, this has a substantially slower response requiring transcription and translation. Here, we present a de novo design of modular, scalable signaling pathways based on proteolysis and designed coiled-coils (CC) implemented in mammalian cells. A set of split proteases with highly specific orthogonal cleavage motifs was constructed and combined with strategically positioned cleavage sites and designed orthogonal CC dimerizing domains of tunable affinity for competitive displacement after proteolytic cleavage. This enabled implementation of Boolean logic functions and signaling cascades in mammalian cells. Designed split proteasecleavable orthogonal CC-based logic (SPOC logic) circuits enable response to chemical or biological signals within minutes rather than hours, useful for diverse medical and nonmedical applications.

Flagellin is a wide-spread bacterial virulence factor sensed by the membrane-bound Toll-like receptor 5 (TLR5) and by the intracellular NAIP5/NLRC4 inflammasome receptor. TLR5 recognizes a conserved region within the D1 domain of flagellin, crucial for the interaction between subunits in the flagellum and for bacterial motility. While it is known that a deletion of the D0 domain of flagellin, which lines the interior of flagella, also completely abrogates activation of TLR5, its functional role remains unknown. Using a protein fusion strategy, we propose a role for the D0 domain in the stabilization of an active dimeric signaling complex of flagellin-TLR5 at a 2:2 stoichiometric ratio. Alanine-scanning mutagenesis of flagellin revealed a previously unidentified region of flagellin, the C-terminal D0 domain, to play a crucial role in TLR5 activation. Interestingly, we show that TLR5 recognizes the same hydrophobic motif of the D0 domain of flagellin as the intracellular NAIP5/NLRC4 inflammasome receptor. Further, we show that residues within the D0 domain play a previously unrecognized role in the evasion of TLR5 recognition by Helicobacter pylori. These findings demonstrate that TLR5 is able to simultaneously sense several spatially separated sites of flagellin that are essential for its functionality, hindering bacterial evasion of immune recognition. Our findings significantly contribute to the understanding of the mechanism of TLR5 activation, which plays an important role in host defense against several pathogens, but also in several diseases, such as Crohn’s disease, cystic fibrosis and rheumatoid arthritis.

The COVID-19 pandemic is one of the most serious medical emergencies since the last century. SARS-CoV-2, which was first reported at the end of 2019, has affected the entire world, and there are still very few therapeutic options. One of the fastest ways toward therapy would be the repurposing of already approved drugs and dietary compounds. Several drugs have been shown to inhibit viral replication by targeting either viral components, such as inhibitors of viral RNA polymerases, 1,2 or compounds that target the human cell proteins that interact with or process viral components, such as protease or kinase inhibitors [reviewed in 3,4]. Approved compounds that might interfere with the binding of spike proteins to human angiotensin-converting enzyme-2 (ACE2) viral receptors have been proposed. 5 For some compounds, the mechanism of inhibition or molecular target is not clear although the effect on viral replication