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.

Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Response of protein secretion based on transcriptional control is rather slow, as it requires transcription, translation and transport from the endoplasmic reticulum (ER) to the plasma membrane via the conventional protein secretion (CPS) pathway. An alternative regulation to provide faster response would be valuable. Here we present two genetically encoded orthogonal regulatory secretion systems, which rely on the retention of pre-synthesized proteins on the ER membrane (membER, released by a cytosolic protease) or inside the ER lumen (lumER, released by an ER-luminal protease), respectively, and their release by the chemical signal-regulated proteolytic removal of an ER-retention signal, without triggering ER stress due to protein aggregates. Design of orthogonal chemically-regulated split proteases enables the combination of signals into logic functions. Its application was demonstrated on a chemically regulated therapeutic protein secretion and regulated membrane translocation of a chimeric antigen receptor (CAR) targeting cancer antigen. Regulation of the ER escape represents a platform for the design of fast-responsive and tightly-controlled modular and scalable protein secretion system for mammalian cells.

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design.

Effective and safe vaccines against SARS-CoV-2 are highly desirable to prevent casualties and societal cost caused by Covid-19 pandemic. The receptor binding domain (RBD) of the surface-exposed spike protein of SARS-CoV-2 represents a suitable target for the induction of neutralizing antibodies upon vaccination. Small protein antigens typically induce weak immune response while particles measuring tens of nanometers are efficiently presented to B cell follicles and subsequently to follicular germinal center B cells in draining lymph nodes, where B cell proliferation and affinity maturation occurs. Here we prepared and analyzed the response to several DNA vaccines based on genetic fusions of RBD to four different scaffolding domains, namely to the foldon peptide, ferritin, lumazine synthase and β-annulus peptide, presenting from 6 to 60 copies of the RBD on each particle. Scaffolding strongly augmented the immune response with production of neutralizing antibodies and T cell response including cytotoxic lymphocytes in mice upon immunization with DNA plasmids. The most potent response was observed for the 24-residue β-annulus peptide scaffold that forms large soluble assemblies, that has the advantage of low immunogenicity in comparison to larger scaffolds. Our results support the advancement of this vaccine platform towards clinical trials.

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The clustering of biosynthetic enzymes is used in nature to channel reaction products and increase the yield of compounds produced by multiple reaction steps. The coupling of multiple enzymes has been shown to increase the biosynthetic product yield. Different clustering strategies have particular advantages as the spatial organization of multiple enzymes creates biocatalytic cascades with a higher efficiency of biochemical reaction. However, there are also some drawbacks, such as misfolding and the variable stability of interaction domains, which may differ between particular biosynthetic reactions and the host organism. Here, we compared different protein-based clustering strategies, including direct fusion, fusion mediated by intein, and noncovalent interactions mediated through small coiled-coil dimer-forming domains. The clustering of enzymes through orthogonally designed coiled-coil interaction domains increased the production of resveratrol in Escherichia coli more than the intein-mediated fusion of biosynthetic enzymes. The improvement of resveratrol production correlated with the stability of the coiled-coil dimers. The coiled-coil fusion-based approach also increased mevalonate production in Saccharomyces cerevisiae, thus demonstrating the wider applicability of this strategy.

Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Protein secretion based on transcriptional control is rather slow, as proteins requires transcription, translation, followed by the transport from the endoplasmic reticulum (ER) through the conventional protein secretion (CPS) pathway towards the plasma membrane. An alternative faster bypass would be valuable. Here we present two genetically encoded orthogonal secretion systems, which rely on the retention of pre-synthesized proteins on the ER membrane (membER, released by cytosolic protease) or inside the ER lumen (lumER, released by ER luminal protease), respectively, and their release by the chemical signal-regulated proteolytic removal of an ER-retention signal, without triggering ER stress due to protein aggregates. Design of orthogonal chemically-regulated split proteases enables precise combination of signals into logic functions and was demonstrated on a chemically regulated insulin secretion. Regulation of ER escape represents a platform for the design of fast responsive and tightly-controlled modular and scalable protein secretion system.Abstract FigureAbstract figure:membER and lumER system.By equipping a protein of interest (POI) with an N-terminal signaling sequence, which initiates the transport of proteins into the endoplasmic reticulum (ER), and a C-terminal KDEL ER retention sequence for luminal proteins or a KKXX sequence for transmembrane proteins, we can retain those proteins inside the ER and cis-Golgi apparatus (GA) through retrograde transport. Insertion of a protease cleavage site adjacent to the retention signal allows for the regulated fast secretion through proteolytic cleavage. The membrane bound, ER membrane (membER) and ER-luminal (lumER) systems allow for the controlled secretion of pre-synthesized protein, stored inside the ER. This platform enables release of target proteins several hours faster than systems relying transcription and translation.

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