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
Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyREM, the agonists glycine, β-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyREM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyREM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyREM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for ICD in pentameric ligand-gated channels.
Highly regulated intracellular calcium entry affects numerous cellular physiological events. External regulation of intracellular calcium signaling presents a great opportunity for the artificial regulation of cellular activity. Calcium entry can be mediated by STIM proteins interacting with Orai calcium channels; therefore, the STIM1–Orai1 pair has become a tool for artificially modulating calcium entry. We report on an innovative genetically engineered protease-activated Orai activator called PACE. CAD self-dimerization and activation were inhibited with a coiled-coil forming peptide pair linked to CAD via a protease cleavage site. PACE generated sustained calcium entry after its activation with a reconstituted split protease. We also generated PACE, whose transcriptional activation of NFAT was triggered by PPV or TEV protease. Using PACE, we successfully activated the native NFAT signaling pathway and the production of cytokines in a T-cell line. PACE represents a useful tool for generating sustained calcium entry to initiate calcium-dependent protein translation. PACE provides a promising template for the construction of links between various protease activation pathways and calcium signaling.
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