Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS‐CoV‐2. However, dual strategies combining virus neutralization and immune ...response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross‐priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT, are first generated by grafting an anti‐RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo‐EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high‐avidity neutralizing interaction. Then, by C‐terminal fusion of an anti‐DNGR‐1 scFv to TNT, the bispecific trimerbody TNTDNGR‐1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross‐priming. Therapeutic administration of TNTDNGR‐1, but not TNT, protects K18‐hACE2 mice from a lethal SARS‐CoV‐2 infection, boosting virus‐specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus‐neutralizing antibody activity and demonstrate the therapeutic potential of the Fc‐free strategy that can be used advantageously to provide both immediate and long‐term protection against SARS‐CoV‐2 and other viral infections.
Here they generate a bispecific Fc‐free trimeric antibody to neutralize and target SARS‐CoV‐2 virions to type 1 conventional dendritic cells and promote T cell cross‐priming. Therapeutic administration protected mice from a lethal infection, boosting virus‐specific humoral and CD8+ T cell immune responses. The results highlight the potential of bispecific trimerbodies with neutralizing and vaccine‐like action to provide both immediate and long‐term protection against SARS‐CoV‐2 and other viral infections.
The human calcium-sensing receptor (hCaR) is a family-3/C G-protein-coupled receptor that regulates Ca2+ homeostasis by controlling parathyroid hormone secretion. Here we investigated the role of ...Rab1, a small GTP-binding protein that specifically regulates protein transport from the endoplasmic reticulum to the Golgi, in cell surface transport of the hCaR. Cell surface expression of hCaR transiently expressed in human embryonic kidney 293 cells was strongly augmented by coexpression of Rab1 and attenuated by disruption of endogenous Rab1 function by expression of the dominant-negative Rab1N124I mutant or depletion of Rab1 with small interfering RNA. Rab1N124I expression also partially attenuated cell surface expression and signaling response to gain-of-function mutants of hCaR with truncated carboxyl-terminal sequences at positions 895 and 903. These carboxyl-tail truncations are similar to a deletion between residues S895 and V1075 found in a patient family causing autosomal dominant hypocalcemia. In addition, coexpression with wild-type Rab1 increased cell surface expression of the loss-of-function missense mutation R185Q, located on the hCaR amino-terminal extracellular ligand-binding domain (ECD), which causes familial hypocalciuric hypercalcemia. Truncated hCaR variants containing either the ECD with the first transmembrane helix or only the ECD also display Rab1-dependent cell surface expression or secretion into the culture medium, respectively. These data reveal a role for Rab1 in hCaR trafficking from the endoplasmic reticulum to the Golgi that regulates receptor cell surface expression and thereby cell signaling responsiveness to extracellular calcium.
A Rab1-dependent mechanism regulates intracellular and cell surface delivery of the human calcium-sensing receptor.
Biochemical and structural studies demonstrate that S100A1 is involved in a Ca
-dependent interaction with the type 2α and type 2β regulatory subunits of protein kinase A (PKA) (RIIα and RIIβ) to ...activate holo-PKA. The interaction was specific for S100A1 because other calcium-binding proteins (i.e., S100B and calmodulin) had no effect. Likewise, a role for S100A1 in PKA-dependent signaling was established because the PKA-dependent subcellular redistribution of HDAC4 was abolished in cells derived from S100A1 knockout mice. Thus, the Ca
-dependent interaction between S100A1 and the type 2 regulatory subunits represents a novel mechanism that provides a link between Ca
and PKA signaling, which is important for the regulation of gene expression in skeletal muscle via HDAC4 cytosolic-nuclear trafficking.
Family 3 G-protein-coupled receptors (GPCRs), which includes metabotropic glutamate receptors (mGluRs), sweet and “umami” taste receptors (T1Rs), and the extracellular calcium-sensing receptor (CaR), ...represent a distinct group among the superfamily of GPCRs characterized by large amino-terminal extracellular ligand-binding domains (ECD) with homology to bacterial periplasmic amino acid-binding proteins that are responsible for signal detection and receptor activation through as yet unresolved mechanism(s) via the seven-transmembrane helical domain (7TMD) common to all GPCRs. To address the mechanism(s) by which ligand-induced conformational changes are conveyed from the ECD to the 7TMD for G-protein activation, we altered the length and composition of a 14-amino acid linker segment common to all family 3 GPCRs except GABAB receptor, in the CaR by insertion, deletion, and site-directed mutagenesis of specific highly conserved residues. Small alterations in the length and composition of the linker impaired cell surface expression and abrogated signaling of the chimeric receptors. The exchange of nine amino acids within the linker of CaR with the homologous sequence of mGluR1, however, preserved receptor function. Ala substitution for the four highly conserved residues within this amino acid sequence identified a Leu at position 606 of the CaR critical for cell surface expression and signaling. Substitution of Leu606 for Ala resulted in impaired cell surface expression. However, Ile and Val substitutions displayed strong activating phenotypes. Disruption of the linker by insertion of nine amino acids of a random-coiled structure uncoupled the ECD from regulating the 7TMD. These data are consistent with a model of receptor activation in which the peptide linker, and particularly Leu606, provides a critical interaction for the CaR signal transmission, a finding likely to be relevant for all family 3 GPCRs containing this conserved motif.
Therapeutic solutions to combat the Clostridioides difficile infection (CDI) are high in‐demand due to antibiotic resistance and limited treatment options. C. difficile transferase (CDT) binary toxin ...is associated with hypervirulence, in addition to the major clostridial toxins. CDT consists of an enzymatic component (CDTa), and a pore‐forming binding component (CDTb). Cytotoxicity of the host cell is initiated by the formation of CDTa‐CDTb binary toxin complex and subsequent conformational shift in the CDTb to enforce translocation of the CDTa into the cell. This results in destruction of the host cell cytoskeleton through ADP‐ribosylation of the actin filaments.
Molecular level characterization of full length active wild‐type CDTb by Cryogenic Electron Microscopy (CryoEM) revealed existence of two novel di‐heptamer units in the absence of full‐length CDTa. The ‘pore’ state is represented by extended β‐barrel assembly in the AsymCDTb structure and the ‘pre‐pore’ state or the SymCDTb form consisted identical heptamer domains. Detailed analysis of the structure depicted the presence of different domains including two receptor binding domains (RBD). The RBD1 domain represents a discrete non‐homologous structure that was not identified in other binary toxins and the RBD2 domain is crucial for di‐heptamer assembly and physiological activity of the toxin.
A unique calcium‐binding site in RBD1 in the X‐ray crystal structure of AsymCDTb pinpointed the significant role of calcium in the structural stability and the protein folding mechanisms. Historically, potent calcium‐containing therapeutic agents have been able to neutralize the activity of large clostridial toxins. Therefore, we explored the structural features of the CDTbD623/734A double mutant which disrupted the calcium binding site in the RBD1. The 2D‐structural details revealed unique conformational changes in the heptameric organization of the mutant which indicated potential disturbances to the formation of β‐barrel. We compared the toxicity of wild type CDTb and CDTbD623/734A in the presence of CDTa using a Vero cell assay. CDTbD623/734A showed a significant decrease in toxicity (TC50 CDTb 80±6pM; CDTbD623/734A 637±20pM), supporting the idea that this novel calcium‐binding site is required for function.
Calcium plays a pivotal role in the structure‐activity relationship and toxin machinery. Therefore, ablation of the divalent interaction and targeting the binding site stand out as promising strategies in the drug development for CDI. Unraveling the distinctive conformational features in different mutant constructs of the binary toxin can be beneficial in fine‐tuning these potential therapeutic agents.
Our understanding of mammalian olfactory coding has been impeded by the paucity of information about the odorant receptors (ORs) that respond to a given odorant ligand in awake, freely behaving ...animals. Identifying the ORs that respond in vivo to a given odorant ligand from among the ∼1100 ORs in mice is intrinsically challenging but critical for our understanding of olfactory coding at the periphery. Here, we report an in vivo assay that is based on a novel gene-targeted mouse strain, S100a5-tauGFP, in which a fluorescent reporter selectively marks olfactory sensory neurons that have been activated recently in vivo. Because each olfactory sensory neuron expresses a single OR gene, multiple ORs responding to a given odorant ligand can be identified simultaneously by capturing the population of activated olfactory sensory neurons and using expression profiling methods to screen the repertoire of mouse OR genes. We used this in vivo assay to re-identify known eugenol- and muscone-responsive mouse ORs. We identified additional ORs responsive to eugenol or muscone. Heterologous expression assays confirmed nine eugenol-responsive ORs (Olfr73, Olfr178, Olfr432, Olfr610, Olfr958, Olfr960, Olfr961, Olfr913, and Olfr1234) and four muscone-responsive ORs (Olfr74, Olfr235, Olfr816, and Olfr1440). We found that the human ortholog of Olfr235 and Olfr1440 responds to macrocyclic ketone and lactone musk odorants but not to polycyclic musk odorants or a macrocyclic diester musk odorant. This novel assay, called the Kentucky in vivo odorant ligand-receptor assay, should facilitate the in vivo identification of mouse ORs for a given odorant ligand of interest.
Family 3 G‐protein‐coupled receptors (GPCRs) consist of the extracellular calcium‐sensing receptor (CaR), metabotropic glutamate receptors (mGluR1‐8), γ‐aminobutyric acid receptor (GABABR), taste ...receptors (TIR1‐3), pheromone receptors (V2Rs) and orphan receptors. These are characterized by a large amino‐terminal extracellular ligand binding domain (ECD) that is responsible for signaling and activation involving the seven‐transmembrane helical domains (7TMD). The solved crystal structure for the closed conformation of mGluR1 indicates the carboxy‐terminal portions of the monomeric units are drawn together but the signaling mechanism remains unsolved. A specific 14‐amino acid linker segment of the mGluR1 connecting the ECD to the 7TMD, present in all Family 3 GPCRs except GABABR, was critical for signaling and is consistent with the peptide‐linker model of receptor activation. To determine the relevance of this conserved motif in other Family 3 GPCRs, we altered the length and composition of the mGluR1 linker via insertion, deletion, and site‐directed mutagenesis. Phosphoinositol hydrolysis assays, ligand binding assays and calcium imaging analyses of chimeric receptors suggest that the mGluR1 linker is critical for signaling from the ECD to the 7TMD and is consistent with our finding for the CaR, supporting the peptide‐linker activation model for receptors containing this conserved motif.
Work supported by the Intramural Research program of the NIDCD, National Institutes of Health.
The ability to detect small volatile molecules in the environment is mediated by the large repertoire of odorant receptors (ORs) in each species. The mammalian OR repertoire is an attractive model to ...study evolution because ORs have been subjected to rapid gene gains and losses between species, presumably caused by changes of the olfactory system to adapt to the environment. Despite the complicated history, clear orthologs—genes related via speciation—can still be identified even in distantly related species. Functional assessment of ORs in related species remains largely untested and sequence similarity is often used as a proxy for functional data. Here I describe the functional properties of primate and rodent ORs to determine how well evolutionary distance predicts functional characteristics. Using human and mouse ORs with previously identified ligands, we cloned 18 OR orthologs from chimpanzee and rhesus macaque and 17 mouse-rat orthologous pairs that are broadly representative of the OR repertoire. Using a heterologous expression system, we functionally characterized the responses of ORs to a wide panel of odors and found similar ligand selectivity but dramatic differences in response magnitude. 87% of human-primate orthologs and 94% of mouse-rat orthologs showed differences in receptor potency (EC50) and/or efficacy (dynamic range) to an individual ligand. Notably dN/dS ratio, an indication of selective pressure during evolution, does not predict functional similarities between orthologs. Additionally, we found that orthologs responded to a common ligand 82% of the time, while human OR paralogs of the same subfamily responded to the common ligand only 33% of the time. Our results suggest that while OR orthologs tend to show conserved ligand selectivity, their potency and/or efficacy dynamically change during evolution, even in closely related species. These functional changes in orthologs provide a platform for examining how the evolution of ORs can meet species-specific demands.
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