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Journal of Medical Virology Feb 2023The development of a safe and effective vaccine is essential to protect populations against coronavirus disease 2019 (COVID-19). There are several vaccine candidates... (Randomized Controlled Trial)
Randomized Controlled Trial
Safety and immunogenicity of a recombinant receptor-binding domain-based protein subunit vaccine (Noora vaccine™) against COVID-19 in adults: A randomized, double-blind, placebo-controlled, Phase 1 trial.
The development of a safe and effective vaccine is essential to protect populations against coronavirus disease 2019 (COVID-19). There are several vaccine candidates under investigation with different mechanisms of action. In the present study, we have evaluated the safety and immunogenicity of a recombinant receptor-binding domain (RBD)-based protein subunit vaccine (Noora vaccine) against COVID-19 in adults. This Phase 1 trial is a randomized, double-blind, placebo-controlled study to evaluate the safety and immunogenicity of the recombinant RBD-based protein subunit vaccine (Noora vaccine) against COVID-19 in healthy adults volunteers. Eligible participants were included in this study after evaluating their health status and considering the exclusion criteria. They were then randomized into three groups and received three doses of vaccine (80 µg, 120 µg, and placebo) on Days 0, 21, and 35. Primary outcomes including solicited, unsolicited, and medically attended adverse events were recorded during this study. Secondary outcomes including the humoral and cellular immunity (including anti-RBD IgG antibody and neutralizing antibody) were measured on Days 0, 21, 28, 35, 42, and 49 by using the ELISA kit and the Virus Neutralization Test (VNT) was performed on day 49. Totally 70 cases were included in this Phase 1 trial and 60 of them completed the study. Safety assessments showed no severe adverse events. Local pain at the vaccine injection site occurred in 80% of the vaccinated volunteers. Induration and redness at the injection site were the other adverse reactions of this vaccine. There was no significant difference between the studied groups regarding adverse reactions. Anti-RBD IgG antibody and neutralizing antibody assessment showed significant seroconversion in comparison to the placebo group (80%, and 100% respectively, p < 0.001). The cellular immunity panel also showed mild to moderate induction of TH1 responses and the VNT showed 78% of seroprotection. The results of this Phase 1 trial showed acceptable safety without serious adverse events and significant seroconversions in the humoral and cellular immunity panel. The dose of 80 µg is an appropriate dose for injection in the next phases of the trial.
Topics: Adult; Humans; COVID-19; Protein Subunits; Antibodies, Neutralizing; Vaccines, Synthetic; Vaccines, Subunit; Immunoglobulin G; Double-Blind Method; Immunogenicity, Vaccine; Antibodies, Viral
PubMed: 36029105
DOI: 10.1002/jmv.28097 -
The Na/K-ATPase α1 and c-Src form signaling complex under native condition: A crosslinking approach.Scientific Reports Apr 2020The protein-protein interactions amongst the Na/K-ATPase α1 subunit, c-Src, and caveolin-1 (cav-1) are essential for the Na/K-ATPase signaling functions. However, there...
The protein-protein interactions amongst the Na/K-ATPase α1 subunit, c-Src, and caveolin-1 (cav-1) are essential for the Na/K-ATPase signaling functions. However, there are arguments concerning the interaction model. The present study aims to clarify the interactions amongst the endogenous native proteins in live cells under native resting condition. Under native condition, Blue Native-PAGE and Blue Native-PAGE/SDS-PAGE 2D analyses demonstrated co-existence of the α1 subunit and c-Src in same protein complex, as well as a direct interaction between the α1 subunit and c-Src. By comparison of cleavable and non-cleavable cysteine-cysteine crosslinked samples, capillary immunoblotting analysis demonstrated that depletion of Src kinase family members (c-Src, Yes, and Fyn) or cav-1 clearly reduced the interactions of the α1 subunit with proteins, but depletion of cav-1 did not affect the interaction of c-Src with the α1 subunit. The data indicated that there are direct interactions between the α1 subunit and c-Src as well as between the α1 subunit and cav-1, but argued about the interaction between c-Src and cav-1 under the condition. Furthermore, the data also indicated the existence of different protein complexes containing the α1 subunit and c-Src, which might have different signaling functions.
Topics: Animals; CSK Tyrosine-Protein Kinase; Caveolin 1; Cross-Linking Reagents; Humans; LLC-PK1 Cells; Protein Interaction Maps; Protein Subunits; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Swine
PubMed: 32265464
DOI: 10.1038/s41598-020-61920-4 -
Proceedings of the National Academy of... Feb 2021Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from four different, but evolutionarily related, subunits. These subunits...
Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from four different, but evolutionarily related, subunits. These subunits assemble with a precise stoichiometry and arrangement such that two chemically distinct agonist-binding sites are formed between specific subunit pairs. How this subunit complexity evolved and became entrenched is unclear. Here we show that a single historical amino acid substitution is able to constrain the subunit stoichiometry of functional acetylcholine receptors. Using a combination of ancestral sequence reconstruction, single-channel electrophysiology, and concatenated subunits, we reveal that an ancestral β-subunit can not only replace the extant β-subunit but can also supplant the neighboring δ-subunit. By forward evolving the ancestral β-subunit with a single amino acid substitution, we restore the requirement for a δ-subunit for functional channels. These findings reveal that a single historical substitution necessitates an increase in acetylcholine receptor complexity and, more generally, that simple stepwise mutations can drive subunit entrenchment in this model heteromeric protein.
Topics: Amino Acid Substitution; Cell Line; Evolution, Molecular; Humans; Protein Binding; Protein Domains; Protein Multimerization; Protein Subunits; Receptors, Nicotinic
PubMed: 33579823
DOI: 10.1073/pnas.2018731118 -
The Journal of Biological Chemistry Oct 2023α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) auxiliary subunits are specialized, nontransient binding partners of AMPARs that modulate AMPAR...
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) auxiliary subunits are specialized, nontransient binding partners of AMPARs that modulate AMPAR channel gating properties and pharmacology, as well as their biogenesis and trafficking. The most well-characterized families of auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs), cornichon homologs (CNIHs), and the more recently discovered GSG1-L. These auxiliary subunits can promote or reduce surface expression of AMPARs (composed of GluA1-4 subunits) in neurons, thereby impacting their functional role in membrane signaling. Here, we show that CNIH-2 enhances the tetramerization of WT and mutant AMPARs, presumably by increasing the overall stability of the tetrameric complex, an effect that is mainly mediated by interactions with the transmembrane domain of the receptor. We also find CNIH-2 and CNIH-3 show receptor subunit-specific actions in this regard with CNIH-2 enhancing both GluA1 and GluA2 tetramerization, whereas CNIH-3 only weakly enhances GluA1 tetramerization. These results are consistent with the proposed role of CNIHs as endoplasmic reticulum cargo transporters for AMPARs. In contrast, TARP γ-2, TARP γ-8, and GSG1-L have no or negligible effect on AMPAR tetramerization. On the other hand, TARP γ-2 can enhance receptor tetramerization but only when directly fused with the receptor at a maximal stoichiometry. Notably, surface expression of functional AMPARs was enhanced by CNIH-2 to a greater extent than TARP γ-2, suggesting that this distinction aids in maturation and membrane expression. These experiments define a functional distinction between CNIHs and other auxiliary subunits in the regulation of AMPAR biogenesis.
Topics: Glutamic Acid; Neurons; Protein Domains; Receptors, AMPA; Signal Transduction; Protein Multimerization; Protein Subunits; HEK293 Cells; Humans
PubMed: 37673338
DOI: 10.1016/j.jbc.2023.105227 -
Journal of Molecular Biology Jun 2021Proper functioning of the precisely controlled endolysosomal system is essential for maintaining the homeostasis of the entire cell. Tethering factors play pivotal roles...
Proper functioning of the precisely controlled endolysosomal system is essential for maintaining the homeostasis of the entire cell. Tethering factors play pivotal roles in mediating the fusion of different transport vesicles, such as endosomes or autophagosomes with each other or with lysosomes. In this work, we uncover several new interactions between the endolysosomal tethering factors Rabenosyn-5 (Rbsn) and the HOPS and CORVET complexes. We find that Rbsn binds to the HOPS/CORVET complexes mainly via their shared subunit Vps18 and we mapped this interaction to the 773-854 region of Vps18. Based on genetic rescue experiments, the binding between Rbsn and Vps18 is required for endosomal transport and is dispensable for autophagy. Moreover, Vps18 seems to be important for β1 integrin recycling by binding to Rbsn and its known partner Vps45.
Topics: Binding Sites; Endosomes; HEK293 Cells; HeLa Cells; Humans; Lipids; Lysosomes; Microtubule-Associated Proteins; Protein Binding; Protein Subunits; Vesicular Transport Proteins
PubMed: 33781757
DOI: 10.1016/j.jmb.2021.166965 -
Journal of Medicinal Chemistry Nov 2020The immunoproteasome (i-20S) has emerged as a therapeutic target for autoimmune and inflammatory disorders and hematological malignancies. Inhibition of the chymotryptic...
The immunoproteasome (i-20S) has emerged as a therapeutic target for autoimmune and inflammatory disorders and hematological malignancies. Inhibition of the chymotryptic β5i subunit of i-20S inhibits T cell activation, B cell proliferation, and dendritic cell differentiation in vitro and suppresses immune responses in animal models of autoimmune disorders and allograft rejection. However, cytotoxicity to immune cells has accompanied the use of covalently reactive β5i inhibitors, whose activity against the constitutive proteasome (c-20S) is cumulative with the time of exposure. Herein, we report a structure-activity relationship study of a class of noncovalent proteasome inhibitors with picomolar potencies and 1000-fold selectivity for i-20S over c-20S. Furthermore, these inhibitors are specific for β5i over the other five active subunits of i-20S and c-20S, providing useful tools to study the functions of β5i in immune responses. The potency of these compounds in inhibiting human T cell activation suggests that they may have therapeutic potential.
Topics: Binding Sites; Cell Proliferation; Dipeptides; HeLa Cells; Humans; Inhibitory Concentration 50; Kinetics; Lymphocyte Activation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Subunits; Structure-Activity Relationship; T-Lymphocytes
PubMed: 33095579
DOI: 10.1021/acs.jmedchem.0c01520 -
Journal of Chemical Theory and... Jan 2021We investigated gramicidin A (gA) subunit dimerization in lipid bilayers using microsecond-long replica-exchange umbrella sampling simulations, millisecond-long unbiased...
We investigated gramicidin A (gA) subunit dimerization in lipid bilayers using microsecond-long replica-exchange umbrella sampling simulations, millisecond-long unbiased molecular dynamics simulations, and machine learning. Our simulations led to a dimer structure that is indistinguishable from the experimentally determined gA channel structures, with the two gA subunits joined by six hydrogen bonds (6HB). The simulations also uncovered two additional dimer structures, with different gA-gA stacking orientations that were stabilized by four or two hydrogen bonds (4HB or 2HB). When examining the temporal evolution of the dimerization, we found that two bilayer-inserted gA subunits can form the 6HB dimer directly, with no discernible intermediate states, as well as through paths that involve the 2HB and 4HB dimers.
Topics: Bacterial Proteins; Brevibacillus; Gramicidin; Hydrogen Bonding; Lipid Bilayers; Molecular Dynamics Simulation; Protein Conformation; Protein Multimerization; Protein Subunits; Thermodynamics
PubMed: 33378617
DOI: 10.1021/acs.jctc.0c00989 -
International Journal of Biological... Jun 2021Protein fusion using a linker plays an important role for protein evolution. However, designing suitable linkers for protein evolution is yet challenging and...
Protein fusion using a linker plays an important role for protein evolution. However, designing suitable linkers for protein evolution is yet challenging and under-explored. To further clarify the regular pattern of suitable type of linker for fusion proteins, one nitrile hydratase (NHase) was used as a target protein and subunit fusion strategy was carried out to improve its efficient catalysis. Subunit-fused variants with three different types of linkers were constructed and characterized. All variants exhibited higher stability than that of the wild type. The longer the linker was, the higher stability NHase showed, however, too long linker affected NHase activity and expression. Among the three types of linkers, the α-helical linker seemed more suitable for NHase than flexible or rigid linkers. Though it is not clear how the linkers affecting the activity, structure analysis indicated that the stability improvement is dependent on the additional salt bridge, H-bond, and the subunit interface area increasing due to the linker insertion, among which the additional salt bridge and interface area were more important factors. The results described here may be useful for redesigning other enzymes through subunit fusion.
Topics: Biocatalysis; Catalytic Domain; Enzyme Stability; Hydro-Lyases; Kinetics; Molecular Dynamics Simulation; Protein Subunits; Recombinant Proteins; Temperature
PubMed: 33753198
DOI: 10.1016/j.ijbiomac.2021.03.103 -
Journal of Neurochemistry Oct 2019N-methyl-d-Aspartate receptors (NMDARs) are abundant postsynaptic proteins that are critical for normal synaptic communication. NMDAR channel function is regulated by...
N-methyl-d-Aspartate receptors (NMDARs) are abundant postsynaptic proteins that are critical for normal synaptic communication. NMDAR channel function is regulated by multiple properties, including phosphorylation. Inhibition of protein phosphatase 1 (PP1) in hippocampal neurons increases NMDAR activity, an effect abrogated by loss of spinophilin, the major PP1-targeting protein in the postsynaptic density. However, how spinophilin regulates PP1-dependent NMDAR function is unclear. We hypothesize that spinophilin regulates PP1 binding to the NMDAR to alter NMDAR phosphorylation. Our data demonstrate that spinophilin interacts with the GluN2B subunit of the NMDAR. In human embryonic kidney 293 FT cells, activation and/or overexpression of protein kinase A increased the association between spinophilin and the GluN2B subunit of the NMDAR. Functionally, we found that spinophilin overexpression decreased PP1 binding to the GluN2B subunit of the NMDAR and attenuated the PP1-dependent dephosphorylation of GluN2B at Ser-1284. Moreover, in P28 hippocampal lysates isolated from spinophilin KO compared to WT mice, there was increased binding of GluN2B to PP1, decreased phosphorylation of GluN2B at Ser-1284, and altered GluN2B protein interactions with postsynaptic density-enriched proteins. Together, our data demonstrate that spinophilin decreases PP1 binding to GluN2B and concomitantly enhances the phosphorylation of GluN2B at Ser-1284. The putative consequences of these spinophilin-dependent alterations in GluN2B phosphorylation and interactions on synaptic GluN2B localization and function are discussed. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.
Topics: Animals; Female; HEK293 Cells; Humans; Male; Mice; Mice, Inbred C57BL; Microfilament Proteins; Nerve Tissue Proteins; Phosphorylation; Protein Binding; Protein Subunits; Receptors, N-Methyl-D-Aspartate
PubMed: 31325175
DOI: 10.1111/jnc.14831 -
Cell Research Dec 2020In Gram-negative bacteria, phospholipids are major components of the inner membrane and the inner leaflet of the outer membrane, playing an essential role in forming the...
In Gram-negative bacteria, phospholipids are major components of the inner membrane and the inner leaflet of the outer membrane, playing an essential role in forming the unique dual-membrane barrier to exclude the entry of most antibiotics. Understanding the mechanisms of phospholipid translocation between the inner and outer membrane represents one of the major challenges surrounding bacterial phospholipid homeostasis. The conserved MlaFEDB complex in the inner membrane functions as an ABC transporter to drive the translocation of phospholipids between the inner membrane and the periplasmic protein MlaC. However, the mechanism of phospholipid translocation remains elusive. Here we determined three cryo-EM structures of MlaFEDB from Escherichia coli in its nucleotide-free and ATP-bound conformations, and performed extensive functional studies to verify and extend our findings from structural analyses. Our work reveals unique structural features of the entire MlaFEDB complex, six well-resolved phospholipids in three distinct cavities, and large-scale conformational changes upon ATP binding. Together, these findings define the cycle of structural rearrangement of MlaFEDB in action, and suggest that MlaFEDB uses an extrusion mechanism to extract and release phospholipids through the central translocation cavity.
Topics: Adenosine Triphosphate; Cryoelectron Microscopy; Escherichia coli; Escherichia coli Proteins; Models, Biological; Models, Molecular; Phospholipids; Protein Binding; Protein Structure, Secondary; Protein Subunits
PubMed: 32884137
DOI: 10.1038/s41422-020-00404-6