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Vaccine Mar 2022A reference standard is needed for quality control of protein subunit SARS-CoV-2 vaccines to meet urgent domestic needs. The Chinese National Institutes for Food and...
A reference standard is needed for quality control of protein subunit SARS-CoV-2 vaccines to meet urgent domestic needs. The Chinese National Institutes for Food and Drug Control (NIFDC) launched a project to establish the first reference material for the protein subunit SARS-CoV-2 vaccine to be used for calibration of antigen testing. The potency and stability of the national candidate standard (CS) were determined by collaborative calibration, and accelerated and freeze-thaw degradation studies. Moreover, a suitability study of the CS was performed. Eight laboratories in mainland China were asked to detect antigen content of CS using a common validated enzyme-linked immunosorbent assay (ELISA) kit established by NIFDC and in-house kits in the collaborative study. Six laboratories returned valid results, which established that the antigen content of the CS was 876,938 YU/mL, with good agreement across laboratories. In the suitability study, the CS exhibited excellent parallelism and a linear relationship with four samples produced by different expression systems and target proteins. In addition, good stability in the accelerated and freeze-thaw degradation study was observed. In conclusion, the CS was approved by the Biological Product Reference Standards Sub-Committee of the National Drug Reference Standards Committee as the first Chinese national standard for determining antigen content of protein subunit SARS-CoV-2 vaccines, with an assigned antigen content of 877,000 U/mL (Lot. 300050-202101). This standard will contribute to a standardized assessment of protein subunit SARS-CoV-2 vaccine in China and may provide experience for developing reference materials for antigen content detection of SARS-CoV-2 vaccine in other countries.
Topics: COVID-19; COVID-19 Vaccines; Humans; Protein Subunits; Reference Standards; SARS-CoV-2
PubMed: 35227521
DOI: 10.1016/j.vaccine.2022.02.048 -
Nature May 2024The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the...
The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the paused elongation complex consisting of Pol II, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) and how it cleaves the nascent RNA transcript, but has not explained how Integrator removes Pol II from the DNA template. Here we present three cryo-electron microscopy structures of the complete Integrator-PP2A complex in different functional states. The structure of the pre-termination complex reveals a previously unresolved, scorpion-tail-shaped INTS10-INTS13-INTS14-INTS15 module that may use its 'sting' to open the DSIF DNA clamp and facilitate termination. The structure of the post-termination complex shows that the previously unresolved subunit INTS3 and associated sensor of single-stranded DNA complex (SOSS) factors prevent Pol II rebinding to Integrator after termination. The structure of the free Integrator-PP2A complex in an inactive closed conformation reveals that INTS6 blocks the PP2A phosphatase active site. These results lead to a model for how Integrator terminates Pol II transcription in three steps that involve major rearrangements.
Topics: RNA Polymerase II; Cryoelectron Microscopy; Models, Molecular; Protein Phosphatase 2; Transcription Termination, Genetic; Humans; Transcription Factors; Protein Binding; Transcriptional Elongation Factors; Nuclear Proteins; Protein Subunits
PubMed: 38570683
DOI: 10.1038/s41586-024-07269-4 -
Annual Review of Biochemistry Jun 2020Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In... (Review)
Review
Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.
Topics: Anti-Bacterial Agents; Antineoplastic Agents; Biocatalysis; Drug Discovery; Enzyme Inhibitors; Escherichia coli; Escherichia coli Infections; Humans; Molecular Docking Simulation; Neoplasms; Nucleotides; Oxidation-Reduction; Protein Structure, Secondary; Protein Subunits; Ribonucleotide Reductases; Small Molecule Libraries; Structure-Activity Relationship
PubMed: 32569524
DOI: 10.1146/annurev-biochem-013118-111843 -
Biochimica Et Biophysica Acta.... Mar 2020Complex I is the largest and most intricate redox-driven proton pump of the respiratory chain. The structure of bacterial and mitochondrial complex I has been determined... (Review)
Review
Complex I is the largest and most intricate redox-driven proton pump of the respiratory chain. The structure of bacterial and mitochondrial complex I has been determined by X-ray crystallography and cryo-EM at increasing resolution. The recent cryo-EM structures of the complex I-like NDH complex and membrane bound hydrogenase open a new and more comprehensive perspective on the complex I superfamily. Functional studies and molecular modeling approaches have greatly advanced our understanding of the catalytic cycle of complex I. However, the molecular mechanism by which energy is extracted from the redox reaction and utilized to drive proton translocation is unresolved and a matter of ongoing debate. Here, we review progress in structure determination and functional characterization of complex I and discuss current mechanistic models.
Topics: Electron Transport Complex I; Hydrophobic and Hydrophilic Interactions; Oxidation-Reduction; Protein Subunits; Protons; Ubiquinone
PubMed: 31935361
DOI: 10.1016/j.bbabio.2020.148153 -
Cell Reports. Medicine Apr 2021The outbreak and spread of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) is a current global health emergency, and effective prophylactic vaccines are...
The outbreak and spread of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) is a current global health emergency, and effective prophylactic vaccines are needed urgently. The spike glycoprotein of SARS-CoV-2 mediates entry into host cells, and thus is the target of neutralizing antibodies. Here, we show that adjuvanted protein immunization with soluble SARS-CoV-2 spike trimers, stabilized in prefusion conformation, results in potent antibody responses in mice and rhesus macaques, with neutralizing antibody titers exceeding those typically measured in SARS-CoV-2 seropositive humans by more than one order of magnitude. Neutralizing antibody responses were observed after a single dose, with exceptionally high titers achieved after boosting. A follow-up to monitor the waning of the neutralizing antibody responses in rhesus macaques demonstrated durable responses that were maintained at high and stable levels at least 4 months after boosting. These data support the development of adjuvanted SARS-CoV-2 prefusion-stabilized spike protein subunit vaccines.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; COVID-19; COVID-19 Vaccines; Female; Macaca mulatta; Male; Memory B Cells; Mice; Mice, Inbred C57BL; Protein Domains; Protein Subunits; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Time Factors; Vaccination
PubMed: 33842900
DOI: 10.1016/j.xcrm.2021.100252 -
Nucleic Acids Research Jul 2021Protein-protein interactions play crucial roles in diverse biological processes, including various disease progressions. Atomistic structural details of protein-protein...
Protein-protein interactions play crucial roles in diverse biological processes, including various disease progressions. Atomistic structural details of protein-protein interactions may provide important information that can facilitate the design of therapeutic agents. GalaxyHeteromer is a freely available automatic web server (http://galaxy.seoklab.org/heteromer) that predicts protein heterodimer complex structures from two subunit protein sequences or structures. When subunit structures are unavailable, they are predicted by template- or distance-prediction-based modelling methods. Heterodimer complex structures can be predicted by both template-based and ab initio docking, depending on the template's availability. Structural templates are detected from the protein structure database based on both the sequence and structure similarities. The templates for heterodimers may be selected from monomer and homo-oligomer structures, as well as from hetero-oligomers, owing to the evolutionary relationships of heterodimers with domains of monomers or subunits of homo-oligomers. In addition, the server employs one of the best ab initio docking methods when heterodimer templates are unavailable. The multiple heterodimer structure models and the associated scores, which are provided by the web server, may be further examined by user to test or develop functional hypotheses or to design new functional molecules.
Topics: Molecular Docking Simulation; Protein Multimerization; Protein Subunits; Sequence Analysis, Protein; Software
PubMed: 34048578
DOI: 10.1093/nar/gkab422 -
Journal of Neurochemistry Jul 2020The N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate the flux of calcium (Ca ) into the post-synaptic compartment. Ca influx... (Review)
Review
The N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate the flux of calcium (Ca ) into the post-synaptic compartment. Ca influx subsequently triggers the activation of various intracellular signalling cascades that underpin multiple forms of synaptic plasticity. Functional NMDARs are assembled as heterotetramers composed of two obligatory GluN1 subunits and two GluN2 or GluN3 subunits. Four different GluN2 subunits (GluN2A-D) are present throughout the central nervous system; however, they are differentially expressed, both developmentally and spatially, in a cell- and synapse-specific manner. Each GluN2 subunit confers NMDARs with distinct ion channel properties and intracellular trafficking pathways. Regulated membrane trafficking of NMDARs is a dynamic process that ultimately determines the number of NMDARs at synapses, and is controlled by subunit-specific interactions with various intracellular regulatory proteins. Here we review recent progress made towards understanding the molecular mechanisms that regulate the trafficking of GluN2-containing NMDARs, focusing on the roles of several key synaptic proteins that interact with NMDARs via their carboxyl termini.
Topics: Animals; Dendritic Spines; Humans; Protein Subunits; Protein Transport; Receptors, N-Methyl-D-Aspartate
PubMed: 31978252
DOI: 10.1111/jnc.14970 -
Cells Dec 2021Cell-mediated immunity is driven by antigenic peptide presentation on major histocompatibility complex (MHC) molecules. Specialized proteasome complexes called... (Review)
Review
Cell-mediated immunity is driven by antigenic peptide presentation on major histocompatibility complex (MHC) molecules. Specialized proteasome complexes called immunoproteasomes process viral, bacterial, and tumor antigens for presentation on MHC class I molecules, which can induce CD8 T cells to mount effective immune responses. Immunoproteasomes are distinguished by three subunits that alter the catalytic activity of the proteasome and are inducible by inflammatory stimuli such as interferon-γ (IFN-γ). This inducible activity places them in central roles in cancer, autoimmunity, and inflammation. While accelerated proteasomal degradation is an important tumorigenic mechanism deployed by several cancers, there is some ambiguity regarding the role of immunoproteasome induction in neoplastic transformation. Understanding the mechanistic and functional relevance of the immunoproteasome provides essential insights into developing targeted therapies, including overcoming resistance to standard proteasome inhibition and immunomodulation of the tumor microenvironment. In this review, we discuss the roles of the immunoproteasome in different cancers.
Topics: Animals; Humans; Models, Biological; Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Subunits
PubMed: 34944095
DOI: 10.3390/cells10123587 -
Emerging Microbes & Infections Dec 2023The SARS-CoV-2 Omicron variants of concern (VOCs) showed severe resistance to the early-approved COVID-19 vaccines-induced immune responses. The breakthrough infections...
The SARS-CoV-2 Omicron variants of concern (VOCs) showed severe resistance to the early-approved COVID-19 vaccines-induced immune responses. The breakthrough infections by the Omicron VOCs are currently the major challenge for pandemic control. Therefore, booster vaccination is crucial to enhance immune responses and protective efficacy. Previously, we developed a protein subunit COVID-19 vaccine ZF2001, based on the immunogen of receptor-binding domain (RBD) homodimer, which was approved in China and other countries. To adapt SARS-CoV-2 variants, we further developed chimeric Delta-Omicron BA.1 RBD-dimer immunogen which induced broad immune responses against SARS-CoV-2 variants. In this study, we tested the boosting effect of this chimeric RBD-dimer vaccine in mice after priming with two doses of inactivated vaccines, compared with a booster of inactivated vaccine or ZF2001. The results demonstrated that boosting with bivalent Delta-Omicron BA.1 vaccine greatly promoted the neutralizing activity of the sera to all tested SARS-CoV-2 variants. Therefore, the Delta-Omicron chimeric RBD-dimer vaccine is a feasible booster for those with prior vaccination of COVID-19 inactivated vaccines.
Topics: Animals; Humans; Mice; Carrier Proteins; COVID-19 Vaccines; SARS-CoV-2; Protein Subunits; COVID-19; Antibodies, Neutralizing; Antibodies, Viral
PubMed: 36803449
DOI: 10.1080/22221751.2023.2179357 -
PLoS Computational Biology Apr 2020Hepatitis B virus (HBV) is a leading cause of liver disease. The capsid is an essential component of the virion and it is therefore of interest how it assembles and...
Hepatitis B virus (HBV) is a leading cause of liver disease. The capsid is an essential component of the virion and it is therefore of interest how it assembles and disassembles. The capsid protein is unusual both for its rare fold and that it polymerizes according to two different icosahedral symmetries, causing the polypeptide chain to exist in seven quasi-equivalent environments: A, B, and C in AB and CC dimers in T = 3 capsids, and A, B, C, and D in AB and CD dimers in T = 4 capsids. We have compared the two capsids by cryo-EM at 3.5 Å resolution. To ensure a valid comparison, the two capsids were prepared and imaged under identical conditions. We find that the chains have different conformations and potential energies, with the T = 3 C chain having the lowest. Three of the four quasi-equivalent dimers are asymmetric with respect to conformation and potential energy; however, the T = 3 CC dimer is symmetrical and has the lowest potential energy although its intra-dimer interface has the least free energy of formation. Of all the inter-dimer interfaces, the CB interface has the least area and free energy, in both capsids. From the calculated energies of higher-order groupings of dimers discernible in the lattices we predict early assembly intermediates, and indeed we observe such structures by negative stain EM of in vitro assembly reactions. By sequence analysis and computational alanine scanning we identify key residues and motifs involved in capsid assembly. Our results explain several previously reported observations on capsid assembly, disassembly, and dimorphism.
Topics: Amino Acid Sequence; Binding Sites; Capsid; Capsid Proteins; Computational Biology; Hepatitis B virus; Protein Conformation; Protein Multimerization; Protein Subunits; Thermodynamics
PubMed: 32310951
DOI: 10.1371/journal.pcbi.1007782