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Neurochemistry International May 2023NMDA-type glutamate receptors (NMDARs) are tetrameric channel complex composed of two subunits of GluN1, which is encoded by a single gene and diversified by alternative...
NMDA-type glutamate receptors (NMDARs) are tetrameric channel complex composed of two subunits of GluN1, which is encoded by a single gene and diversified by alternative splicing, and two subunits from four subtypes of GluN2, leading to various combinations of subunits and channel specificities. However, there is no comprehensive quantitative analysis of GluN subunit proteins for relative comparison, and their compositional ratios at various regions and developmental stages have not been clarified. Here we prepared six chimeric subunits, by fusing an N-terminal side of the GluA1 subunit with a C-terminal side of each of two splicing isoforms of GluN1 subunit and four GluN2 subunits, with which titers of respective NMDAR subunit antibodies could be standardized using common GluA1 antibody, thus enabling quantification of relative protein levels of each NMDAR subunit by western blotting. We determined relative protein amounts of NMDAR subunits in crude, membrane (P2) and microsomal fractions prepared from the cerebral cortex, hippocampus and cerebellum in adult mice. We also examined amount changes in the three brain regions during developmental stages. Their relative amounts in the cortical crude fraction were almost parallel to those of mRNA expression, except for some subunits. Interestingly, a considerable amount of GluN2D protein existed in adult brains, although its transcription level declines after early postnatal stages. GluN1 was larger in quantity than GluN2 in the crude fraction, whereas GluN2 increased in the membrane component-enriched P2 fraction, except in the cerebellum. These data will provide the basic spatio-temporal information on the amount and composition of NMDARs.
Topics: Animals; Mice; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Cerebellum; Brain; Glutamic Acid; Protein Subunits
PubMed: 36913980
DOI: 10.1016/j.neuint.2023.105517 -
Scientific Reports May 2023The optimal booster vaccine schedule against COVID-19 is still being explored. The present study aimed at assessment of the immunogenicity and antibody persistency of...
The optimal booster vaccine schedule against COVID-19 is still being explored. The present study aimed at assessment of the immunogenicity and antibody persistency of inactivated-virus based vaccine, BBIP-CorV and protein-subunit based vaccines, PastoCovac/Plus through heterologous and homologous prime-boost vaccination. Totally, 214 individuals who were previously primed with BBIBP-CorV vaccines were divided into three arms on their choice as heterologous regimens BBIBP-CorV/PastoCovac (n = 68), BBIBP-CorV/PastoCovac Plus (n = 72) and homologous BBIBP-CorV (n = 74). PastoCovac booster recipients achieved the highest rate of anti-Spike IgG titer rise with a fourfold rise in 50% of the group. Anti-RBD IgG and neutralizing antibody mean rise and fold rise were almost similar between the PastoCovac and PastoCovac Plus booster receivers. The antibody durability results indicated that the generated antibodies were persistent until day 180 in all three groups. Nevertheless, a higher rate of antibody titer was seen in the heterologous regimen compared to BBIP-CorV group. Furthermore, no serious adverse event was recorded. The protein subunit-based booster led to a stronger humoral immune response in comparison with the BBIP-CorV booster receivers. Both the protein subunit boosters neutralized SARS-CoV-2 significantly more than BBIP-CorV. Notably, PastoCovac protein subunit-based vaccine could be successfully applied as a booster with convenient immunogenicity and safety profile.
Topics: Humans; COVID-19 Vaccines; Immunity, Humoral; Protein Subunits; COVID-19; SARS-CoV-2; Antibodies, Neutralizing; Immunoglobulin G; Antibodies, Viral
PubMed: 37202438
DOI: 10.1038/s41598-023-35147-y -
Expert Review of Vaccines 2023Vaccines prevent disease and disability; save lives and represent a good assessment of health interventions. Several systematic reviews on the efficacy and effectiveness...
INTRODUCTION
Vaccines prevent disease and disability; save lives and represent a good assessment of health interventions. Several systematic reviews on the efficacy and effectiveness of COVID-19 vaccines have been published, but the immunogenicity and safety of these vaccines should also be addressed.
AREAS COVERED
This systemic investigation sought to explain the efficacy, immunogenicity, and safety of new vaccination technologies against SARS-CoV-2 in people over 18 years old. Original research studying the effectiveness on mRNA, protein subunit vaccines, and viral vector vaccines against SARS-CoV-2 in people over 18 years old was analyzed. Several databases (Web of Science, Scopus, MEDLINE and EMBASE) were searched between 2012 and November 2022 for English-language papers using text and MeSH terms related to SARS-CoV-2, mechanism, protein subunit vaccine, viral vector, and mRNA. The protocol was registered on PROSPERO, CRD42022341952. Study quality was assessed using the NICE methodology. We looked at a total of six original articles. All studies gathered and presented quantitative data.
EXPERT OPINION
Our results suggest that new vaccinations could have more than 90% efficacy against SARS-CoV-2, regardless of the technology used. Furthermore, adverse reactions go from mild to moderate, and good immunogenicity can be observed for all vaccine types.
Topics: Humans; Adolescent; Viral Vaccines; Protein Subunits; COVID-19 Vaccines; SARS-CoV-2; RNA, Messenger; COVID-19; Vaccines, Subunit; Antibodies, Viral; Immunogenicity, Vaccine
PubMed: 36484136
DOI: 10.1080/14760584.2023.2156861 -
Proceedings of the National Academy of... Jun 2022Structural maintenance of chromosomes (SMC) complexes are essential for chromatin organization and functions throughout the cell cycle. The cohesin and condensin SMCs...
Structural maintenance of chromosomes (SMC) complexes are essential for chromatin organization and functions throughout the cell cycle. The cohesin and condensin SMCs fold and tether DNA, while Smc5/6 directly promotes DNA replication and repair. The functions of SMCs rely on their abilities to engage DNA, but how Smc5/6 binds and translocates on DNA remains largely unknown. Here, we present a 3.8 Å cryogenic electron microscopy (cryo-EM) structure of DNA-bound Saccharomyces cerevisiae Smc5/6 complex containing five of its core subunits, including Smc5, Smc6, and the Nse1-3-4 subcomplex. Intricate interactions among these subunits support the formation of a clamp that encircles the DNA double helix. The positively charged inner surface of the clamp contacts DNA in a nonsequence-specific manner involving numerous DNA binding residues from four subunits. The DNA duplex is held up by Smc5 and 6 head regions and positioned between their coiled-coil arm regions, reflecting an engaged-head and open-arm configuration. The Nse3 subunit secures the DNA from above, while the hook-shaped Nse4 kleisin forms a scaffold connecting DNA and all other subunits. The Smc5/6 DNA clamp shares similarities with DNA-clamps formed by other SMCs but also exhibits differences that reflect its unique functions. Mapping cross-linking mass spectrometry data derived from DNA-free Smc5/6 to the DNA-bound Smc5/6 structure identifies multi-subunit conformational changes that enable DNA capture. Finally, mutational data from cells reveal distinct DNA binding contributions from each subunit to Smc5/6 chromatin association and cell fitness. In summary, our integrative study illuminates how a unique SMC complex engages DNA in supporting genome regulation.
Topics: Cell Cycle Proteins; Cryoelectron Microscopy; DNA Replication; DNA, Fungal; Nucleic Acid Conformation; Protein Binding; Protein Conformation; Protein Subunits; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35648833
DOI: 10.1073/pnas.2202799119 -
Cellular & Molecular Immunology May 2022Neutrophil extracellular traps (NETs) can capture and kill viruses, such as influenza viruses, human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV),...
Neutrophil extracellular traps (NETs) can capture and kill viruses, such as influenza viruses, human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV), thus contributing to host defense. Contrary to our expectation, we show here that the histones released by NETosis enhance the infectivity of SARS-CoV-2, as found by using live SARS-CoV-2 and two pseudovirus systems as well as a mouse model. The histone H3 or H4 selectively binds to subunit 2 of the spike (S) protein, as shown by a biochemical binding assay, surface plasmon resonance and binding energy calculation as well as the construction of a mutant S protein by replacing four acidic amino acids. Sialic acid on the host cell surface is the key molecule to which histones bridge subunit 2 of the S protein. Moreover, histones enhance cell-cell fusion. Finally, treatment with an inhibitor of NETosis, histone H3 or H4, or sialic acid notably affected the levels of sgRNA copies and the number of apoptotic cells in a mouse model. These findings suggest that SARS-CoV-2 could hijack histones from neutrophil NETosis to promote its host cell attachment and entry process and may be important in exploring pathogenesis and possible strategies to develop new effective therapies for COVID-19.
Topics: Animals; COVID-19; Histones; Mice; N-Acetylneuraminic Acid; Protein Subunits; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Virus Internalization
PubMed: 35273357
DOI: 10.1038/s41423-022-00845-6 -
Journal of Cell Science Jul 2021Neurodevelopmental disorders (NDDs), including intellectual disability (ID), autism and schizophrenia, have high socioeconomic impact, yet poorly understood etiologies.... (Review)
Review
Neurodevelopmental disorders (NDDs), including intellectual disability (ID), autism and schizophrenia, have high socioeconomic impact, yet poorly understood etiologies. A recent surge of large-scale genome or exome sequencing studies has identified a multitude of mostly de novo mutations in subunits of the protein phosphatase 2A (PP2A) holoenzyme that are strongly associated with NDDs. PP2A is responsible for at least 50% of total Ser/Thr dephosphorylation in most cell types and is predominantly found as trimeric holoenzymes composed of catalytic (C), scaffolding (A) and variable regulatory (B) subunits. PP2A can exist in nearly 100 different subunit combinations in mammalian cells, dictating distinct localizations, substrates and regulatory mechanisms. PP2A is well established as a regulator of cell division, growth, and differentiation, and the roles of PP2A in cancer and various neurodegenerative disorders, such as Alzheimer's disease, have been reviewed in detail. This Review summarizes and discusses recent reports on NDDs associated with mutations of PP2A subunits and PP2A-associated proteins. We also discuss the potential impact of these mutations on the structure and function of the PP2A holoenzymes and the etiology of NDDs.
Topics: Animals; Humans; Intellectual Disability; Mutation; Phosphorylation; Protein Phosphatase 2; Protein Subunits
PubMed: 34228795
DOI: 10.1242/jcs.248187 -
Nature Nov 2021Glycine receptors (GlyRs) are pentameric, 'Cys-loop' receptors that form chloride-permeable channels and mediate fast inhibitory signalling throughout the central...
Glycine receptors (GlyRs) are pentameric, 'Cys-loop' receptors that form chloride-permeable channels and mediate fast inhibitory signalling throughout the central nervous system. In the spinal cord and brainstem, GlyRs regulate locomotion and cause movement disorders when mutated. However, the stoichiometry of native GlyRs and the mechanism by which they are assembled remain unclear, despite extensive investigation. Here we report cryo-electron microscopy structures of native GlyRs from pig spinal cord and brainstem, revealing structural insights into heteromeric receptors and their predominant subunit stoichiometry of 4α:1β. Within the heteromeric pentamer, the β(+)-α(-) interface adopts a structure that is distinct from the α(+)-α(-) and α(+)-β(-) interfaces. Furthermore, the β-subunit contains a unique phenylalanine residue that resides within the pore and disrupts the canonical picrotoxin site. These results explain why inclusion of the β-subunit breaks receptor symmetry and alters ion channel pharmacology. We also find incomplete receptor complexes and, by elucidating their structures, reveal the architectures of partially assembled α-trimers and α-tetramers.
Topics: Animals; Brain Stem; Cryoelectron Microscopy; Models, Molecular; Phenylalanine; Picrotoxin; Protein Subunits; Receptors, Glycine; Spinal Cord; Swine
PubMed: 34555840
DOI: 10.1038/s41586-021-04022-z -
Biochemical Society Transactions Oct 2020Phosphoprotein Phosphatases (PPPs) are enzymes highly conserved from yeast and human and catalyze the majority of the serine and threonine dephosphorylation in cells. To... (Review)
Review
Phosphoprotein Phosphatases (PPPs) are enzymes highly conserved from yeast and human and catalyze the majority of the serine and threonine dephosphorylation in cells. To achieve substrate specificity and selectivity, PPPs form multimeric holoenzymes consisting of catalytic, structural/scaffolding, and regulatory subunits. For the Protein Phosphatase 2A (PP2A)-subfamily of PPPs, holoenzyme assembly is at least in part regulated by an unusual carboxyl-terminal methyl-esterification, commonly referred to as 'methylation'. Carboxyl-terminal methylation is catalyzed by Leucine carboxyl methyltransferase-1 (LCMT1) that utilizes S-adenosyl-methionine (SAM) as the methyl donor and removed by protein phosphatase methylesterase 1 (PME1). For PP2A, methylation dictates regulatory subunit selection and thereby downstream phosphorylation signaling. Intriguingly, there are four families of PP2A regulatory subunits, each exhibiting different levels of methylation sensitivity. Thus, changes in PP2A methylation stoichiometry alters the complement of PP2A holoenzymes in cells and creates distinct modes of kinase opposition. Importantly, selective inactivation of PP2A signaling through the deregulation of methylation is observed in several diseases, most prominently Alzheimer's disease (AD). In this review, we focus on how carboxyl-terminal methylation of the PP2A subfamily (PP2A, PP4, and PP6) regulates holoenzyme function and thereby phosphorylation signaling, with an emphasis on AD.
Topics: Alzheimer Disease; Animals; Catalysis; Catalytic Domain; Dimerization; Enzymes; Gene Expression Regulation; Holoenzymes; Humans; Methylation; Mice; Mutation; Phosphoproteins; Phosphorylation; Protein Conformation; Protein Domains; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Subunits; Saccharomyces cerevisiae; Signal Transduction; Substrate Specificity
PubMed: 33125487
DOI: 10.1042/BST20200177 -
Nature Communications Aug 2021The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic...
The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics.
Topics: Bacterial Proteins; Biocatalysis; Carboxy-Lyases; Catalytic Domain; Cycloaddition Reaction; Decarboxylation; Flavin Mononucleotide; Kinetics; Models, Molecular; Oxygen; Protein Domains; Protein Subunits; Solvents; Structure-Activity Relationship; Substrate Specificity; Viscosity
PubMed: 34417452
DOI: 10.1038/s41467-021-25278-z -
The Journal of Biological Chemistry Sep 2023Voltage-gated sodium (Na) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory β-subunits. The β-subunits...
Voltage-gated sodium (Na) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory β-subunits. The β-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous β-subunits. To better reveal β-subunit functions, we engineered a human cell line devoid of endogenous Na β-subunits and their immediate phylogenetic relatives. This new cell line, β-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the β-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac Na α-subunit, Na1.5, was highly sensitive to β-subunit modulation and revealed that each β-subunit and even MPZ imparted unique gating properties. Furthermore, combining β1 and β2 with Na1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of β-subunits to regulate Na1.5 activity and can be used to improve the characterization of other α/β Na complexes.
Topics: Humans; Action Potentials; Cell Line; Intracellular Signaling Peptides and Proteins; NAV1.5 Voltage-Gated Sodium Channel; Phosphoproteins; Protein Subunits; Voltage-Gated Sodium Channel beta Subunits; Mutation
PubMed: 37544648
DOI: 10.1016/j.jbc.2023.105132