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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 -
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 -
Endocrine Reviews Apr 2016The TSH receptor (TSHR) on the surface of thyrocytes is unique among the glycoprotein hormone receptors in comprising two subunits: an extracellular A-subunit, and a... (Review)
Review
The TSH receptor (TSHR) on the surface of thyrocytes is unique among the glycoprotein hormone receptors in comprising two subunits: an extracellular A-subunit, and a largely transmembrane and cytosolic B-subunit. Unlike its ligand TSH, whose subunits are encoded by two genes, the TSHR is expressed as a single polypeptide that subsequently undergoes intramolecular cleavage into disulfide-linked subunits. Cleavage is associated with removal of a C-peptide region, a mechanism similar in some respects to insulin cleavage into disulfide linked A- and B-subunits with loss of a C-peptide region. The potential pathophysiological importance of TSHR cleavage into A- and B-subunits is that some A-subunits are shed from the cell surface. Considerable experimental evidence supports the concept that A-subunit shedding in genetically susceptible individuals is a factor contributing to the induction and/or affinity maturation of pathogenic thyroid-stimulating autoantibodies, the direct cause of Graves' disease. The noncleaving gonadotropin receptors are not associated with autoantibodies that induce a "Graves' disease of the gonads." We also review herein current information on the location of the cleavage sites, the enzyme(s) responsible for cleavage, the mechanism by which A-subunits are shed, and the effects of cleavage on receptor signaling.
Topics: Animals; Humans; Protein Multimerization; Protein Subunits; Protein Transport; Proteolysis; Receptors, Thyrotropin; Thyroid Epithelial Cells
PubMed: 26799472
DOI: 10.1210/er.2015-1098 -
Current Topics in Membranes 2016Voltage-gated Na(+) channels (VGSCs) isolated from mammalian neurons are heterotrimeric complexes containing one pore-forming α subunit and two non-pore-forming β... (Review)
Review
Voltage-gated Na(+) channels (VGSCs) isolated from mammalian neurons are heterotrimeric complexes containing one pore-forming α subunit and two non-pore-forming β subunits. In excitable cells, VGSCs are responsible for the initiation of action potentials. VGSC β subunits are type I topology glycoproteins, containing an extracellular amino-terminal immunoglobulin (Ig) domain with homology to many neural cell adhesion molecules (CAMs), a single transmembrane segment, and an intracellular carboxyl-terminal domain. VGSC β subunits are encoded by a gene family that is distinct from the α subunits. While α subunits are expressed in prokaryotes, β subunit orthologs did not arise until after the emergence of vertebrates. β subunits regulate the cell surface expression, subcellular localization, and gating properties of their associated α subunits. In addition, like many other Ig-CAMs, β subunits are involved in cell migration, neurite outgrowth, and axon pathfinding and may function in these roles in the absence of associated α subunits. In sum, these multifunctional proteins are critical for both channel regulation and central nervous system development.
Topics: Animals; Arrhythmias, Cardiac; Cell Adhesion Molecules; Central Nervous System; Epilepsy; Evolution, Molecular; Myocytes, Cardiac; Neurons; Protein Subunits; Voltage-Gated Sodium Channels
PubMed: 27586289
DOI: 10.1016/bs.ctm.2016.07.003 -
The Journal of Physiology Jan 2015NMDA receptors (NMDARs) are a class of ionotropic glutamate receptors (iGluRs) that are essential for neuronal development, synaptic plasticity, learning and cell... (Review)
Review
NMDA receptors (NMDARs) are a class of ionotropic glutamate receptors (iGluRs) that are essential for neuronal development, synaptic plasticity, learning and cell survival. Several features distinguish NMDARs from other iGluRs and underlie the crucial roles NMDARs play in nervous system physiology. NMDARs display slow deactivation kinetics, are highly Ca(2+) permeable, and require depolarization to relieve channel block by external Mg(2+) ions, thereby making them effective coincidence detectors. These properties and others differ among NMDAR subtypes, which are defined by the subunits that compose the receptor. NMDARs, which are heterotetrameric, commonly are composed of two GluN1 subunits and two GluN2 subunits, of which there are four types, GluN2A-D. 'Diheteromeric' NMDARs contain two identical GluN2 subunits. Gating and ligand-binding properties (e.g. deactivation kinetics) and channel properties (e.g. channel block by Mg(2+)) depend strongly on the GluN2 subunit contained in diheteromeric NMDARs. Recent work shows that two distinct regions of GluN2 subunits control most diheteromeric NMDAR subtype-dependent properties: the N-terminal domain is responsible for most subtype dependence of gating and ligand-binding properties; a single residue difference between GluN2 subunits at a site termed the GluN2 S/L site is responsible for most subtype dependence of channel properties. Thus, two structurally and functionally distinct regions underlie the majority of subtype dependence of NMDAR properties. This topical review highlights recent studies of recombinant diheteromeric NMDARs that uncovered the involvement of the N-terminal domain and of the GluN2 S/L site in the subtype dependence of NMDAR properties.
Topics: Protein Subunits; Receptors, N-Methyl-D-Aspartate
PubMed: 25556790
DOI: 10.1113/jphysiol.2014.273763 -
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 -
Nature Communications Nov 2018Transcription preinitiation complex assembly on the promoters of protein encoding genes is nucleated in vivo by TFIID composed of the TATA-box Binding Protein (TBP) and...
Transcription preinitiation complex assembly on the promoters of protein encoding genes is nucleated in vivo by TFIID composed of the TATA-box Binding Protein (TBP) and 13 TBP-associate factors (Tafs) providing regulatory and chromatin binding functions. Here we present the cryo-electron microscopy structure of promoter-bound yeast TFIID at a resolution better than 5 Å, except for a flexible domain. We position the crystal structures of several subunits and, in combination with cross-linking studies, describe the quaternary organization of TFIID. The compact tri lobed architecture is stabilized by a topologically closed Taf5-Taf6 tetramer. We confirm the unique subunit stoichiometry prevailing in TFIID and uncover a hexameric arrangement of Tafs containing a histone fold domain in the Twin lobe.
Topics: DNA, Fungal; Models, Molecular; Promoter Regions, Genetic; Protein Binding; Protein Subunits; Saccharomyces cerevisiae; Transcription Factor TFIID; Yeasts
PubMed: 30405110
DOI: 10.1038/s41467-018-07096-y -
Sub-cellular Biochemistry 2017The assembly of individual protein subunits into large-scale structures is important in many biological contexts. Proteins may assemble into geometrical cages or... (Review)
Review
The assembly of individual protein subunits into large-scale structures is important in many biological contexts. Proteins may assemble into geometrical cages or extended lattices that are characterized by a high degree of symmetry; examples include viral capsids and bacterial S-layers. The precisely defined higher order structure exhibited by these assemblies has inspired efforts to design such structures de novo by applying the principles of symmetry evident in natural protein assemblies. Here we discuss progress towards this goal and also examples of natural protein cages and lattices that have been engineered to repurpose them towards a diverse range of applications in materials science and nano-medicine.
Topics: Materials Science; Nanomedicine; Protein Subunits; Proteins
PubMed: 28271478
DOI: 10.1007/978-3-319-46503-6_8 -
Pharmacological Research Jul 2018GABA receptors play a dominant role in mediating inhibition in the mature mammalian brain, and defects of GABAergic neurotransmission contribute to the pathogenesis of a... (Comparative Study)
Comparative Study Review
GABA receptors play a dominant role in mediating inhibition in the mature mammalian brain, and defects of GABAergic neurotransmission contribute to the pathogenesis of a variety of neurological and psychiatric disorders. Two types of GABAergic inhibition have been described: αβγ receptors mediate phasic inhibition in response to transient high-concentrations of synaptic GABA release, and αβδ receptors produce tonic inhibitory currents activated by low-concentration extrasynaptic GABA. Both αβδ and αβγ receptors are important targets for general anesthetics, which induce apparently different changes both in GABA-dependent receptor activation and in desensitization in currents mediated by αβγ vs. αβδ receptors. Many of these differences are explained by correcting for the high agonist efficacy of GABA at most αβγ receptors vs. much lower efficacy at αβδ receptors. The stoichiometry and subunit arrangement of recombinant αβγ receptors are well established as β-α-γ-β-α, while those of αβδ receptors remain controversial. Importantly, some potent general anesthetics selectively bind in transmembrane inter-subunit pockets of αβγ receptors: etomidate acts at β/α interfaces, and the barbiturate R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB) acts at α/β and γ/β interfaces. Thus, these drugs are useful as structural probes in αβδ receptors formed from free subunits or concatenated subunit assemblies designed to constrain subunit arrangement. Although a definite conclusion cannot be drawn, studies using etomidate and R-mTFD-MPAB support the idea that recombinant α1β3δ receptors may share stoichiometry and subunit arrangement with α1β3γ2 receptors.
Topics: Anesthetics, General; Animals; Binding Sites; Humans; Protein Subunits; Receptors, GABA-A; Synapses
PubMed: 29294355
DOI: 10.1016/j.phrs.2017.12.031