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Molecules (Basel, Switzerland) Jun 2020It is said that "hindsight is 20-20", so, given the current year, it is an opportune time to review and learn from experiences studying long noncoding RNAs.... (Review)
Review
It is said that "hindsight is 20-20", so, given the current year, it is an opportune time to review and learn from experiences studying long noncoding RNAs. Investigation of the telomerase RNA, TLC1, has unveiled striking flexibility in terms of both structural and functional features. Results support the "flexible scaffold" hypothesis for this 1157-nt telomerase RNA. This model describes TLC1 acting as a tether for holoenzyme protein subunits, and it also may apply to a plethora of RNAs beyond telomerase, such as types of lncRNAs. In this short perspective review, I summarize findings from studying the large yeast telomerase ribonucleoprotein (RNP) complex in the hope that this hindsight will sharpen foresight as so many of us seek to mechanistically understand noncoding RNA molecules from vast transcriptomes.
Topics: Protein Subunits; RNA; RNA, Fungal; Ribonucleoproteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Telomerase
PubMed: 32545864
DOI: 10.3390/molecules25122750 -
Molecular Pharmacology Oct 2020Epibatidine is a potent analgetic agent with very high affinity for brain nicotinic acetylcholine receptors (nAChR). We determined the activity profiles of three...
Epibatidine is a potent analgetic agent with very high affinity for brain nicotinic acetylcholine receptors (nAChR). We determined the activity profiles of three epibatidine derivatives, RTI-36, RTI-76, and RTI-102, which have affinity for brain nAChR equivalent to that of epibatidine but reduced analgetic activity. RNAs coding for nAChR monomeric subunits and/or concatamers were injected into oocytes to obtain receptors of defined subunit composition and stoichiometry. The epibatidine analogs produced protracted activation of high sensitivity (HS) 4- and 2-containing receptors with the stoichiometry of 2alpha:3beta subunits but not low sensitivity (LS) receptors with the reverse ratio of alpha and beta subunits. Although not strongly activated by the epibatidine analogs, LS 4- and 2-containing receptors were potently desensitized by the epibatidine analogs. In general, the responses of 4(2)2(2)5 and 34262 receptors were similar to those of the HS 42 receptors. RTI-36, the analog closest in structure to epibatidine, was the most efficacious of the three compounds, also effectively activating 7 and 34 receptors, albeit with lower potency and less desensitizing effect. Although not the most efficacious agonist, RTI-76 was the most potent desensitizer of 4- and 2-containing receptors. RTI-102, a strong partial agonist for HS 42 receptors, was effectively an antagonist for LS 42 receptors. Our results highlight the importance of subunit stoichiometry and the presence or absence of specific accessory subunits for determining the activity of these drugs on brain nAChR, affecting the interpretation of in vivo studies since in most cases these structural details are not known. SIGNIFICANCE STATEMENT: Epibatidine and related compounds are potent ligands for the high-affinity nicotine receptors of the brain, which are therapeutic targets and mediators of nicotine addiction. Far from being a homogeneous population, these receptors are diverse in subunit composition and vary in subunit stoichiometry. We show the importance of these structural details for drug activity profiles, which present a challenge for the interpretation of in vivo experiments since conventional methods, such as in situ hybridization and immunohistochemistry, cannot illuminate these details.
Topics: Animals; Animals, Genetically Modified; Brain; Bridged Bicyclo Compounds, Heterocyclic; Humans; Molecular Structure; Multiprotein Complexes; Nicotinic Agonists; Protein Subunits; Pyridines; Receptors, Nicotinic; Tropanes; Xenopus
PubMed: 32690626
DOI: 10.1124/molpharm.120.000037 -
Biochemical and Biophysical Research... Nov 2023Targeted cytokine delivery has been gaining popularity in cancer immunotherapy since systemic recombinant cytokine treatment has not been successful due to low response...
Targeted cytokine delivery has been gaining popularity in cancer immunotherapy since systemic recombinant cytokine treatment has not been successful due to low response rate and systemic toxicities in the clinical studies. In order to address these issues, we propose a new concept that cytokine signal is specifically activated at tumor-micro-environment (TME) by delivering two protein subunits of heterodimeric cytokine fused with a tumor targeting antibody respectively to TME and by bridging the two subunits into active heterodimeric form.Interleukin-12 (IL-12) is one of the major cytokines which can induce immune activation. IL-12 consists of two protein subunits which are p35 and p40. IL-12 signaling is initiated when it forms as the heterodimeric protein and binds to IL-12 receptor complex. We made fusion proteins of both IL-12p35 and IL-12p40 targeting specific tumor associated antigens (TAAs) and demonstrated the formation of bioactive IL12p70 with TME targeting antibody toward both p35 and p40 to form as the active molecule. We describe our concept validation in an in vitro based functional assay.
Topics: Humans; Cytokines; Protein Subunits; Interleukin-12; Recombinant Proteins; Neoplasms; Interleukin-12 Subunit p40; Tumor Microenvironment
PubMed: 37890200
DOI: 10.1016/j.bbrc.2023.10.009 -
Microbiology Spectrum Oct 2022Confronted with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, such as Delta and Omicron, with high infectivity and immune evasion capacity,...
Boosting with Multiple Doses of mRNA Vaccine after Priming with Two Doses of Protein Subunit Vaccine MVC-COV1901 Elicited Robust Humoral and Cellular Immune Responses against Emerging SARS-CoV-2 Variants.
Confronted with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, such as Delta and Omicron, with high infectivity and immune evasion capacity, vaccination remains the most effective tool to prevent infection and severe illness. However, heterologous vaccination of mRNA vaccines primed with protein subunit vaccines had not been evaluated before the current study. Since subunit vaccine MVC-COV1901 (MVC) has been granted emergency use authorization in Taiwan, in this study, we explored the humoral and cellular immune responses to additional third (2× MVC/Mod) and fourth (2× MVC/2× Mod) doses of mRNA-1273 (Mod) after priming with two doses of subunit vaccine MVC against the emerging variants. We found a 12.3- to 16.1-fold increase in antibodies targeting the receptor binding domain (RBD) of the Delta variant with 2× MVC/Mod compared to two doses of MVC (2× MVC) or AZD1222 (2× AZ) regimens and a 26- to 32.2-fold improvement in neutralizing potency against the Omicron variant (BA.1). Besides, the numbers of gamma interferon (IFN-γ)-secreting T cells induced by 2× MVC/Mod were also elevated 3.5-fold and 3.7- to 4.3-fold for the wild type and Delta variant. However, boosting with a fourth dose of Mod (2× MVC/2× Mod) after the 2× MVC/Mod regimen failed to significantly improve the immune responses. Moreover, all vaccination schedules showed reduced neutralizing activity against the Omicron variant. Collectively, our results suggested that the third or fourth dose booster vaccination with mRNA vaccine after priming with two doses of protein subunit vaccine could elicit stronger humoral and cellular immune responses. These findings could provide a future global heterologous boosting strategy against COVID-19. Vaccination is the most important strategy to combat the COVID-19 outbreak; however, it remains to be determined whether heterologous prime-boost regimens could induce equal or even stronger immune responses against SARS-CoV-2. Here, we showed that boosting the additional doses of mRNA-1273 (Mod) priming with two doses of MVC-COV1901 (MVC) (2× MVC/Mod) improved humoral and cellular immunity compared to two doses of AZD1222 (2× AZ) or MVC (2× MVC) against SARS-CoV-2 variants. However, the Omicron variant showed strong immune evasion ability for all vaccination schedules. Our findings provided evidence supporting that heterologous vaccination by boosting with mRNA vaccine after priming with two doses of protein subunit vaccine could strongly promote humoral and cellular immune responses against the emerging SARS-CoV-2 variants.
Topics: Humans; SARS-CoV-2; Protein Subunits; Viral Vaccines; Interferon-gamma; COVID-19; ChAdOx1 nCoV-19; Immunity, Cellular; Vaccination; Vaccines, Subunit; Antibodies, Viral; Antibodies, Neutralizing; mRNA Vaccines
PubMed: 36005765
DOI: 10.1128/spectrum.00609-22 -
Biochemistry Jun 2021Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme capable of catalyzing the fissure of the C-H bond of methane and the insertion of one atom of...
Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme capable of catalyzing the fissure of the C-H bond of methane and the insertion of one atom of oxygen from O to yield methanol. Efficient multiple-turnover catalysis occurs only in the presence of all three sMMO protein components: hydroxylase (MMOH), reductase (MMOR), and regulatory protein (MMOB). The complex series of sMMO protein component interactions that regulate the formation and decay of sMMO reaction cycle intermediates is not fully understood. Here, the two tryptophan residues in MMOB and the single tryptophan residue in MMOR are converted to 5-fluorotryptophan (5FW) by expression in defined media containing 5-fluoroindole. In addition, the mechanistically significant N-terminal region of MMOB is F-labeled by reaction of the K15C variant with 3-bromo-1,1,1-trifluoroacetone (BTFA). The 5FW and BTFA modifications cause minimal structural perturbation, allowing detailed studies of the interactions with sMMOH using F NMR. Resonances from the 275 kDa complexes of sMMOH with 5FW-MMOB and BTFA-K15C-5FW-MMOB are readily detected at 5 μM labeled protein concentration. This approach shows directly that MMOR and MMOB competitively bind to sMMOH with similar values, independent of the oxidation state of the sMMOH diiron cluster. These findings suggest a new model for regulation in which the dynamic equilibration of MMOR and MMOB with sMMOH allows a transient formation of key reactive complexes that irreversibly pull the reaction cycle forward. The slow kinetics of exchange of the sMMOH:MMOB complex is proposed to prevent MMOR-mediated reductive quenching of the high-valent reaction cycle intermediate before it can react with methane.
Topics: Bacterial Proteins; Fluorine; Kinetics; Methylosinus trichosporium; Nuclear Magnetic Resonance, Biomolecular; Oxygenases; Protein Binding; Protein Structure, Quaternary; Protein Subunits; Tryptophan
PubMed: 34100595
DOI: 10.1021/acs.biochem.1c00293 -
Nature Communications Sep 2021GABA receptors are vital for controlling neuronal excitability and can display significant levels of constitutive activity that contributes to tonic inhibition. However,...
GABA receptors are vital for controlling neuronal excitability and can display significant levels of constitutive activity that contributes to tonic inhibition. However, the mechanisms underlying spontaneity are poorly understood. Here we demonstrate a strict requirement for β3 subunit incorporation into receptors for spontaneous gating, facilitated by α4, α6 and δ subunits. The crucial molecular determinant involves four amino acids (GKER) in the β3 subunit's extracellular domain, which interacts with adjacent receptor subunits to promote transition to activated, open channel conformations. Spontaneous activity is further regulated by β3 subunit phosphorylation and by allosteric modulators including neurosteroids and benzodiazepines. Promoting spontaneous activity reduced neuronal excitability, indicating that spontaneous currents will alter neural network activity. This study demonstrates how regional diversity in GABA receptor isoform, protein kinase activity, and neurosteroid levels, can impact on tonic inhibition through the modulation of spontaneous GABA receptor gating.
Topics: Algorithms; Amino Acid Sequence; Animals; Cells, Cultured; HEK293 Cells; Hippocampus; Humans; Ion Channel Gating; Mice; Models, Molecular; Models, Neurological; Neurons; Patch-Clamp Techniques; Protein Conformation; Protein Subunits; Rats, Sprague-Dawley; Receptors, GABA-A; Sequence Homology, Amino Acid; gamma-Aminobutyric Acid; Rats
PubMed: 34526505
DOI: 10.1038/s41467-021-25633-0 -
International Journal of Molecular... Sep 2022The NMDA receptor is a Ca-permeant glutamate receptor which plays key roles in health and disease. Canonical NMDARs contain two GluN2 subunits, of which 2A and 2B are... (Review)
Review
The NMDA receptor is a Ca-permeant glutamate receptor which plays key roles in health and disease. Canonical NMDARs contain two GluN2 subunits, of which 2A and 2B are predominant in the forebrain. Moreover, the relative contribution of 2A vs. 2B is controlled both developmentally and in an activity-dependent manner. The GluN2 subtype influences the biophysical properties of the receptor through difference in their N-terminal extracellular domain and transmembrane regions, but they also have large cytoplasmic Carboxyl (C)-terminal domains (CTDs) which have diverged substantially during evolution. While the CTD identity does not influence NMDAR subunit specific channel properties, it determines the nature of CTD-associated signalling molecules and has been implicated in mediating the control of subunit composition (2A vs. 2B) at the synapse. Historically, much of the research into the differential function of GluN2 CTDs has been conducted in vitro by over-expressing mutant subunits, but more recently, the generation of knock-in (KI) mouse models have allowed CTD function to be probed in vivo and in ex vivo systems without heterologous expression of GluN2 mutants. In some instances, findings involving KI mice have been in disagreement with models that were proposed based on earlier approaches. This review will examine the current research with the aim of addressing these controversies and how methodology may contribute to differences between studies. We will also discuss the outstanding questions regarding the role of GluN2 CTD sequences in regulating NMDAR subunit composition, as well as their relevance to neurodegenerative disease and neurodevelopmental disorders.
Topics: Animals; Disease Models, Animal; Growth and Development; Mice; Neurodegenerative Diseases; Neurodevelopmental Disorders; Protein Subunits; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Synapses
PubMed: 36232696
DOI: 10.3390/ijms231911392 -
Proceedings of the National Academy of... Feb 2021Gene duplication is ubiquitous and a major driver of phenotypic diversity across the tree of life, but its immediate consequences are not fully understood. Deleterious...
Gene duplication is ubiquitous and a major driver of phenotypic diversity across the tree of life, but its immediate consequences are not fully understood. Deleterious effects would decrease the probability of retention of duplicates and prevent their contribution to long-term evolution. One possible detrimental effect of duplication is the perturbation of the stoichiometry of protein complexes. Here, we measured the fitness effects of the duplication of 899 essential genes in the budding yeast using high-resolution competition assays. At least 10% of genes caused a fitness disadvantage when duplicated. Intriguingly, the duplication of most protein complex subunits had small to nondetectable effects on fitness, with few exceptions. We selected four complexes with subunits that had an impact on fitness when duplicated and measured the impact of individual gene duplications on their protein-protein interactions. We found that very few duplications affect both fitness and interactions. Furthermore, large complexes such as the 26S proteasome are protected from gene duplication by attenuation of protein abundance. Regulatory mechanisms that maintain the stoichiometric balance of protein complexes may protect from the immediate effects of gene duplication. Our results show that a better understanding of protein regulation and assembly in complexes is required for the refinement of current models of gene duplication.
Topics: Gene Duplication; Gene Expression Regulation, Fungal; Genes, Essential; Genetic Fitness; Proteasome Endopeptidase Complex; Protein Interaction Maps; Protein Subunits; Saccharomycetales
PubMed: 33526669
DOI: 10.1073/pnas.2014345118 -
Nature Apr 2020R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics. Each bactericidal complex uses a collar to bridge a hollow tube with...
R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics. Each bactericidal complex uses a collar to bridge a hollow tube with a contractile sheath loaded in a metastable state by a baseplate scaffold. Fine-tuning of such nucleic acid-free protein machines for precision medicine calls for an atomic description of the entire complex and contraction mechanism, which is not available from baseplate structures of the (DNA-containing) T4 bacteriophage. Here we report the atomic model of the complete R2 pyocin in its pre-contraction and post-contraction states, each containing 384 subunits of 11 unique atomic models of 10 gene products. Comparison of these structures suggests the following sequence of events during pyocin contraction: tail fibres trigger lateral dissociation of baseplate triplexes; the dissociation then initiates a cascade of events leading to sheath contraction; and this contraction converts chemical energy into mechanical force to drive the iron-tipped tube across the bacterial cell surface, killing the bacterium.
Topics: Bacteriophage T4; Cryoelectron Microscopy; Crystallography, X-Ray; Genes, Bacterial; Models, Molecular; Protein Subunits; Pseudomonas aeruginosa; Pyocins; Substrate Specificity; Type VI Secretion Systems
PubMed: 32350467
DOI: 10.1038/s41586-020-2186-z -
Channels (Austin, Tex.) Dec 2020The KCNH family comprises the ERG, EAG, and ELK voltage-activated, potassium-selective channels. Distinct from other K channels, KCNH channels contain unique structural... (Review)
Review
The KCNH family comprises the ERG, EAG, and ELK voltage-activated, potassium-selective channels. Distinct from other K channels, KCNH channels contain unique structural domains, including a PAS (Per-Arnt-Sim) domain in the N-terminal region and a CNBHD (cyclic nucleotide-binding homology domain) in the C-terminal region. The intracellular PAS domains and CNBHDs interact directly and regulate some of the characteristic gating properties of each type of KCNH channel. The PAS-CNBHD interaction regulates slow closing (deactivation) of hERG channels, the kinetics of activation and pre-pulse dependent population of closed states (the Cole-Moore shift) in EAG channels and voltage-dependent potentiation in ELK channels. KCNH channels are all regulated by an intrinsic ligand motif in the C-terminal region which binds to the CNBHD. Here, we focus on some recent advances regarding the PAS-CNBHD interaction and the intrinsic ligand.
Topics: Animals; Humans; Intracellular Space; Ion Channel Gating; Potassium Channels, Voltage-Gated; Protein Domains; Protein Subunits
PubMed: 32924766
DOI: 10.1080/19336950.2020.1816107