Unraveling the molecular interactions between α7 nicotinic receptor and a RIC3 variant associated with backward speech.

Recent work putatively linked a rare genetic variant of the chaperone Resistant to Inhibitors of acetylcholinesterase (RIC3) (NM_024557.4:c.262G > A, NP_078833.3:p.G88R) to a unique ability to speak backwards, a language skill that is associated with exceptional working memory capacity. RIC3 is important for the folding, maturation, and functional expression of α7 nicotinic acetylcholine receptors (nAChR). We compared and contrasted the effects of RIC3G88R on assembly, cell surface expression, and function of human α7 receptors using fluorescent protein tagged α7 nAChR and Förster resonance energy transfer (FRET) microscopy imaging in combination with functional assays and 125I-α-bungarotoxin binding. As expected, the wild-type RIC3 protein was found to increase both cell surface and functional expression of α7 receptors. In contrast, the variant form of RIC3 decreased both. FRET analysis showed that RICG88R increased the interactions between RIC3 and α7 protein in the endoplasmic reticulum. These results provide interesting and novel data to show that a RIC3 variant alters the interaction of RIC3 and α7, which translates to decreased cell surface and functional expression of α7 nAChR.

How Plants Recalibrate Cellular Iron Homeostasis.

Insufficient iron supply poses severe constraints on plants, restricting species with inefficient iron uptake mechanisms from habitats with low iron availability and causing yield losses in agricultural ecosystems. Iron deficiency also poses a severe threat on human health. Anemia resulting from insufficient iron intake is affecting one of four people in the world. It is, therefore, imperative to understand the mechanisms by which plants acquire iron against a huge soil-cell gradient and how iron is distributed within the plant to develop strategies that increase its concentration in edible plant parts. Research into the processes that are employed by plants to adjust cellular iron homeostasis revealed an astonishingly complex puzzle of signaling nodes and circuits, which are intertwined with the perception and communication of other environmental cues such as pathogens, light, nutrient availability and edaphic factors such as pH. In a recent Spotlight issue in this journal, a collection of review articles summarized the state-of-the-art in plant iron research, covering the most active and, debatably, most important topics in this field. Here, we highlight breakthroughs that were reported after the publication date of this review collection, focusing on exciting and potentially influential studies that have changed our understanding of plant iron nutrition.

The Apis mellifera alpha 5 nicotinic acetylcholine receptor subunit expresses as a homomeric receptor that is sensitive to serotonin.

Insect nicotinic acetylcholine receptors (nAChRs) are molecular targets of highly effective insecticides such as neonicotinoids. Functional expression of these receptors provides useful insights into their functional and pharmacological properties. Here, we report that the α5 nAChR subunit of the honey bee, Apis mellifera, functionally expresses in Xenopus laevis oocytes, which is the first time a homomeric insect nAChR has been robustly expressed in a heterologous system without the need for chaperone proteins. Using two-electrode voltage-clamp electrophysiology we show that the α5 receptor has low sensitivity to acetylcholine with an EC50 of 2.37 mM. However, serotonin acts as an agonist with a considerably lower EC50 at 119 µM that is also more efficacious than acetylcholine in activating the receptor. Molecular modelling indicates that residues in the complementary binding site may be involved in the selectivity towards serotonin. This is the first report of a ligand-gated ion channel activated by serotonin from an insect and phylogenetic analysis shows that the α5 subunit of A. mellifera and other non-Dipteran insects, including pest species, belong to a distinct subgroup of subunits, which may represent targets for the development of novel classes of insecticides.

A potential interaction between the SARS-CoV-2 spike protein and nicotinic acetylcholine receptors.

Changeux et al. (Changeux et al. C. R. Biol. 343:33-39.) recently suggested that the SARS-CoV-2 spike protein may interact with nicotinic acetylcholine receptors (nAChRs) and that such interactions may be involved in pathology and infectivity. This hypothesis is based on the fact that the SARS-CoV-2 spike protein contains a sequence motif similar to known nAChR antagonists. Here, we use molecular simulations of validated atomically detailed structures of nAChRs and of the spike to investigate the possible binding of the Y674-R685 region of the spike to nAChRs. We examine the binding of the Y674-R685 loop to three nAChRs, namely the human α4ß2 and α7 subtypes and the muscle-like αßγδ receptor from Tetronarce californica. Our results predict that Y674-R685 has affinity for nAChRs. The region of the spike responsible for binding contains a PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. The conformational behavior of the bound Y674-R685 is highly dependent on the receptor subtype; it adopts extended conformations in the α4ß2 and α7 complexes but is more compact when bound to the muscle-like receptor. In the α4ß2 and αßγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation, similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket in which it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1, and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of simulations of the glycosylated spike show that the Y674-R685 region is accessible for binding. We suggest a potential binding orientation of the spike protein with nAChRs, in which they are in a nonparallel arrangement to one another.

Chromatin enrichment for proteomics in plants (ChEP-P) implicates the histone reader ALFIN-LIKE 6 in jasmonate signalling.

BACKGROUND: Covalent modifications of core histones govern downstream DNA-templated processes such as transcription by altering chromatin structure and function. Previously, we reported that the plant homeodomain protein ALFIN-LIKE 6 (AL6), a bona fide histone reader that preferentially binds trimethylated lysin 4 on histone 3 (H3K4me3), is critical for recalibration of cellular phosphate (Pi) homeostasis and root hair elongation under Pi-deficient conditions. RESULTS: Here, we demonstrate that AL6 is also involved in the response of Arabidopsis seedlings to jasmonic acid (JA) during skotomorphogenesis, possibly by modulating chromatin dynamics that affect the transcriptional regulation of JA-responsive genes. Dark-grown al6 seedlings showed a compromised reduction in hypocotyl elongation upon exogenously supplied JA, a response that was calibrated by the availability of Pi in the growth medium. A comparison of protein profiles between wild-type and al6 mutant seedlings using a quantitative Chromatin Enrichment for Proteomics (ChEP) approach, that we modified for plant tissue and designated ChEP-P (ChEP in Plants), yielded a comprehensive suite of chromatin-associated proteins and candidates that may be causative for the mutant phenotype. CONCLUSIONS: Altered abundance of proteins involved in chromatin organization in al6 seedlings suggests a role of AL6 in coordinating the deposition of histone variants upon perception of internal or environmental stimuli. Our study shows that ChEP-P is well suited to gain holistic insights into chromatin-related processes in plants. Data are available via ProteomeXchange with identifier PXD026541.

Inadequate use of antibiotics in the covid-19 era: effectiveness of antibiotic therapy.

BACKGROUND: Since December 2019, the COVID-19 pandemic has changed the concept of medicine. This work aims to analyze the use of antibiotics in patients admitted to the hospital due to SARS-CoV-2 infection. METHODS: This work analyzes the use and effectiveness of antibiotics in hospitalized patients with COVID-19 based on data from the SEMI-COVID-19 registry, an initiative to generate knowledge about this disease using data from electronic medical records. Our primary endpoint was all-cause in-hospital mortality according to antibiotic use. The secondary endpoint was the effect of macrolides on mortality. RESULTS: Of 13,932 patients, antibiotics were used in 12,238. The overall death rate was 20.7% and higher among those taking antibiotics (87.8%). Higher mortality was observed with use of all antibiotics (OR 1.40, 95% CI 1.21-1.62; p < .001) except macrolides, which had a higher survival rate (OR 0.70, 95% CI 0.64-0.76; p < .001). The decision to start antibiotics was influenced by presence of increased inflammatory markers and any kind of infiltrate on an x-ray. Patients receiving antibiotics required respiratory support and were transferred to intensive care units more often. CONCLUSIONS: Bacterial co-infection was uncommon among COVID-19 patients, yet use of antibiotics was high. There is insufficient evidence to support widespread use of empiric antibiotics in these patients. Most may not require empiric treatment and if they do, there is promising evidence regarding azithromycin as a potential COVID-19 treatment.

Potentiation of a neuronal nicotinic receptor via pseudo-agonist site.

Neuronal nicotinic receptors containing α4 and ß2 subunits assemble in two pentameric stoichiometries, (α4)3(ß2)2 and (α4)2(ß2)3, each with distinct pharmacological signatures; (α4)3(ß2)2 receptors are strongly potentiated by the drug NS9283, whereas (α4)2(ß2)3 receptors are unaffected. Despite this stoichiometry-selective pharmacology, the molecular identity of the target for NS9283 remains elusive. Here, studying (α4)3(ß2)2 receptors, we show that mutations at either the principal face of the ß2 subunit or the complementary face of the α4 subunit prevent NS9283 potentiation of ACh-elicited single-channel currents, suggesting the drug targets the ß2-α4 pseudo-agonist sites, the α4-α4 agonist site, or both sites. To distinguish among these possibilities, we generated concatemeric receptors with mutations at specified subunit interfaces, and monitored the ability of NS9283 to potentiate ACh-elicited single-channel currents. We find that a mutation at the principal face of the ß2 subunit at either ß2-α4 pseudo-agonist site suppresses potentiation, whereas mutation at the complementary face of the α4 subunit at the α4-α4 agonist site allows a significant potentiation. Thus, monitoring potentiation of single concatemeric receptor channels reveals that the ß2-α4 pseudo-agonist sites are required for stoichiometry-selective drug action. Together with the recently determined structure of the (α4)3(ß2)2 receptor, the findings have implications for structure-guided drug design.

Nicotinic Antagonist UFR2709 Inhibits Nicotine Reward and Decreases Anxiety in Zebrafish.

Zebrafish is becoming a popular animal model in neuropharmacology and drug discovery, mainly due to its ease of handling and low costs involved in maintenance and experimental work. This animal displays a series of complex behaviours that makes it useful for assessing the effects of psychoactive drugs. Here, adult zebrafish were used for assessment of the anxiolytic and anti-addictive properties of UFR2709, a nicotinic receptor (nAChR) antagonist, using two behavioural paradigms to test for addiction, the novel tank diving test to assess anxiety and the conditioned place preference (CPP). Furthermore, the expression of nAChR subunits α4 and α7 was measured in the zebrafish brain. The results show that UFR2709 exhibits an anxiolytic effect on zebrafish and blocks the effect evoked by nicotine on CPP. Moreover, UFR2709 significantly decreased the expression of α4 nicotinic receptor subunit. This indicates that UFR2709 might be a useful drug for the treatment of nicotine addiction.

A General Mechanism for Signal Propagation in the Nicotinic Acetylcholine Receptor Family.

Nicotinic acetylcholine receptors (nAChRs) modulate synaptic activity in the central nervous system. The α7 subtype, in particular, has attracted considerable interest in drug discovery as a target for several conditions, including Alzheimer's disease and schizophrenia. Identifying agonist-induced structural changes underlying nAChR activation is fundamentally important for understanding biological function and rational drug design. Here, extensive equilibrium and nonequilibrium molecular dynamics simulations, enabled by cloud-based high-performance computing, reveal the molecular mechanism by which structural changes induced by agonist unbinding are transmitted within the human α7 nAChR. The simulations reveal the sequence of coupled structural changes involved in driving conformational change responsible for biological function. Comparison with simulations of the α4ß2 nAChR subtype identifies features of the dynamical architecture common to both receptors, suggesting a general structural mechanism for signal propagation in this important family of receptors.

The regulation and plasticity of root hair patterning and morphogenesis.

Root hairs are highly specialized cells found in the epidermis of plant roots that play a key role in providing the plant with water and mineral nutrients. Root hairs have been used as a model system for understanding both cell fate determination and the morphogenetic plasticity of cell differentiation. Indeed, many studies have shown that the fate of root epidermal cells, which differentiate into either root hair or non-hair cells, is determined by a complex interplay of intrinsic and extrinsic cues that results in a predictable but highly plastic pattern of epidermal cells that can vary in shape, size and function. Here, we review these studies and discuss recent evidence suggesting that environmental information can be integrated at multiple points in the root hair morphogenetic pathway and affects multifaceted processes at the chromatin, transcriptional and post-transcriptional levels.

Scopoletin 8-Hydroxylase-Mediated Fraxetin Production Is Crucial for Iron Mobilization.

Iron (Fe) is an essential mineral nutrient and an important factor for the composition of natural plant communities. Low Fe availability in aerated soils with neutral or alkaline pH has led to the evolution of elaborate mechanisms that extract Fe from the soil solution. In Arabidopsis (Arabidopsis thaliana), Fe is acquired by an orchestrated strategy that comprises mobilization, chelation, and reduction of Fe3+ prior to its uptake. Here, we show that At3g12900, previously annotated as scopoletin 8-hydroxylase (S8H), participates in Fe acquisition by mediating the biosynthesis of fraxetin (7,8-dihydroxy-6-methoxycoumarin), a coumarin derived from the scopoletin pathway. S8H is highly induced in roots of Fe-deficient plants both at the transcript and protein levels. Mutants defective in the expression of S8H showed increased sensitivity to growth on pH 7.0 media supplemented with an immobile source of Fe and reduced secretion of fraxetin. Transgenic lines overexpressing S8H exhibited an opposite phenotype. Homozygous s8h mutants grown on media with immobilized Fe accumulated significantly more scopolin, the storage form of scopoletin, supporting the designated function of S8H in scopoletin hydroxylation. Fraxetin exhibited Fe-reducing properties in vitro with higher rates being observed at neutral relative to acidic pH. Supplementing the media containing immobile Fe with fraxetin partially rescued the s8h mutants. In natural Arabidopsis accessions differing in their performance on media containing immobilized Fe, the amount of secreted fraxetin was highly correlated with growth and Fe and chlorophyll content, indicating that fraxetin secretion is a decisive factor for calcicole-calcifuge behavior (i.e. the ability/inability to thrive on alkaline soils) of plants.

Ethyl-for-methyl substitution enhances the subtype specificity of mecamylamine analogues.

The synthesis of novel mecamylamine analogues is described in which one, two or three of the methyl groups of mecamylamine have been systematically replaced with ethyl groups. Assessment of the compounds highlights that simple ethyl for methyl changes changes to the parent structure can dramatically enhance activity and selectivity towards either the α4ß2 (at the expense of α3ß4) or the α3ß4 (at the expense of α4ß2) nicotinic acetylcholine receptor sub-type as compared to the parent compound.

Molecular function of the novel α7ß2 nicotinic receptor.

The α7 nicotinic receptor is a promising drug target for neurological and inflammatory disorders. Although it is the homomeric member of the family, a novel α7ß2 heteromeric receptor has been discovered. To decipher the functional contribution of the ß2 subunit, we generated heteromeric receptors with fixed stoichiometry by two different approaches comprising concatenated and unlinked subunits. Receptors containing up to three ß2 subunits are functional. As the number of ß2 subunits increases in the pentameric arrangement, the durations of channel openings and activation episodes increase progressively probably due to decreased desensitization. The prolonged activation episodes conform the kinetic signature of α7ß2 and may have an impact on neuronal excitability. For activation of α7ß2 receptors, an α7/α7 binding-site interface is required, thus indicating that the three ß2 subunits are located consecutively in the pentameric arrangement. α7-positive allosteric modulators (PAMs) are emerging as novel therapeutic drugs. The presence of ß2 in the pentamer affects neither type II PAM potentiation nor activation by an allosteric agonist whereas it impairs type I PAM potentiation. This first single-channel study provides fundamental basis required to decipher the role and function of the novel α7ß2 receptor and opens doors to develop selective therapeutic drugs.

Minimal Structural Changes Determine Full and Partial Nicotinic Receptor Agonist Activity for Nicotine Analogues.

Neuronal α4ß2 nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels (LGIC) that have been implicated in nicotine addiction, reward, cognition, pain disorders, anxiety, and depression. Nicotine has been widely used as a template for the synthesis of ligands that prefer α4ß2 nAChRs subtypes. The most important therapeutic use for α4ß2 nAChRs is as replacement therapy for smoking cessation and withdrawal and the most successful therapeutic ligands are partial agonists. In this case, we use the N-methylpyrrolidine moiety of nicotine to design and synthesize new α4ß2 nicotinic derivatives, coupling the pyrrolidine moiety to an aromatic group by introducing an ether-bonded functionality. Meta-substituted phenolic derivatives were used for these goals. Radioligand binding assays were performed on clonal cell lines of hα4ß2 nAChR and two electrode voltage-clamp experiments were used for functional assays. Molecular docking was performed in the open state of the nAChR in order to rationalize the agonist activity shown by our compounds.

α4ß2 Nicotinic Acetylcholine Receptors: RELATIONSHIPS BETWEEN SUBUNIT STOICHIOMETRY AND FUNCTION AT THE SINGLE CHANNEL LEVEL.

Acetylcholine receptors comprising α4 and ß2 subunits are the most abundant class of nicotinic acetylcholine receptor in the brain. They contribute to cognition, reward, mood, and nociception and are implicated in a range of neurological disorders. Previous measurements of whole-cell macroscopic currents showed that α4 and ß2 subunits assemble in two predominant pentameric stoichiometries, which differ in their sensitivity to agonists, antagonists, and allosteric modulators. Here we compare agonist-elicited single channel currents from receptors assembled with an excess of either the α4 or ß2 subunit, forming receptor populations biased toward one or the other stoichiometry, with currents from receptors composed of five concatemeric subunits in which the subunit stoichiometry is predetermined. Our results associate each subunit stoichiometry with a unique single channel conductance, mean open channel lifetime, and sensitivity to the allosteric potentiator 3-[3-(3-pyridinyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS-9283). Receptors with the composition (α4ß2)2α4 exhibit high single channel conductance, brief mean open lifetime, and strong potentiation by NS-9283, whereas receptors with the composition (α4ß2)2ß2 exhibit low single channel conductance and long mean open lifetime and are not potentiated by NS-9283. Thus single channel current measurements reveal bases for the distinct functional and pharmacological properties endowed by different stoichiometries of α4 and ß2 subunits and establish pentameric concatemers as a means to delineate interactions between subunits that confer these properties.

Role of the Cys Loop and Transmembrane Domain in the Allosteric Modulation of α4ß2 Nicotinic Acetylcholine Receptors.

Allosteric modulators of pentameric ligand-gated ion channels are thought to act on elements of the pathways that couple agonist binding to channel gating. Using α4ß2 nicotinic acetylcholine receptors and the α4ß2-selective positive modulators 17ß-estradiol (ßEST) and desformylflustrabromine (dFBr), we have identified pathways that link the binding sites for these modulators to the Cys loop, a region that is critical for channel gating in all pentameric ligand-gated ion channels. Previous studies have shown that the binding site for potentiating ßEST is in the C-terminal (post-M4) region of the α4 subunit. Here, using homology modeling in combination with mutagenesis and electrophysiology, we identified the binding site for potentiating dFBr on the top half of a cavity between the third (M3) and fourth transmembrane (M4) α-helices of the α4 subunit. We found that the binding sites for ßEST and dFBr communicate with the Cys loop, through interactions between the last residue of post-M4 and Phe170 of the conserved FPF sequence of the Cys loop, and that these interactions affect potentiating efficacy. In addition, interactions between a residue in M3 (Tyr309) and Phe167, a residue adjacent to the Cys loop FPF motif, also affect dFBr potentiating efficacy. Thus, the Cys loop acts as a key control element in the allosteric transduction pathway for potentiating ßEST and dFBr. Overall, we propose that positive allosteric modulators that bind the M3-M4 cavity or post-M4 region increase the efficacy of channel gating through interactions with the Cys loop.

A MYB/ZML Complex Regulates Wound-Induced Lignin Genes in Maize.

Lignin is an essential polymer in vascular plants that plays key structural roles in vessels and fibers. Lignification is induced by external inputs such as wounding, but the molecular mechanisms that link this stress to lignification remain largely unknown. In this work, we provide evidence that three maize (Zea mays) lignin repressors, MYB11, MYB31, and MYB42, participate in wound-induced lignification by interacting with ZML2, a protein belonging to the TIFY family. We determined that the three R2R3-MYB factors and ZML2 bind in vivo to AC-rich and GAT(A/C) cis-elements, respectively, present in a set of lignin genes. In particular, we show that MYB11 and ZML2 bind simultaneously to the AC-rich and GAT(A/C) cis-elements present in the promoter of the caffeic acid O-methyl transferase (comt) gene. We show that, like the R2R3-MYB factors, ZML2 also acts as a transcriptional repressor. We found that upon wounding and methyl jasmonate treatments, MYB11 and ZML2 proteins are degraded and comt transcription is induced. Based on these results, we propose a molecular regulatory mechanism involving a MYB/ZML complex in which wound-induced lignification can be achieved by the derepression of a set of lignin genes.

Crucial role of nicotinic α5 subunit variants for Ca2+ fluxes in ventral midbrain neurons.

Neuronal nicotinic acetylcholine receptors (nAChRs) containing the α5 subunit modulate nicotine consumption, and the human CHRNA5 rs16969968 polymorphism, causing the replacement of the aspartic acid residue at position 398 with an asparagine (α5DN), has recently been associated with increased use of tobacco and higher incidence of lung cancer. We show that in ventral midbrain neurons, the α5 subunit is essential for heteromeric nAChR-induced intracellular-free Ca(2+) concentration elevations and that in α5(-/-) mice, a class of large-amplitude nicotine-evoked currents is lost. Furthermore, the expression of the α5DN subunit is not able to restore nicotinic responses, indicating a loss of function by this subunit in native neurons. To understand how α5DN impairs heteromeric nAChR functions, we coexpressed α4, α5, or α5DN subunits with a dimeric concatemer (ß2α4) in a heterologous system, to obtain nAChRs with fixed stoichiometry. Both α5(ß2α4)2 and α5DN(ß2α4)2 nAChRs yielded similar levels of functional expression and Ca(2+) permeability, measured as fractional Ca(2+) currents (8.2 ± 0.7% and 8.0 ± 1.9%, respectively), 2-fold higher than α4(ß2α4)2. Our results indicate that the loss of function of nicotinic responses observed in α5DN-expressing ventral midbrain neurons is neither due to an intrinsic inability of this subunit to form functional nAChRs nor to an altered Ca(2+) permeability but likely to intracellular modulation.

A new synthesis and preliminary evaluation of some analogues of mecamylamine - a compound with anti-addiction properties.

A new synthesis of mecamylamine - a known anti-hypertensive drug with anti-addictive properties is described. The new route allowed access to two novel analogues whose activity at two nicotinic acetylcholine receptor subtypes was assessed.

Non-equivalent ligand selectivity of agonist sites in (α4ß2)2α4 nicotinic acetylcholine receptors: a key determinant of agonist efficacy.

The α4ß2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR type in the brain, and this receptor type exists in alternate (α4ß2)2α4 and (α4ß2)2ß2 forms, which are activated by agonists with strikingly differing efficacies. Recent breakthroughs have identified an additional operational agonist binding site in the (α4ß2)2α4 nAChR that is responsible for the signature sensitivity of this receptor to activation by agonists, yet the structural mechanisms determining agonist efficacy at this receptor type are not yet fully understood. In this study, we characterized the ligand selectivity of the individual agonist sites of the (α4ß2)2α4 nAChR to determine whether differences in agonist selectivity influence agonist efficacy. Applying the substituted cysteine accessibility method to individual agonist sites in concatenated (α4ß2)2α4 receptors, we determined the agonist selectivity of the agonist sites of the (α4ß2)2α4 receptor. We show that (a) accessibility of substituted cysteines to covalent modification by methanesulfonate reagent depends on the agonist site at which the modification occurs and (b) that agonists such as sazetidine-A and TC-2559 are excluded from the site at the α4/α4 interface. Given that additional binding to the agonist site in the α4/α4 interface increases acetylcholine efficacy and that agonists excluded from the agonist site at the α4/α4 interface behave as partial agonists, we conclude that the ability to engage all agonist sites in (α4ß2)2α4 nAChRs is a key determinant of agonist efficacy. The findings add another level of complexity to the structural mechanisms that govern agonist efficacy in heteromeric nAChRs and related ligand-gated ion channels.