Dynamin-2 R465W mutation induces long range perturbation in highly ordered oligomeric structures

High order oligomers are crucial for normal cell physiology, and protein function perturbed by missense mutations underlies several autosomal dominant diseases. Dynamin-2 is one of such protein forming helical oligomers that catalyze membrane fission. Mutations in this protein, where R465W is the most frequent, cause dominant centronuclear myopathy, but the molecular mechanisms underpinning the functional modifications remain to be investigated. To unveil the structural impact of this mutation in dynamin-2, we used full-atom molecular dynamics simulations and coarse-grained models and built dimers and helices of wild-type (WT) monomers, mutant monomers, or both WT and mutant monomers combined. Our results show that the mutation R465W causes changes in the interactions with neighbor amino acids that propagate through the oligomer. These new interactions perturb the contact between monomers and favor an extended conformation of the bundle signaling element (BSE), a dynamin region that transmits the conformational changes from the GTPase domain to the rest of the protein. This extended configuration of the BSE that is only relevant in the helices illustrates how a small change in the microenvironment surrounding a single residue can propagate through the oligomer structures of dynamin explaining how dominance emerges in large protein complexes. © 2020, The Author(s).Indexación Scopushttps://www-nature-com.recursosbiblioteca.unab.cl/articles/s41598-020-75216-

El proceso de articulación entre educación parvularia y educación básica y su aplicación al aula

Tesis (Educación Parvularia, Licenciado en Educación)... El problema de este trabajo se orienta a describir como se está produciendo el proceso de articulación entre ambos niveles, específicamente la pregunta que dirige el trabajo de investigación de este seminario puede plantearse de la siguiente manera. ¿Cómo está ocurriendo el proceso de articulación entre el nivel de Educación Parvularia y la Educación Básica?. ¿cuáles son sus fortalezas y debilidades? ¿cómo mejorar este proceso y cuáles son los elementos pedagógicos de la Educación Parvularia que son útiles en considerar en el cambio de un nivel a otro

Identification and Functional Expression of a Glutamate- and Avermectin-Gated Chloride Channel from Caligus rogercresseyi, a Southern Hemisphere Sea Louse Affecting Farmed Fish

International audienceParasitic sea lice represent a major sanitary threat to marine salmonid aquaculture, an industry accounting for 7% of world fish production. Caligus rogercresseyi is the principal sea louse species infesting farmed salmon and trout in the southern hemisphere. Most effective control of Caligus has been obtained with macrocyclic lactones (MLs) ivermectin and emamectin. These drugs target glutamate-gated chloride channels (GluCl) and act as irreversible non-competitive agonists causing neuronal inhibition, paralysis and death of the parasite. Here we report the cloning of a full-length CrGluCla receptor from Caligus rogercresseyi. Expression in Xenopus oocytes and electrophysiological assays show that CrGluCla is activated by glutamate and mediates chloride currents blocked by the ligand-gated anion channel inhibitor picrotoxin. Both ivermectin and emamectin activate CrGluCla in the absence of glutamate. The effects are irreversible and occur with an EC 50 value of around 200 nM, being cooperative (n H = 2) for ivermectin but not for emamectin. Using the three-dimensional structure of a GluCla from Caenorabditis elegans, the only available for any eukaryotic ligand-gated anion channel, we have constructed a homology model for CrGluCla. Docking and molecular dynamics calculations reveal the way in which ivermectin and emamectin interact with CrGluCla. Both drugs intercalate between transmembrane domains M1 and M3 of neighbouring subunits of a pentameric structure. The structure displays three H-bonds involved in this interaction, but despite similarity in structure only of two these are conserved from the C. elegans crystal binding site. Our data strongly suggest that CrGluCla is an important target for avermectins used in the treatment of sea louse infestation in farmed salmonids and open the way for ascertaining a possible mechanism of increasing resistance to MLs in aquaculture industry. Molecular modeling could help in the design of new, more efficient drugs whilst functional expression of the receptor allows a first stage of testing of their efficacy

Energy and affinity values for the best pose of ivermectin and emamectin in docking assays with CrGluClα molecular model.

<p>Energy and affinity values for the best pose of ivermectin and emamectin in docking assays with CrGluClα molecular model.</p

Variations in deduced CrGluClα primary structure.

<p>The rooted phylogenetic tree was done by using the UPGMA method after aligning twelve representative clones with CLUSTALW software (<a href="http://www.genome.jp/tools/clustalw" target="_blank">http://www.genome.jp/tools/clustalw</a>). The positions of amino acid changes (V27I, D73G, R387K and L411Q), or deletions (isoleucine 20 (I or Δ), and alanine 376 plus serine 377 (AS or Δ)) are shown. The clone name is composed by CrGluClα, source and Arabic number. Source: Valdivia (Vald), Darwin (Dw) and Errázuriz (Err). Arabic number is the number assigned during the cloning process.</p

Conservation of residues involved in drug binding in <i>Caligus</i> and <i>C. elegans</i> GluClα.

<p>Residues involved in hydrogen-bonding (green) and van der Waals (grey) interactions of ivermectin or emamectin with CrGluClα (CrGluClα-IVM, CrGluClα-EMA, see text) are compared with those identified in the CeGluClα-ivermectin complex (CeGluClα-IVM) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004402#ppat.1004402-Hibbs1" target="_blank">[16]</a>. Only those interactions existing during >70 ns of the 140 ns MD runs are shown. Residues highlighted in red are those apparently involved in creating a hydrophobic seal in the channel pore in the absence of agonists <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004402#ppat.1004402-Cheng1" target="_blank">[31]</a>.</p

Effect of ivermectin or emamectin upon CrGluClα-T318A-dependent currents.

<p>A and B. Effect of increasing concentrations of emamectin and ivermectin on WT CrGluClα-dependent currents. The currents were first activated by addition of 50 µM glutamate and during continuous exposure to glutamate increasing concentrations of emamemctin or ivermectin were added. Reduction of [Cl⁻] to 7.6 mM or addition of 100 mM PTX are also indicated. C and D. Similar experiments performed using the CrGluClα-T318A mutant. E and F. Concentration dependence of the effects of avermectins on WT and T318A mutant CrGluClα receptors. Normalised average responses to emamectin and ivermectin are shown (means±SEM, n = 6 for all experiments shown). Fits of Hill decay functions to the average values are shown by the solid lines.</p

Irreversible activation by emamectin of CrGluClα receptor expressed in <i>Xenopus</i> oocytes.

<p>A. Current trace obtained at 60 mV during bath application of emamectin at concentrations going from 5 nM to 10 µM during the times shown in the boxes. The effect of picrotoxin addition at 100 µM is also shown immediately after removal of emamectin. Wash out of picrotoxin in the absence of emamectin returns current to the levels attained in the presence of high concentration of emamectin. B: Current-voltage relations taken from voltage-ramps applied during experiment in A. Control denotes current prior to emamectin addition. C. Dose-response relationship of the emamectin-sensitive currents. Data are normalized (mean ± SEM) to the maximal effect of emamectin and originate in seven separate experiments. The line is a Hill equation fitted simultaneously to measurements taken at −80 and 60 mV giving a value for EC₅₀ of 202±21 nM and n_H 1.1±0.11.</p

A. Channel pore radius along the z-axis in the receptor alone (black) and in the CrGluClα-emamectin (green) and CrGluClα-ivermectin (red) systems.

<p>A length, mainly intramembrane, of the pore is shown. The discontinuous lines show the pore segment lined by M2 α-helices. Values have been taken at the end of MD trajectories. B and C. Lateral views of the pore at time zero (C) and at the end (B) of the MD trajectory for CrGluClα in absence of drugs. Only four M2 helices are shown with the fifth removed for clarity. Residues P288, A292 and L299 are shown in licorice. Water occupancy is shown as licorice and surface.</p

Irreversible activation of CrGluClα by ivermectin.

<p>A. Current trace obtained at 60 mV during bath application of 50 µM glutamate and then to ivermectin at concentrations going from 1 to 3000 nM during the times shown in the boxes. The effect of picrotoxin addition at 100 µM is shown at the end of the trace. The trace in panel B shows that whilst ivermectin-induced current blockade by picrotoxin is reversible, the current evoked by the ivermectin is persistent even after prolonged washing. As for the glutamate response, ivermectin-induced outward current was greatly decreased upon decreasing [Cl⁻]_o to 7.6 mM. In C and D current voltage relations taken from voltage-ramps applied during experiment in B are shown. Those in D have been corrected by subtracting the current remaining in the presence of 100 µM PTX. E. Dose-response relationship of the Ivermectin-sensitive currents. Data are normalized (mean ± SEM) to the maximal effect of ivermectin and originate in five separate experiments. The line is a Hill equation fitted simultaneously to measurements taken at −80 and 60 mV giving a value for EC₅₀ of 181±10 nM, with corresponding n_H value of 2.1±0.26.</p