PID 6088 Estudio de la influencia de las hormonas tiroideas en el control de los sistemas de adhesión cadherinas-cateninas durante el desarrollo de vertebrados

El desarrollo de organismos pluricelulares depende en gran medida del establecimiento y mantenimiento de contactos adhesivos fuertes, pero a la vez dinámicos para posibilitar el remodelamiento tisular frente a señales específicas, tanto durante el desarrollo como en el estado adulto. Interesados en evaluar mecanismos de control hormonal de estos contactos, estudiamos si las hormonas tiroideas eran capaces de controlar el desarrollo animal a través de la regulación de la expresión de moléculas de adhesión celular (CAMs), las cuales influencian la adhesión celular y la morfología celular y tisular. Para ello, en una primera etapa, se implementaron bioensayos de bloqueo e inducción de la metamorfosis de Rhinella arenarum y se analizó por inmunohistoquímica cuantitativa el patrón de expresión de las CAMs cadherina E, b- y a-catenina en el intestino anterior o estómago larval de esta especie. En una segunda etapa se utilizaron técnicas de inmunofluorescencia y microscopia de desconvolución digital tridimensional cuantitativa para analizar la influencia de los niveles de fosforilación de las proteínas de los complejos de unión cadherina-catenina in vivo. En una tercera etapa, se analizaron los niveles de expresión de los ARNm de cadherina E y β-catenina y de sus proteínas para correlacionar los estudios morfométricos realizados con estudios moleculares, empleando retrotranscripción y amplificación de ADNcopia (ADNc) por reacción en cadena de la polimerasa (RT-PCR) y western blotting, para, respectivamente. Para ello, se realizaron análisis bioinformáticos de las secuencias y estructuras de las moléculas bajo estudio. Los resultados obtenidos permiten postular por primera vez en forma cuantitativa, un control positivo espacial y temporal de cadherina E, b- y a-catenina por la hormona T3 durante el desarrollo metamórfico del estómago larval de Rhinella arenarum. La alteración de los niveles de fosforilación de las proteínas de los complejos de unión cadherina E-β-catenina, produce una drástica pérdida de estas moléculas en los contactos célula-célula y el incremento citoplasmático y nuclear de β-catenina en las células epidérmicas, sugiriendo la activación de la ruta de señalización nuclear mediada por β-catenina. Sorprendentemente, no se detectan cambios en la forma celular o en la arquitectura de la piel, sugiriendo que la cadherina E epidérmica estaría involucrada en la señalización celular más que en el mantenimiento de los contactos intercelulares durante el mantenimiento de la arquitectura epitelial in vivo. Finalmente, se aislaron, secuenciaron y caracterizaron filogenéticamente secuencias de nucleótidos de cadherina E y de b-catenina de Rhinella arenarum, que resultaron estar altamente conservadas rentre 8 especies de vertebrados.

Correction: Pockets as structural descriptors of EGFR kinase conformations.

[This corrects the article DOI: 10.1371/journal.pone.0189147.]

Pockets as structural descriptors of EGFR kinase conformations

<div><p>Epidermal Growth Factor Receptor (EGFR), a tyrosine kinase receptor, is one of the main tumor markers in different types of cancers. The kinase native state is mainly composed of two populations of conformers: <i>active</i> and <i>inactive</i>. Several sequence variations in EGFR kinase region promote the differential enrichment of conformers with higher activity. Some structural characteristics have been proposed to differentiate kinase conformations, but these considerations could lead to ambiguous classifications. We present a structural characterisation of EGFR kinase conformers, focused on active site pocket comparisons, and the mapping of known pathological sequence variations. A structural based clustering of this pocket accurately discriminates active from inactive, well-characterised conformations. Furthermore, this main pocket contains, or is in close contact with, ≈65% of cancer-related variation positions. Although the relevance of protein dynamics to explain biological function has been extensively recognised, the usage of the ensemble of conformations in dynamic equilibrium to represent the functional state of proteins and the importance of pockets, cavities and/or tunnels was often neglected in previous studies. These functional structures and the equilibrium between them could be structurally analysed in wild type as well as in sequence variants. Our results indicate that biologically important pockets, as well as their shape and dynamics, are central to understanding protein function in wild-type, polymorphic or disease-related variations.</p></div

Positions belonging to and in close contact with the main pocket.

<p>Exon: shows the limits of each exon. Region: informs the conserved regions involved in catalytic control. Abbreviations: CS: catalytic spine; ADi, SDi: assymetric/symmetric dimer interface; RS: regulatory spine; GK: gatekeeper. Position, list number and residue for each position in the main pocket and close contact. P/C: depicts whether a position is considered as part of the main pocket (P) or in close contact (C). Position and P/C rows are coloured, according to the considerations in P/C, for those sites with defined coordinates in all the structures used in the present work. Those in white correspond to positions that are found as missing in at least one of these structures.</p

Variant site mapping over kinase domain conformers.

<p>The main pocket is represented as a surface. From left to right and from top to bottom: active monomer with ATP analog–peptide conjugate (PDB id 2GS6), inactive monomer with pyrimido[4,5-b]azepine-derived inhibitor (PDB id 3W32), active chain in asymmetric dimer with WZ4002 irreversible inhibitor (PDB id 3IKA_A), and inactive chain in asymmetric dimer (PDB id 3IKA_B). The most frequently affected sites (those in red, violet and dark blue) map in the catalytic pocket and are found in particular regions: the activation segment (with the classical L858R), the Gly-rich loop, and the region that connects both lobes of the kinase. Interestingly, most of the residues that serve as the docking site for the substrate peptide are not affected by variations (circled with a dashed black line). The total number of missense substitutions observed in COSMIC for each position is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189147#pone.0189147.s004" target="_blank">S2 Table</a>, column <i>Gen_var_count_ocurrence</i>. Missing regions are connected with straight dashed lines.</p

Main pocket comparison.

<p>Main pocket comparison between active (left, PDB 2GS6 with ATP analog–peptide conjugate) and inactive (right, PDB 3W32 with pyrimido [4,5-b]azepine-derived inhibitor) monomers, following the same colouring scheme as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189147#pone.0189147.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189147#pone.0189147.g002" target="_blank">2</a> (active: A colours, inactive: I colours). The 53 main pocket positions are represented as surfaces in both monomers. For the symmetric and asymmetric interface residues, only those included or in close contact to the main pocket are depicted in black and bright green, respectively.</p