Nuevos hallazgos en la relación estructura-función del hemicanal de conexina 26 humana
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Fecha
2013
Autores
Profesor/a Guía
Facultad/escuela
Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Las conexinas constituyen una familia de proteínas que forman los canales de
unión de hendidura. Los canales de unión de hendidura conectan el citoplasma
de células adyacentes mediante el acoplamiento cabeza a cabeza de dos
hemicanales. Cada hemicanal esta formado por un anillo simétrico formado por
el arreglo hexamérico de monómeros de conexina. Mutaciones asociadas a
enfermedades han sido ubicadas dentro de la región codificante de los genes
de conexina produciendo anomalías en las diferentes etapas del ciclo de vida
de estas proteínas, incluyendo, síntesis, ensamblaje, transporte, función del
canal y degradación. Mutaciones en conexina 26 (Cx26), expresada en la
cóclea, han sido asociadas con sordera hereditaria. Se ha sugerido que estas
mutaciones pueden alterar las propiedades de los canales de unión de
hendidura que forman, incluyendo permeabilidad, selectividad y mecanismos de
apretura y cierre.
A pesar del amplio interés por el estudio de la estructura y función de conexinas
generado durante los últimos años, preguntas claves respecto al efecto que
mutaciones asociadas a enfermedades producen en la estructura y función de
estos canales permanecen sin respuesta. En este contexto, esta tesis tiene
como objetivo encontrar e inferir nuevas relaciones entre la estructura y función
presentes en el hemicanal de Cx26 mediante el estudio comparativo de Cx26-
WT y un grupo de mutantes que se sabe afecta la función de esta proteína.
Se realizaron simulaciones de dinámica molecular, seguidas de la aplicación de
una serie de análisis comparativos para caracterizar la estructura y dinámica de
Cx26-WT y los mutantes seleccionados. Los resultados obtenidos para Cx26-
WT proveen nueva información respecto a la dinámica de la estructura de esta
conexina revelando un nuevo grupo de interacciones intra e inter monoméricas
que no se pueden observar a partir de la estructura cristalográfica de Cx26. El
análisis de la dinámica y ordenamiento de las moléculas de agua dentro del
hemicanal produjo una detallada imagen de cómo estas moléculas difunden y
se ordenan de maneras diferentes en las distintas zonas del hemicanal. Estas
diferencias no dependerían sólo a la geometría del poro sino a las propiedades
de los aminoácidos expuestos. Este estudio permitió caracterizar una nueva
cavidad hidratada presente en cada uno de los seis monómeros de Cx26. El
estudio detallado de esta cavidad en términos de la dinámica del agua y de los
residuos que la forman nos permitió hipotetizar acerca de un nuevo participante
en los mecanismos de respuesta a voltaje de Cx26. Por último, la comparación
del hemicanal de Cx26-WT con las mutaciones en estudio revelo que existe un
delicado balance entre interacciones intra e ínter monoméricas que se supone
serian responsables por la respuesta coordinada del hemicanal frente a
cambios de voltaje. Este balance seria alterado por las mutaciones estudiadas
mediante perturbaciones locales y no-locales sobre la estructura de Cx26.
Connexins are a family of proteins that constitute gap junction channels (GJCs). Gap junction channels connect the cytoplasm of adjacent cells by the end-toend docking of two hemi-channels. A six-fold symmetric ring of connexin monomers forms each hemi-channel. Disease-causing mutations in the connexin genes have been located within the protein coding region , producing abnormalities at various steps in the protein life cycle, including synthesis, assembly, transport, channel function , and degradation. Mutations in connexin 26 (Cx26), found in the cochlea, have been associated with hereditary deafness. lt has been suggested that these mutations can alter several properties of gap junction channels including permeability, selectivity and gating. Despite the broad interest that the study of the structure and function of connexins has raised during the last years, key questions regarding the effect that disease-related mutations produce on the channel structure and function remain to be answered. In this context, this thesis aims to find and infer new structure/function relationships present in the Cx26 hemichannel by means of a comparative study between Cx26-WT and a group of mutations known to alter the function of th is protein. Molecular dynamics simulations were performed followed by a series of comparative analyzes to characterize the structure and dynamics of Cx26-WT and the selected mutations. The results obtained for Cx26-WT provide new information regarding the dynamics of connexin structure revealing a new set of intra- and inter-monomeric interactions not observable from the crystallographic structure alone. Analysis of the dynamics and ordering of water molecules provided a detailed picture of how they differential diffuse and order in different zones inside the hemichannel. These differences are predicted to be not only due to the geometry of the pore but also to the nature of the exposed residues. From this combined approach, a new hydrated cavity was predicted. A detailed study of this cavity in terms of water dynamics and of the residues that form it, allowed us to hypothesize a new participant in the voltage sensing/gating mechanisms of Cx26. Finally, the comparison of the Cx26-WT hemichannel with the mutations under study revealed that a delicate balance exists between intraand inter-monomeric interactions that are hypothesized to be responsible for the coordinated response to voltage changes. This balance is predicted to be altered by the mutations by means of local and non-local perturbations of the structure of Cx26.
Connexins are a family of proteins that constitute gap junction channels (GJCs). Gap junction channels connect the cytoplasm of adjacent cells by the end-toend docking of two hemi-channels. A six-fold symmetric ring of connexin monomers forms each hemi-channel. Disease-causing mutations in the connexin genes have been located within the protein coding region , producing abnormalities at various steps in the protein life cycle, including synthesis, assembly, transport, channel function , and degradation. Mutations in connexin 26 (Cx26), found in the cochlea, have been associated with hereditary deafness. lt has been suggested that these mutations can alter several properties of gap junction channels including permeability, selectivity and gating. Despite the broad interest that the study of the structure and function of connexins has raised during the last years, key questions regarding the effect that disease-related mutations produce on the channel structure and function remain to be answered. In this context, this thesis aims to find and infer new structure/function relationships present in the Cx26 hemichannel by means of a comparative study between Cx26-WT and a group of mutations known to alter the function of th is protein. Molecular dynamics simulations were performed followed by a series of comparative analyzes to characterize the structure and dynamics of Cx26-WT and the selected mutations. The results obtained for Cx26-WT provide new information regarding the dynamics of connexin structure revealing a new set of intra- and inter-monomeric interactions not observable from the crystallographic structure alone. Analysis of the dynamics and ordering of water molecules provided a detailed picture of how they differential diffuse and order in different zones inside the hemichannel. These differences are predicted to be not only due to the geometry of the pore but also to the nature of the exposed residues. From this combined approach, a new hydrated cavity was predicted. A detailed study of this cavity in terms of water dynamics and of the residues that form it, allowed us to hypothesize a new participant in the voltage sensing/gating mechanisms of Cx26. Finally, the comparison of the Cx26-WT hemichannel with the mutations under study revealed that a delicate balance exists between intraand inter-monomeric interactions that are hypothesized to be responsible for the coordinated response to voltage changes. This balance is predicted to be altered by the mutations by means of local and non-local perturbations of the structure of Cx26.
Notas
Tesis (Doctor en Biotecnología)
Proyecto financiado por Becas de Doctorado de CONICYT y los proyectos FONDECYT- 1130652, Basal PFB-16
Aporte del Centro lnterdisciplinario de Neurociencias de Valparaiso (ICM-ECONOMIA P09-022-F).
Proyecto financiado por Becas de Doctorado de CONICYT y los proyectos FONDECYT- 1130652, Basal PFB-16
Aporte del Centro lnterdisciplinario de Neurociencias de Valparaiso (ICM-ECONOMIA P09-022-F).
Palabras clave
Conexinas, Proteínas de Transporte de Membrana