Tubulin code eraser CCP5 binds branch glutamates by substrate deformation.

Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes1. Glutamylation-the addition of branched (isopeptide-linked) glutamate chains-is the most evolutionarily widespread tubulin modification2. It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family1. Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function3-9, and mutations in CCPs lead to human disease10-13. Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant ß-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax.

Physiological role of extracellular nucleotides at the central nervous system: signalling through P2X and P2Y receptors

En los últimos años los receptores de nucleótidos, receptores ionotrópicosP2X1-7 y metabotrópicos P2Y1, 2, 4, 6, 11, 12, 13, 14, han adquirido una importancia excepcionaldebido a su localización estratégica en órganos y tejidos, a su gran variedadjunto con la complejidad de vías de señalización a las que están asociados y a lasprimeras evidencias de importantes alteraciones debidas a su mal funcionamiento.Nuestro grupo ha sido pionero en la caracterización estos receptores en el sistemanervioso, donde definimos su localización y su funcionalidad. La abundante presencia,a nivel presináptico, de las subunidades P2X3 y P2X7 debe ser resaltada,donde gracias a la entrada de calcio inducen la exocitosis de varios neurotransmisores,como glutamato, GABA, catecolaminas y acetilcolina entre otros, como hasido descrito por nuestro grupo en trabajos previos. Además, estos receptores inducenuna profunda remodelación del citoesqueleto de las terminales nerviosas yde los mecanismos exocitóticos a través de la CaMKII y pueden interactuar conotros receptores ionotrópicos y metabotrópicos co-existentes en sus cercanías. Lamayoría de los receptores P2Y también están presentes en las células nerviosas,activando vías de señalización a través de una gran variedad de cascadas intracelulares.Recientemente hemos demostrado que los receptores metabotrópicos P2Ypertenecientes a la sub-familia de receptores activados por ADP, especialmente elP2Y13, están conectados con la señalización hacia GSK3 y β-catenina, lo que abrenuevas vías para la comprensión de la función de los nucleótidos en la supervivenciay el mantenimiento de las células nerviosas. Además, tanto los receptores P2Xcomo los P2Y juegan un papel en los estadíos iniciales del desarrollo y en lamaduración neuronal donde su función aún ha de ser plenamente comprendida.Los receptores de nucleótidos son también muy abundantes en las células gliales, y nuestro grupo ha demostrado que la mayoría de los receptores P2Y están presentesy son plenamente funcionales en astrocitos en cultivo, donde, dependiendo delsubtipo de receptor, activan una gran variedad de cascadas de señalización

In the last few years nucleotide receptors, the ionotropic P2X1-7 subunits andthe metabotropic P2Y1, 2, 4, 6, 11, 12, 13, 14, have acquired an excepcional importance dueto their strategic location in organs and tissues, their great variety along with thecomplexity of the associated signalling pathways and the first evidence of theserious alterations entailed in their dysfunctions. Our group has been pioneer inthe characterization of these receptors in the nervous system, where we definedtheir location and functionality. The abundant presence, at a presynaptic level, ofP2X3 and P2X7 should be emphasized, where by means of calcium intake theyinduce neurotransmitter exocytosis, such as glutamate, GABA, catecholamines andacetylcholine among others, as described in previous works by our group. In addition, they induce an extensive remodeling of the terminal’s cytoskeleton and exocytoticmechanisms through CaMKII and they can interact widely with other ionotropicand metabotropic receptors co-existing in nearby areas. Neural cells alsoexhibit the presence of most P2Y receptors signalling through a large variety ofintracellular cascades. Recently we have demostrated that P2Y metabotropic receptorsof the sub-family activated by ADP, especially P2Y13, are connected withthe signalling towards GSK3 and Beta-catenin, opening new ways of understading thenucleotide function in survival and maintenance of neural cells. In addition bothP2X and P2Y receptors play a role in early developmental stages and neural maturationwhere their function has to be fully understanded. Nucleotide receptorsare also very abundant in glial cells, and our group has shown that most P2Yreceptors are present and fully functional in cultured astrocytes, where, dependingon the subtype receptor they activate a large variety of signalling cascades