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Interneurons navigate along multiple tangential paths to settle into appropriate cortical layers. They undergo a saltatory migration paced by intermittent nuclear jumps whose regulation relies on interplay between extracellular cues and genetic-encoded information. It remains unclear how cycles of pause and movement are coordinated at the molecular level. Post-translational modification of proteins contributes to cell migration regulation. The present study uncovers that carboxypeptidase 1, which promotes post-translational protein deglutamylation, controls the pausing of migrating cortical interneurons. Moreover, we demonstrate that pausing during migration attenuates movement simultaneity at the population level, thereby controlling the flow of interneurons invading the cortex. Interfering with the regulation of pausing not only affects the size of the cortical interneuron cohort but also impairs the generation of age-matched projection neurons of the upper layers.

Metallocarboxypeptidases (MCPs) are zinc-dependent exopeptidases that catalyze the hydrolysis of C-terminal amide bonds in proteins and peptides. MCPs are involved in a wide range of physiological processes and have recently emerged as relevant drug targets in biomedicine (Arolas et. al., 2007). In higher plants, small proteinaceous protease inhibitors are wound-induced molecules produced as a part of its defense system against insect attack (Graham et. al., 1981; Villanueva et. al. 1998). Among such inhibitors, only two are specific for MCPs, i.e. the potato carboxypeptidase inhibitor (PCI) and its close homologue found in tomato plants. In humans, MCP action is exquisitely regulated and dysregulation of their function might lead to disease or even to cell death (Arolas et. al., 2007). In this context, the discovery and characterization of new MCPs inhibitors constitute a valuable approach for the development of new therapeutic strategies. Over the last few decades, the presence of MCPs inhibitors in Solanaceae has been extensively reported, revealing potato (Solanum tuberosum) as one of the most important sources of MCPs inhibitors (Hass et. al.,1979; Lufrano et. al., 2015). In this context, potatos are native from the Andean region of South America, where thousands of different potato varieties coexist, constituting a natural reservoir for the discovery of novel MCP inhibitors (Figure 1). In this protocol, we describe an optimized, simple and accessible microplate method for the measure of the specific and dose-response carboxypeptidase A inhibitory activities present in Andean potatoes tubers., Centro de Investigación de Proteínas Vegetales

Aquesta tesis s’emmarca en el camp de les carboxipeptidases citosòliques de mamífers, centrant-­‐se particularment en els membres 2 i 3 d’aquesta subfamília de metalocarboxipeptidases. Aquest treball constitueix el primer estudi sobre la funció d’aquests enzims a través d’aproximacions in silico, in vitro i in vivo. Aquesta tesis consta de les parts descrites a continuació. El primer capítol introdueix el camp de les matalocarboxipeptidases, els microtúbuls, les modificacions post-­‐traduccionals de la tubulina i estableix els objectius de la tesi. El segon capítol és una compilació dels procediments experimentals i materials utilitzats en el treball experimental. El tercer capítol presenta la caracterització funcional de la carboxipeptidasa citosòlica 3 humana. Els resultats d’aquest estudi han estat la base pel quart capítol, on es descriu la funció de la CCP2. Ambdues CCP2 i CCP3 isoformes de ratolí s’estudien mitjançant enginyeria de proteïnes i utilitzant els models de ratolí knockout de CCP2 i CCP3. Els capítols III i IV es subdivideixen en un resum del capítol, els resultats que descriuen el detall dels descobriments i una discussió. A continuació es presenta una discussió general sobre CCP2 i CCP3, el seu rol en la cèl·∙lula i en el context de la subfamília de carboxipeptidases que, en mamífers, consta de sis gens codificants per enzims amb activitat deglutamilasa. La tesis finalitza amb un resum de les conclusions. El capítol IV juntament amb els experiments de modelat estructural del capítol III són la base de la següent publicació científica: Tort O, Tanco S, Rocha C, Bièche I, Seixas C, Bosc C, Andrieux A, Moutin MJ, Avilés FX, Lorenzo J, Janke C. The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids. MBoC. doi:10.1091/mbc.E14-­‐06-­‐1072, This thesis is situated in the field of the mammalian cytosolic carboxypeptidases, particularly focusing in members 2 and 3 from this subfamily of metallocarboxypeptidases. This work gives insight for the first time into the role of those enzymes by in silico, in vitro and in vivo approaches. The thesis is divided in parts as described below. The first chapter introduces the fields of metallocarboxypeptidases, microtubules, tubulin posttranslational modifications and settle the objectives of the thesis. The second chapter is a compilation of the experimental procedures and materials used for the experimental work. The third chapter presents the functional characterization of human cytosolic carboxypeptidase 3. The results from this study were the basis for chapter four, where the function of CCP2 is described and both CCP2 and CCP3 mouse isoforms are studied by protein engineering and using CCP2 and CCP3 knock-­‐out mouse models. Chapters III and IV are subdivided into a summary of the chapter, the results describing the findings in detail and a discussion. The following chapter sets a general discussion on CCP2 and CCP3, their role in the cell and in the context of a family, in mammals, of six genes coding deglutamylating enzymes. A final section includes a summary of the conclusions. Chapter IV together with the modeling experiments form Chapter III is the basis of the following scientific paper: Tort O, Tanco S, Rocha C, Bièche I, Seixas C, Bosc C, Andrieux A, Moutin MJ, Avilés FX, Lorenzo J, Janke C. The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids. MBoC. doi:10.1091/mbc.E14-­‐06-­‐1072