Metallo-carboxypeptidases (MCPs) are exopeptidases, which contain a zinc atom in the active site, involved in the hydrolysis of peptide bonds at the C-terminus of peptides and proteins. Carboxypeptidase A1 (CPA1) was the first M14 peptidase identified. This enzyme was isolated more than 80 years ago from pancreatic extracts. Since then, the number of pancreatic and extra-pancreatic MCPs belonging to this family has been increasing. In mammals, these enzymes are related to a wide range of biological processes, such as protein digestion, regulation of fibrinolysis in the blood, maturation of neuropeptides, or specific processing of tubulin, among other functions._x000D_ In this thesis, we have carried out the biochemical, functional and structural characterization of two M14 MCPs with an acidic substrate specificity: cytosolic carboxypeptidase 6 (hCCP6) and human carboxypeptidase O (hCPO). These MCPs have been poorly studied in the past due to their recent discovery, and due to the difficulty of their production using conventional expression systems. For these reasons, there is a lack of knowledge regarding the structural determinants that define the substrate specificity for C-t acidic residues in the M14 family MCPs. The main goal of our work was the study of the structural characteristics that determine the substrate preference for C-terminal acid residues in hCCP6 and hCPO, as well as the study of other aspects related to its particular function and inhibition. _x000D_ In chapter I we present the biochemical and functional characterization of hCCP6, an intracellular exopeptidase which participate in the post-translational modification of the C-t of tubulin. In particular, this enzyme is involved in the removal of C-t Glu residues in tubulin from the microtubules of the cell, thus participating in the regulation of its stability and functionality. In addition to tubulin, hCCP6 can cleave other intracellular substrates such the myosin light-chain kinase (MLCK1) and telokin. The current work is an important contribution with the aim to expand the current knowledge about the function and structure of hCCP6, to stablish the basis for future lines of research which will help us to understand the mechanisms of regulation of the polyglutamylation in tubulin, and to identify new endogenous substrates for hCCP6._x000D_ In Chapter II we present for the first time the three-dimensional structure of hCPO in complex with the Nerita versicolor carboxypeptidase inhibitor (NvCI). hCPO is a novel digestive MCP of the M14 family which acts complementing the actions of pancreatic MCPs by hydrolyzing C-t acidic residues of dietary proteins and peptides. Thanks to this work, we have been able to increase the understanding of the structural features that determine the substrate preference for acidic residues in hCPO. Moreover, the analysis of the structure of hCPO in complex with NvCI revealed important molecular details about the mechanism of action of this inhibitor. Finally, in this chapter we applied a set of different biochemical approaches to characterize the substrate specificity of this enzyme, and to investigate the functional role of this protease in the intestinal tract in humans.
CARACTERIZACIÓN ESTRUCTURAL Y FUNCIONAL DE DOS METALO-CARBOXIPEPTIDASAS DE LA FAMILIA M14 CON ESPECIFICIDAD DE SUSTRATO TIPO ACÍDICO: CARBOXIPEPTIDASA CITOSÓLICA 6 Y CARBOXIPEPTIDASA O HUMANAS.
García Guerrero, M. D. C. (Author). 29 Sept 2017
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis