Metallocarboxypeptidases (MCPs) are zinc-dependent enzymes that cleave single amino acids from the C termini of proteins and peptides. The first MCP to be identified was carboxypeptidase A1 (CPA1), a pancreatic enzyme that removes C-terminal hydrophobic residues. In the ensuing decades since the discovery of CPA1, dozens of additional MCPs have been found in different extra-pancreatic tissues and fluids, comprising a wide range of physiological roles ranging from digestion of food to the production of neuropeptides and peptide hormones and the selective processing of tubulin. The present thesis has the aim to gain insights into the knowledge of the structure and biological functions of two regulatory MCPs. For this purpose, we applied a wide range of biochemical approaches to elucidate biological activities of human carboxypeptidases D and Z. Furthermore, we decided to study for the first time the structure and roles of the transthyretin-like (TTL) domains found in all members of this subfamily of proteases, taking as example the first TTL domain belonging to the first catalytic domain of human carboxypeptidase D (termed here as h-TTL). The first chapter describes the amyloid formation under physiological conditions by h-TTL and unravels that the monomeric transthyretin fold has an inherent propensity to aggregate due to the presence of preformed amyloidogenic structural elements. The aggregation mechanism described in this work for a natively monomeric transthyretin-like protein, is being found also in a number of initially soluble globular proteins associated with protein deposition diseases and might be in fact quite generic for folds displaying preformed amyloidogenic elements in their structures, essentially β-sheets. The second chapter presents the crystal structure solved at ultra-high resolution of the h-TTL described in the first chapter. The information derived in the present study might facilitate the understanding of the biological roles of the TTL domains found in M14B subfamily members and would be an interesting tool to analyze in detail the structural properties and the folding mechanisms of these domains. The third chapter comprises the characterization of the substrate specificity of human carboxypeptidase D by using a combination of quantitative peptidomic approaches. This unique enzyme with multiple catalytic sites might be implicated in the processing of neuropeptides and growth factors. Thereby, the study of its mechanism of action is of significant importance for biomedicine. The fourth chapter describes de development of a simple and inexpensive method to improve protein production of heparin-affinity carboxypeptidases using mammalian cells, taking as example the case of carboxypeptidase Z. The purified protein is enzymatically active and can be used for high-throughput functional and structural studies. The fifth chapter applies several quantitative peptidomic approaches to characterize the substrate specificity of the human carboxypeptidase Z. Furthermore, this work provides the modelling of its catalytic domain, as well as of their frizzled-like domain, in order to analyze their role in Wnt signaling.