Proteases are enzymes that irreversibly cleave proteins by the catalysis of peptide-bond hydrolysis. With all proteins undergoing proteolysis at any point of their life cycle, proteases regulate virtually every biological process. Carboxypeptidases are proteolytic enzymes that catalyze the hydrolisis of peptidic bonds at the C-terminus of peptides and proteins. In this thesis, we studied carboxypeptidases from the M14 family (according to the MEROPS database classification), hereafter described as metallocarboxypeptidases. Metallocarboxypeptidases play key roles in controlling various biological processes, including blood coagulation/fibrinolysis, blood pressure regulation, pro-hormone and neuropeptide processing; and are also implicated in various pathological conditions such as cancer or neurodegenerative disorders. Despite the important functions performed by these enzymes, there is usually limited knowledge about their in vivo biological roles. The present thesis was aimed to gain insights into the understanding of the biological functions of different metallocarboxypeptidases. For this purpose, we applied different approaches that included kinetic studies, cell biological studies and structural characterization. In addition, we participated in the development of different proteomic tools for protease/carboxypeptidase substrate determination (degradomics). The present thesis consists of four independent research works that focus in the structural and functional characterization of different metallocarboxypeptidases. The first work presents the crystal structure of a short isoform of Drosophila melanogaster silver gene, which corresponds to the first repeat of a mammalian CPD. Silver gene is responsible for the silver mutation, characterized for adult flies that display cuticles that are pale and silvery in color, and pointed wings. This three-dimensional structure was solved in presence of an inhibitor (GEMSA) at 2.7 Å resolution and overall corresponds with the structure of other members of its subfamily of metallocarboxypeptidases. A unique structural element in the here presented structure is a surface hotspot targetable by peptidases, suggesting that this enzyme might be regulated (i.e., inactivated) by proteolysis. The second work comprises the biochemical and functional characterization of human carboxypeptidase A4 (CPA4), an enzyme that has been associated with prostate cancer aggressiveness. We found that this enzyme is secreted outside the cells and displays a neutral pH optimum that is compatible with a function in the extracellular environment. A peptidomic study identified several biologically relevant putative peptidic substrates of CPA4: neurotensin, granins, and opioid peptides such as Met- and Leu-enkephalin. These peptides are involved in the proliferation and differentiation of prostate cancer cells, potentially explaining the link between CPA4 and cancer aggressiveness. Altogether, CPA4 would function in the extracellular neuropeptide processing. The third work comprises the development of a novel proteomic approach to study the substrate preferences of carboxypeptidases. This technology can be described as a COFRADIC-based proteome-derived peptide library approach and allows for the enrichment of thousands of carboxypeptidase products from natural, proteome-derived peptide libraries. This approach served to delineate the previously little studied substrate specificity profile of mouse mast cell carboxypeptidase. In the fourth work we applied a proteomic tool (C-terminal COFRADIC) that allows searching for natural substrates of carboxypeptidases. Particularly, we searched for natural substrates of cytosolic carboxypeptidase 1 (CCP1) in a cellular system. This enzyme is considered to be a molecular link between neuronal degeneration and regeneration, although the molecular pathways in which CCP1 is implicated remain undefined. It has been proposed that CCP1 would posttranslationally modify tubulin and other proteins that present glutamate-stretches in their C-terminus. Here, we were able to identify seven new putative CCP1 protein substrates.
Insights into the specificity and function of M14 metallocarboxypeptidases from structural and degradomic studies
Tanco, S. (Author). 22 Oct 2012
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis