Insights into the functional role of Cytosolic Carboxypeptidases 1 and 6: from interactomics to cell biology

Abstract

Proteases are presumed to regulate nearly every biological function, as they represent a considerable percentage of the genome; nevertheless, little is known about the in vivo biological roles for many proteases. This is especially true for cytosolic carboxypeptidases, which have suggested functions and implications but are often unknown in terms of their true biological activities and implication in complex cellular processes. The present thesis work reports on the general functional characterisation of CCP1 and CCP6, two deglutamylating enzymes members of the M14D subfamily of metallocarboxypeptidases which have been related with relevant cellular processes. Thus, our research has specifically focused on determining the functional role of CCPs under a biomedical point of view, trying to arise new insights into their implication in health and disease. Cell biology studies, CRISPR/Cas9 knock out generation, high-resolution imaging, and enzymatic activity assessing were all carried out with this goal in mind. Furthermore, a proteomic technique was developed and applied for the study of the carboxypeptidase interactomic landscape. This thesis work is composed by three interconnected chapters were the cytosolic carboxypeptidases 1 and 6 are functionally characterized. In the first chapter we aimed for the general characterisation of human CCP1, first focusing on how cells are affected by its loss of activity. CCP1 is the largest member of the CCP family, being the one with the largest number of predicted domains. The elucidation organelle-specific functional roles have long been a factor that has contributed significatively to the study of CCPs. In this chapter have determined general considerations of CCP1 regarding its functional role using the knocking out of the gene in specific cell lines, focusing on its implications in general deglutamylation processes, describing its subcellular localisation, and its specific nuclear functional role. The second chapter was aimed for the identification of the human CCP1 and CCP6 interactomic landscapes under a biomedical point of view. Unveiling the protein-protein interactions can reveal how they are involved in different processes, providing hints or aid in determining their implication in disease. In this chapter the interactomic landscapes of CCP1 and CCP6 were generated from in vitro human cells, identifying new potential interactors and substrates which have allowed us to gain insight into their functional role. The proteomic work performed in this chapter was performed at the Bioscope Group (Universidade Nova de Lisboa, Portugal) headed by Professor Jose Luis Capelo under Dr. Hugo Santos surveillance. In last chapter we aimed for the characterisation of the conserved N-domain of cytosolic carboxypeptidases, focusing on CCP6 as the minimal CCP expression unit. This characteristic domain is conserved along the evolutionary line, being present in all identified CCPs, but not in other metallocarboxypeptidases. Although an extensive work has been conducted to describe the conserved catalytic domain, little is known about the N-domain and its implication in CCP activity. At present, the only information regarding the N-domain has been obtained from Caenorhabditis elegans. In this chapter, we evaluated the effects of this point mutation on the enzymatic activity of human CCP6 while assessing their implications in protein stability, subcellular localisation, and substrate specificity. The experimental work of this chapter was performed in collaboration with Paula Alfonso.

Date of Award	10 Feb 2022
Original language	English
Supervisor	Julia Lorenzo Rivera (Director)