Obtention, biochemical and structural characterization of a hALDH1A3 mutant and OCX32, and field improvements.

Student thesis: Doctoral thesis


Proteins are one of the most abundant biological groups of macromolecules and are involved in almost all the processes taking place in organisms. They can be classified into many categories, but one of the most important criteria is based on the function that they display in the cell. Enzymes are proteins that increase the reaction rate by lowering the activation energy of a reaction. The presence of enzymes allows biochemical reactions that could not be feasible under physiological conditions and/or in the time required. The projects developed in this thesis involves two types of enzymes: oxidoreductases and proteases. Oxidoreductases are proteins that catalyse the transfer of electrons from the reductant molecule to the oxidant molecule. This group of enzymes usually utilizes NADP+ or NAD+ as cofactors. Oxidoreductases is a very large family, which englobes different types of enzymes that can be classified according to their functional mechanism used to transport electrons. Aldehyde dehydrogenases (ALDHs) are one of the most important types of oxidoreductases, responsible for the elimination of toxic aldehydes by the cell. In recent years, its medical significance has been demonstrated by the determination that various human pathologies are caused by mutations in ALDH genes that cause malfunction, absence, inactivation, or protein deficiency. The pathologies related are various: many cancers, neurological abnormalities, and metabolic diseases, among others. ALDH1A3 participates in the synthesis of retinoic acid from retinaldehyde, and it has been established as an interesting target for the treatment of different types of cancers. With the aim to obtain useful information about this protein, a mutant variant involving the catalytic residue of ALDH1A3 has been designed. The objective of this mutation was to realize a deep study not only at the kinetical level but also at the structural level. Our objective was to try to obtain structural information on the ALDH1A3 variant alone or by interacting with the substrate, cofactor, and some inhibitors. This information could become a useful tool for the design of new drugs to mitigate the effects of ALDH1A3 in the pathologies in which it is involved. Proteases are the enzymes responsible for peptide bond cleavage and protein degradation. They are involved in different biological processes playing a crucial role in organism survival. Proteases encompass a large family of proteins with the ability to hydrolase peptide bounds at different position. This thesis focuses on metallocarboxypeptidases (MCPs), a subfamily of proteases responsible for the hydrolysis of the peptide bond of the residue located at the C-terminus of the protein. In recent years, MCPs have become more relevant due to their involvement in various pathologies such as cancer and epilepsy, among others. Because of their importance at both biotechnological and biomedical levels, the need for developing methodologies that allows us their recombinant obtention for their study has increased. For this reason, in this thesis we suggest a new improved and economical protocol, for the recombinant production of MCPs that have affinity for heparin. Other important molecules for the study of MCPs are the inhibitors. MCPs inhibitors are usually small proteins that are responsible for blocking the activity of the enzyme, and can be used as regulators or even as drugs. High molecular weight inhibitors such as Latexin have also been described, and in this thesis, we describe an inhibitor homologous to latexine, extracted from eggshell. This inhibitor is called Ovocalyxin-32 and shows a strong interaction with some members of the MCPs family.
Date of Award4 Feb 2021
Original languageEnglish
SupervisorFrancesc Xavier Aviles Puigvert (Director), Julia Lorenzo Rivera (Director) & Julia Lorenzo Rivera (Tutor)

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