Biochemical studies of proteins involved in the vitamin A metabolism: from the structural investigation to the development of potential tools for cancer treatment and diagnosis

Vitamin A is a large group of essential micronutrients divided in preformed or provitamin A, based on their origin. These molecules exert roles in several physiological pathway, in particular for the sight, tissue development and cellular differentiation. The vitamin A is stocked in the liver, and then there is the necessity, is complexed with the retinol binding protein 4 (RBP4) and led to target tissues as the brain, the retina and the placenta. Here is internalized by STRA6, the only know specific vitamin A receptor. This is a transmembrane receptor, composed by two promoters, with a lid peptide at the N-terminal domain and two calmodulin bound at the C-terminal domain of each monomer. The only published structure is from the D. Rerio orthologs so is demanding to obtain a complete biochemical and structural characterization of a mammalian version of STRA6 to better understand the possible role of the calcium efflux in the process of vitamin A internalization. In this thesis we started to optimize the protocol for the expression and purification of the B. Taurus version of STRA6 and we were able to obtain a final model of the protein incorporated in nanodisk at 10 Å, using the single particle CRYO-EM technique. When it is incorporated in the cytosol, the retinol is converted to retinaldehyde by the retinol dehydrogenase and then to retinoic acid by the retinaldehyde dehydrogenases (ALDH), composed by ALDH1A1, 1A2 and 1A3. The three members of the ALDH1A subfamily belong to an enzymatic superfamily composed by 19 isoforms and involved in the oxidation of several aldehydes, in the reduction of oxidative stress and in the xenobiotic metabolism. Many scientific evidences highlighted the overexpression and overactivity of the ALDH1As in many solid tumour as the ALDH1A3 in glioblastoma. In this thesis we developed a series of selective and potent inhibitor and fluorescent tool to target ALDH1A3, characterized from the in silico, in vitro and in vivo perspective.