Aromatic amino acids decarboxylase and histidine decarboxylase: deep functional investigations give insights into pathophysiological mechanisms with possible therapeutic implications

Aromatic amino acids decarboxylase and histidine decarboxylase (AADC and HDC) are two homologous enzymes responsible for the synthesis of dopamine/serotonin and histamine, respectively, and other minor signalling aromatic amines. All these molecules are main protagonists or regulators of several physiological pathways, which are fundamental both in central nervous system and in peripheral tissues. Alterations of their homeostasis, indeed, as well as of AADC and HDC functioning or expression, cause and/or participate in the development and progression of several often severe and disabling pathological conditions, such as AADC Deficiency and cholangiocarcinoma. Consequently, AADC and HDC characterization might be useful in the pathophysiological understanding of several diseases and in improving/developing new therapeutic strategies. However, the knowledge of the biochemical features of these two crucial enzymes is still rather limited. Thus, the aim of this thesis is to biochemically characterise human HDC, mostly unknown, and to individuate some possible regulative mechanisms for both HDC and AADC. In addition, a neuronal AADC Deficiency cell model, derived from patient induced pluripotent stem cells (iPSCs), was used to evaluate endogenous AADC features, as well as to research further alterations in dopaminergic pathway. Investigations on human recombinant HDC allowed to discover that, surprisingly, its conformation and catalytic efficiency are influenced by redox state: increasing oxidizing conditions, indeed, favour a more stable and active form of the dimeric enzyme, due to the presence of an intermolecular reversible disulphide bridge involving residue Cys180 of both subunits. Then, in solution analyses of a possible phosphorylation of AADC identified Ser193 as protein kinase A target site, and allowed the detection of an effect on enzyme kinetic parameters, in particular an increased affinity for its substrates. Finally, endogenous AADC levels analyses in dopaminergic neurons derived from AADC Deficiency patients suggested a possible positive feedback mechanism that could tend to increase AADC expression, and the same cell model showed alterations in other cell types besides neurons, in particular glia cells, suggesting that variations in neurons-glia cells Abstract 5 interplay could participate in the pathophysiology mechanisms of AADC Deficiency. Altogether, data and information obtained from the performed experiments have increased AADC and HDC knowledge, as well as paved the way for new hypothesis regarding possible efforts in the development of new disease treatments.