Taste receptors in the gut: a chemosensitive mechanism from fish to human

The ingestion of a meal evokes a series of digestive processes, which consist of the essential functions of the digestive system: food transport, secretory activity, absorption of nutrients and the expulsion of undigested residues do not absorbed. The gastrointestinal chemosensitivity is characterized by cellular elements of the endocrine gastrointestinal mucosa and nerve fibers, in particular of vagal nature. A wide range of mediators endocrine and/or paracrine can be released from various endocrine cells in response to nutrients in the diet. These hormones, in addition to their direct activity, act through specific receptors activating some of the most important functions in the control of energy intake and energy homeostasis in the body. For integration of this complex system of control of gastrointestinal chemosensitivity, recent evidence demonstrates the presence of taste receptors (TR) belonging to the family of G proteins coupled receptor expressed in the mucosa of the gastrointestinal tract of different mammals and human. This thesis is divided into several research projects that have been conceived in order to clarify the relationship between TR and nutrients. To define this relationship I have used various scientific approaches, which have gone on to evaluate changes in signal molecules of TR, in particular of the α-transducin in the fasting state and after refeeding with standard diet in the gastrointestinal tract of the pig, the mapping of the same molecule signal in the gastrointestinal tract of fish (Dicentrarchus labrax), the signaling pathway of bitter TR in the STC-1 endocrine cell line and finally the involvement of bitter TR in particular of T2R38 in patients with an excessive caloric intake. The results showed how there is a close correlation between nutrients, TR and hormonal release and how they are useful both in taste perception but also likely to be involved in chronic diseases such as obesity.

Computational Investigation of the Molecular Mechanisms Regulating Nucleic Acid Processing Metalloenzymes

Polymerases and nucleases are enzymes processing DNA and RNA. They are involved in crucial processes for cell life by performing the extension and the cleavage of nucleic acid chains during genome replication and maintenance. Additionally, both enzymes are often associated to several diseases, including cancer. In order to catalyze the reaction, most of them operate via the two-metal-ion mechanism. For this, despite showing relevant differences in structure, function and catalytic properties, they share common catalytic elements, which comprise the two catalytic ions and their first-shell acidic residues. Notably, recent studies of different metalloenzymes revealed the recurrent presence of additional elements surrounding the active site, thus suggesting an extended two-metal-ion-centered architecture. However, whether these elements have a catalytic function and what is their role is still unclear. In this work, using state-of-the-art computational techniques, second- and third-shell elements are showed to act in metallonucleases favoring the substrate positioning and leaving group release. In particular, in hExo1 a transient third metal ion is recruited and positioned near the two-metal-ion site by a structurally conserved acidic residue to assist the leaving group departure. Similarly, in hFEN1 second- and third-shell Arg/Lys residues operate the phosphate steering mechanism through (i) substrate recruitment, (ii) precise cleavage localization, and (iii) leaving group release. Importantly, structural comparisons of hExo1, hFEN1 and other metallonucleases suggest that similar catalytic mechanisms may be shared by other enzymes. Overall, the results obtained provide an extended vision on parallel strategies adopted by metalloenzymes, which employ divalent metal ion or positively charged residues to ensure efficient and specific catalysis. Furthermore, these outcomes may have implications for de novo enzyme engineering and/or drug design to modulate nucleic acid processing.