ricerca sulle strutture di proteine di ancoraggio della active zone presinaptica

The communication between excitable cells occurs to specialized sites called synapses. Synapses use two different kind of signals: chemical or electric. When an action potential reaches the presynaptic cell, the consequent depolarization causes the release of neurotransmitter through specific synaptic vesicles, resulting in the activation of postsynaptic receptors. The exocytosis of vesicles is associated with endocytosis and this cycle ensures a rapid neurotransmission also in case of sustained nervous activity. The active zone is a highly specialized region of the presynaptic terminal where a pool of different protein factors controls exocytosis. These proteins form a protein scaffold, which is mainly constituted by RIM, Munc13, RIM-BP, α-liprin and ELKS proteins. These proteins are conserved in Homo sapiens, Drosophila melanogaster and Caenorhabditis elegans and this fact allows to use for instance the neuromuscular junction of Drosophila as a model system. Biochemical and biophysical techniques such as cloning, protein purification, crystallization and protein X-ray crystallography allows to obtain information on components structure leading to a detailed understanding of their function in exocytosis- endocytosis coupling. The thesis reaches the goal to obtain the structural information of proteins belonging to RIM-BP and ELKS/CAST family. These results will create a substrate for a solid basement to study and understand deeply the physiological role of these proteins and the possibility of an implication of these factors in the manifestation of pathological events. As a matter of fact, it is observed that in some pathologies of the nervous system the first cause of these diseases is the neurodegeneration. Neurodegeneration can be caused by defects in the mechanisms of release or re-uptake of neurotransmitters. Therefore depth studies about structure- activity relation of factors regulating these processes can represent an excellent base to understand physiological mechanisms of the nervous system. The developed study is about the protein Bruchpilot of D.melanogaster and about the SH3I, SH3II and SH3III domains of RIM-BP protein. The protein purification of different expression constructs and subsequent crystallization experiments have been performed. In particular, it was possible to obtain crystals of SH3II domain with different peptides, derived of other active zone proteins, under different experimental conditions. This was the basis for a more detailed research of the two other SH3 domains of RIM-BP. In conclusion, the work represents a part of the complex research process necessary to understand the active zone. The results represent a small part of the puzzle showing the protein-protein interaction of different active zone proteins. The project is certainly a good starting point for further structural and pathophysiological investigations of active zone proteins.