DEVELOPMENT OF MOLECULAR PROBES ACTING ON aSYN/TUBULIN AND TAU/TUBULIN INTERACTION

Microtubules (MTs) are ubiquitous structures playing many roles in all eukaryotic cell types from fungi to mammals mainly consisting of conserved α/β tubulin heterodimers. Neurons are an impressive example of cells in which the contribution of MTs is fundamental to achieve their sophisticated cell architecture and to sustain their functional complexity. A growing body of evidence indicates that microtubule defects are linked to neurodegenerative diseases, such as Parkinson’s Disease (PD). Several independent studies report a genome-wide crucial association with PD of single nucleotide polymorphisms in the MAPT locus, containing the gene coding for the Tau protein, a microtubule-binding protein that is predominantly expressed in neurons. The evidence of Tau pathology in PD is striking and contributes to neuronal dysfunction, impairment of axonal transport and cell death underlying PD. Recently another protein, α-Synuclein (αSyn), was put in the spotlight of various research groups since it was found aggregated in PD patients’ brains. αSyn, composed of 140 amino acids, is in fact deeply involved in PD, as i) it is the main component of Lewy bodies, which are well-known as the histopathological hallmark of PD and ii) the gene that encodes it, SCNA, is mutated in familial PD. Even though, lots of new information regarding both αSyn/tubulin and Tau/tubulin have been obtained, their relevance over neuronal dysfunction remains unclear. For these reasons, their interaction is set at the center of my research project. The first main aim of this PhD is the development of peptide based molecular probes able to modulate the Tau and α-Synuclein interaction with Tubulin. A library of peptides, both native and mutated sequences of αSyn, were synthesized and were used as molecular ‘tools’ for the conduction of in vitro experiments. The secondary structure of the peptides was characterized so to have a better idea of their possible activities during the in vitro experiments. Microscale Thermophoresis (MST) and the tubulin polymerization assay were employed using these peptides, in conditions which were approximate to the physiological ones. From preliminary data, two mutants, seemed to affect the polymerization of tubulin in vitro and in a specific cell line. Moreover, we also aimed to stabilize a secondary conformation in the previously synthesized peptides, as a result a library of conformationally stable peptides was synthesized in order to obtain α-helically stable peptides. Their secondary structure was evaluated via CD and AT-IR experiments. In conclusion we could highlight that αSyn sequences tend to be difficult to be characterized conformationally since it is an intrinsically disordered protein, and in our case stapling strategies were completely successful. We, therefore, explored a less common stapling approach, by also designing a novel linker to afford the stapling. Unfortunately, our linker was not stable in standard SPPS conditions. Finally, Two Tau-deriving peptides were synthesized and used as molecular tools to detect their binding site on tubulin. The two peptides were then examined by our co-workers in PSI, Switzerland in soaking and co-crystallization experiments. The results have not been reported.