Analisi funzionale e comparata delle Longine VAMP7 e Ykt6

Longins are proteins characterized by a conserved N-terminal extension of 120-140 amino acids, named longin domain (LD). They belong to SNARE that are crucial proteins for subcellular trafficking control, and they are divided into three main families: VAMP7, Sec22 and Ykt6. The research activity performed during this PhD is linked to a project (funded by CA.RI.PA.RO. Fondation) that aims at studying functional modulation and interactions of VAMP7 and Ykt6 LDs. Therefore the distribution of LD and longin proteins in eukaryots has been studied, by means of a bioinformatic screening on proteomes and genomes. This allowed for highlighting differencial roles of Ykt6 and VAMP7 longin families. In addition, Phytolongins have been discovered: they are representing the first example of non-SNARE longins family, showing a variable central region that replaced the SNARE motif. By means of phylogenetic trees, longins evolution between and inside the families has been analyzed and has shown interesting data, particularly as regards the relationships between Ykt6, VAMP7 and Phytolongins. We performed tridimensional models of LDs in order to infer functional insights from the conservation or divergence of surface specific patches, and also to unravel key amino acids for the interactions with other vesicular proteins. A hydrophobic surface of ? helix 1 seems to mediate the autoinhibitory mechanism in yeast Ykt6 LD, but our data on conserved hydrophobic or polar surfaces in ?1 indicate that this mechanism is family-specific and not always dependent on hydrophobicity. We have performed site-directed mutagenesis of particular residues that are conserved in Ykt6 and VAMP7, in order to obtain indications about their value in future two-hybrid assays.