Abstract
Abstract
Protein-protein interactions and mutiprotein complexes formation are very important in a lot of physiological processes, such as signal transduction, cellular adhesion, development and communication between cells. Specific proteins have a crucial role in these interactions and recruit receptors, enzymes, and adapters in particular cellular compartments.
This thesis project intend to characterize VSP protein (Ventral Eye Staining Protein). The gene was originally identified in Danio rerio and it is expressed specifically in the neural ventral region, in the optic stalk during early stage of development of eye and in dorsal ganglia; then the expression is extended to others regions of nervous system. A putative interactor of VSP is the protein ARMS (Ankiryn Rich-repeat Membrane
Spanning), previously isolated in our laboratory by means of phage display
experiments. Also this protein is expressed specifically in dorsal ganglia and in a lot of regions of nervous system. Moreover it is expressed in neuroendocrine cells, such as PC12 cells, where it concentrates at the tip of neurites upon differentiation with NGF (Bracale et al., 2006).
During my thesis project I analyzed the expression of recombinant VSP and ARMS tagged fusion proteins using as model systems to study the interactions PC12 neuroendocrine rat cells and Hek293 human embryonic kidney cells.
Thanks to transfection, electrophoresis analysis and Western blotting I verified the correct production of the recombinant proteins. Subsequently I performed co-immunoprecipitation and co-localization assays. These experiments permitted to demonstrate a direct interaction between the two proteins. In detail, VSP protein binds
Abstract
ARMS C-terminal region, thanks to a PDZ structural domain. This PDZ domain recognizes specifically the last four residues in the target protein.
