Biochemical characterization of proteins involved in visual function and their association with ocular diseases

Eyes are responsible for detection of light in the retina and the conversion of a light stimulus into an electrical signal that is ultimately conveyed to the visual cortex of the brain. The first step of vision occurs during the phototransduction cascade, which takes place in specialized cells in the retina, namely rods and cones. In the complex mechanism of phototransduction, a cascade of biochemical events is regulated by variations of the concentration of two second messengers, cGMP and calcium. Their intracellular concentrations are strictly interconnected and finely regulated by guanylate cyclase-activating proteins (GCAPs). GCAPs are neuronal calcium sensor proteins that detect subtle variations of calcium concentration and thereby regulate the enzymatic activity of membrane-bound Guanylate Cyclases (GCs), that synthetize cGMP, through a Ca2+- dependent feedback mechanism. Three isoforms of GCAPs have been described in the human retina (GCAP1, 2 and 3) with a different distribution in the photoreceptor cells; however, the physiological meaning of this apparent redundancy is not well understood. The best-known human isoform is the GCAP1, whereas no thorough biochemical investigation of the molecular proprieties of human GCAP2 and GCAP3 has been performed so far. My PhD research is focused on the biochemical and biophysical characterization of human GCAP2 and GCAP3 with the goal to understand their structural properties and their role in the phototransduction cascade. Beside focusing on their physiological role, I also investigated the pathological determinants of these two proteins by evaluating the effects of single point mutations in their associated variant, G157R-GCAP2 and D101H-GCAP3 respectively, which have been associated with inherited retinal diseases. A promising approach for treating these genetic disorders is the use of liposomes as potential delivery system for therapeutic molecules, such as functional proteins or genes. To probe the potential of liposomes as a tool to replace mutated variants with functional and active proteins, initial experiments have been performed with cultured HEK293 cells, with the aim to understand the mechanism of liposomes up-take and the release of physiologically active biomolecules, such as DNA and functional GCAP. A common disease that affects vision, with fare less harmful effects compared to retinal dystrophies, is Dry Eye disease (DED). A key role in DED is played by mucins. These are glycoproteins secreted by conjunctival goblet cells that are responsible to create a stable tear film and to protect and maintain the integrity of the ocular surface. It has been reported that the gene expression of mucins, and in particular mucin-4, were significantly lower in conjunctival epithelium of patients affected by DED and altered levels of these glycoproteins could be restored by the topical administration of eyedrops. In the second part of my PhD I investigated the effects of the treatment with hyaluronic acid cross-linked with urea (U-HACL) eyedrop and characterized the expression levels of mucins before and after treatment in patients affected by DED.