Kruczek, PM; (2015) Characterisation of interacting partner(s) for EYS, a major gene implicated in autosomal recessive retinitis pigmentosa. Doctoral thesis , UCL (University College London).

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Abstract

Mutations in EYS are a common cause of autosomal recessive retinitis pigmentosa (arRP). EYS is one of the largest genes expressed in the retina and the role of the protein it encodes is presently unclear. It has been shown, however, that EYS localises to the outer segments of porcine photoreceptors and that the Drosophila orthologue of EYS is essential in the biogenesis of the ommatidium, where it interacts with prominin, a highly conserved protein implicated in retinopathies. The aim of this project was to examine the role of EYS in the retina by investigating its subcellular localisation and by identifying its interacting partners. Characterisation of the genetic structure of EYS has revealed that it has at least four isoforms expressed in the retina and testis. Immunocytochemistry studies have shown that EYS isoforms predominantly localise to the cytoplasm of cultured cells whereas immunohistochemistry studies in the primate retina have revealed that it localises to the photoreceptor ciliary axoneme. Yeast 2-hybrid screening has resulted in identification of one potential interacting partner of EYS, AIPL1, which is a molecular chaperone required for the proteostasis of the retina. Further analysis by co-immunoprecipitation and immunofluorescence has confirmed that AIPL1 interacts with the N terminal fragment of EYS isoforms 1 and 4 as well as EYS isoforms 2 and 3. Furthermore, co-immunoprecpietation assays and immunofluorescence studies have suggested that the human orthologues of EYS and Prominin-1 do not interact. The mutation screening of PROM1 has resulted in identification of seven heterozygous novel variants in six unrelated arRP patients; however, pathogenicity of the changes could not be established. Altogether, the results of the study have demonstrated that EYS may be a novel protein associated with the photoreceptor ciliary axoneme, where it could play a role in maintenance of the photoreceptor outer segments. Furthermore, the analysis of the interacome of EYS has demonstrated that it may require the activity of AIPL1 for correct folding and trafficking, and that the functional link of EYS and Prominin-1 described in Drosophila is unlikely to be conserved in humans. The knowledge gained from the study presented herein has brought us closer to unravelling the molecular mechanisms underlying arRP and adds to the overall understanding of the physiology of the retina in health and disease.

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