PurposeMutations in the EYS gene are a common cause of autosomal recessive retinitis pigmentosa (arRP), yet the role of the EYS protein in humans is presently unclear. The aim of this study was to investigate the isoform structure, expression and potential function of EYS in the mammalian retina in order to better understand its involvement in the pathogenesis of arRP.MethodsTo achieve the objective, we examined the expression of mRNA transcripts of EYS isoforms in human tissues and cell lines by RT-PCR. We also investigated the localisation of EYS in cultured cells and retinal cryo-sections by confocal fluorescence microscopy and Western blot analysis.ResultsRT-PCR analysis confirmed that EYS has at least four isoforms. In addition to the previously reported EYS isoforms 1 and 4, we present the experimental validation of two smaller variants referred to as EYS isoforms 2 and 3. All four isoforms are expressed in the human retina and Y79 cells and the short variants were additionally detected in the testis. Immunofluorescent confocal microscopy and Western blot analysis revealed that all EYS isoforms preferentially localise to the cytoplasm of Y79 and HeLa cells. Moreover, an enrichment of the endogenous protein was observed near the centrosomes in Y79 cells. Interestingly, EYS was observed at the ciliary axoneme in Y79 ciliated cells. In macaque retinal cryosections, EYS was found to localise in the region of the photoreceptor ciliary axoneme in both rods and cones as well as in the cytoplasm of the ganglion cells.ConclusionThe results obtained in this study lead us to speculate that, in photoreceptor cells, EYS could be a protein involved in maintaining the stability of the ciliary axoneme in both rods and cones. The variability of its isoform structure suggests that other roles are also possible and yet to be established.
The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/ PSMC1), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS.
ObjectiveProstaglandin-D2 synthase (PTGDS; MIM#176803) is a novel protein-partner of TOPORS (TOPORS; MIM# 609507), a ubiquitously expressed nuclear and ciliary protein, implicated in retinitis pigmentosa. This study investigated the localisation of PTGDS and its potential mechanism-of-association with TOPORS. MethodsYeast two-hybrid screens, using TOPORS as bait, were performed against human retinal cDNA libraries. Validation and interaction-characterisation were performed in yeast, and by co-immunoprecipitation (co-IP) from HeLa cell extracts. Co-localisation studies were performed in hTERT-RPE1 cell line, and in murine retina cryo-sections. PTGDS expression was validated by RT-PCR. ResultsCo-IP demonstrated PTGDS was found in endogenous protein complexes with TOPORS, whereas in yeast PTGDS interacted most strongly with TOPORS' residues 1-380, comprising the RING-domain conferring its E3-ubiquitin-ligase activity. PTGDS co-localised with TOPORS, and centriolar markers in dividing cells, and was observed at basal body and along ciliary axoneme in ciliated cells. In mouse retina PTGDS was observed in several cell layers, partly overlapping with TOPORS in the photoreceptor layer. In human retina, RT-PCR studies demonstrated expression of several PTGDS isoforms. ConclusionPTGDS, a novel component of the primary cilium, could be involved in centriolar-ciliary homeostasis. This putative role of prostaglandin synthases, is additionally supported by independent findings on the role of prostaglandin-E2 in ciliogenesis. Results suggest TOPORS could regulate PTGDS levels at the cilium by marking it for degradation by the ubiquitin-proteasome system, providing a basis for understanding the retinal ciliopathy associated with TOPORS mutations.
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