PURPOSE. Retinitis pigmentosa is a form of inherited retinal degeneration, whereby the rod photoreceptors are gradually lost. Patients usually manifest symptoms in the third decade of life, beginning with night blindness and loss of peripheral vision gradually progressing to tunnel vision and eventually complete blindness, as cone photoreceptors die as a secondary effect. Mutations causing non-syndromic, autosomal dominant retinitis pigmentosa (adRP) were recently identified in TOPORS (RP31 locus, chromosome 9p21.1). TOPORS is a ubiquitously expressed gene, encoding a protein showing multi-functional character. It is currently the only known protein to demonstrate E3 ligase activity for both ubiquitin and SUMO-1 (small ubiquitin-like modifier). It also shows tumor-suppressor activity. The purpose of this study is to characterize the role of TOPORS in the retina, in particular in the photoreceptor cells, which may explain the retinal degeneration seen with mutations in this gene. METHODS. To identify the functional consequences of the mutations TOPORS was cloned from human retinal cDNA into FLAG-tagged vector pCATCH, identified mutations were introduced by site-directed mutagenesis and transfected into MDCK cells. Over-expressed TOPORS was detected using an antibody directed against the FLAG-tag. Functional analyses of WT lymphoblastoid cell lines and specifically generated patient cell lines was also conducted using the TOPORS antibody. A commercially available antibody was obtained and used for immunofluorescent staining of endogenous topors in a number of cell lines, and in mouse, pig and human retinal sections. Tissue sections were costained for TOPORS with several proteins of known localisation in the retina (for example, RP1 and γ-tubulin), to ascertain a more detailed localisation of TOPORS and to indicate potential interactants. Cellular localisation experiments were carried out in cell lines on a single cell level to determine the exact sub-cellular localisation. To identify functional partners of TOPORS, immuno-precipitation experiments were carried out to substantiate data obtained from localisation studies in retinal sections. RESULTS. Both mutations in TOPORS result in frameshift, prematurely terminating the protein; however this appeared to make no difference to the nuclear localization of the over-expressed WT and mutants proteins. Work on the patient lymphoblastoid cell lines showed that the mutant protein is not expressed although the mutant mRNA is detectable by RT-PCR. Immunoblot analysis confirmed the expression of TOPORS as a 150kDa band in cell lines and retinal tissue extracts. Localisation studies showed a novel localisation for TOPORS in the retina and in confluent, non-diving cells. TOPORS localised to the connecting cilium of the photoreceptors in retinal sections, and to the primary cilia of cells grown at confluence. Further analyses showed that it co-localizes with γ-tubulin to the base of the cilium, to the basal body. Co-immunoprecipitation experiments showed that TOPORS does not interact with many known connecting cilium proteins although it has a similar localization pattern. It did not show interaction with proteins involved in anterograde transport. However, TOPORS does show interaction with some of the protein subunits involved in retrograde transport. CONCLUSIONS. Topors is a multi-functional, ubiquitously expressed gene however mutations in it only cause retinal degeneration (adRP). Results suggest that the mutant proteins are not expressed in patients, hence haplo-insuffiency is likely to be the cause of retinal degeneration. TOPORS is expressed in many cell and tissue types, and is primarily a nuclear protein. However, it shows a unique localisation in the retina - to the base of the connecting cilium in the photoreceptors. Similarly, in ciliated cells, topors localised to the base of the primary cilium which was confirmed by co-localisation with γ-tubulin. The co-IP experiments confirmed its interaction with γ-tubulin and also showed that topors associates with protein involved in retrograde transport. This together suggests it plays a novel role in the retina, possibly related to signal transduction and/or trafficking, that when compromised, results in specific photoreceptor degeneration
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