Ophthalmic research · 2009
Optineurin and its mutants: molecules associated with some forms of glaucoma.
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Chalasani Madhavi Latha, Swarup Ghanshyam, Balasubramanian Dorairajan
Аннотация
AIMS: Optineurin is a gene (OPTN) associated with normal-tension glaucoma and primary open-angle glaucoma. The aim of our study is to understand the functions of the protein optineurin in retinal ganglion cells, and the modifications that come about in its functions when mutations occur in its sequence.
METHODS: We have worked with the rat retinal ganglion cell line RGC5, as well as with some others such as HeLa (human cancer cell line), COS-1 (monkey kidney cell line), IMR-32 (human neuroblastoma cell line), and D407 (human retinal pigment epithelial cell line). The cDNAs of wild-type OPTN, and of its mutants H26D, E50K, R454Q and H486R, were transfected into each of these cell lines and the effects studied. Localization of the proteins in cells was monitored by confocal microscopy and cell imaging, and their interaction with other cellular proteins by yeast two-hybrid assay and co-immunoprecipitation.
RESULTS: We found optineurin to interact with several proteins, which are involved in NF-kappaB regulation, vesicular trafficking at recycling endosomes, immune response and transcription regulation. Some of these proteins showed altered/reduced interactions with E50K and H486R mutants. E50K, a severe phenotype-related mutant of optineurin, was found to selectively kill RGC5 cells through oxytosis and apoptosis, while none of the other mutants did so.
CONCLUSIONS: Our results indicate that several independent mechanisms are likely to be involved in the pathogenesis of glaucoma caused by mutations in optineurin.
METHODS: We have worked with the rat retinal ganglion cell line RGC5, as well as with some others such as HeLa (human cancer cell line), COS-1 (monkey kidney cell line), IMR-32 (human neuroblastoma cell line), and D407 (human retinal pigment epithelial cell line). The cDNAs of wild-type OPTN, and of its mutants H26D, E50K, R454Q and H486R, were transfected into each of these cell lines and the effects studied. Localization of the proteins in cells was monitored by confocal microscopy and cell imaging, and their interaction with other cellular proteins by yeast two-hybrid assay and co-immunoprecipitation.
RESULTS: We found optineurin to interact with several proteins, which are involved in NF-kappaB regulation, vesicular trafficking at recycling endosomes, immune response and transcription regulation. Some of these proteins showed altered/reduced interactions with E50K and H486R mutants. E50K, a severe phenotype-related mutant of optineurin, was found to selectively kill RGC5 cells through oxytosis and apoptosis, while none of the other mutants did so.
CONCLUSIONS: Our results indicate that several independent mechanisms are likely to be involved in the pathogenesis of glaucoma caused by mutations in optineurin.