Interaction Between Optineurin and bZIP Transcription Factor NRL

Abstract

Purpose: : To investigate the function of optineurin protein (OPTN) and the molecular mechanism of open-angle glaucoma (OAG) caused by mutations in OPTN gene, we have screened genes for OPTN-interacting proteins. Of these, bZIP transcription factor NRL (Neural Retina Leucine Zipper) was found which is one of causative genes for retinitis pigmentosa. In this study, the interaction of OPTN and NRL was experimentally proven.

Methods: : NRL and OPTN cDNAs were isolated from human retina cDNA library and inserted into N-terminal HA-tag and FLAG-tag vectors, respectively. Both constructs were co-expressed in HeLa S3 cells. Immunoprecipitation and Western blot with anti-tag antibodies were carried out to detect the interaction between them in the cells. NRL and OPTN expression in rat and mouse eyes were observed by immunofluorescent staining using anti-NRL and anti-OPTN antibodies. To detect their interaction in rat retina, whole neural retina lysate or its nuclear fraction was analyzed with the same immunoprecipitation procedure.

Results: : In HeLa S3 cells, the interaction between HA-tagged NRL and FLAG-tagged OPTN was detected. In rat and mouse eyes, NRL was observed in nuclei of photoreceptor cells. Meanwhile, OPTN was seen mainly in cytoplasm of photoreceptor cells but some cells showed higher expression in nuclei. Interaction between NRL and OPTN in rat retina was examined. Immunoprecipitation with anti-OPTN failed to detect OPTN-bound NRL from whole cell lysate but succeeded from nuclear fraction. This was considered because contribution of cytoplasmic OPTN is rather large and only nuclear OPTN is bound to NRL.

Conclusions: : Expression of OPTN in photoreceptor cells was described as the first report, here. The interaction of OPTN and NRL in living eye tissue was revealed. NRL is the first eye-specific protein which was shown to interact with OPTN. The meaning of their interaction in the pathogenesis of glaucoma and retinitis pigmentosa remains to be analyzed.

Keywords: proteins encoded by disease genes • retina