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Keywords:

  • co-immunoprecipitation;
  • NRL;
  • optineurin;
  • photoreceptor;
  • protein interaction;
  • proximity ligation assay

Abstract

Although the gene encoding optineurin (OPTN) is a causative gene for glaucoma and amyotrophic lateral sclerosis, it is ubiquitously expressed in all body tissues, including the retina. To study the function of OPTN in retinal ganglion cells as well as the whole retina, we previously isolated OPTN-interacting proteins and identified the gene encoding the bZIP transcription factor neural retina leucine zipper (NRL), which is a causative gene for retinitis pigmentosa. Herein, we investigated the binding between OPTN and NRL proteins in HeLaS3 cells. Co-expression of HA-tagged NRL and FLAG-tagged OPTN in HeLaS3 cells followed by immunoprecipitation and Western blotting with anti-tag antibodies demonstrated the binding of these proteins in HeLaS3 cells, which was confirmed by proximity ligation assay. NRL is the first OPTN-binding protein to show eye-specific expression. A series of partial-deletion OPTN plasmids demonstrated that the tail region (423–577 amino acids [aa]) of OPTN was necessary for binding with NRL. Immunostaining showed that Optn (rat homologue of OPTN) was expressed in rat photoreceptors and localised in the cytoplasm of photoreceptor cells. This is a novel demonstration of Optn expression in photoreceptor cells. OPTN was not detected in photoreceptor nuclei under our experimental conditions. Further analyses are necessary to elucidate the function of OPTN and the significance of its possible binding with NRL in photoreceptor cells.