Identification of the binding region for interaction of optineurin with neural retina leucine

Abstract

Background Gene encoding optineurin (OPTN) is a causative gene for glaucoma and amyotrophic lateral sclerosis,with a more expression in retina.Our previous study isolated OPTN-interacting proteins and identified that the gene encode the basic leucine zipper (bZIP) transcription factor neural retina leucine (NRL) zipper,a causative gene for retinitis pigmentosa,and further study demonstrated the interaction between OPTN and NRL proteins in nuclei of cultured HeLaS3 cells.Objective This study was to determine the protein binding site of OPTN necessary for NRL binding.Methods A deletion series of OPTN-expression plasmids were constructed and co-expressed with hemagglutinin (HA)-tagged NRL in HeLaS3 cells,respectively.The cytoplasmic and nuclear fractions were used to perform co-immunoprecipitate (CoIP) and Western blot with anti-tag antibodies.Results In the nuclear fractions of cells transfected with the del1 st,del2nd or del3rd plasmid,a band of coimmunoprecipitated HA-labelled NRL (HA-NRL) was detected.However,the del4th plasmid did not produce a band.The NRL band was not found in cytoplasmic fractions from transfected cells with any of the deletion plasmids or with the whole-length OPTN plasmid.Conclusions The protein binding site of OPTN necessary for NRL binding is determined.This result demonstrates the binding of Flag-OPTN and HA-NRL in HeLaS3 cells.A series of partial-deletion OPTN plasmids demonstrated that the tail region (423-577 amino acids) of OPTN was necessary for binding with NRL. Key words: Optineurin; Neural retina leucine zipper; Protein interaction; Co-immunoprecipitation; Protein binding site