The Preliminary Research On The Molecular Mechanisms Of Optineurin As A Autophagy Receptor

Objective:we try to preliminarily investigate the roles Optineurin plays in autophagy as aautophagy receptor and how Optineurin influences. NF-kB signaling pathway as well asOptineurin’s function in resisting virus and clearing bacterial. To study these aspects,GST pull-down, immunofluorescence and Yeast two-hybrid were employed for theresearch. And the investigation provides further insights how Optinerin participates inGlaucoma and neurodegenerative disease.Methods:pET28a-OPTN-HA, pGEX4t-1-LC3andpCDGFP-OPTN were constructed andsequenced to confirm the correctness of reading frame. GST-LC3and HA-tagged OPTNwere expressed in BL21strain then purified fallowing by GST pull-down to detectwhether OPTN interacts with LC3in vitro. Immunofluorescence then was employed toobserve the localization of OPTN. Cy5(goat-anti-mouse, red) coupled secondaryantibody was used to combine OPTN primary antibody so that the localization ofEndogenous OPTN could be observed. pRFP-C-RS-shOPTN were transfected in to293FT cells to knockdown OPTN and the knockdown efficiency was checked viaWestern-blotting. The proliferation of Salmonella was tested when OPTN wasknock-down in order to study the functions of OPTN in autophagy. Bait vectorpGBKT7-OPTN was constructed and employed to identify the E3(s) for OPTN viascreening human E3s library. Results:Restriction digestion and sequencing results showed that all the cloning construct arecorrect.GST pull-down assays showed that OPTN interacted with LC3invitro.Immunofluorescence assays showed that OPTN localized mainly in cytoplasma inHEK293FT cells. E3ligases were identified via yeast two-hybrid.ConclusionOPTN participated in apoptosis, NF-kB signaling pathway and autophagy as well asresisting infections of virus and bacterial. And our work laid the foundation of thefurther study how Optinerin participates in Glaucoma and neurodegenerative disease.