The optineurin oligomer: its existence as a major form in cells and cross-linking upon oxidative stimuli through ubiquitin binding domain
Year: 2013
Type: dissertation
Abstract: Optineurin gene, OPTN, is one of the causative genes of primary open-angle glaucoma. Although the oligomerization of optineurin in cultured cells was previously observed by gel filtration analysis and blue native gel electrophoresis (BNE), there is no detailed information about the feature of optineurin oligomer. The present study aims to analyze the oligomeric state of optineurin and its affecting factors, such as environmental stimuli or the mutations of optineurin. Here, we demonstrated that both endogenous and transfected optineurin exist as oligomer, rather than monomer in NIH3T3 cells using BNE or immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We also applied in situ proximity ligation assay to visualize the self-interaction of optineurin in fixed HeLaS3 cells and reveal that the optineurin oligomer was localized diffusely in cytoplasm. Optineurin oligomer was usually detected as a single band at the size equal to optineurin monomer by SDS-PAGE, while there was an additional protein band with larger size observed when cells were treated with H2O2 before cell lysis followed by SDS-PAGE. The protein complex with larger size was proved to be optineurin oligomer by immunoprecipitation and therefore called covalent optineurin oligomer. Interestingly, in most common glaucoma-associated mutation of optineurin, E50K expressing cells, the covalent oligomer was formed even without H2O2 stimulation. Anti-oxidants inhibited the formation of E50K-induced covalent oligomer in different degrees. A series of truncated constructs of optineurin was used to reveal that covalent oligomer might be optineurin trimer and the ubiquitin binding domain is essential for this trimer formation. Our results indicates that optineurin trimer might be the basic unit of the oligomer. The oligomeric state can be affected by many factors, like demonstrated here that some covalent bond(s) is induced by H2O2 or transfection of E50K, which provides a novel insight into E50K pathogenesis. Furthermore, the regulation of oligomeric state becomes important worth further studies.
Author: Jie Gao
Institution: Hamamatsu University School of Medicine
Language: English
Subfield: Ophthalmology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2277322635