Oligomerization of Optineurin and Its Oxidative Stress- or E50K Mutation-Driven Covalent Cross-Linking: Possible Relationship with Glaucoma Pathology
Minoshima S (2014) Oligomerization of Optineurin and Its Oxidative Stress- or E50K Mutation-Driven Covalent Cross-Linking:
Possible Relationship with Glaucoma Pathology. PLoS ONE 9(7): e101206. doi:10.1371/journal.pone.0101206
Oligomerization of Optineurin and Its Oxidative Stress- or E50K Mutation-Driven Covalent Cross-Linking: Possible Relationship with Glaucoma Pathology
Jie Gao 0
Masafumi Ohtsubo 0
Yoshihiro Hotta 0
Shinsei Minoshima 0
Michael G. Anderson, University of Iowa, United States of America
0 1 Department of Photomedical Genomics, Basic Medical Photonics Laboratory, Medical Photonics Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan, 2 Department of Ophthalmology, Hamamatsu University School of Medicine , Hamamatsu , Japan
The optineurin gene, OPTN, is one of the causative genes of primary open-angle glaucoma. Although oligomerization of optineurin in cultured cells was previously observed by gel filtration analysis and blue native gel electrophoresis (BNE), little is known about the characteristics of optineurin oligomers. Here, we aimed to analyze the oligomeric state of optineurin and factors affecting oligomerization, such as environmental stimuli or mutations in OPTN. Using BNE or immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we demonstrated that both endogenous and transfected optineurin exist as oligomers, rather than monomers, in NIH3T3 cells. We also applied an in situ proximity ligation assay to visualize the self-interaction of optineurin in fixed HeLaS3 cells and found that the optineurin oligomers were localized diffusely in the cytoplasm. Optineurin oligomers were usually detected as a single band of a size equal to that of the optineurin monomer upon SDS-PAGE, while an additional protein band of a larger size was observed when cells were treated with H2O2. We showed that larger protein complex is optineurin oligomers by immunoprecipitation and termed it covalent optineurin oligomers. In cells expressing OPTN bearing the most common glaucoma-associated mutation, E50K, covalent oligomers were formed even without H2O2 stimulation. Antioxidants inhibited the formation of E50K-induced covalent oligomers to various degrees. A series of truncated constructs of OPTN was used to reveal that covalent oligomers may be optineurin trimers and that the ubiquitin-binding domain is essential for formation of these trimers. Our results indicated that optineurin trimers may be the basic unit of these oligomers. The oligomeric state can be affected by many factors that induce covalent bonds, such as H2O2 or E50K, as demonstrated here; this provides novel insights into the pathogenicity of E50K. Furthermore, regulation of the oligomeric state should be studied in the future.
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Funding: This study was supported by the JSPS KAKENHI Grant Number 24592622. The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The optineurin gene, OPTN, has been identified as a causative
gene of adult-onset primary open-angle glaucoma (POAG) [1] and
was named after optic neuropathy-inducing protein. Glaucoma is
the second leading cause of blindness worldwide [24]. It is a
neurodegenerative disease of the optic nerve, characterized by the
progressive loss of retinal ganglion cells (RGCs) and their axons.
The most common form of glaucoma is POAG, affecting over 33
million people throughout the world [2]. One subgroup of POAG
is normal tension glaucoma (NTG). The molecular mechanism of
glaucoma is largely unknown; however, numerous studies have
established a genetic etiology for this disorder.
It is a common view that glaucoma is a multifactorial disease
resulting from the involvement of genetic variation in one or more
genes and/or various environment factors. The environment
factors include higher intraocular pressure (IOP) and other stimuli
mainly associated with IOP or age, such as oxidative stress [5] and
cytokines [6]. Sequence alterations in OPTN were found in 16.7%
of families with autosomal dominantly inherited NTG. The most
prevalent mutation was the missense p.Glu50Lys (E50K)
mutation, which occurred in 13.5% of these families [1]. In addition,
E50K-bearing glaucoma patients appear to have more progressive
and severe phenotypes [7,8].
Optineurin (OPTN) is a 577-amino-acid protein, which
contains several putative functional domains, including at least
one leucine zipper, multiple coiled-coil domains, a
ubiquitinbinding domain (UBD), and a zinc finger domain; the latter two
domains are located at the C terminal region [9]. Public databases
show that OPTN homologs from mouse, rat, pig, and bovine have
a substantial degree of similarity to human OPTN. Moreover,
OPTN shares 53% sequence similarity with the NF-kB essential
modulator (NEMO), and was therefore also named
NEMOrelat (...truncated)