Oligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.

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 olig...

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Autores principales:	Jie Gao, Masafumi Ohtsubo, Yoshihiro Hotta, Shinsei Minoshima
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/344e3f916477431cbb4f69070e76463f
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spelling	oai:doaj.org-article:344e3f916477431cbb4f69070e76463f2021-11-25T06:10:08ZOligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.1932-620310.1371/journal.pone.0101206https://doaj.org/article/344e3f916477431cbb4f69070e76463f2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24983867/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203The 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.Jie GaoMasafumi OhtsuboYoshihiro HottaShinsei MinoshimaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 7, p e101206 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jie Gao Masafumi Ohtsubo Yoshihiro Hotta Shinsei Minoshima Oligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.
description	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.
format	article
author	Jie Gao Masafumi Ohtsubo Yoshihiro Hotta Shinsei Minoshima
author_facet	Jie Gao Masafumi Ohtsubo Yoshihiro Hotta Shinsei Minoshima
author_sort	Jie Gao
title	Oligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.
title_short	Oligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.
title_full	Oligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.
title_fullStr	Oligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.
title_full_unstemmed	Oligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.
title_sort	oligomerization of optineurin and its oxidative stress- or e50k mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/344e3f916477431cbb4f69070e76463f
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Oligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.

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