Oligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.
Oligomerization of optineurin and its oxidative stress- or E50K mutation-driven covalent cross-linking: possible relationship with glaucoma pathology.
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DOI：
10.1371/journal.pone.0101206
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Minoshima S
中科院分区：
文献类型：
--
作者：
Gao J;Ohtsubo M;Hotta Y;Minoshima S
OPTN基因是原发性开角型青光眼的致病基因之一。虽然以前通过凝胶过滤分析和蓝色天然凝胶电泳（BNE）观察到培养细胞中优神经蛋白的低聚化，但对优神经蛋白低聚物的特性知之甚少。在这里，我们旨在分析OPTN的寡聚状态和影响寡聚的因素，如环境刺激或OPTN突变。使用BNE或免疫沉淀法，然后使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），我们证明内源性和转染的opineurin在NIH3T3细胞中以低聚物而不是单体的形式存在。我们还应用原位接近结扎实验来观察固定HeLaS3细胞中optinineurin的自相互作用，发现optinineurin低聚物在细胞质中弥漫性定位。在SDS-PAGE上，通常可以检测到Optineurin低聚物为与Optineurin单体相同大小的单个条带，而当细胞用H2O2处理时，可以观察到另一个更大大小的蛋白质条带。我们通过免疫沉淀发现较大的蛋白复合物是优神经蛋白低聚物，并将其命名为共价优神经蛋白低聚物。在携带最常见青光眼相关突变E50K的表达OPTN的细胞中，即使没有H2O2刺激，共价低聚物也会形成。抗氧化剂对e50k诱导的共价低聚物的形成有不同程度的抑制作用。OPTN的一系列截短结构揭示了共价低聚物可能是优神经蛋白三聚体，泛素结合结构域对这些三聚体的形成至关重要。我们的结果表明，优神经蛋白三聚体可能是这些低聚物的基本单位。低聚态可以受到许多诱导共价键的因素的影响，如H2O2或E50K，如图所示；这为E50K的致病性提供了新的见解。此外，寡聚物状态的调控还需进一步研究。
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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