Turturro Sanja, Shen Xiang, Shyam Rajalekshmy, Yue Beatrice Yjt, Ying Hongyu
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, 1855 W Taylor Street, Chicago, IL 60612 USA.
Springerplus. 2014 Feb 19;3:99. doi: 10.1186/2193-1801-3-99. eCollection 2014.
Optineurin is a gene associated with normal tension glaucoma (NTG) and amyotrophic lateral sclerosis (ALS). Foci formation and functional consequences including Golgi fragmentation, impairment of vesicle trafficking and apoptosis were observed previously upon overexpression and/or mutation of optineurin. In the current study, a total of 15 GFP tagged constructs that included NTG (E50K and 2 bp-AG insertion), ALS (exon 5 deletion, R96L, Q398X, and E478G) and non-disease (L157A and D474N) associated mutants and a series of deletion fragments were cloned into mammalian expression vectors and transfected into RGC5 and/or Neuro2A cells to evaluate whether their expression confer the optineurin phenotypes. The cells were monitored for foci formation and stained by immunofluorescence with anti-GM130 to analyze the Golgi integrity. Transferrin uptake experiments were performed to evaluate the protein trafficking process and apoptosis was assessed with the active caspase 3/7 detection kit. We demonstrated that cells expressing E50K and R96L optineurin exhibited all of the optineurin phenotypes. Q398X mutant did not induce foci formation, but triggered Golgi fragmentation, impairment of transferrin uptake and increase in apoptosis. The 2 bp-AG insertion mutant had a nuclear localization, compromised the transferrin uptake and strongly induced apoptosis. The foci formation, which might not predict the rest of the phenotypes, appeared to require both the leucine zipper and ubiquitin binding domains of the optineurin sequence. Interactions of optineurin with proteins including Rab8, myosin VI, huntingtin and transferrin receptor might directly determine whether the Golgi and protein trafficking phenotypes would be manifested. Examination of mutants and deletion fragments located at various sites of optineurin gene provide clues as to what regions of the gene may play a critical role in the development of pathologic consequences.
视紫质是一种与正常眼压性青光眼（NTG）和肌萎缩侧索硬化症（ALS）相关的基因。先前在视紫质过表达和/或突变时观察到了病灶形成以及包括高尔基体碎片化、囊泡运输受损和细胞凋亡在内的功能后果。在本研究中，总共15个绿色荧光蛋白标记的构建体被克隆到哺乳动物表达载体中，这些构建体包括与NTG（E50K和2bp-AG插入）、ALS（外显子5缺失、R96L、Q398X和E478G）以及非疾病（L157A和D474N）相关的突变体和一系列缺失片段，然后将其转染到RGC5和/或Neuro2A细胞中，以评估它们的表达是否会赋予视紫质表型。监测细胞的病灶形成情况，并用抗GM130进行免疫荧光染色以分析高尔基体的完整性。进行转铁蛋白摄取实验以评估蛋白质运输过程，并用活性半胱天冬酶3/7检测试剂盒评估细胞凋亡情况。我们证明，表达E50K和R96L视紫质的细胞表现出所有视紫质表型。Q398X突变体未诱导病灶形成，但引发了高尔基体碎片化、转铁蛋白摄取受损和细胞凋亡增加。2bp-AG插入突变体具有核定位，损害了转铁蛋白摄取并强烈诱导细胞凋亡。病灶形成似乎需要视紫质序列的亮氨酸拉链和泛素结合结构域，而这可能无法预测其余的表型。视紫质与包括Rab8、肌球蛋白VI、亨廷顿蛋白和转铁蛋白受体在内的蛋白质之间的相互作用可能直接决定高尔基体和蛋白质运输表型是否会显现。对视紫质基因不同位点的突变体和缺失片段进行检查，为该基因的哪些区域可能在病理后果的发展中起关键作用提供了线索。
