Analysis of glaucoma-related protein interaction

青光眼是影响全球7000万人失明的主要原因之一。青光眼常伴有眼压升高，导致视网膜神经节细胞(RGC)特异性细胞死亡。然而，正常眼压性青光眼(NTG)是原发性开角型青光眼(POAG)的一种亚型，患者受影响时眼压不会升高。在日本，98%以上的POAG患者被诊断为NTG。导致该病发病的分子途径仍不清楚，主要是由于缺乏与该病相关的蛋白质的信息。我们将重点放在视神经磷酸酶(OPTN)上，它被确认为NTG的第一个基因。为了确定导致严重NTG的突变E50K的位置，我们决定分析OPTN和Rab8之间的蛋白质-蛋白质相互作用。用石英晶体微天平(QCM)和GST-Pull-down法测定相互作用。所有的OPTN突变体和Rab8都在细菌中表达，并通过亲和层析和凝胶过滤纯化。将每个突变体加入反应室，将野生型Rab8固定在QCM传感器芯片上。仅E50K与Rab8无相互作用。这一结果后来被下拉实验证实。Rab8属于Rab8 GTP结合蛋白家族，其功能已知调节细胞内的蛋白质运输。OPTN-Rab8之间相互作用的中断可能解释了视网膜神经节细胞(RGC)特异性细胞死亡的原因。我们目前正在过度表达突变体E50K以观察RGC中细胞器的变化。

Glaucoma is one of the leading causes of blindness affecting 70 million people world wide. Glaucoma is often associated with elevated intraocular pressure (IOP) which leads to specific cell death of the retinal ganglion cells (RGC). However, patients with normal tension glaucoma (NTG), a subtype of primary open angle glaucoma (POAG), are affected without the elevation of IOP. In Japan, more than 98% of patients affected by POAG are diagnosed as NTG. Molecular pathways leading to the pathology of the disease are still unclear mainly due to the lack of information of proteins responsible for the disease.We have focused on optineurin (OPTN), which was identified as the first gene for NTG. Do to the location of the mutation E50K, which is responsible for severe NTG, we decided to analyze the protein-protein interaction between OPTN and Rab8. Interaction was measured by Quartz-Crystal Microbalance (QCM) and GST-pull down assay. All OPTN mutants and Rab8 where expressed in bacteria and purified by affinity chromatography and gel filtration. Each mutant was added to the reaction chamber where wild type Rab8 was fixed on QCM sensor chip. Only E50K showed no interaction with Rab8. This result was later confirmed by the pull down assay.Rab8 belongs to the family of Rab8 GTP-binding proteins which is functionally known to regulate protein trafficking in the cell. Disruption of interaction between OPTN-Rab8 may explain the cause of specific cell death of retinal ganglion cells (RGC). We are currently over expressing the mutant E50K to observe organelle change in RGC.