Analysis of glaucoma-related protein interaction

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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.