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Yuriko Minegishi, Takeshi Iwata; Distinct protein complex formation evokes insolubility of OPTN and mislocalization in iPSC-derived neural cells from E50K-POAG patients. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1142.
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© ARVO (1962-2015); The Authors (2016-present)
Optineurin (OPTN) is a multifunctional protein and its E50K mutation responsible for primary open angle glaucoma (POAG) and amyotrophic lateral sclerosis (ALS). We have previously reported our E50K transgenic mouse (E50K-tg) exhibits thinner retina and retinal ganglion cell loss, while the underlying molecular mechanisms have been unclear. Together with additional phenotypes of E50K-tg retina and over expression studies, the endogenous OPTN and E50K mutant protein dynamics was investigated by iPS Cell (iPSC) from E50K-POAG patients to elucidate the fundamental pathoetiology.
E50K-tg retina were examined by using anti-GFAP, anti-OPTN, and anti-HA antibodies. The intracellular localization and protein property of endogenous OPTN in wild type control and in E50K carrier were also examined by iPSCs and iPSC-derived neural cells. FLAG-tagged OPTN and E50K were expressed in HEK293T cells and protein complex formation/oligimerization was examined by Native-PAGE and LC-MS/MS proteomics. Interaction with obtained candidates and further biochemical analysis were examined by general molecular biological techniques.
E50K-tg retina exhibited significant reactive gliosis. In E50K-tg retina, E50K mutant protein is accumulated in OPL where exhibited the severe cell death and atrophy. Overexpressed OPTN and E50K exhibited different hydrophobicity and only E50K is accumulated in endoplasmic reticulum (ER) prior to Golgi transition. These distinct protein properties were also consistent with under endogenous condition in iPSCs and iPSC-derived neural cells from E50K-POAG patient. Proteomics revealed distinct complex formation between OPTN and E50K. The treatment with specific inhibitor for E50K specific binding partner rescued the abnormal insolubility.
Underlying pathoetiology of E50K-POAG originates from the alteration of protein complex formation that deteriorates the OPTN/E50K intracellular dynamics. This alteration is the probable cause of other terminal E50K phenotypes such as Golgi deformation, intracellular transport failure and cell deaths. We further investigate E50K phenotypes with endogenous conditions and E50K knock-in mouse, as well as the possibility of plasticity of E50K-glaucomatous phenotypes.
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