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Songhua Li, Marijana Samardzija, Zhihui Yang, Christian Grimm, Minghao Jin; Attempts to increase protein level, membrane-association, and isomerase activity of R91W RPE65 in mouse model. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5454. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
R91W substitution, the most frequent RPE65 mutation associated with Leber’s congenital amaurosis, causes a severe decrease in protein level and retinoid isomerase activity of RPE65 by unknown mechanisms in mouse model. Through in vitro studies, we recently found that the 26S proteasome non-ATPase regulatory subunit 13 (PSMD13), sodium 4-phenylbutyrate (PBA), and low temperature play critical roles in regulating stability and activity of some pathogenic mutant RPE65s. The purpose of this study is to test if these factors exhibit similar effects on mouse RPE with R91W knock-in mutation.
Protein and mRNA expression levels of wild-type (WT) and R91W RPE65s in mice eyecups treated with protease inhibitors or maintained at 30°C were measured by quantitative immunoblot analysis or real-time RT-PCR. Over-expression and siRNA-mediated knockdown were applied to test whether PSMD13 mediates degradation of R91W RPE65. Retinoid isomerase activity was measured by monitoring synthesis of 11-cis retinol from all-trans retinol incubated with mice eyecups at 37°C or 30°C in the presence or absence of PBA. Retinoids were analyzed by high-performance liquid chromatography. Membrane fractions of mice RPE were prepared by ultracentrifugation. Distribution of RPE65 in mice RPE treated with PBA was determined by immunohistochemistry and confocal microscopy.
R91W RPE65’s protein level, but not mRNA level, was significantly lower than that of WT RPE65 and was increased at least 2-fold in eyecups maintained at 30°C or treated with proteasome inhibitor. Co-expression of PSMD13 further decreased expression levels of R91W RPE65, whereas knockdown of PSMD13 increased protein level of the mutant. Isomerase activity and membrane-association of R91W RPE65 were significantly increased in eyecups incubated at 30°C. WT RPE65 was evenly distributed in RPE cell body whereas R91W RPE65 was mainly localized to the basal site of RPE. In PBA-treated mice, however, R91W RPE65 was observed in both basal and apical sites, and was significantly increased in RPE treated with PBA.
R91W RPE65 undergoes PSMD13-mediated proteasomal degradation due to misfolding; low temperature and PBA treatments can increase isomerase activity by helping proper folding, which increases protein level and membrane-association of the mutant RPE65.
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