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David Kuai, Molly Smith, Jessica Martin, Wei Shen, Amelia Verhoeven, Kyle Wallace, David Gamm, Ruchira Singh; The effect of BEST1 mutations on cellular protein degradation pathways in a BD hiPSC-RPE model. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3844.
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© ARVO (1962-2015); The Authors (2016-present)
Best disease (BD) is an inherited macular degeneration caused by mutations in the BEST1 gene. We have recently shown perturbed calcium homeostasis and delayed degradation of photoreceptor outer segment (POS) in a human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) model of BD (BD hiPSC-RPE). Given that intracellular calcium signaling modulates multiple protein degradation processes, in this study using a BD hiPSC-RPE model, we sought to determine if alterations in specific protein degradation pathways contribute to the pathophysiology of BD.
Monolayers of hiPSC-RPE were derived from two BD patients and unaffected siblings. To assess whether the rate of global protein degradation was delayed in BD, we compared the amount of oxidized and poly-ubiquitinated proteins in BD and control hiPSC-RPE using ELISA and Western blot analyses. To determine if a specific protein degradation pathway was affected in BD hiPSC-RPE, we evaluated the expression and activity of key enzymes involved in lysosomal, ubiquitin-proteasomal and autophagy-mediated protein degradation. We also quantified lysosomal pH in BD and control hiPSC-RPE using lysosensor yellow/blue dye. Furthermore, to investigate whether lipofuscin accumulation in BD is due to altered exocytosis, we quantified the amount of exosome release and its protein composition in BD and control hiPSC-RPE.
An increased amount of oxidized proteins, but not polyubiquitinated proteins, was seen in BD hiPSC-RPE compared to control hiPSC-RPE. Of the lysosomal and proteasomal enzymes tested, the protein levels of cathepsin-D and mono-ubiquitin were lower in BD hiPSC-RPE compared to control hiPSC-RPE. Baseline levels of autophagy markers (LC3 I, LC3 II, P62) were at the lower limit of detection in both control and BD hiPSC-RPE, and no difference in lysosomal pH was observed between them. However, differences were seen in the amount of exosome released from BD vs. control hiPSC-RPE.
Our current evidence suggests that defective protein processing and degradation in the lysosomal and/or proteasomal pathways contributes to delayed degradation of POS and subsequent lipofuscin accumulation in the BD hiPSC-RPE model.
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