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Minghao Jin, Songhua Li, Jane Hu, Robin J. Jin, Samuel G Jacobson, Dean Bok; Low temperature-dependent functional rescue of RPE65 mutants associated with retinal degeneration. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5455. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The mechanisms by which many pathogenic mutations in RPE65 cause a severe decrease in protein level and enzymatic activity of RPE65 remain poorly understood. We recently showed that the 26S proteasome non-ATPase regulatory subunit 13 (PSMD13) and low temperature play important roles in regulating expression levels and isomerase activities of some disease-causing mutant RPE65s. The purpose of this study is to test whether PSMD13 and low temperature have similar effects on other pathogenic mutants of RPE65.
Expression levels and degradation pathways of three mutant RPE65s (Y249C, Y318N, and R515W) whose mutation sites mapped on non-active sites were analyzed in cultured human RPE and ARPE-19 cells treated with proteasome and lysosome inhibitors. Over-expression and siRNA-mediated knockdown were applied to test whether PSMD13 mediates degradation of the mutant proteins. Formation of disulfide bond-mediated protein complexes was analyzed using an ubiquitin-activating enzyme inhibitor and a reducing reagent. Cytoplasmic inclusion body-formation, membrane-association, and isomerase activities of the mutant RPE65s in cells grown at 37°C or 30°C were compared to test if misfolding is the molecular basis for their degradation and loss of function.
Expression levels of the mutant proteins were less than 30% of wild-type RPE65 in the RPE cells. Proteasome inhibitors, but not lysosome inhibitor, significantly increased expression levels of the mutant proteins. Co-expression of PSMD13 further decreased protein levels of the mutants, whereas knockdown of PSMD13 increased expression levels of the mutant proteins. Inhibition of protein ubiquitination promoted formation of high molecular weight complexes (HMC) containing mutant RPE65. This HMC formation was significant in the absence of reducing reagent, but decreased in the presence of DTT. Inclusion body-formation of the mutant proteins was drastically reduced in cells grown at 30°C compared to the cells maintained at 37°C. Isomerase activity and membrane-association of the mutant RPE65s were increased several fold when cells were maintained at 30°C.
Misfolding is the molecular basis for the PSMD13-mediated proteasomal degradation and loss of enzymatic function of the mutant RPE65s and low temperature could be a “protein repair” approach for rescuing the enzymatic function for pathogenic RPE65s with non-active site mutations.
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