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Songhua Li, Tadahide Izumi, Jane Hu, Samuel G Jacobson, Dean Bok, Minghao Jin; PSMD13 Promotes Degradation of Disease-Causing RPE65s via the Ubiquitin-Proteasome Pathway. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3475.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the RPE65 gene have been associated with Leber's congenital amaurosis (LCA). Many LCA-associated missense mutations result in rapid degradation of mutant RPE65 by an unknown mechanism. Pathogenicity of these mutations may depend on the degradation rate of mutant RPE65. The purpose of this study is to identify the molecular mechanism underlying rapid degradation of mutant RPE65s and to determine whether the 26S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, mediates degradation of mutant RPE65s.
LCA-associated RPE65s generated by site directed mutagenesis were expressed in cultured human primary RPE and ARPE19 cells. Protein and mRNA contents of wild-type (WT) and mutant RPE65s in the cells were determined by quantitative immunoblotting and RT-PCR, respectively. To determine if mutant RPE65s are degraded in the proteasome, cells were treated with proteasome inhibitors (MG115 & MG132) or pepstatin A, a lysosome inhibitor. Over-expression and siRNA-mediated knockdown were applied to test whether PSMD13 mediates degradation of WT and mutant RPE65s. RPE65s ubiquitinated with ubiquitin-His6 were purified with Ni-NTA beads and subjected to immunoblot analysis. Inhibition of protein ubiquitination was carried out using an inhibitor of ubiquitin-activating enzyme E1. Formation of disulfide bond-mediated protein complexes was determined using reducing agents. Aggresomes of mutant RPE65s in the cells were observed by confocal microscopy.
Expression levels of mutant RPE65s were 70-90% lower than that of WT RPE65 in the cells whereas the mRNA contents of mutant RPE65s were similar to that of WT RPE65. Proteasome inhibitors, but not pepstatin A, significantly increased expression levels of mutant RPE65s. Co-expression of PSMD13 resulted in a decrease in protein content of mutant RPE65s whereas knock down of PSMD13 rescued expression levels of the mutant RPE65s. WT and mutant RPE65s were ubiquitinated in ARPE19 cells. Inhibition of protein ubiquitination promoted formation of high molecular weight complexes (HMC) that contain mutant RPE65s. This HMC formation was significant in the absence of reducing reagents, but decreased in the presence of reducing reagents. Mutant RPE65s formed aggresomes in the RPE cells.
PSMD13 regulates pathogenicity of RPE65 mutations by mediating degradation of mutant RPE65 in the proteasome.
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