Rescue of enzymatic function for disease-associated RPE65s containing various missense mutations in non-active sites.

Over 70 different missense mutations, including a dominant mutation, in RPE65 retinoid isomerase are associated with distinct forms of retinal degeneration; yet the disease mechanisms for most of these mutations have not been studied. Although some mutations have been shown to abolish enzyme activity, the molecular mechanisms leading to the ...
loss of enzymatic function and retinal degeneration remain poorly understood. Here we show that the 26S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, plays a critical role in regulating pathogenicity of three mutations (L22P, T101I, and L408P) by mediating rapid degradation of mutated RPE65s via an ubiquitination- and proteasome-dependent non-lysosomal pathway. These mutant RPE65s are misfolded and formed aggregates or high molecular complexes via disulfide bonds. Interaction of PSMD13 with mutant RPE65s promotes degradation of misfolded, but not properly folded, mutant RPE65s. Many mutations, including the three mutations, are mapped on non-active sites. Although their activities are very low, these mutant RPE65s are catalytically active and can be significantly rescued at low temperature while mutant RPE65s with a distinct active site mutation cannot be rescued under the same conditions. Sodium 4-phenylbutyrate (PBA) and glycerol displayed a significant synergistic effect on the low temperature rescue of the mutant RPE65s by promoting proper folding, reducing aggregation, and increasing membrane-association. Our results suggest that low temperature eye mask and PBA, a FDA-approved oral medicine, may provide a promising "protein repair therapy," which can enhance the efficacy of gene therapy by reducing cytotoxic effect of misfolded mutant RPE65s.
J. Biol. Chem.
Date: May. 21, 2014
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