title: Functional Rescue of Retinal Degeneration-Associated Mutant RPE65 Proteins.
abstract: More than 100 different mutations in the RPE65 gene are associated with inherited retinal degeneration. Although some missense mutations have been shown to abolish isomerase activity of RPE65, the molecular bases leading to loss of function and retinal degeneration remain incompletely understood. Here we show that several missense mutations resulted in significant decrease in expression level of RPE65 in the human retinal pigment epithelium cells. The 26S proteasome non-ATPase regulatory subunit 13, a newly identified negative regulator of RPE65, mediated degradation of mutant RPE65s, which were misfolded and formed aggregates in the cells. Many mutations, including L22P, T101I, and L408P, were mapped on nonactive sites of RPE65. Enzyme activities of these mutant RPE65s were significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. 4-phenylbutyrate (PBA) displayed a significant synergistic effect on the low temperature-mediated rescue of the mutant RPE65s. Our results suggest that a low temperature eye mask and PBA, a FDA-approved oral medicine, may provide a promising "protein repair therapy" that can enhance the efficacy of gene therapy for delaying retinal degeneration caused by RPE65 mutations.
authors.affiliation: Department of Ophthalmology and Neuroscience Center, Louisiana State University Health Sciences Center, 2020 Gravier St. Suite D, 70112, New Orleans, LA, USA. mjin@lsuhsc.edu.
authors.forename: Minghao
authors.initials: M
authors.lastname: Jin
authors.affiliation: Department of Ophthalmology and Neuroscience Center, Louisiana State University Health Sciences Center, 2020 Gravier St. Suite D, 70112, New Orleans, LA, USA. sli@lsuhsc.edu.
authors.forename: Songhua
authors.initials: S
authors.lastname: Li
authors.affiliation: Jules Stein Eye Institute, University of California, 90095, Los Angeles, CA, USA. hu@jsei.ucla.edu.
authors.forename: Jane
authors.initials: J
authors.lastname: Hu
authors.affiliation: Department of Biology, Washington University, 63130, St. Louis, MO, USA. jin.hui@wustl.edu.
authors.forename: Heather H
authors.initials: HH
authors.lastname: Jin
authors.affiliation: Scheie Eye Institute, Department of Ophthalmology, Perelman School of Medicine, University of Pennsylvania, 19104, Philadelphia, PA, USA. jacobsos@mail.med.upenn.edu.
authors.forename: Samuel G
authors.initials: SG
authors.lastname: Jacobson
authors.affiliation: Jules Stein Eye Institute, University of California, 90095, Los Angeles, CA, USA. bok@jsei.ucla.edu.
authors.forename: Dean
authors.initials: D
authors.lastname: Bok
mesh_terms: D015153:Blotting, Western; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D003080:Cold Temperature; D057809:HEK293 Cells; D006801:Humans; D018613:Microscopy, Confocal; D050505:Mutant Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010654:Phenylbutyrates / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012162:Retinal Degeneration / Q000235:genetics*; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D019745:cis-trans-Isomerases / Q000235:genetics* / Q000378:metabolism; D000099178:Retinoid Isomerohydrolase
chemical_list: D050505:Mutant Proteins; D010654:Phenylbutyrates; D046988:Proteasome Endopeptidase Complex; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C075773:4-phenylbutyric acid; C119011:26S proteasome non-ATPase regulatory subunit 13
keywords: Chemical chaperone; Gene therapy; Leber congenital amaurosis; Low temperature; PSMD13; Proteasome; RPE65; Retina; Retinitis pigmentosa; Retinoid; Visual cycle