Rescue of enzymatic function for disease-associated RPE65 proteins 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; however, 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 26 S 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 a ubiquitination- and proteasome-dependent non-lysosomal pathway. These mutant RPE65s were misfolded and formed aggregates or high molecular complexes via disulfide bonds. Interaction of PSMD13 with mutant RPE65s promoted degradation of misfolded but not properly folded mutant RPE65s. Many mutations, including L22P, T101I, and L408P, were mapped on non-active sites. Although their activities were very low, these mutant RPE65s were catalytically active and could be significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. Sodium 4-phenylbutyrate 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 a low temperature eye mask and sodium 4-phenylbutyrate, a United States Food and Drug Administration-approved oral medicine, may provide a promising "protein repair therapy" that can enhance the efficacy of gene therapy by reducing the cytotoxic effect of misfolded mutant RPE65s.
From the Department of Ophthalmology and Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112.
Songhua
S
Li
Graduate Center for Toxicology, University of Kentucky College of Medicine, Lexington, Kentucky 40536.
Tadahide
T
Izumi
Jules Stein Eye Institute and Department of Neurobiology, University of California, Los Angeles, California 90095.
Jane
J
Hu
Department of Biology, Washington University, St. Louis, Missouri 63130.
Heather H
HH
Jin
Department of Neuroscience, Tulane University, New Orleans, Louisiana 70118, and.
Ahmed-Abdul A
AA
Siddiqui
Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
Samuel G
SG
Jacobson
Jules Stein Eye Institute and Department of Neurobiology, University of California, Los Angeles, California 90095.
Dean
D
Bok
From the Department of Ophthalmology and Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, mjin@lsuhsc.edu.
Minghao
M
Jin
D000818:Animals; D020134:Catalytic Domain; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004194:Disease / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005990:Glycerol / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020125:Mutation, Missense; D010654:Phenylbutyrates / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017510:Protein Folding / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D055213:Retinal Pigment Epithelium / Q000166:cytology; D013696:Temperature; D054875:Ubiquitination / Q000187:drug effects; D019745:cis-trans-Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000099178:Retinoid Isomerohydrolase
D005990:Glycerol; D050505:Mutant Proteins; D010654:Phenylbutyrates; D046988:Proteasome Endopeptidase Complex; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C075773:4-phenylbutyric acid; C061553:ATP dependent 26S protease; C119011:26S proteasome non-ATPase regulatory subunit 13
Aggregation; Leber Congenital Amaurosis; Membrane; PSMD13; Photoreceptor; Proteasome; RPE65; Retinal Degeneration; Retinoid; Ubiquitination