Temperature-sensitive retinoid isomerase activity of RPE65 mutants associated with Leber Congenital Amaurosis.
RPE65 is a membrane-associated retinoid isomerase involved in the visual cycle responsible for sustaining vision. Many mutations in the human RPE65 gene are associated with distinct forms of retinal degenerative diseases. The pathogenic mechanisms for most of these mutations remain poorly understood. Here, we show that three Leber congenital amaurosis -associated RPE65 mutants (R91W, Y249C and R515W) undergo rapid proteasomal degradation mediated by the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13) in cultured human retinal pigment epithelium (RPE) cells. These mutant proteins formed cytosolic inclusion bodies or high molecular weight complexes via disulfide bonds. The mutations are mapped on non-active sites but severely reduced isomerase activity of RPE65. At 30°C, however, the enzymatic function and membrane-association of the mutant RPE65s are significantly rescued possibly due to proper folding. In addition, PSMD13 displayed a drastically decreased effect on degradation of the mutant proteins in the cells grown at 30°C. These results suggest that PSMD13 plays a critical role in regulating pathogenicity of the mutations and the molecular basis for the PSMD13-mediated rapid degradation and loss of function of the mutants is misfolding of RPE65.
Department of Ophthalmology and Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112 USA;
Songhua
S
Li
Jules Stein Eye Institute and Department of Neurobiology, University of California, Los Angeles, CA 90095 USA;
Jane
J
Hu
State University of New York at Buffalo, Buffalo, NY 14214 USA;
Robin J
RJ
Jin
Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA 70112 USA; and.
Ashok
A
Aiyar
Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA 19104 USA.
Samuel G
SG
Jacobson
Jules Stein Eye Institute and Department of Neurobiology, University of California, Los Angeles, CA 90095 USA;
Dean
D
Bok
Department of Ophthalmology and Neuroscience Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112 USA; mjin@lsuhsc.edu.
Minghao
M
Jin
D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D020022:Genetic Predisposition to Disease; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D057130:Leber Congenital Amaurosis / Q000201:enzymology* / Q000235:genetics*; D008958:Models, Molecular; D008970:Molecular Weight; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D059748:Proteolysis; D013696:Temperature; D019745:cis-trans-Isomerases / Q000235:genetics*; D000099178:Retinoid Isomerohydrolase
D004220:Disulfides; D050505:Mutant Proteins; D046988:Proteasome Endopeptidase Complex; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C119011:26S proteasome non-ATPase regulatory subunit 13
26S proteasome non-ATPase regulatory subunit 13 (PSMD13); RPE65; retinal degeneration; retinal pigment epithelium; retinoid isomerise