M-opsin protein degradation is inhibited by MG-132 in Rpe65⁻/⁻ retinal explant culture.
OBJECTIVE
The 65 kDa retinal pigment epithelium-specific protein, RPE65, is an essential enzyme for 11-cis-retinal synthesis in the eye. Mutations of the RPE65 gene in humans result in severe vision loss, and Rpe65(-/-) mice show early cone photoreceptor degeneration. We used an explant culture system to evaluate whether posttranslational downregulation of M-opsin protein in Rpe65(-/-) mice is caused by proteolytic degradation.


METHODS
The eyes of three-week-old Rpe65(-/-) mice were incubated in culture medium. Western blot analysis was used to evaluate the level of M-opsin protein, and immunofluorescence was used for protein localization. The transcriptional level of M-opsin was evaluated with real-time reverse-transcriptase-PCR.


RESULTS
Degradation of the M-opsin protein in Rpe65(-/-) mouse retina was inhibited by the proteasome inhibitor MG-132 but not by the lysosomal inhibitor pepstatin A and E64d. 9-cis-retinal, used as an analog of 11-cis-retinal, increased M-opsin protein but did not increase M-opsin mRNA. Moreover, 9-cis-retinal did not change the transcriptional levels of photoreceptor specific genes.


CONCLUSIONS
Our data suggest that M-opsin protein was degraded through a proteasome pathway and that M-opsin degradation was suppressed with 9-cis-retinal treatment in Rpe65(-/-) mice to some extent.
Department of Ophthalmology, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori, Japan.
Kota
K
Sato
Taku
T
Ozaki
Sei-ichi
S
Ishiguro
Mitsuru
M
Nakazawa
D000818:Animals; D055358:Cone Opsins / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004224:Diterpenes; D005123:Eye / Q000187:drug effects* / Q000378:metabolism; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology*; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009924:Organ Culture Techniques; D010436:Pepstatins / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D059748:Proteolysis / Q000187:drug effects; D060888:Real-Time Polymerase Chain Reaction; D012172:Retinaldehyde / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D019745:cis-trans-Isomerases / Q000172:deficiency / Q000235:genetics*; D000099178:Retinoid Isomerohydrolase
D055358:Cone Opsins; D015853:Cysteine Proteinase Inhibitors; D004224:Diterpenes; D007930:Leucine; D007976:Leupeptins; D010436:Pepstatins; D046988:Proteasome Endopeptidase Complex; D061988:Proteasome Inhibitors; D012172:Retinaldehyde; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C031390:9-cis-retinal; C108192:aloxistatin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C031375:pepstatin