Cleavage of the retinal pigment epithelium-specific protein RPE65 under oxidative stress.
The regeneration of the 11-cis-retinyl imine chromophore of rhodopsin during the visual cycle and mechanisms that control this process are central questions in the field of vision research. The retinal pigment epithelium (RPE)-specific protein RPE65 is centrally involved in the isomerization and hydrolysis of all-trans-retinyl esters. In this study, we investigated RPE65 cleavage and potential regulatory mechanisms under oxidative stress conditions. The D407 RPE cell cultures were exposed to H(2)O(2) (100-1000 microM). Changes in the levels of RPE65 and proteins related to apoptosis were investigated using gel electrophoresis and western blotting. Mass spectrometry was used to confirm the identity of RPE65. C57BL/6J (M450) and C3HeB/FeJ (L450) mice were used for in vivo experiments. We found that a novel 45kDa truncated fragment of the RPE65 protein, designated RPE45, appears in RPE cells upon light exposure or oxidative stress. RPE45 is generated in vitro by recombinant caspases via an ubiquitination-dependent mechanism. Collectively, our results indicate that oxidative stress during the visual cycle results in cleavage of RPE65.
Department of Ophthalmology, University of South Carolina, Columbia, SC 29208, USA.
Hyunju
H
Lee
Hyewon
H
Chung
Hilal
H
Arnouk
Folami
F
Lamoke
Richard C
RC
Hunt
William J M
WJ
Hrushesky
Patricia A
PA
Wood
Sung Haeng
SH
Lee
Wan Jin
WJ
Jahng
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D018384:Oxidative Stress; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D013379:Substrate Specificity; D054875:Ubiquitination; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D002352:Carrier Proteins; D020169:Caspases; D005136:Eye Proteins; D010446:Peptide Fragments; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase