A New Retinoid Signaling Molecule in the Retinal Pigment Epithelium: RPE45

Purpose:The control elements that regulate the overall biosynthetic pathway to 11-cis-retinal under light and oxidative stress are not well understood. In order to study this important isomerization/hydrolysis step, we used a differential proteomics approach with bovine, murine and human retinal pigment epithelial cells to study the effects of the light and oxidative stress on RPE65 expression and RPE45, a caspase-mediated cleavage form of RPE65. Methods:RPE65 expression levels were tested in various conditions in vitro (RPE D407), ex vivo (bovine, mouse), and in vivo (mouse). Proteomics tools such as 2D SDS-PAGE, MALDITOF-TOF mass spectrometry analysis and biochemical methods including Western blot, immunohistochemistry, retinoid binding assay were employed to study functional role of RPE45. Results:We found that a new 45kDa protein, designated as RPE45, a truncated fragment of RPE65, appears upon light exposure and oxidative stress. RPE45 is formed by recombinant caspases using a ubiquitination mechanism. We show that RPE45 results from an interaction of specific proteases with RPE65 through ubiquitination induced by light and ROS. RT-PCR revealed that the differential levels of stress-induced RPE65 expression are regulated at a posttranscriptional level. Conclusions:In this study, comparative proteomics using 2D gel electrophoresis and mass spectrometry analysis reveal that RPE65 is one of the major proteins regulated by light and ROS in RPE cells. We find that the level of RPE65 is increased in light compared to dark, and RPE45, a 45kDa fragment of RPE65, is present only in the light or in high oxidative stress. Therefore, RPE45 can serve as a biomarker for oxidative stress and light exposure in the eye. These findings demonstrate the role of RPE65 as a key regulator that responds to light and ROS in the visual cycle.