Department of Ophthalmology, University of South Carolina, Columbia, SC 29208, USA.
Int J Biol Macromol. 2010 Aug 1;47(2):104-8. doi: 10.1016/j.ijbiomac.2010.05.014. Epub 2010 May 25.
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.
视循环中 11-顺式视黄醛亚胺发色团的再生和控制这一过程的机制是视觉研究领域的核心问题。视网膜色素上皮（RPE）特异性蛋白 RPE65 参与全反式视黄醇酯的异构化和水解。在这项研究中，我们研究了视循环中氧化应激条件下 RPE65 的切割和潜在的调控机制。D407 RPE 细胞培养物暴露于 H₂O₂（100-1000μM）中。使用凝胶电泳和 Western blot 检测 RPE65 和与细胞凋亡相关的蛋白水平的变化。使用质谱法确认 RPE65 的身份。C57BL/6J（M450）和 C3HeB/FeJ（L450）小鼠用于体内实验。我们发现，在光照或氧化应激下，RPE 细胞中出现了一种新型的 45kDa 截断片段的 RPE65 蛋白，命名为 RPE45。RPE45 在体外通过重组半胱天冬酶通过泛素化依赖的机制产生。总之，我们的结果表明，视循环中的氧化应激导致 RPE65 的切割。
