Two point mutations of RPE65 from patients with retinal dystrophies decrease the stability of RPE65 protein and abolish its isomerohydrolase activity
Two point mutations of RPE65 from patients with retinal dystrophies decrease the stability of RPE65 protein and abolish its isomerohydrolase activity
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DOI：
10.1074/jbc.m603725200
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发表时间：
2006-08-04
影响因子：
4.8
通讯作者：
Ma, Jian-Xing
中科院分区：
文献类型：
--
作者：
Takahashi, Yusuke;Chen, Ying;Ma, Jian-Xing
RPE 65是类维生素A视觉循环中的异构水解酶，其对于11-顺式视黄醛的再循环至关重要，11-顺式视黄醛是视杆和视锥光感受器中的视色素的发色团。RPE 65基因的突变与遗传性视网膜营养不良有关，其机制尚不清楚。在这里，我们表明，两个点突变的RPE 65，R91 W和Y368 H，在视网膜营养不良患者中发现，都取消了RPE 65的异构水解酶活性后，视网膜下注射到Rpe 65（-/-）小鼠和在体外异构水解酶活性测定，独立于其蛋白质水平。此外，当与野生型RPE 65（wtRPE 65）相比时，R91 W和Y368 H突变体显示出显著降低的蛋白质水平，但mRNA水平不变。蛋白质稳定性分析表明，wtRPE 65是一个相当稳定的蛋白质，具有明显的半衰期长于10小时，当在293 A细胞中表达。在相同的条件下，突变体R91 W和Y368 H都表现出显著降低的蛋白质稳定性，半衰期分别小于2和6小时。亚细胞分级分离和Western印迹分析表明，wtRPE 65主要存在于膜组分，而这两个突变体主要分布在胞质组分，表明这些突变破坏RPE 65的膜缔合。棕榈酰化实验表明，wtRPE 65和两个突变体均发生棕榈酰化。这些结果表明，这些突变可能导致RPE 65蛋白的关键结构改变，破坏其膜结合，从而损害其异构水解酶活性，导致视网膜变性。
RPE65 is the isomerohydrolase in the retinoid visual cycle essential for recycling of 11-cis retinal, the chromophore for visual pigments in both rod and cone photoreceptors. Mutations in the RPE65 gene are associated with inherited retinal dystrophies with unknown mechanisms. Here we show that two point mutations of RPE65, R91W and Y368H, identified in patients with retinal dystrophies both abolished the isomerohydrolase activity of RPE65 after a subretinal injection into the Rpe65(-/-) mice and in the in vitro isomerohydrolase activity assay, independent of their protein levels. Further, the R91W and Y368H mutants showed significantly decreased protein levels but unchanged mRNA levels when compared with the wild-type RPE65 (wtRPE65). Protein stability analysis showed that wtRPE65 is a fairly stable protein, with an apparent half-life longer than 10 h, when expressed in 293A cells. Under the same conditions, mutants R91W and Y368H both showed substantially decreased protein stabilities, with half-lives less than 2 and 6 h, respectively. Subcellular fractionation and Western blot analysis demonstrated that wtRPE65 predominantly exists in the membrane fraction, while both of the mutants are primarily distributed in the cytosolic fraction, suggesting that these mutations disrupt the membrane association of RPE65. However, palmitoylation assay showed that wtRPE65 and both of the mutants were palmitoylated. These results suggest that these mutations may result in critical structural alterations of RPE65 protein, disrupt its membrane association, and consequently impair its isomerohydrolase activity, leading to retinal degeneration.