Retinal pigment epithelium lipofuscin proteomics
Retinal pigment epithelium lipofuscin proteomics
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DOI：
10.1074/mcp.m700525-mcp200
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发表时间：
2008-07-01
影响因子：
7
通讯作者：
Crabb, John W.
中科院分区：
文献类型：
--
作者：
Ng, Kwok-Peng;Gugiu, Bogdan;Crabb, John W.
随着年龄的增长，脂褐素在视网膜色素上皮（RPE）中以离散的颗粒细胞器积累，并可能导致年龄相关性黄斑变性。由于以往的研究表明，脂褐素包含的蛋白质，可能会影响致病机制，我们追求脂褐素的蛋白质组学分析。RPE脂褐素的组成及其发病机制的部分原因是由于分离制剂的异质性而知之甚少。我们纯化的RPE脂褐素颗粒与蛋白酶K或SDS的处理，并通过光，共聚焦，和透射电子显微镜显示，纯化的颗粒是免费的颗粒外材料和相关的膜。通过（i）LC MS/MS蛋白质组学分析，（ii）氧化蛋白质修饰的免疫分析，（iii）氨基酸分析，（iv）双视黄酸的HPLC，和（v）测定对RPE细胞的光毒性，定量比较粗品和纯化的脂褐素制剂。从粗脂褐质制剂中鉴定出186种蛋白质，其中许多似乎被修饰。相比之下，在纯化的颗粒中发现非常少的蛋白质（通过氨基酸分析类似于2%（w/w））并且没有可识别的蛋白质，其保留对培养的RPE细胞的完全光毒性。我们的分析表明，在纯化和粗脂褐质制剂颗粒表现出没有统计学上的显着差异，直径或圆度或在含量的bisretinoids A2 E，isoA 2 E，和全反式视网膜二聚体磷脂酰乙醇胺。纯化的颗粒含有最少的蛋白质，但保留光毒性活性的发现表明，RPE脂褐质的发病机制在很大程度上是独立的相关蛋白质。纯化的颗粒还表现出氧化蛋白质修饰，包括由反应性氮氧化物物质产生的硝基酪氨酸和由反应性脂质片段产生的羧乙基吡咯和异[4]左旋酮E-2加合物。这一发现与先前的研究一致，证明RPE脂褐素是活性氧物质的有效产生者，并支持这样的物质，包括来自脂质和类维生素A的反应性片段，有助于RPE脂褐素发病机制的假设。
Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein (similar to 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4] levuglandin E-2 adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.