Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate.
Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate.
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DOI：
10.1007/s00417-011-1856-9
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发表时间：
2012-03
影响因子：
2.7
通讯作者：
Stitt, Alan W.
中科院分区：
文献类型：
--
作者：
Glenn, J. V.;Mahaffy, H.;Dasari, S.;Oliver, M.;Chen, M.;Boulton, M. E.;Xu, H.;Curry, W. J.;Stitt, Alan W.
视网膜色素上皮(RPE)和Bruchs膜在衰老过程中经历了显著的变化。细胞-基质界面的晚期糖基化终末产物(AGEs)对脂质和蛋白质的渐进性、年龄相关的修饰与RPE功能障碍和老年性黄斑变性(AMD)的进展有关。Bruch膜中这些加合物的致病性质及其对覆盖的RPE的影响尚不清楚。本研究旨在确定暴露于AGE修饰基底膜(AGE-BM)的细胞中RPE蛋白表达的变化，确定这种“老化”底物如何影响RPE功能，并定位已识别的蛋白质在老化视网膜中的定位。将融合的ARPE-19单层培养在AGE-BM和天然、未修饰的BM上。孵育28天后，用双向凝胶电泳法(2D)分析蛋白质组，用密度计量学和图像分析技术对电喷雾电离质谱仪(ESIMS/MS)鉴定的目的蛋白质进行图谱分析。用免疫细胞化学方法定位培养在未修饰和AGE-BM上的ARPE-19单层中已鉴定的蛋白质，并分析老化的人视网膜。图像分析检测到处理组之间蛋白质斑点密度的变化，并通过LC ESI MS/MS鉴定了感兴趣的蛋白质，包括热休克蛋白、细胞骨架和代谢调节因子。免疫细胞化学显示去泛素化酶泛素羧基末端水解酶-1(UCH-L1)在接触AGE的RPE中表达上调，在人视网膜切片中也定位于RPE。本研究表明，基底膜的AGE修饰改变了RPE蛋白质组。在这种老化模型中，许多蛋白质发生了变化，包括UCHL-1，这可能会影响RPE的降解能力。S膜上AGEs的积聚可能在增龄性视网膜色素上皮功能障碍中起重要作用。
The retinal pigment epithelium (RPE) and underlying Bruch’s membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell–substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch’s membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this “aged” substrate impacts RPE function and to map the localisation of identified proteins in ageing retina. Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina. Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections. This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch”s membrane could play a significant role in age-related dysfunction of the RPE.
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DOI：
10.1007/bf00941767
发表时间：
1997-04-01
影响因子：
2.7
作者：
Loeffler, KU;Mangini, NJ
通讯作者：
Mangini, NJ
影响因子：
4.8
作者：
Ko, HS;Uehara, T;Nomura, Y
通讯作者：
Nomura, Y
DOI：
10.1073/pnas.222551899
发表时间：
2002-11-12
影响因子：
11.1
作者：
Crabb, JW;Miyagi, M;Hollyfield, JG
通讯作者：
Hollyfield, JG
影响因子：
7.3
作者：
LOWE, J;MCDERMOTT, H;WILKINSON, KD
通讯作者：
WILKINSON, KD
影响因子：
4.1
作者：
Chen, YS;Hackett, SF;Campochiaro, PA
通讯作者：
Campochiaro, PA