Proteomic Analysis of the Retina

视网膜的蛋白质组学分析

• 批准号: 7061195
• 负责人: MONICA M JABLONSKI
• 金额: $ 14.26万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-02 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7061195
• 关键词: Muller' s cell; Xenopus; cell adhesion molecules; cytoskeletal proteins; developmental neurobiology; embryo /fetus; gene expression; genetic manipulation; histogenesis; mass spectrometry; matrix assisted laser desorption ionization; molecular assembly /self assembly; nonmammalian vertebrate embryology; protein purification; protein quantitation /detection; protein signal sequence; proteomics; retina; tissue /cell culture; two dimensional gel electrophoresis; visual photoreceptor

DESCRIPTION (provided by applicant): Despite several decades of studies, the mechanisms that regulate photoreceptor outer segment assembly remain largely unknown. The focus of my laboratory is to understand how the interactions between the retinal pigment epithelium (RPE) and the neural retina regulate outer segment assembly. In our previous studies of the Xenopus laevis embryonic retina, we have shown that removal of the RPE not only disrupts outer segment assembly, but also differentially regulates the protein expression profiles of both photoreceptors and Muller cells. We have also identified factors that, when added to culture, can mimic the presence of the RPE, allowing for normal outer segment assembly and normal retinal protein expression profiles. To facilitate and expedite the delineation of the mechanisms underlying the complex process of photoreceptor outer segment assembly, during the requested grant period, we will incorporate two-dimensional (2-D) separation of proteins followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry into our experimental strategies. We propose to use these powerful proteomic tools to compare the protein expression profiles of the retina, i.e., the retinal proteome, of Xenopus laevis eyes under four well-characterized experimental conditions (differential proteomics): the control whole RPE-neuroretinal proteome; that of RPEdeprived retinas with disorganized outer segments; that of retinas cultured in the presence of a nonmetabolizable glycan that supports normal outer segment assembly ("permissive" glycan) as a positive control; and that of retinas exposed to a "non permissive" glycan, to control for false positives. Inter-gel variability will be minimized and reproducibility enhanced by using 2D Differential In-Gel Electrophoresis (2D-DIGE) and the ETTANtwelve multicasting gel system. Proteins that are up- or downregulated under the different conditions will be identified MALDI-ToF MS, categorized by cluster analysis, and subsequently characterized. We predict that: (1) the majority of the differentially regulated proteins will cluster within three functional groups (cell adhesion molecules, cytoskeletal proteins, and intracellular signaling pathways); and (2) that the majority of the involved proteins will be expressed in photoreceptors and Muller cells. Therefore, we will use these criteria to prioritize our analysis of the differentially regulated proteins. We will then determine if selected target proteins that fulfill these criteria are necessary and sufficient to support outer segment assembly by down regulating their expression using a Morpholino gene knockdown approach followed by quantification of outer segment organization. Our approach will allow us to identify any functional group and/or novel protein that are sufficient for photoreceptor outer segment assembly. These studies will also generate the framework for future project periods in which the precise molecular mechanisms and detailed pathways that control photoreceptor outer segment assembly will be determined.

描述（由申请人提供）：尽管经过了几十年的研究，调节光感受器外节组装的机制仍然很大程度上未知。我实验室的重点是了解视网膜色素上皮 (RPE) 和神经视网膜之间的相互作用如何调节外节组装。在我们之前对非洲爪蟾胚胎视网膜的研究中，我们发现去除 RPE 不仅会破坏外节的组装，还会差异性地调节光感受器和 Muller 细胞的蛋白质表达谱。我们还发现了一些因素，当添加到培养物中时，可以模拟 RPE 的存在，从而实现正常的外节组装和正常的视网膜蛋白表达谱。为了促进和加快描述光感受器外节组装复杂过程的机制，在请求的资助期内，我们将结合蛋白质的二维（2-D）分离，然后进行基质辅助激光解吸电离时间-飞行（MALDI-ToF）质谱分析进入我们的实验策略。我们建议使用这些强大的蛋白质组学工具来比较非洲爪蟾眼睛视网膜的蛋白质表达谱，即视网膜蛋白质组，在四种充分表征的实验条件下（差异蛋白质组学）：对照整个 RPE-神经视网膜蛋白质组；视网膜色素上皮（RPE）缺失，外部节段混乱；在支持正常外节组装（“许可”聚糖）的不可代谢聚糖存在下培养的视网膜作为阳性对照；以及暴露于“非允许”聚糖的视网膜，以控制假阳性。通过使用二维微分凝胶内电泳 (2D-DIGE) 和 ETTANtwelve 多播凝胶系统，可以最大限度地减少凝胶间的变异性并提高重现性。在不同条件下上调或下调的蛋白质将被 MALDI-ToF MS 识别，通过聚类分析进行分类，并随后进行表征。我们预测：（1）大多数差异调节蛋白将聚集在三个功能组（细胞粘附分子、细胞骨架蛋白和细胞内信号通路）内； (2)大多数相关蛋白质将在光感受器和米勒细胞中表达。因此，我们将使用这些标准来优先分析差异调节的蛋白质。然后，我们将使用 Morpholino 基因敲低方法下调其表达，然后量化外节组织，从而确定满足这些标准的所选靶蛋白是否必要且足以支持外节组装。我们的方法将使我们能够识别足以进行光感受器外节组装的任何官能团和/或新型蛋白质。这些研究还将为未来的项目阶段构建框架，其中将确定控制光感受器外节组装的精确分子机制和详细途径。