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）：尽管进行了数十年的研究，但调节光感受器外部段组件的机制在很大程度上仍然未知。我的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部段组件。在我们先前对Laevis胚胎视网膜的研究中，我们已经表明，RPE的去除不仅会破坏外部段组装，而且还差异化调节光感受器和Muller细胞的蛋白质表达谱。我们还确定了在培养中添加的因素，可以模仿RPE的存在，从而允许正常的外部段组装和正常的视网膜蛋白表达谱。为了促进并加快划定光感受器外部段组件复杂过程的基础机制，在要求的赠款期内，我们将结合蛋白质的二维（2-D）分离，然后将矩阵辅助激光剂吸收电离时间验证时间 - 飞行时间（MALDI-TOF）质量构造质量图。我们建议使用这些强大的蛋白质组学工具比较视网膜的蛋白质表达谱，即在四个特征良好的实验条件（差异蛋白质组学）下，Xenopus laevis Eyes的视网膜蛋白质组，即视网膜蛋白质组：控制整个RPE-NERERETINAL PROTERETINAL PROTETINAL PROTEMOME；带有杂乱无章的外部细分市场的剥皮视网膜；在存在不可代谢的聚糖的存在下培养的视网膜的，该聚糖支持正常的外部段组装（“允许”聚糖）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2D差异内电泳（2D-DIGE）和EttantWelve多播凝胶系统，将最小化凝胶间的变异性，并提高可重复性。在不同条件下向上或下调的蛋白质将被鉴定出MALDI-TOF MS，通过聚类分析进行分类，随后表征。我们预测：（1）大多数差异调节的蛋白将聚集在三个官能团（细胞粘附分子，细胞骨架蛋白和细胞内信号通路）中； （2）大多数所涉及的蛋白质将在感光细胞和穆勒细胞中表达。因此，我们将使用这些标准来确定对差异调节蛋白的分析。然后，我们将通过使用morpholino基因敲低方法来调节其表达，然后确定其表达方式，然后确定满足外部段组件的必要且足以支持外部段的组装蛋白，然后进行量化外部段组织的量化。我们的方法将使我们能够识别任何功能群和/或新型蛋白质，这些蛋白质足以用于光感受器外部段组装。这些研究还将生成未来项目时期的框架，在该项目期间，将确定控制光感受器外部段组装的精确分子机制和详细途径。