Molecular Mechanisms of Tubby and Tulp1 Mediated RPE Phagocytosis

项目摘要/摘要： 我们最近开发了开放阅读框架(ORF)噬菌体展示，这项技术具有 加入酵母双杂交系统和质谱学作为一项主要功能技术的潜力 蛋白质组学。为了探索ORF噬菌体展示的广泛应用，我们使用了功能克隆策略 分离视网膜色素上皮(RPE)细胞中的Eat-Me信号或吞噬刺激分子 鉴定了9个可能的Eat-Me信号，包括Tubby-like Protein 1(Tulp1)和Tubby。基因的缺失突变 Tulp1-DC44和Tubby-DC44中的44个氨基酸(AA)与视网膜相关 机制不明确的退化。此外，Tubby-DC44和Tulp1-DC44取消了它们对 RPE吞噬功能。该项目的长期目标是确定Tubby和Tulp1的发病机制 视网膜退行性变。本申请的目的是阐明高度保守的C- RPE吞噬过程中Tubby和Tulp1的末端结构域。这项研究的中心假设是，胖子和 Tulp1是桥联分子，通过同时与Mer酪氨酸激酶结合来促进RPE的吞噬 (MerTK)在RPE细胞和脱落的光感受器外段(POS)小泡上。这一假设将是 通过囊泡下拉实验表征Tubby和Tulp1的C-末端结构域 以及突变分析，以定位它们与光感受器外部片段(POS)结合的最小结构域。 此外，Tubby和Tulp1C-末端结构域的功能将通过鉴定其蛋白质来确定 结合合作伙伴使用新开发的ORF噬菌体展示技术。这项研究不仅将阐明 Tubby和Tulp1C末端突变的病理机制，但也将有助于促进和 ORF噬菌体展示作为一种高效、灵敏、 功能蛋白质组学在生物医学研究中的通用和便捷技术。

Project Summary/Abstract: We have recently developed open-reading-frame (ORF) phage display, a technology which has the potential to join yeast two-hybrid system and mass spectrometry as a major technology of functional proteomics. To explore the versatile applications of ORF phage display, we used a functional cloning strategy to isolate eat-me signals or phagocytosis stimulating molecules in retinal pigment epithelium (RPE) cells and identified 9 putative eat-me signals including tubby-like protein 1 (Tulp1) and tubby. Deletion mutations of the C-terminal 44 amino acids (aa) in tubby (tubby-DC44) and Tulp1 (Tulp1-DC44) associate with retinal degeneration with undefined mechanisms. Moreover, tubby-DC44 and Tulp1-DC44 abolish their stimulation of RPE phagocytosis. The long term goal of this project is to define the disease mechanisms of tubby and Tulp1 in retinal degeneration. The objective of this application is to elucidate the role of the highly conserved C- terminal domain of tubby and Tulp1 in RPE phagocytosis. The central hypothesis of this study is that tubby and Tulp1 are bridging molecules to facilitate RPE phagocytosis by simultaneously binding to Mer tyrosine kinase (MerTK) on RPE cells and the shed photoreceptor outer segments (POS) vesicles. This hypothesis will be investigated by characterizing the C-terminal domains of tubby and Tulp1 through vesicle pull-down assays and mutational analysis to map their minimum domains that bind to photoreceptor outer segments (POS). Moreover, the function of tubby and Tulp1 C-terminal domains will be defined by identifying their protein binding partners using the newly-developed ORF phage display technology. This study will not only elucidate the pathological mechanisms of tubby and Tulp1 C-terminal mutations, but will also help promote and disseminate the new technology of ORF phage display for its broad application as an efficient, sensitive, versatile and convenient technology of functional proteomics in biomedical research.