Molecular Mechanisms of Tubby and Tulp1 Mediated RPE Phagocytosis

项目概要/摘要： 我们最近开发了开放阅读框（ORF）噬菌体展示技术， 有潜力加入酵母双杂交系统和质谱作为功能的主要技术 蛋白质组学为了探索ORF噬菌体展示的广泛应用，我们使用了功能性克隆策略 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， 确定了9个假定的eat-me信号，包括tubby样蛋白1（Tulp 1）和tubby。基因的缺失突变 tubby（tubby-DC 44）和Tulp 1（Tulp 1-DC 44）中的C末端44个氨基酸（aa）与视网膜相关 不确定机制的退化。此外，tubby-DC 44和Tulp 1-DC 44消除了它们对 RPE吞噬作用。本项目的长期目标是确定tubby和Tulp的发病机制1 视网膜退化本申请的目的是阐明高度保守的C- Tubby和Tulp 1末端结构域在RPE吞噬中作用。这项研究的中心假设是，tubby和 Tulp 1是通过同时结合Mer酪氨酸激酶促进RPE吞噬的桥接分子 （MerTK）对RPE细胞和脱落的光感受器外节（POS）囊泡的作用。这一假设将是 通过囊泡下拉试验表征tubby和Tulp 1的C-末端结构域进行研究 和突变分析以绘制它们与光感受器外节（POS）结合的最小结构域。 此外，tubby和Tulp 1 C-末端结构域的功能将通过鉴定它们的蛋白质来确定 结合伙伴使用新开发的ORF噬菌体展示技术。这项研究不仅将阐明 tubby和Tulp 1 C末端突变的病理机制，但也将有助于促进和 推广ORF噬菌体展示新技术，使其作为一种高效、灵敏、 功能蛋白质组学是生物医学研究中一种通用、便捷的技术。

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.