Molecular Mechanisms of Tubby and Tulp1 Mediated RPE Phagocytosis

描述(申请人提供)：我们最近开发了开放阅读框架(ORF)噬菌体展示技术，这项技术有可能加入酵母双杂交系统和质谱学，成为功能蛋白质组学的主要技术。为了探索ORF噬菌体展示的广泛应用，我们利用功能克隆策略分离了视网膜色素上皮(RPE)细胞中的Eat-me信号或吞噬刺激分子，并鉴定了9个可能的Eat-Me信号，包括Tubby-like Protein 1(Tulp1)和Tubby。Tubby(Tubby-DC44)和Tulp1(Tulp1-DC44)C末端44个氨基酸(AA)的缺失突变与视网膜变性相关，机制不明。此外，Tubby-DC44和Tulp1-DC44取消了它们对RPE吞噬功能的刺激。该项目的长期目标是明确视网膜退行性变中Tubby和Tulp1的发病机制。本应用的目的是阐明Tubby和Tulp1的高度保守的C-末端结构域在RPE吞噬中的作用。这项研究的中心假设是Tubby和Tulp1是桥联分子，通过同时结合RPE细胞上的Mer酪氨酸激酶(MerTK)和脱落的光感受器外段(POS)小泡来促进RPE的吞噬。这一假设将通过囊泡下拉分析和突变分析来表征Tubby和Tulp1的C-末端结构域，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使用新开发的ORF噬菌体展示技术确定其蛋白质结合伙伴来定义。本研究不仅将阐明Tubby和Tulp1C末端突变的病理机制，而且将有助于ORF噬菌体展示技术作为一种高效、灵敏、通用和方便的功能蛋白质组学技术在生物医学研究中的广泛应用而促进和推广这一新技术。 公共卫生相关性：Tubby和Tulp1高度保守的C-末端结构域的突变与RPE吞噬功能缺陷和视网膜变性有关。了解Tubby和Tulp1的分子作用将大大增加我们对RPE吞噬在视网膜变性中的生理和病理作用的了解。这将为视网膜退化提供新的见解，并将帮助我们开发预防和治疗失明的治疗策略。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Mutations in the highly conserved C-terminal domains of tubby and Tulp1 are associated with defective RPE phagocytosis and retinal degeneration. Understanding the molecular role of tubby and Tulp1 will substantially increase our knowledge on the physiological and pathological roles of RPE phagocytosis in retinal degeneration. This will provide novel insights on retinal degeneration and will help us develop therapeutic strategies to prevent and treat blindness.