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Molecular Mechanisms of Tubby and Tulp1 Mediated RPE Phagocytosis

Tubby 和 Tulp1 介导的 RPE 吞噬作用的分子机制

基本信息

批准号：
8111580
负责人：
Nora Blanca Caberoy
金额：
$ 9万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2013-07-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：我们最近开发了开放阅读框（ORF）噬菌体展示技术，该技术有可能将酵母双杂交系统和质谱技术结合起来，成为功能蛋白质组学的主要技术。为了探索ORF噬菌体展示的多功能应用，我们使用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，并鉴定了9个推定的eat-me信号，包括tubby样蛋白1（Tulp 1）和tubby。tubby（tubby-DC 44）和Tulp 1（Tulp 1-DC 44）中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-DC 44和Tulp 1-DC 44消除了它们对RPE吞噬作用的刺激。该项目的长期目标是确定tubby和Tulp 1在视网膜变性中的疾病机制。本申请的目的是阐明高度保守的tubby和Tulp 1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Tulp 1是桥接分子，以促进RPE吞噬作用，同时结合到视网膜色素上皮细胞和脱落的感光细胞外节（POS）囊泡上的Mer酪氨酸激酶（MerTK）。这一假设将通过表征的C-末端结构域的tubby和Tulp 1通过囊泡下拉试验和突变分析，以映射其最小域结合到感光细胞外节（POS）进行研究。此外，tubby和Tulp 1的C-末端结构域的功能将通过使用新开发的ORF噬菌体展示技术鉴定它们的蛋白质结合伴侣来定义。本研究不仅有助于阐明tubby和Tulp 1 C端突变的病理机制，而且有助于推广和推广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。公共卫生关系：tubby和Tulp 1高度保守的C-末端结构域的突变与缺陷性RPE吞噬作用和视网膜变性相关。了解tubby和Tulp 1的分子作用将大大增加我们对RPE吞噬作用在视网膜变性中的生理和病理作用的认识。这将为视网膜变性提供新的见解，并将帮助我们制定预防和治疗失明的治疗策略。