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

• 批准号: 8523889
• 负责人: Nora Blanca Caberoy
• 金额: $ 23.66万
• 依托单位: UNIVERSITY OF NEVADA LAS VEGAS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8523889
• 关键词: Address; Amino Acids; Apoptotic; Binding; Biological; Biological Assay; Biological Process; Biomedical Research; Blindness; C-terminal; C-terminal binding protein; Cells; Cloning; Deletion Mutation; Discipline; Disease; Eating; Goals; Immunohistochemistry; In Situ Hybridization; Knowledge; Maps; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Mentors; Molecular; Mutation; N-terminal; Open Reading Frames; Pathogenesis; Phage Display; Phagocytes; Phagocytosis; Phase; Photoreceptors; Physiological; Protein Binding; Protein Family; Protein S; Protein Tyrosine Kinase; Proteins; Proteomics; Research; Retina; Retinal Degeneration; Role; Signal Transduction; Structure of retinal pigment epithelium; System; Technology; Therapeutic; Trypsin; Vesicle; abstracting; base; deletion analysis; insight; member; new technology; novel; novel strategies; prevent; programs; yeast two hybrid system

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.

项目摘要/摘要： 我们最近开发了开放阅读框（ORF）噬菌体展示技术，该技术具有 有潜力加入酵母双杂交系统和质谱作为功能性检测的主要技术 蛋白质组学。为了探索 ORF 噬菌体展示的多功能应用，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“吃我”信号或吞噬刺激分子 识别出 9 个假定的“吃我”信号，包括管状蛋白 1 (Tulp1) 和管状蛋白。的缺失突变 tuberby (tubby-DC44) 和 Tulp1 (Tulp1-DC44) 的 C 端 44 个氨基酸 (aa) 与视网膜相关 机制不明的退化。此外，tubby-DC44 和 Tulp1-DC44 消除了它们对 RPE 吞噬作用。该项目的长期目标是明确 tubeby 和 Tulp1 的疾病机制 在视网膜变性中。本申请的目的是阐明高度保守的 C- RPE 吞噬作用中 tuberby 和 Tulp1 的末端结构域。这项研究的中心假设是矮胖和 Tulp1 是桥接分子，通过同时结合 Mer 酪氨酸激酶来促进 RPE 吞噬作用 (MerTK) 对 RPE 细胞和​​脱落的光感受器外节 (POS) 囊泡的影响。这个假设将是 通过囊泡下拉分析表征 tubeby 和 Tulp1 的 C 末端结构域进行研究 和突变分析来绘制与光感受器外节（POS）结合的最小结构域。 此外，tubby 和 Tulp1 C 端结构域的功能将通过鉴定其蛋白质来定义 使用新开发的 ORF 噬菌体展示技术结合伴侣。这项研究不仅将阐明 tuberby 和 Tulp1 C 端突变的病理机制，但也将有助于促进和 传播 ORF 噬菌体展示新技术，以其作为一种高效、灵敏、 生物医学研究中功能蛋白质组学的通用且方便的技术。