Molecular Mechanisms Controlling Endocytic Recycling

描述（由申请方提供）：内吞再循环对于控制哺乳动物细胞质膜上的受体至关重要。因此，再循环通过调节关键的细胞事件如信号转导和增殖、胆固醇稳态、营养摄取和胰岛素依赖性葡萄糖转运来影响健康。因此，了解内吞事件的调节对于包括恶性肿瘤、心脏病和糖尿病在内的多种疾病至关重要。内吞转运和再循环由许多小的Rab GTP结合蛋白控制。最近，一种称为EHD 1的非Rab蛋白被认为在调节再循环室的再循环中起作用。然而，EHD 1与Rab-family蛋白协调其调节活性的模式尚不清楚。一种生物化学方法已经鉴定了二价Rab 4/Rab 5效应蛋白Rabenosyn-5作为EHD 1的结合伴侣，并确定了它在早期内体再循环中的作用。目前的关键任务是了解早期内体的运输事件如何与再循环区室的运输事件相关联，特别是调节早期内体衍生的囊泡与再循环区室的运输和融合的因素。第一个目标将集中于确定蛋白质从早期内体转运到内吞再循环区室，途中到质膜的机制。工作假设是EHD 1和Rabenosyn-5之间的相互作用对于将内化的蛋白质从早期内体转运到再循环隔室是至关重要的。第二个目标是基于新的数据阐明EHD蛋白和Rab 11效应蛋白之间的物理连接，并提出确定EHD蛋白与Rab 11和SNARE蛋白协调内吞回收和运输的机制。工作假设是EHD蛋白与Rab 11及其效应物协调运输步骤，并且SNARE蛋白Syntaxin 13和SNAP 29在回收室的早期内体衍生囊泡的融合中起关键作用。这些目标将使用来自EHD 1敲除小鼠的新型成纤维细胞、基于RNAi的“敲低/敲入”策略以及一系列生物化学、流式细胞术和显微镜测定来实现。这些研究将大大提高我们对调节循环机制的基本理解，并对癌症、动脉粥样硬化和糖尿病等多种疾病产生重要影响。

DESCRIPTION (provided by applicant): Endocytic recycling is essential for the control of receptors on the plasma membrane in mammalian cells. Consequently, recycling impacts health by regulating crucial cellular events such as signal transduction and proliferation, cholesterol homeostasis, nutrient uptake, and insulin-dependent glucose transport. Thus, understanding the regulation of endocytic events is critical for a wide range of diseases, including malignancies, heart disease and diabetes. Endocytic transport and recycling are controlled by a number of small Rab GTP-binding proteins. Recently, a non-Rab protein called EHD1 has been ascribed a role in regulating recycling at the recycling compartment. However, the mode by which EHD1 coordinates its regulatory activity with Rab-family proteins is not understood. A biochemical approach has identified the divalent Rab4/Rab5 effector prtoein, Rabenosyn-5, as a binding partner for EHD1, and defined a role for it in recycling at the early endosome. The critical task at hand is to understand how trafficking events at the early endosome are linked to those at the recycling compartment, particularly what regulates transport and fusion of early endosome-derived vesicles with the recycling compartment. The first aim will focus on identifying the mechanisms by which proteins are transported from early endosomes to the endocytic recycling compartment, en route to the plasma membrane. The working hypothesis is that the interaction between EHD1 and Rabenosyn-5 is critical for transport of internalized proteins from early endosomes to the recycling compartment. The second aim is based on new data elucidating a physical connection between EHD proteins and a Rab11 effector protein, and proposes to determine the mechanisms by which EHD proteins coordinate endocytic recycling and transport with Rab11 and SNARE proteins. The working hypothesis is that EHD proteins coordinate transport steps with Rab11 and its effectors, and that the SNARE proteins Syntaxin13 and SNAP29 play a critical role in fusion of early endosome-derived vesicles at the recycling compartment. These aims will be accomplished using novel fibroblasts from EHD1-knock-out mice, RNAi- based 'knock-down/knock-in' strategy, and a series of biochemical, flow cytometry and microscopic assays. These studies will significantly enhance our fundamental understanding of the mechanisms regulating recycling and have an important bearing on diseases as diverse as cancer and atherosclerosis and diabetes.