Molecular Mechanisms Controlling Endocytic Recycling

描述（由申请人提供）：内吞循环对于控制哺乳动物细胞质膜上的受体是必不可少的。因此，循环利用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的葡萄糖运输，影响健康。因此，了解内吞事件的调控对包括恶性肿瘤、心脏病和糖尿病在内的多种疾病至关重要。内吞运输和再循环是由一些小的Rab gtp结合蛋白控制的。最近，一种名为EHD1的非拉布蛋白被认为在调节回收室的回收中起作用。然而，EHD1与rabb家族蛋白协调其调节活性的模式尚不清楚。生化方法鉴定出二价Rab4/Rab5效应蛋白rabensyn -5是EHD1的结合伙伴，并确定了其在早期核内体循环中的作用。当前的关键任务是了解早期核内体的运输事件如何与回收室的运输事件联系在一起，特别是如何调节早期核内体衍生囊泡与回收室的运输和融合。第一个目标将集中在确定蛋白质从早期核内体转运到内吞循环室的机制，并在通往质膜的途中。工作假设是EHD1和rabensyn -5之间的相互作用对于内化蛋白质从早期核内体运输到回收室至关重要。第二个目标是基于阐明EHD蛋白与Rab11效应蛋白之间的物理联系的新数据，并提出确定EHD蛋白与Rab11和SNARE蛋白协调内吞循环和运输的机制。工作假设是EHD蛋白与Rab11及其效应物协调转运步骤，SNARE蛋白Syntaxin13和SNAP29在回收室早期内核体衍生囊泡融合中发挥关键作用。这些目标将通过来自ehd1敲除小鼠的新型成纤维细胞、基于RNAi的“敲除/敲入”策略以及一系列生化、流式细胞术和显微分析来实现。这些研究将大大提高我们对循环调节机制的基本理解，并对癌症、动脉粥样硬化和糖尿病等多种疾病具有重要意义。

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.