Molecular Mechanisms Controlling Endocytic Recycling

描述（由申请人提供）：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，回收通过调节关键的细胞事件（例如信号转导和增殖、胆固醇稳态、营养吸收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多种疾病至关重要，包括恶性肿瘤、心脏病和糖尿病。内吞转运和再循环由许多小型 Rab GTP 结合蛋白控制。最近，一种称为 EHD1 的非 Rab 蛋白被认为在调节回收室的回收中发挥着作用。然而，EHD1 与 Rab 家族蛋白协调其调节活性的模式尚不清楚。生化方法已鉴定出二价 Rab4/Rab5 效应蛋白 Rabenosyn-5 作为 EHD1 的结合伴侣，并确定了它在早期内体回收中的作用。当前的关键任务是了解早期内体的运输事件如何与回收室的运输事件相关，特别是调节早期内体衍生囊泡与回收室的运输和融合的因素。第一个目标将重点是确定蛋白质从早期内体转运到内吞回收室，再到达质膜的机制。目前的假设是，EHD1 和 Rabenosyn-5 之间的相互作用对于内化蛋白从早期内体转运到回收室至关重要。第二个目标是基于阐明 EHD 蛋白和 Rab11 效应蛋白之间物理联系的新数据，并提出确定 EHD 蛋白与 Rab11 和 SNARE 蛋白协调内吞回收和运输的机制。工作假设是 EHD 蛋白与 Rab11 及其效应子协调运输步骤，并且 SNARE 蛋白 Syntaxin13 和 SNAP29 在回收室中早期内体衍生囊泡的融合中发挥关键作用。这些目标将通过使用 EHD1 敲除小鼠的新型成纤维细胞、基于 RNAi 的“敲低/敲入”策略以及一系列生化、流式细胞术和显微镜检测来实现。这些研究将显着增强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.