Molecular Mechanisms Controlling Endocytic Recycling

描述(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，循环通过调节关键的细胞事件来影响健康，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。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都是至关重要的。胞内转运和循环是由许多小分子Rab GTP结合蛋白控制的。最近，一种名为EHD1的非Rab蛋白被认为在调节回收隔间的回收方面发挥了作用。然而，EHD1与Rab家族蛋白协调其调节活性的方式尚不清楚。一种生化方法已经确定了二价Rab4/Rab5效应器原，Rabenosyn-5，作为EHD1的结合伙伴，并确定了它在早期内吞体内循环中的作用。手头的关键任务是了解早期内吞体内的贩运事件与回收隔间的贩运事件是如何联系在一起的，特别是是什么调节了早期内体衍生的囊泡与回收隔间的运输和融合。第一个目标将集中在确定蛋白质从早期的内吞体内运输到内吞再循环隔间，再到质膜的机制。工作假说是，EHD1和Rabenosyn-5之间的相互作用对于内化蛋白从早期内体到循环室的运输至关重要。第二个目标是基于新的数据阐明EHD蛋白和Rab11效应蛋白之间的物理联系，并建议确定EHD蛋白与Rab11和SNARE蛋白协调细胞内循环和运输的机制。工作假说是EHD蛋白与Rab11及其效应器协调运输步骤，SNARE蛋白Synaxin13和SNAP29在回收隔室早期内体衍生的囊泡融合中发挥关键作用。这些目标将通过使用来自EHD1基因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、流式细胞术和显微镜分析来实现。这些研究将极大地增强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各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.