Molecular mechanisms controlling endocytic recycling

描述(申请人提供)：控制受体内化和质膜再循环是正常细胞功能的核心，而这些过程的失调是导致动脉粥样硬化、糖尿病和癌症等各种疾病的根本原因。在关键的内吞调节蛋白中，有50多种Rab-GTP结合蛋白及其效应物，它们控制着囊泡的运输和融合事件。在过去的五年里，我们一直专注于了解一个新的内吞调节蛋白家族的作用，被称为C-末端Eps15同源结构域蛋白(EHD)。尽管越来越多的研究表明EHD蛋白在调节多种受体的运输中所起的作用，但到目前为止，对于EHD蛋白的实际机制功能的统一假说仍然难以捉摸。EH结构域与含有天冬酰胺-脯氨酸-苯丙氨酸(NPF)三肽基序的蛋白质相互作用。对于这次更新应用，最重要的是，我们发现与CASL-like 1相互作用的分子(Mical-L1)是EHD1的一个新的含NPF的相互作用伙伴。Mical-L1是一种特殊的Rab效应器，因为它对Rab8a和EHD1在富含磷脂酰肌醇-4-磷酸和磷脂酰肌醇-4，5-二磷酸的管状循环内小体的募集至关重要。Mical-L1的耗尽模拟EHD1的耗尽，导致质膜再循环的延迟和内胞再循环室(ERC)内化受体的积累。此外，新的初步数据表明，Mical-L1还与膜弯曲的杆状结构域蛋白Syndapin II相互作用。总的来说，我们的数据表明Mical-L1在调节管状内小体的生成和招募EHD1方面发挥了关键作用，而EHD1似乎对随后的囊泡形成负责。我们的中心假设是，EHD1在管膜断裂中起关键作用，并促进膜和蛋白质向质膜的循环。我们的第一个特定目标是确定EHD1在小管膜断裂和再循环中的作用机制。我们的工作假设是，EHD1对ATP的水解促进了囊泡从含有EHD1的小管上分离出来，从而支持回收到质膜上。我们的第二个特定目标是研究选择的EHD1相互作用伙伴在小管膜形成和EHD1功能调节中的作用。我们假设含有BAR结构域的EHD1相互作用伙伴(如Syndapin II和/或BIN1)产生招募EHD1的管状膜。我们进一步假设，Mical-L1通过维持EHD1在小管膜上的功能，在调节EHD1功能中发挥关键作用。最终，这些研究将促进新策略的开发，以治疗由于异常内吞事件而产生的许多疾病。 公共卫生相关性：控制受体定位到质膜是正常细胞功能的核心，而调节失调是导致动脉粥样硬化、糖尿病和癌症等各种疾病的根本原因。全面了解控制膜形成、转运和分裂的机制是至关重要的，并可能导致新的治疗靶点的开发。

DESCRIPTION (provided by applicant): Control of receptor internalization and recycling to the plasma membrane is central to normal cell function, and dysregulation of these processes is the underlying cause for diseases as diverse as atherosclerosis, diabetes, and cancer. Among the key endocytic regulatory proteins are more than 50 Rab-GTP binding proteins and their effectors that control vesicle transport and fusion events. Over the past five years, we have focused on understanding the role of a novel endocytic regulatory protein family, known as the C-terminal Eps15 Homology Domain proteins (EHD). Despite a growing number of studies demonstrating roles for EHD proteins in the regulation of transport of a variety of receptors, thus far a unifying hypothesis for the actual mechanistic function of EHD proteins has remained elusive. EH-domains interact with proteins containing the tripeptide motif asparagine-proline- phenylalanine (NPF). Most significantly for this renewal application, we have discovered that the protein known as Molecule Interacting with CasL-Like 1 (MICAL-L1) is a novel NPF-containing interaction partner of EHD1. MICAL-L1 acts as unusual Rab effectors because it is critical for the recruitment of both Rab8a and EHD1 to tubular recycling endosomes that are enriched in phosphatidylinositol-4-phosphate and phosphatidylinositol-4, 5-bisphosphate. Depletion of MICAL-L1 simulates EHD1 depletion, causing a delay in recycling to the plasma membrane and an accumulation of internalized receptors at the endocytic recycling compartment (ERC). Moreover, new preliminary data show that MICAL-L1 also interacts with the membrane-bending BAR-domain protein, Syndapin II. Collectively, our data indicate a key role for MICAL-L1 in regulating the generation of tubular endosomes, and in recruiting EHD1, which appears to be responsible for their subsequent vesiculation. Our central hypothesis is that EHD1 plays a critical role in tubular membrane scission and facilitates recycling of membrane and proteins to the plasma membrane. Our first specific aim is to determine the mechanism by which EHD1 functions in tubule membrane scission and recycling. Our working hypothesis is that ATP hydrolysis by EHD1 promotes scission of vesicles from EHD1- containing tubules, thus supporting recycling to the plasma membrane. Our second Specific Aim is to characterize the roles of select EHD1 interaction partners in tubule membrane generation and in the modulation of EHD1 function. We hypothesize that BAR-domain-containing EHD1 interaction partners (such as Syndapin II and/or Bin1) generate the tubular membranes to which EHD1 is recruited. We further hypothesize that MICAL-L1 plays a crucial role in regulating EHD1 function by maintaining it on tubular membranes. Ultimately, these studies will facilitate the development of new strategies to treat the many diseases that arise as a result of aberrant endocytic events. PUBLIC HEALTH RELEVANCE: Control of receptor localization to the plasma membrane is central to normal cell function, and dysregulation is the underlying cause for diseases as diverse as atherosclerosis, diabetes and cancer. A comprehensive understanding of the mechanisms that control membrane tubulation, transport and fission is of fundamental importance and will likely lead to the development of new therapeutic targets.