Molecular mechanisms controlling endocytic recycling

描述（由申请人提供）：受体内化和再循环到质膜的控制是正常细胞功能的核心，这些过程的失调是动脉粥样硬化、糖尿病和癌症等多种疾病的潜在原因。在关键的内吞调节蛋白中，有超过50种rabb - gtp结合蛋白及其效应蛋白控制囊泡运输和融合事件。在过去的五年中，我们专注于了解一个新的内吞调节蛋白家族的作用，称为c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转运中发挥作用，但迄今为止，关于EHD蛋白的实际机制功能的统一假设仍然难以捉摸。eh结构域与含有三肽基序天冬氨酸-脯氨酸-苯丙氨酸（NPF）的蛋白质相互作用。对于这种更新应用最重要的是，我们发现被称为与CasL-Like 1分子相互作用（MICAL-L1）的蛋白质是EHD1的一种新的含有npf的相互作用伙伴。MICAL-L1作为一种不寻常的Rab效应物，因为它对Rab8a和EHD1募集到富含磷脂酰肌醇-4-磷酸和磷脂酰肌醇- 4,5 -二磷酸的管状循环内体至关重要。MICAL-L1的耗竭模拟了EHD1的耗竭，导致质膜回收的延迟和内吞回收室（ERC）内化受体的积累。此外，新的初步数据显示MICAL-L1还与膜弯曲bar结构域蛋白Syndapin II相互作用。总的来说，我们的数据表明MICAL-L1在调节管状核内体的产生和募集EHD1中起关键作用，EHD1似乎负责管状核内体随后的泡化。我们的中心假设是EHD1在管状膜分裂中起关键作用，并促进膜和蛋白质向质膜的再循环。我们的第一个具体目标是确定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，ATP被EHD1水解促进了含有EHD1的小管的囊泡断裂，从而支持了质膜的再循环。我们的第二个特定目标是描述选定的EHD1相互作用伙伴在小管膜生成和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）产生了EHD1被募集到的管状膜。我们进一步假设MICAL-L1通过维持管膜上的EHD1功能在调节EHD1功能中起关键作用。最终，这些研究将促进新策略的发展，以治疗由于异常内吞事件而引起的许多疾病。

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.