EH DOMAIN IN COMPLEX WITH NPF MOTIF

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 胞内转运是控制游离胆固醇在细胞内分布的关键机制，而胞内循环室是储存胆固醇并调节其转运的重要细胞器。Rab11通过ERC调节转运，调节胆固醇从这个细胞器中排出。EHD1是C末端EH结构域蛋白家族的成员，通过Rab11途径调节循环和协调运输，也有报道称EHD1在通过ERC的游离胆固醇运输中发挥作用。为了直接评估EHD1在胆固醇稳态和细胞分布中的作用，我们使用了来自EHD1基因敲除小鼠的小鼠胚胎成纤维细胞(MEF-/-)。令人惊讶的是，这些细胞显示出细胞内游离胆固醇和酯化胆固醇水平的降低，这一效应可以通过过度表达野生型EHD1来挽救。为了了解在没有EHD1的情况下细胞内胆固醇的减少，我们将重点转向低密度脂蛋白(LDL)及其受体LDLR，这是细胞胆固醇摄取的主要来源。我们观察到MEF-/-细胞质膜上的低密度脂蛋白受体水平较高，但低密度脂蛋白本身在这些细胞中的内化速度较慢。此外，在缺乏EHD1的细胞中，脂滴的大小似乎大大减小，这表明脂滴中的酯化胆固醇和甘油三酯较少。双杂交结合分析和核磁共振波谱表明，EHD1的EH结构域与Epsin相互作用，Epsin是内吞途径中一个具有良好特征的成分，包含NPF基序并调节受体内化。我们的数据表明，EHD1通过控制低密度脂蛋白受体的内化而影响胆固醇的动态平衡和脂滴的生物发生，可能是通过与含有NPF的内吞调节因子(如epsin)相互作用来实现的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Endocytic transport is a key mechanism for controlling the subcellular distribution of free cholesterol and the endocytic recycling compartment (ERC) is an important organelle that stores cholesterol and regulates its transport. Rab11, which regulates transport via the ERC, regulates the exit of cholesterol from this organelle. EHD1, a member of the C-terminal EH-domain family of proteins that regulates recycling and coordinates transport via the Rab11 pathway, has also been reported to play a role in free cholesterol transport through the ERC. To directly assess the role of EHD1 on cholesterol homeostasis and cellular distribution, we utilized mouse embryonic fibroblasts derived from EHD1 knockout mice (MEF -/-). Surprisingly, these cells displayed reduced levels of cellular free and esterified cholesterol, an effect that could be rescued by overexpression of wild-type EHD1. To understand the reduction in intracellular cholesterol in the absence of EHD1, we turned our focus to low density lipoprotein (LDL) and its receptor, LDLR, a major source of cellular cholesterol intake. We observed higher levels of LDLR on the plasma membrane of MEF -/- cells, yet LDL itself was internalized at a slower rate in these cells. Furthermore, in cells lacking EHD1, lipid droplets appeared greatly reduced in size, suggesting that less esterified cholesterol and triglycerides were packaged in lipid droplets. Two-hybrid binding assays and NMR spectroscopy suggest that the EH-domain of EHD1 interacts with Epsin, a well-characterized component of the endocytic pathway that contains NPF motifs and regulates receptor internalization. Our data indicates EHD1 affects cholesterol homeostasis and lipid droplet biogenesis by controlling internalization of LDL receptor, possibly through interactions with NPF-containing endocytic regulators such as epsin.