EH DOMAIN IN COMPLEX WITH NPF MOTIF

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 内吞转运是控制游离胆固醇亚细胞分布的关键机制，而内吞再循环室（ERC）是储存胆固醇并调节其转运的重要细胞器。Rab 11通过ERC调节运输，调节胆固醇从该细胞器的退出。EHD 1是C-末端EH-结构域蛋白家族的成员，通过Rab 11途径调节再循环和协调转运，也有报道在通过ERC的游离胆固醇转运中发挥作用。为了直接评估EHD 1对胆固醇稳态和细胞分布的作用，我们利用来自EHD 1敲除小鼠（MEF -/-）的小鼠胚胎成纤维细胞。令人惊讶的是，这些细胞显示出细胞游离胆固醇和酯化胆固醇水平降低，这种效果可以通过野生型EHD 1的过表达来挽救。为了了解在缺乏EHD 1的情况下细胞内胆固醇的减少，我们将注意力转向低密度脂蛋白（LDL）及其受体LDLR，这是细胞胆固醇摄入的主要来源。我们在MEF -/-细胞的质膜上观察到更高水平的LDLR，但LDL本身在这些细胞中以较慢的速率内化。此外，在缺乏EHD 1的细胞中，脂滴的大小似乎大大减少，这表明脂滴中包装的酯化胆固醇和甘油三酯较少。双杂交结合试验和NMR光谱表明，EH-域的EHD 1相互作用与Epsin，一个很好的特点，包含NPF基序和调节受体内化的内吞途径的组成部分。我们的数据表明EHD 1通过控制LDL受体的内化，可能通过与含NPF的内吞调节因子如epsin相互作用，影响胆固醇稳态和脂滴生物合成。

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.