Department of Biochemistry, Stanford University School of Medicine, Stanford, United States.
Department of Chemistry, Technische Universität Berlin, Berlin, Germany.
Elife. 2020 May 15;9:e57089. doi: 10.7554/eLife.57089.
Transport of LDL-derived cholesterol from lysosomes into the cytoplasm requires NPC1 protein; NPC1L1 mediates uptake of dietary cholesterol. We introduced single disulfide bonds into NPC1 and NPC1L1 to explore the importance of inter-domain dynamics in cholesterol transport. Using a sensitive method to monitor lysosomal cholesterol efflux, we found that NPC1's N-terminal domain need not release from the rest of the protein for efficient cholesterol export. Either introducing single disulfide bonds to constrain lumenal/extracellular domains or shortening a cytoplasmic loop abolishes transport activity by both NPC1 and NPC1L1. The widely prescribed cholesterol uptake inhibitor, ezetimibe, blocks NPC1L1; we show that residues that lie at the interface between NPC1L1's three extracellular domains comprise the drug's binding site. These data support a model in which cholesterol passes through the cores of NPC1/NPC1L1 proteins; concerted movement of various domains is needed for transfer and ezetimibe blocks transport by binding to multiple domains simultaneously.
将 LDL 衍生的胆固醇从溶酶体转运到细胞质需要 NPC1 蛋白；NPC1L1 介导膳食胆固醇的摄取。我们在 NPC1 和 NPC1L1 中引入了单个二硫键，以探讨在胆固醇转运过程中结构域间动力学的重要性。使用一种灵敏的方法来监测溶酶体胆固醇外排，我们发现 NPC1 的 N 端结构域不需要从蛋白质的其余部分释放，就可以有效地进行胆固醇输出。引入单个二硫键来限制腔/细胞外结构域，或缩短细胞质环，都会使 NPC1 和 NPC1L1 的转运活性丧失。广泛使用的胆固醇摄取抑制剂依折麦布会抑制 NPC1L1；我们发现位于 NPC1L1 的三个细胞外结构域之间的界面的残基构成了药物的结合位点。这些数据支持这样一种模型，即胆固醇穿过 NPC1/NPC1L1 蛋白的核心；各种结构域的协调运动对于转移是必要的，依折麦布通过同时与多个结构域结合来阻断转运。
