PDF(Pubmed)
Abstract：
BACKGROUND: Cholesterol plays a vital role in multiple physiological processes. Cellular uptake of cholesterol is mediated primarily through endocytosis of low-density lipoprotein (LDL) receptor. New modifiers of this process remain to be characterized. Particularly, the role of fasting- and CREB-H-induced (FACI) protein in cholesterol homeostasis merits further investigation.
METHODS: Interactome profiling by proximity labeling and affinity purification - mass spectrometry was performed. Total internal reflection fluorescence microscopy and confocal immunofluorescence microscopy were used to analyze protein co-localization and interaction. Mutational analysis was carried out to define the domain and residues required for FACI localization and function. Endocytosis was traced by fluorescent cargos. LDL uptake in cultured cells and diet-induced hypercholesterolemia in mice were assessed.
RESULTS: FACI interacted with proteins critically involved in clathrin-mediated endocytosis, vesicle trafficking, and membrane cytoskeleton. FACI localized to clathrin-coated pits (CCP) on plasma membranes. FACI contains a conserved DxxxLI motif, which mediates its binding with the adaptor protein 2 (AP2) complex. Disruption of this motif of FACI abolished its CCP localization but didn\'t affect its association with plasma membrane. Cholesterol was found to facilitate FACI transport from plasma membrane to endocytic recycling compartment in a clathrin- and cytoskeleton-dependent manner. LDL endocytosis was enhanced in FACI-overexpressed AML12 cells but impaired in FACI-depleted HeLa cells. In vivo study indicated that hepatic FACI overexpression alleviated diet-induced hypercholesterolemia in mice.
CONCLUSIONS: FACI facilitates LDL endocytosis through its interaction with the AP2 complex.
METHODS: Interactome profiling by proximity labeling and affinity purification - mass spectrometry was performed. Total internal reflection fluorescence microscopy and confocal immunofluorescence microscopy were used to analyze protein co-localization and interaction. Mutational analysis was carried out to define the domain and residues required for FACI localization and function. Endocytosis was traced by fluorescent cargos. LDL uptake in cultured cells and diet-induced hypercholesterolemia in mice were assessed.
RESULTS: FACI interacted with proteins critically involved in clathrin-mediated endocytosis, vesicle trafficking, and membrane cytoskeleton. FACI localized to clathrin-coated pits (CCP) on plasma membranes. FACI contains a conserved DxxxLI motif, which mediates its binding with the adaptor protein 2 (AP2) complex. Disruption of this motif of FACI abolished its CCP localization but didn\'t affect its association with plasma membrane. Cholesterol was found to facilitate FACI transport from plasma membrane to endocytic recycling compartment in a clathrin- and cytoskeleton-dependent manner. LDL endocytosis was enhanced in FACI-overexpressed AML12 cells but impaired in FACI-depleted HeLa cells. In vivo study indicated that hepatic FACI overexpression alleviated diet-induced hypercholesterolemia in mice.
CONCLUSIONS: FACI facilitates LDL endocytosis through its interaction with the AP2 complex.
摘要：
背景：胆固醇在多个生理过程中起着至关重要的作用。胆固醇的细胞摄取主要通过低密度脂蛋白(LDL)受体的内吞作用介导。该过程的新改性剂仍有待表征。特别是,Fasting和CREB-H诱导（FACI）蛋白在胆固醇稳态中的作用值得进一步研究。
方法：通过邻近标记和亲和纯化-进行质谱分析。全内反射荧光显微镜和共聚焦免疫荧光显微镜用于分析蛋白质共定位和相互作用。进行突变分析以确定FACI定位和功能所需的结构域和残基。通过荧光货物追踪细胞增多。评估了培养细胞中LDL的摄取和饮食诱导的小鼠高胆固醇血症。
结果：FACI与网格蛋白介导的内吞作用密切相关的蛋白质相互作用，囊泡贩运，和膜细胞骨架。FACI定位于质膜上网格蛋白涂层的凹坑（CCP）。FACI包含保守的DxxxLI图案，介导其与衔接蛋白2（AP2）复合物的结合。FACI的该基序的破坏取消了其CCP定位，但不影响其与质膜的关联。发现胆固醇以笼状和细胞骨架依赖性方式促进FACI从质膜向内吞再循环室的转运。LDL内吞作用在FACI过表达的AML12细胞中增强，但在FACI耗尽的HeLa细胞中受损。体内研究表明，肝脏FACI过表达减轻了饮食诱导的小鼠高胆固醇血症。
结论：FACI通过与AP2复合物的相互作用促进LDL内吞作用。
方法：通过邻近标记和亲和纯化-进行质谱分析。全内反射荧光显微镜和共聚焦免疫荧光显微镜用于分析蛋白质共定位和相互作用。进行突变分析以确定FACI定位和功能所需的结构域和残基。通过荧光货物追踪细胞增多。评估了培养细胞中LDL的摄取和饮食诱导的小鼠高胆固醇血症。
结果：FACI与网格蛋白介导的内吞作用密切相关的蛋白质相互作用，囊泡贩运，和膜细胞骨架。FACI定位于质膜上网格蛋白涂层的凹坑（CCP）。FACI包含保守的DxxxLI图案，介导其与衔接蛋白2（AP2）复合物的结合。FACI的该基序的破坏取消了其CCP定位，但不影响其与质膜的关联。发现胆固醇以笼状和细胞骨架依赖性方式促进FACI从质膜向内吞再循环室的转运。LDL内吞作用在FACI过表达的AML12细胞中增强，但在FACI耗尽的HeLa细胞中受损。体内研究表明，肝脏FACI过表达减轻了饮食诱导的小鼠高胆固醇血症。
结论：FACI通过与AP2复合物的相互作用促进LDL内吞作用。
