Jelinek David, Heidenreich Randy A, Orlando Robert A, Garver William S
Department of Biochemistry and Molecular Biology, The University of New Mexico Health Sciences Center, Albuquerque, New Mexico, US.
Department of Pediatrics, School of Medicine, The University of New Mexico Health Sciences Center, Albuquerque, New Mexico, US.
J Mol Biochem. 2014 Feb 28;3(1):14-26.
The Niemann-Pick C1 (NPC1) protein has a central role in regulating the efflux of lipoprotein-derived cholesterol from late endosomes/lysosomes and transport to other cellular compartments. Since the NPC1 protein has been shown to regulate the transport of cholesterol to cellular compartments enriched with the ubiquitous cholesterol-binding and transport protein caveolin-1, the present study was performed to determine whether the NPC1 and caveolin-1 proteins interact and function to modulate efflux of low density lipoprotein (LDL)-derived cholesterol from endocytic compartments. To perform these studies, normal human fibroblasts were grown in media with lipoprotein-deficient serum (LPDS) or media with LPDS supplemented with purified human LDL. The results indicated reciprocal co-immunoprecipitation and partial co-localization of the NPC1 and caveolin-1 proteins that was decreased when fibroblasts were grown in media with LDL. Consistent with interaction of the NPC1 and caveolin-1 proteins, a highly conserved caveolin-binding motif was identified within a cytoplasmic loop located adjacent to the sterol-sensing domain (SSD) of the NPC1 protein. To examine the functional relevance of this interaction, fibroblasts were transfected with caveolin-1 siRNA and found to accumulate increased amounts of LDL-derived cholesterol within late endosomes/ lysosomes. Together, this report presents novel results demonstrating that the NPC1 and caveolin-1 proteins interact to modulate efflux of LDL-derived cholesterol from late endocytic compartments.
尼曼-皮克C1（NPC1）蛋白在调节晚期内体/溶酶体中脂蛋白衍生胆固醇的流出并将其转运至其他细胞区室方面发挥着核心作用。由于NPC1蛋白已被证明可调节胆固醇向富含普遍存在的胆固醇结合和转运蛋白小窝蛋白-1的细胞区室的转运，因此进行了本研究以确定NPC1和小窝蛋白-1蛋白是否相互作用并发挥功能，从而调节内吞区室中低密度脂蛋白（LDL）衍生胆固醇的流出。为了进行这些研究，将正常人成纤维细胞培养在含脂蛋白缺乏血清（LPDS）的培养基中，或培养在含LPDS并补充纯化人LDL的培养基中。结果表明，NPC1和小窝蛋白-1蛋白存在相互免疫共沉淀和部分共定位现象，当成纤维细胞在含LDL的培养基中生长时，这种现象会减弱。与NPC1和小窝蛋白-1蛋白的相互作用一致，在NPC1蛋白的固醇感应结构域（SSD）附近的一个细胞质环内鉴定出了一个高度保守的小窝蛋白结合基序。为了研究这种相互作用的功能相关性，用小窝蛋白-1的小干扰RNA（siRNA）转染成纤维细胞，发现晚期内体/溶酶体内LDL衍生胆固醇的积累量增加。总之，本报告呈现了新的结果，证明NPC1和小窝蛋白-1蛋白相互作用以调节晚期内吞区室中LDL衍生胆固醇的流出。
