Mechanism of cholesterol transfer from the Niemann-Pick type C2 protein to model membranes supports a role in lysosomal cholesterol transport
Mechanism of cholesterol transfer from the Niemann-Pick type C2 protein to model membranes supports a role in lysosomal cholesterol transport
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DOI：
10.1074/jbc.m602765200
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发表时间：
2006-10-20
影响因子：
4.8
通讯作者：
Storch, Judith
中科院分区：
文献类型：
--
作者：
Cheruku, Sunita R.;Xu, Zhi;Storch, Judith
细胞通过内质网的从头合成或通过受体介导的内吞作用内化含胆固醇的脂蛋白，特别是低密度脂蛋白(LDL)来获得胆固醇。Niemann-Pick C型遗传性疾病(NPC)是由NPC1和NPC2两个编码蛋白质的基因中的任何一个缺陷引起的，在这种疾病中，低密度脂蛋白从内切/溶酶体转运异常导致溶酶体内大量胆固醇和糖脂积聚。NPC2是一种小的溶酶体内蛋白，其生化特征是胆固醇结合蛋白。我们确定了NPC2运送胆固醇来模拟磷脂膜的速度和机制。用荧光去猝灭实验监测了胆固醇从蛋白质到膜的转移动力学。NPC2的内源性色氨酸荧光在与胆固醇结合时被猝灭，随后受体小泡的加入导致色氨酸信号的消亡，从而能够监测胆固醇向膜的转移。胆固醇转移率作为受体囊泡浓度、受体囊泡磷脂头基组成和水相性质的函数进行评估。结果表明，NPC2通过与受体膜直接相互作用的碰撞机制将胆固醇快速转运到磷脂囊泡中。在酸性环境中，胆固醇转移到膜上的速度更快，而且独特的溶酶体/晚期内体磷脂-二磷脂酸(LBPA)(也称为双单酰基甘油磷酸)的存在大大增强了胆固醇向膜的转移。最后，我们发现在供体囊泡中加入溶二磷脂酸后，胆固醇从囊泡到NPC2的转移速率显著增加。这些结果支持NPC2蛋白在将低密度脂蛋白衍生的胆固醇排出内小体/溶酶体小室的过程中发挥作用。
Cells acquire cholesterol either by de novo synthesis in the endoplasmic reticulum or by internalization of cholesterol-containing lipoproteins, particularly low density lipoprotein (LDL), via receptor-mediated endocytosis. The inherited disorder Niemann-Pick type C (NPC), in which abnormal LDL-cholesterol trafficking from the endo/lysosomal compartment leads to substantial cholesterol and glycolipid accumulation in lysosomes, is caused by defects in either of two genes that encode for proteins designated as NPC1 and NPC2. NPC2 is a small intralysosomal protein that has been characterized biochemically as a cholesterol binding protein. We determined the rate and mechanism by which NPC2 delivers cholesterol to model phospholipid membranes. A fluorescence dequenching assay was used to monitor the kinetics of cholesterol transfer from the protein to membranes. The endogenous tryptophan fluorescence of the NPC2 was quenched upon binding of cholesterol, and the subsequent addition of acceptor vesicles resulted in dequenching of the tryptophan signal, enabling the monitoring of cholesterol transfer to membranes. The rates of cholesterol transfer were evaluated as a function of acceptor vesicle concentration, acceptor vesicle phospholipid headgroup composition, and aqueous phase properties. The results suggest that NPC2 rapidly transports cholesterol to phospholipid vesicles via a collisional mechanism which involves a direct interaction with the acceptor membrane. Transfer of cholesterol to membranes is faster in an acidic environment and is greatly enhanced by the presence of the unique lysosomal/late endosomal phospholipid lyso-bisphosphatidic acid (LBPA) (also known as bismonoacylglycerol phosphate). Finally, we found that the rate of transfer of cholesterol from vesicles to NPC2 was dramatically increased by the presence of lyso-bisphosphatidic acid in the donor vesicles. These results support a role for the NPC2 protein in the egress of LDL derived cholesterol out of the endosomal/lysosomal compartment.