Purified NPC1 protein - I. Binding of cholesterol and oxysterols to a 1278-amino acid membrane protein
Purified NPC1 protein - I. Binding of cholesterol and oxysterols to a 1278-amino acid membrane protein
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DOI：
10.1074/jbc.m707943200
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发表时间：
2008-01-11
影响因子：
4.8
通讯作者：
Goldstein, Joseph L.
中科院分区：
文献类型：
--
作者：
Infante, Rodney E.;Abi-Mosleh, Lina;Goldstein, Joseph L.
尼曼-匹克C1型蛋白（NPC 1）是将脂蛋白衍生的胆固醇从溶酶体转运到内质网所必需的。该1278个氨基酸的多位膜蛋白尚未纯化，其作用机制尚不清楚。出乎意料的是，我们在寻找结合25-羟基胆固醇（25-HC）和其他氧化固醇的膜蛋白时遇到了NPC 1。从兔肝膜中纯化了超过14，000倍的25-HC结合蛋白，并通过质谱鉴定为NPC 1。我们制备了重组人NPC 1，并证实其结合氧固醇的能力，包括在24、25或27位上具有羟基的那些。7、19或20位上的羟基不能赋予结合。重组人NPC 1也结合[H-3]胆固醇的反应抑制Nonidet P-40高于其临界胶束浓度。低浓度的未标记的25-HC消除了[H-3]胆固醇的结合，但匡威则不然，即即使在高浓度下，未标记的胆固醇也不会消除[H-3]25-HC的结合。NPC 1不需要氧固醇的已知调节作用。因此，在NPC 1缺陷的成纤维细胞中，25-HC阻断了固醇调节元件结合蛋白的加工，并以正常方式激活酰基辅酶A：胆固醇酰基转移酶。测定NPC 1体外结合的可用性可能会进一步了解氧化固醇调节细胞内脂质转运的方式。
The Niemann-Pick, Type C1 protein (NPC1) is required for the transport of lipoprotein-derived cholesterol from lysosomes to endoplasmic reticulum. The 1278-amino acid, polytopic membrane protein has not been purified, and its mechanism of action is unknown. Unexpectedly, we encountered NPC1 in a search for a membrane protein that binds 25-hydroxycholesterol (25-HC) and other oxysterols. A 25-HC-binding protein was purified more than 14,000-fold from rabbit liver membranes and identified as NPC1 by mass spectroscopy. We prepared recombinant human NPC1 and confirmed its ability to bind oxysterols, including those with a hydroxyl group on the 24, 25, or 27 positions. Hydroxyl groups on the 7, 19, or 20 positions failed to confer binding. Recombinant human NPC1 also bound [H-3] cholesterol in a reaction inhibited by Nonidet P-40 above its critical micellar concentration. Low concentrations of unlabeled 25-HC abolished binding of [H-3] cholesterol, but the converse was not true, i.e. unlabeled cholesterol, even at high concentrations, did not abolish binding of [H-3]25-HC. NPC1 is not required for the known regulatory actions of oxysterols. Thus, in NPC1-deficient fibroblasts 25-HC blocked the processing of sterol regulatory element-binding proteins and activated acyl-CoA: cholesterol acyltransferase in a normal fashion. The availability of assays to measure NPC1 binding in vitro may further the understanding of ways in which oxysterols regulate intracellular lipid transport.