Binding between the Niemann-Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain
Binding between the Niemann-Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain
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DOI：
10.1073/pnas.0405255101
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发表时间：
2004-08-24
影响因子：
11.1
通讯作者：
Chang, TY
中科院分区：
文献类型：
--
作者：
Ohgami, N;Ko, DC;Chang, TY
Niemann-Pick C型（NPC）1蛋白在通过囊泡运输将胆固醇和其他脂质移出晚期内体中起重要作用，但不知道NPC 1是否直接与胆固醇相互作用。我们使用[H-3] 7，7-偶氮胆甾烷醇（[H-3]AC）对表达荧光蛋白（FP）标记的NPC 1的完整细胞进行光亲和标记。免疫沉淀后，H-3标记的NPC 1-GFP呈现为单一条带。包括过量的未标记的甾醇的标记反应显着减少了标记。用功能丧失突变（P692 S和Y 635 C）改变NPC 1固醇敏感结构域（SSD）严重降低了标记的程度。为了进一步证明标记的特异性，我们表明，NPC 2，一种晚期内体/溶酶体蛋白，结合胆固醇具有高亲和力，被标记，而突变NPC 2蛋白结合胆固醇无活性。Vamp 7是一种丰富的晚期内体膜蛋白，没有SSD，但有一个跨膜结构域，不能被标记。[H-3]AC和NPC 1之间的结合不需要NPC 2。用U-18666 A（一种产生NPC样表型的化合物）或巴弗洛霉素All（一种提高晚期内体pH的化合物）处理细胞对NPC 1-YFP的标记没有影响，这表明这两种药物影响NPC 1与胆固醇结合以外的过程。我们还开发了一种在体外用[H-3]AC标记NPC 1-YFP的方法，并表明胆固醇比其类似物表胆固醇或5-α-胆甾烷更有效地保护NPC 1-YFP免受标记。总体而言，结果表明NPC 1和偶氮胆甾烷醇之间存在直接结合;该结合不需要NPC 2，但需要NPC 1内的功能性SSD。
Niemann-Pick type C (NPC) 1 protein plays important roles in moving cholesterol and other lipids out of late endosomes by means of vesicular trafficking, but it is not known whether NPC1 directly interacts with cholesterol. We performed photoaffinity labeling of intact cells expressing fluorescent protein (FP)-tagged NPC1 by using [H-3]7,7-azocholestanol ([H-3]AC). After immunoprecipitation, H-3-labled NPC1-GFP appeared as a single band. Including excess unlabeled sterol to the labeling reaction significantly diminished the labeling. Altering the NPC1 sterol-sensing domain (SSD) with loss-of-function mutations (P692S and Y635C) severely reduced the extent of labeling. To further demonstrate the specificity of labeling, we show that NPC2, a late endosomal/lysosomal protein that binds to cholesterol with high affinity, is labeled, whereas mutant NPC2 proteins inactive in binding cholesterol are not. Vamp7, an abundant late endosomal membrane protein without an SSD but with one transmembrane domain, cannot be labeled. Binding between [H-3]AC and NPC1 does not require NPC2. Treating cells with either U-18666A, a compound that creates an NPC-like phenotype, or with bafilomycin All, a compound that raises late endosomal pH, has no effect on labeling of NPC1-YFP, suggesting that both drugs affect processes other than NPC1 binding to cholesterol. We also developed a procedure to label the NPC1-YFP by [H-3]AC in vitro and showed that cholesterol is more effective in protection against labeling than its analogs epicholesterol or 5-alpha-cholestan. Overall, the results demonstrate that there is direct binding between NPC1 and azocholestanol; the binding does not require NPC2 but requires a functional SSD within NPC1.