Synthesis and characterization of diazirine alkyne probes for the study of intracellular cholesterol trafficking
Synthesis and characterization of diazirine alkyne probes for the study of intracellular cholesterol trafficking
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DOI：
10.1194/jlr.d091470
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发表时间：
2019-03-01
影响因子：
6.5
通讯作者：
Schaffer, Jean E.
中科院分区：
文献类型：
--
作者：
Feltes, McKenna;Moores, Samantha;Schaffer, Jean E.
胆固醇是细胞膜的基本结构成分，也是合成甾醇、胆汁酸和激素的前体分子。细胞内胆固醇运输途径的研究受到限制，部分原因是缺乏合适的胆固醇类似物。在此，我们开发了三种新型的重氮嘧啶炔胆固醇探针：LKM38， KK174和KK175。我们评估了这些探针以及先前描述的二氮嘧啶炔胆固醇类似物反式甾醇，因为它们作为胆固醇模拟物的保真度和胆固醇运输的研究。LKM38是一种很有前途的胆固醇模拟物，因为它既能维持胆固醇营养不良细胞的生长，又能适当地调节关键的胆固醇稳态途径。当LKM38以酯形式出现在脂蛋白颗粒中时，它最初定位于溶酶体，随后转运到质膜和内质网。LKM38与多种已建立的胆固醇结合蛋白结合。通过使用细胞生物学、生化运输测定和免疫荧光方法对一组重氮嘧啶炔探针的细胞行为进行详细表征，我们得出结论，LKM38可以作为研究胆固醇蛋白相互作用和运输的有力工具。
Cholesterol is an essential structural component of cellular membranes and precursor molecule for oxysterol, bile acid, and hormone synthesis. The study of intracellular cholesterol trafficking pathways has been limited in part due to a lack of suitable cholesterol analogues. Herein, we developed three novel diazirine alkyne cholesterol probes: LKM38, KK174, and KK175. We evaluated these probes as well as a previously described diazirine alkyne cholesterol analogue, trans-sterol, for their fidelity as cholesterol mimics and for study of cholesterol trafficking. LKM38 emerged as a promising cholesterol mimic because it both sustained the growth of cholesterol-auxotrophic cells and appropriately regulated key cholesterol homeostatic pathways. When presented as an ester in lipoprotein particles, LKM38 initially localized to the lysosome and subsequently trafficked to the plasma membrane and endoplasmic reticulum. LKM38 bound to diverse, established cholesterol binding proteins. Through a detailed characterization of the cellular behavior of a panel of diazirine alkyne probes using cell biological, biochemical trafficking assays and immunofluorescence approaches, we conclude that LKM38 can serve as a powerful tool for the study of cholesterol protein interactions and trafficking.