Departments of Medicine Washington University School of Medicine, St. Louis, MO 63110.
Developmental Biology Washington University School of Medicine, St. Louis, MO 63110.
J Lipid Res. 2019 Mar;60(3):707-716. doi: 10.1194/jlr.D091470. Epub 2019 Jan 7.
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, -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.
胆固醇是细胞膜的重要结构组成部分，也是氧化固醇、胆汁酸和激素合成的前体分子。由于缺乏合适的胆固醇类似物，细胞内胆固醇运输途径的研究在一定程度上受到限制。本文开发了三种新型叠氮-炔胆固醇探针：LKM38、KK174 和 KK175。我们评估了这些探针以及之前描述的叠氮-炔胆固醇类似物-甾醇作为胆固醇类似物的准确性，以及用于研究胆固醇运输的用途。LKM38 作为一种有前途的胆固醇类似物脱颖而出，因为它既能维持胆固醇营养缺陷型细胞的生长，又能适当调节关键的胆固醇动态平衡途径。当以脂蛋白颗粒中的酯形式呈现时，LKM38 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系列叠氮-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-蛋白质相互作用和运输的有力工具。
